Preparation and application of anti-GPRC5D / BCMA / CD3 trispecific antibody

Abstract

The invention discloses a tri-specific antigen binding protein, and relates to a tri-specific antibody specifically binding two tumor antigens GPRC5D and BCMA and a T cell surface antigen CD3, a pharmaceutical composition containing the tri-specific antibody, a preparation method and application.

Description

Preparation and use of anti-GPRC5D / BCMA / CD3 trispecific antibodies

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202310664900.5 and application date June 6, 2023, and the Chinese patent application with application number 202410713270.0 and application date June 4, 2024, and claims the priority of the Chinese patent application. The entire contents of the Chinese patent application are hereby introduced into this application as a reference. Technical Field

[0003] The present invention relates to a trispecific antigen-binding protein, and more specifically, to a trispecific antibody that specifically binds to two tumor antigens, GPRC5D and BCMA, and the T cell surface antigen CD3, as well as a pharmaceutical composition, preparation method, and use thereof. Background Art

[0004] T cell bispecific antibodies (BsAbs) have been used in tumor treatment. Such antibodies can form synapses between cytotoxic T lymphocytes and tumor cells, inducing tumor cell destruction. BsAbs generally involve dual targeting of tumor-associated antigens (TAAs) and T cell surface antigens (also known as T cell engagement antigens, TEAs). However, BsAb-based treatment strategies typically rely on the distribution of tumor-associated antigens on the tumor cells to be treated. This leads to selectivity of treatment for patient populations and limitations of treatment. In addition, studies have also shown that treatment modalities targeting a single TAA site may cause recurrence of the disease due to tumor escape mechanisms, thereby limiting the effectiveness of treatment.

[0005] It has been proposed to develop trispecific and / or tetraspecific antibodies to effectively recruit immune cells to the tumor site and improve the efficacy of treatment. To this end, in recent years, a variety of multivalent, multispecific antibody formats have been described. However, the requirements of different therapeutic products in terms of therapeutic functionality and therapeutic behavior are diverse, which determines that there is no "optimal format" that can be applied to most different desired molecular combinations. Therefore, when achieving multispecificity, it is necessary to consider a variety of factors, including, for example, the spatial distribution or size of different target antigens, and the expression density of tumor-associated antigens on the surface of tumor cells. In many cases, antibody formats that are effective for specific target antigen combinations must be identified by generating and comparing the functionality of different antibody formats.

[0006] B cell maturation antigen (BCMA, also known as CD269, 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) characteristic of TNFR family members in its extracellular domain (ECD), which forms a ligand-binding motif. BCMA ligands include B cell-activating factor (BAFF) and B cell proliferation-inducing ligand (APRIL). APRIL binds to BCMA with higher affinity, promoting tumor cell proliferation.

[0007] BCMA has been found to be overexpressed in multiple myeloma (MM) cells in preclinical models and human tumors. It upregulates canonical and non-canonical NF-κB signaling, promoting MM cell growth, survival, and adhesion, inducing osteoclast activation, angiogenesis, metastasis, and immunosuppression. BCMA expression has become an important biomarker for the diagnosis of MM. Furthermore, elevated levels of sBCMA in the serum of MM patients are positively correlated with the number of MM cells in the bone marrow, and changes in sBCMA concentration are closely associated with MM prognosis and treatment response. Multiple myeloma, also known as plasmacytoma or Culler's disease, is a difficult-to-treat B-cell malignancy characterized by abnormal proliferation of plasma cells. Given that BCMA is restricted to plasma cells and absent in naive and memory B cells, it has become a popular target for the treatment of B-cell malignancies, particularly multiple myeloma. There is a need in the art for the development of antibodies against BCMA, particularly multispecific antibodies.

[0008] Although prior art, such as WO2022174813A1, discloses a trispecific antibody that specifically binds CD3, BCMA, and GPRC5D, the BCMA antigen-binding region in this molecular structure is in the form of a scFv and is located at the C-terminus of the Fc. This presents the following challenges: low antibody purification yield and high aggregate formation; the scFv BCMA antigen-binding region has low affinity for the antigen and is poorly positioned, resulting in weak BCMA-mediated tumor cell killing; and the BCMA antigen-binding region contains some hotspot sites with poor druggability.

[0009] Therefore, a new anti-GPRC5D / BCMA / CD3 trispecific antibody is needed that can overcome the above shortcomings, has better tumor killing effect, and is easier to purify and develop into a drug.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides a trispecific antibody comprising a first antigen-binding region that specifically binds to BCMA, and second and third antigen-binding regions that specifically bind to other antigens.

[0012] In some embodiments, the second antigen binding region specifically binds CD3, and the third antigen binding region specifically binds GPRC5D.

[0013] In some embodiments, the trispecific antibodies of the present invention prevent light chain mispairing by applying charge mutations and disulfide bond mutations. In some embodiments, in the trispecific antibodies of the present invention, the first antigen-binding region that specifically binds to BCMA is a Fab.

[0014] In some embodiments, the second antigen binding region that specifically binds CD3 is a Fab.

[0015] In some embodiments, the antigen binding region that specifically binds to BCMA, such as Fab, is located at the N-terminus of the antigen binding region that specifically binds to CD3, such as Fab. In some embodiments, when the trispecific antibody forms an immune synapse for T cell killing, the T cell is closer to the tumor cell than the trispecific antibodies known in the prior art (such as the trispecific antibodies disclosed in WO2022174813A1, in particular the scFv that specifically binds to BCMA is located at the C-terminus of Fc), so the trispecific antibody of the present invention has stronger T cell killing. In some embodiments, in the structure of the trispecific antibody of the present invention, the first antigen binding region that specifically binds to BCMA can block the function of CD3, thereby avoiding the activation of nonspecific T cells throughout the body and the subsequent toxicity in the absence of tumor antigens.

[0016] In some embodiments, the trispecific antibodies of the present invention have a specific antigen-binding region that specifically binds to BCMA, thereby having improved drugability and / or reducing the generation of antibody immunogenicity.

[0017] Therefore, the trispecific antibodies of the present invention have one or more or all of the following advantages, in particular compared to the trispecific antibodies disclosed in WO2022174813A1:

[0018] i) having improved affinity for BCMA;

[0019] ii) have enhanced BCMA-mediated tumor killing;

[0020] iii) having a reduced aggregation tendency of the antibody molecules, and / or an improved purification yield;

[0021] iv) have stronger T cell killing ability;

[0022] v) having reduced nonspecific cytotoxicity, for example, being able to avoid the activation of nonspecific T cells in the body and the subsequent toxicity in the absence of tumor antigens;

[0023] vi) have reduced antibody immunogenicity; and / or

[0024] vii) having improved drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0026] Figure 1 shows exemplary structures of trispecific antibodies, wherein Figure 1A corresponds to the B3 and B3b formats, Figure 1B corresponds to the B5 structure, and Figure 1C corresponds to the F2 format.

[0027] FIG2A shows the cell-based affinity of exemplary antibodies to human BCMA (GS-CHO-hBCMA)-1.

[0028] FIG2B shows the cell-based affinity of exemplary antibodies to human BCMA (GS-CHO-hBCMA)-2.

[0029] FIG2C shows the cell-based affinity of exemplary antibodies to cynomolgus monkey BCMA (GS-CHO-cynoBCMA)-1.

[0030] FIG2D shows the cell-based affinity of exemplary antibodies to human GPRC5D (GS-CHO-hGPRC5D)-1.

[0031] FIG2E shows the cell-based affinity of exemplary antibodies to human GPRC5D (GS-CHO-hGPRC5D)-2.

[0032] FIG2F shows the cell-based affinity of exemplary antibodies to cynomolgus monkey GPRC5D (GS-CHO-cynoGPRC5D)-1.

[0033] Figure 2G shows the cell-based affinity of exemplary antibodies to human CD3 (Jurkat)-1.

[0034] Figure 2H shows the binding of exemplary antibodies to human CD3 (CD8 + T cells, CD4 + T cells) cell-based affinity.

[0035] FIG2I shows the cellular affinity of exemplary antibodies to 293T cells expressing mutant BCMA antigens.

[0036] FIG2J shows the cellular affinity of exemplary antibodies to GS CHO cells expressing mutant BCMA antigens.

[0037] FIG3A shows exemplary antibody-mediated killing of H929 ko GPRC5D cells by PBMC-1.

[0038] FIG3B shows exemplary antibody-mediated killing of H929 ko GPRC5D cells by PBMC-2.

[0039] FIG3C shows exemplary antibody-mediated killing of H929 ko GPRC5D cells by PBMC-3.

[0040] FIG3D shows exemplary antibody-mediated killing of H929 ko BCMA cells by PBMC-1.

[0041] FIG3E shows exemplary antibody-mediated killing of H929 ko BCMA cells by PBMC-2.

[0042] FIG3F shows exemplary antibody-mediated killing of H929 ko BCMA cells by PBMC-3.

[0043] FIG3G shows exemplary antibody-mediated killing of H929 cells by PBMC-1.

[0044] FIG3H shows an example of antibody-mediated killing of H929 cells by PBMC-2.

[0045] FIG. 3I shows exemplary antibody-mediated killing of H929 cells by PBMC-3.

[0046] FIG3J shows exemplary antibody-mediated killing of L363 cells by PBMC-1.

[0047] FIG3K shows exemplary antibody-mediated killing of L363 cells by PBMC-2.

[0048] FIG. 3L shows exemplary antibody-mediated killing of L363 cells by PBMC-3.

[0049] FIG3M shows exemplary antibody-mediated killing of L363 cells by PBMC-4.

[0050] FIG3N shows exemplary antibody-mediated killing of mixed cells by PBMC-1.

[0051] FIG. 3O shows exemplary antibody-mediated killing of mixed cells by PBMC-2.

[0052] FIG3P shows exemplary antibody-mediated killing of non-target Calu-6 cells by PBMCs.

[0053] FIG3Q shows the release of three cytokines accompanying the killing of H929 cells by PBMC mediated by an exemplary antibody (three antibody concentrations) −1 .

[0054] FIG3R shows the release of three cytokines accompanying the killing of H929 cells by PBMC mediated by an exemplary antibody (three antibody concentrations) −2 .

[0055] FIG3S shows exemplary antibody-mediated release of non-antigen-specific cytokines from PBMCs (no target cells, three antibody concentrations) −1 .

[0056] FIG3T shows exemplary antibody-mediated release of non-antigen-specific cytokines from PBMCs (no target cells, three antibody concentrations) −2.

[0057] FIG3U shows exemplary antibody-mediated killing of PBMCs against GS CHO cells overexpressing BCMA mutants.

[0058] FIG4A shows exemplary antibody-mediated activation of CD4 T cells in PBMCs in the presence of sBCMA.

[0059] FIG4B shows exemplary antibody-mediated activation of CD8 T cells in PBMCs in the presence of sBCMA.

[0060] FIG4C shows exemplary antibody-mediated killing of H929 cells by PBMCs in the presence of sBCMA.

[0061] Figure 5A shows the tumor inhibitory effect of the exemplary antibodies in the H929 ko GPRC5D tumor-bearing humanized mouse model. The dosage and statistical methods are indicated in the legend. The administration times were 7 days, 14 days, and 21 days after tumor cell inoculation, respectively.

[0062] Figure 5B shows the tumor inhibitory effect of the exemplary antibodies in the H929 ko BCMA tumor-bearing humanized mouse model. The dosage and statistical methods are indicated in the figure legends. The administration times were 7 days and 14 days after tumor cell inoculation, respectively.

[0063] Figure 5C shows the tumor inhibitory effect of the example antibody in the MM1S tumor-bearing humanized mouse model. The dosage is indicated in the figure legend, and the administration time is 7 days and 14 days after tumor cell inoculation, respectively.

[0064] Figure 5D shows the tumor inhibitory effect of the exemplary antibodies in the L363 tumor-bearing humanized mouse model, with administration times at 7 days and 18 days after tumor cell inoculation, respectively.

[0065] FIG6 shows the results of flow cytometry analysis of BCMA and GPRC5D expression in H929, H929 ko GPRC5D, and H929 ko BCMA cell lines.

[0066] FIG7 shows the difference in aggregation between the B3(24) molecule and the comparative molecule (F2(24)).

[0067] Detailed Description of the Invention

[0068] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0069] definition

[0070] It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0071] To interpret this specification, the following definitions will apply and, wherever appropriate, terms used in the singular may also include the plural, and vice versa.

[0072] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0073] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0074] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0075] The term "GPRC5D" refers to the tumor-associated antigen G protein-coupled receptor family C group 5 member D (e.g., human GPRC5D protein under accession number UniProt Q9NZD1). In one embodiment, the antigen-binding region of the antibody molecule of the present invention that binds to GPRC5D can have high affinity binding activity to cells expressing GPRC5D, and has an EC of, for example, 1-150 nM, such as 20-135 nM, for cells expressing human GPRC5D, as detected by flow cytometry. 50 In one embodiment, the antigen binding specificity is cross-reactive for human and monkey GPRC5D.

[0076] The term "BCMA" refers to a tumor-associated antigen B cell maturation antigen, also known as BCMA, TR17_human, TNFRSF17 (e.g., human BCMA protein under accession number UniProt Q02223). "BCMA" encompasses not only wild-type proteins but also mutants, such as natural variants or variants that have mutated in a disease (particularly variants with mutations that lead to drug resistance). In one embodiment, the antigen-binding region that binds to BCMA in the antibody molecule of the present invention can have a high affinity binding activity to BCMA, for example, a K of 0.1-10 nM, such as 0.125 nM, for human BCMA, as detected by biofilm optical interferometry. D In one embodiment, the antigen binding specificity is cross-reactive for human and monkey BCMA.

[0077] The term "CD3" refers to the T cell engagement antigen T cell surface glycoprotein CD3 (e.g., the human CD3 protein under accession number UniProt P07766). In one embodiment, the antigen binding region of the antibody molecule of the present invention that binds to CD3 can have high affinity binding activity for CD3 (e.g., CD3 E&G) as detected by biofilm optical interferometry, with a KD value for human CD3 E&G of, for example, 10-100 nM, such as 10-70 nM. In one embodiment, the antigen binding specificity has cross-reactivity to human and monkey CD3.

[0078] When "first", "second" and "third" are mentioned herein, it is only to distinguish the three domains or three chains or three antigen-binding regions, but does not indicate the positions of the three domains or three antigen-binding regions in any way.

[0079] When describing the structure of the antibody of the present invention, the term "N-terminus" refers to the last amino acid at the N-terminus, and the term "C-terminus" refers to the last amino acid at the C-terminus.

[0080] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0081] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay described herein.

[0082] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, or it can be a ligand or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled person will understand that any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the 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 occurrence and progression of a tumor) or a T cell adaptor.

[0083] As used herein, the term "target binding region" refers to the portion of a multispecific binding molecule, such as a trispecific binding molecule, that binds to a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the remainder of the multispecific antibody molecule and may bind or not bind to its target as a separate entity. The target binding region can also be a receptor or ligand, or a domain of a receptor that is capable of binding to a ligand. In the case of multispecific antibodies, the "target binding region" is also referred to as an "antigen binding region." In one embodiment, the antigen binding region for the multispecific antibody molecule of the present invention comprises a VH / VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), and the VH / VL pair can be contained in two separated polypeptide chains (e.g., in a Fab heavy chain and a Fab light chain, respectively). In one embodiment, the antigen binding region for the trispecific antibody molecule of the present invention can be a Fab.

[0084] The “IgG-like multispecific antibody” described herein refers to a multispecific antibody comprising an Fc dimer.

[0085] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, such as a trispecific binding molecule, i.e., the molecule comprises at least a first target binding region, a second target binding region, and a third target binding region, wherein the first target binding region binds one target and the second target binding region binds another target and the third target binding region binds a third target. Thus, a multispecific binding molecule according to the invention comprises specificities for at least two different targets. In some embodiments, where the binding molecule is an antibody, the target is an antigen. In some embodiments, the multispecific binding molecule of the invention is a multispecific antibody, such as a trispecific antibody.

[0086] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens. A multispecific antibody is an antibody having binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein are trispecific antibodies having binding specificity for a first antigen, a second antigen, and a third antigen. For example, the present invention provides trispecific antibodies for BCMA, GPRC5D, and CD3.

[0087] When referring to a "first antigen-binding region" in a multispecific antibody or a trispecific antibody, it 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, a 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 and 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 comprises 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 comprises 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.

[0088] When referring to "the antigen-binding region is derived from an antibody", it means that the binding domain constituting the antigen-binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, a fragment of the antigen-binding region that specifically binds to the antigen, such as Fab, is or is derived from a corresponding fragment of the antibody, such as Fab, or the heavy chain variable region and / or light chain variable region of the antigen-binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen-binding region are CDRs of the antibody. The term "derived from" means that the fragment in the antigen-binding region is substantially the same as the fragment of the antibody from which it is derived, but has a mutation at one or more sites, such as a substitution, deletion or addition. In a specific embodiment, the mutation is not in the CDR of the antibody. In a specific embodiment, the mutation is not in the variable region of the antibody.

[0089] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0090] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of the full-length antibody, but is 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 is derived) for antigen binding. Antigen-binding fragments can 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, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full-length antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but can also be produced by recombinant methods using a synthetic linker peptide to link the two domains as a single protein chain in which the VL region and VH region are paired to form a single-chain Fv (scFv).

[0091] The term "variable region" refers to the domain of the antibody heavy or light chain that is involved in binding the antibody to the antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions. In some cases, a single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0092] "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment composed 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, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen binding site. Herein, the Fab polypeptide chain comprising the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; accordingly, the Fab polypeptide chain comprising the light chain constant region CL is also referred to as the "Fab light chain."

[0093] "Complementarity determining region" or "CDR region" or "CDR" is a region in the variable region of an antibody that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the variable region of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable region of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0094] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not exist. Two Fc regions can achieve dimerization to form a dimeric Fc, and two different Fc heterodimerizations form heterodimeric Fc. In this article, the terms "Fc region", "Fc portion" and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin and heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, a human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231, to the carboxyl-terminus of the heavy chain.

[0095] In some embodiments, the multispecific antibodies of the present invention comprise an Fc region. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgG1 subclass.

[0096] In one embodiment, the human IgG1 Fc region polypeptide (including the hinge region) comprises the following amino acid sequence:

[0097] The human IgG4 Fc region polypeptide (including the hinge region) comprises the following amino acid sequence:

[0098] As used herein, "heterodimeric Fc or Fc heterodimer" refers to a scaffold comprising two different Fc regions or formed by dimerization of two different Fc regions, which can be connected at its N-terminus or C-terminus to an antigen-binding domain (e.g., an antibody heavy chain and / or light chain variable region or an antibody antigen-binding fragment that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) to form a multispecific antibody such as a bispecific antibody.

[0099] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 82). In one embodiment, internal disulfide bonds are removed by mutating cysteine ​​residues in CH1 to non-cysteine ​​residues, or new disulfide bonds are remodeled by mutating non-cysteine ​​residues to cysteine ​​residues.

[0100] The term "CH2 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 231 to EU position 340 (EU numbering system). In one embodiment, the CH2 domain comprises

[0101] Amino acid sequence of APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 83).

[0102] The term "CH3 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 341 to EU position 447. In one embodiment, the CH3 domain comprises the amino acid sequence of GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84).

[0103] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in a wild-type antibody heavy chain, for example, the IgG1 hinge region, such as the sequence from D221 to P230 according to EU numbering. Hinge regions of other IgG subclasses can be identified by aligning the hinge region cysteine ​​residues with the IgG1 subclass sequence. The hinge region is typically a dimer composed of two polypeptides with 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 comprises the amino acid sequence HTCPXCP (SEQ ID NO:19), where X is S or P. In one embodiment, the hinge region comprises the amino acid sequence CPXCP (SEQ ID NO:81), where X is S or P. In one embodiment, internal disulfide bonds in the hinge region are removed by mutating the cysteines in the hinge region to non-cysteine ​​residues.

[0104] As used herein, amino acid positions throughout the variable regions of 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) and are referred to herein as “Kabat numbers.”

[0105] As used herein, when referring to amino acid positions in antibody domains other than the variable region (e.g., constant region, e.g., Fc region), the positions are numbered according to the EU numbering system described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and are referred to herein as "EU numbering." When position numbers and / or amino acid residues are assigned to a specific antibody isotype, it is intended to apply to the corresponding positions and / or amino acid residues of any other antibody isotype, as known to those skilled in the art.

[0106] The term "linker" as used herein refers to any molecule that enables the direct connection of the different parts of a multispecific antibody. Examples of linkers for establishing covalent linkages between the different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to a sequence of amino acids, wherein the sequence connects the amino acid sequences of the various parts of the multispecific antibody together. Preferably, the peptide linker has a length that is sufficient to connect the two entities in a manner that allows them to maintain their conformation relative to each other so as not to interfere with the desired activity. The peptide linker may or may not primarily include the following amino acid residues: Gly, Ser, Ala or Thr. Useful linkers include glycine-serine polymers, including, for example (GS) n (SEQ ID NO: 112), (GSGGS) n (SEQ ID NO: 113), (GGGGS) n (SEQ ID NO: 114), (GGGS) n (SEQ ID NO: 115) and (GGGGS) n G (SEQ ID NO: 116), wherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0107] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0108] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An "isolated antibody-encoding nucleic acid" refers to one or more nucleic acid molecules that encode an antibody chain or fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

[0109] Calculation of sequence identity between sequences is performed as follows.

[0110] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0111] The term "amino acid substitution" or "amino acid mutation" refers to the replacement of at least one amino acid residue in a predetermined parent amino acid sequence with a different "substituted" amino acid residue. The replacement residue or residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from the group consisting of: 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). The definition of amino acid substitution herein also encompasses replacement with one or more non-naturally occurring amino acid residues. "Non-natural amino acid residues" refer to residues that are capable of covalently binding to adjacent amino acid residues in a polypeptide chain, in addition to those naturally occurring amino acid residues listed above. Examples of non-natural amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib and other amino acid residue analogs.

[0112] The term "conservative alteration" or "conservative modification" refers to an amino acid modification or change that does not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include conservative amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0113] Amino acid mutations are represented by (original amino acid, amino acid position, mutated amino acid). For example, when the mutation site is located in the V region, "Q39D" means that the glutamine amino acid at Kabat position 39 is replaced by aspartic acid (D); when the mutation site is located in the C region, "Q124C" means that the glutamine at EU position 124 is replaced by cysteine ​​(C). When referring to amino acid positions in the present invention, unless otherwise specified, it refers to the amino acid position numbered according to the IgG1 heavy chain or kappa light chain, that is, it covers the amino acid position numbered based on the IgG1 heavy chain or kappa light chain, as well as the amino acid position corresponding to the amino acid position on other heavy chains or light chains. It should be noted that when describing a mutation, the original amino acid at a specific position can be the described amino acid, or it can be another amino acid at the corresponding position.

[0114] "Knob-in-hole" mutations or "knob-in-knob" mutations are used herein to refer to the introduction of mutations into a first Fc polypeptide and a second Fc polypeptide, respectively, using the "knob-in-knob" technique to form a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and at the interface of the second Fc polypeptide. It is known in the art that the "knob-in-hole" technique can be used to modify the interface between different chains of an antibody molecule to promote the correct association of the chains of the antibody molecule. Typically, the technique involves introducing a "protrusion" at the interface of one chain and a corresponding "hole" at the interface of the other chain to be paired with it, so that the protrusion can be placed in the cavity. A preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired with it. The protrusion can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Another optional interface is the CL domain of the light chain and the CH1 domain of the heavy chain of the Fab fragment described above, which promotes correct heterodimerization between the two chains of the Fab fragment by establishing a protrusion-cavity interaction.

[0115] The term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, C1q binding and complement-dependent cytotoxicity (CDC), downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0116] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" is one of the main mechanisms by which certain cytotoxic effector cells (e.g., natural killer (NK) cells) mediate the killing of target cells and foreign host cells. In some embodiments, the antibodies of the present invention provide antibody-dependent cellular cytotoxicity of T lymphocytes and enhance the antibody-dependent cellular cytotoxicity of NK cells.

[0117] The term "antibody-dependent cellular phagocytosis (ADCP)" refers to a cellular response in which antibodies bound to target cells bind to FcγRIIIa on the surface of macrophages, inducing macrophage activation, leading to internalization of the target cell and its degradation by phagosome acidification. ADCP can also be mediated by FcγRIIa and FcγRI, but to a lesser extent.

[0118] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0119] The term "pharmaceutical excipient" refers to a pharmaceutically acceptable carrier, diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), vehicle, buffer, stabilizer, etc., which is administered together with the active substance.

[0120] A "conjugate" is an antibody conjugated to one or more other substances, including but not limited to a therapeutic agent or a label.

[0121] The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0122] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The term "tumor" encompasses solid tumors and liquid tumors. In some embodiments, the tumor is cancer. The terms "cancer" and "cancerous" refer to the physiological condition in mammals in which cell growth is unregulated. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0123] As used herein, "treat" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay disease progression or to slow the progression of a disease.

[0124] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly before the development of symptoms of cancer in a subject at risk for cancer.

[0125] The term "effective amount" refers to an amount or dosage of an antibody or composition of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses.

[0126] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic result, at dosages and for periods of time necessary. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition thereof are outweighed by the therapeutically beneficial effects.

[0127] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0128] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit. The term "non-fixed combination" means that the active ingredients (e.g., (i) antibodies of the present invention and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in sequence as separate entities, wherein such administration provides two or more active agents at effective levels of prevention or treatment in the patient's body. In some embodiments, the antibodies of the present invention used in the drug combination are administered at a level no greater than when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0129] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0130] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0131] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and the progeny derived therefrom, without regard to the number of passages. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0132] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as an antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope labeling or fluorescent labeling) or can catalyze the chemical alteration of a detectable substrate compound or composition in the case of an enzymatic labeling. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled. The example of indirect labeling includes the detection of the first antibody using a fluorescently labeled second antibody and the end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0133] "Subject / patient sample" refers to a collection of cells, tissues, or body fluids obtained from a patient or subject. The source of the tissue or cell sample can be a solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen, and / or preserved organ or tissue sample; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

[0134] The term "detection" as used herein includes quantitative or qualitative detection. Exemplary detection methods include, but are not limited to, immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR). In some embodiments, biological samples include body fluids, cells, or tissues. In certain embodiments, biological samples are other liquid samples of blood, serum, or biological origin.

[0135] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. Any or all features discussed above and throughout this application can be combined in various embodiments of the present invention. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will become apparent from this specification and accompanying drawings and from the appended claims.

[0136] I. Multispecific Antibodies

[0137] One aspect of the present invention relates to a multispecific antibody, such as a trispecific antibody, comprising a first antigen-binding region that specifically binds to BCMA, and a second antigen-binding region and a third antigen-binding region that specifically bind to a second and a third antigen.

[0138] One aspect of the present invention relates to a trispecific 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.

[0139] In some embodiments, the first antigen-binding region is from an anti-BCMA antibody or an antigen-binding fragment thereof, for example, a Fab fragment of an anti-BCMA antibody. In some embodiments, the first antigen-binding region is from an anti-BCMA antibody or an antigen-binding fragment thereof described herein.

[0140] In some embodiments, the second antigen-binding region is from an anti-CD3 antibody or an antigen-binding fragment thereof, such as a Fab fragment of an anti-CD3 antibody. In some embodiments, the second antigen-binding region is from an anti-CD3 antibody or an antigen-binding fragment thereof described herein.

[0141] In some embodiments, the third antigen binding region is from an anti-GPRC5D antibody or an antigen binding fragment thereof, such as a Fab fragment of an anti-GPRC5D antibody. In some embodiments, the third antigen binding region is from an anti-GPRC5D antibody or an antigen binding fragment thereof described herein.

[0142] The first antigen-binding region of the trispecific antibodies suitable for use in the present invention may comprise or consist of a full-length anti-BCMA antibody or an antigen-binding fragment thereof of the present invention, as long as it can specifically bind to BCMA, including but not limited to, full-length antibodies, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH, or heavy-chain antibodies that specifically bind to BCMA. Preferably, the first antigen-binding region is a Fab that specifically binds to BCMA.

[0143] The second antigen-binding region of the trispecific antibodies suitable for use in the present invention may comprise or consist of an anti-CD3 full-length antibody or an antigen-binding fragment thereof, as long as it can specifically bind to CD3, including but not limited to, full-length antibodies, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH, or heavy-chain antibodies that specifically bind to CD3. Preferably, the second antigen-binding region is a Fab that specifically binds to CD3.

[0144] The third antigen-binding region of the trispecific antibody suitable for use in the present invention may comprise or consist of an anti-GPRC5D full-length antibody or an antigen-binding fragment thereof, as long as it can specifically bind to GPRC5D, including but not limited to, for example, a full-length antibody, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH, or heavy chain antibody that specifically binds to GPRC5D. Preferably, the third antigen-binding region is a Fab that specifically binds to GPRC5D.

[0145] In some embodiments, the trispecific antibodies of the invention are IgG-like trispecific antibodies.In some embodiments, the trispecific antibodies of the invention comprise an Fc dimer, for example, comprise an Fc heterodimer.

[0146] In some embodiments, the trispecific antibodies of the present invention comprise a Fab that specifically binds BCMA as the first antigen-binding region.

[0147] In some embodiments, the three antigen-binding regions of the trispecific antibodies of the invention are all Fab fragments.

[0148] Antigen binding regions of trispecific antibodies suitable for use in the present invention

[0149] In some embodiments, the antigen binding region of the trispecific antibodies of the invention is a Fab fragment.

[0150] The Fab fragment suitable for the antigen-binding region of a trispecific antibody is composed of two polypeptide chains comprising the VH, CH1, VL, and CL domains of the antibody, wherein the VH is paired with the VL and the CH1 is paired with the CL to form an antigen-binding region. In some embodiments, in the Fab, one chain comprises VH and CH1 from N-terminus to C-terminus (i.e., VH-CH1), and the other chain comprises VL and CL from N-terminus to C-terminus (i.e., VL-CL).

[0151] In some embodiments, in the trispecific antibody, the Fab may be fused to the N-terminus of the antibody's Fc domain via the C-terminus of the chain comprising VH; or fused to the C-terminus of the antibody's Fc domain via the N-terminus of the chain comprising VH, wherein the Fc domain may or may not contain a hinge region. In some embodiments, the Fab comprises a VH-CH1 chain and a VL-CL chain, and is fused to the N-terminus of the antibody's Fc domain via the C-terminus of CH1 of the VH-CH1 chain, or fused to the C-terminus of the antibody's Fc domain via the N-terminus of the VH linked to the VH-CH1 chain.

[0152] In some embodiments, the fusion is direct, or fused through a linker. In some embodiments, the linker is (GGGGS)n, where n=1, 2, 3, or 4.

[0153] In some embodiments, the antigen binding region, eg, Fab, of the trispecific antibodies of the invention is mutated to reduce mispairing.

[0154] In some embodiments, one antigen binding region, eg, a Fab fragment, of a trispecific antibody of the invention comprises a disulfide bond remodeling mutation.

[0155] In some embodiments, one antigen-binding region, such as a Fab fragment, of the trispecific antibody of the present invention comprises a charge mutation, wherein the charge mutation is to mutate the two amino acids in the amino acid pair to amino acids with opposite charges.

[0156] In some embodiments, one antigen-binding region, eg, a Fab fragment, of a trispecific antibody of the invention comprises a charge mutation and a disulfide bond remodeling mutation.

[0157] In some embodiments, in the two antigen-binding regions of the trispecific antibodies of the present invention, such as two Fab fragments, one antigen-binding region comprises a charge mutation, and the other antigen-binding region comprises a charge mutation and a disulfide bond remodeling mutation. In some embodiments, the two antigen-binding regions each comprise a charge mutation at a corresponding position, and preferably, the charge mutations comprised in the two antigen-binding regions are opposite.

[0158] Disulfide bond remodeling mutations:

[0159] In some embodiments, the disulfide bond remodeling mutation is in the CH1-CL of the antigen binding region, (e.g., IgG1, IgG2, IgG3, or IgG4) heavy chain CH1 has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 126 (EU numbering), and the light chain CL has a substitution of a non-cysteine ​​amino acid to a cysteine ​​amino acid at position 124 (EU numbering). In some embodiments, the disulfide bond remodeling mutation is to include 126C (e.g., F126C) in the CH1 in the antigen binding region and 124C (e.g., Q124C) (EU numbering) in the CL. In some embodiments, the disulfide bond remodeling mutation also includes replacing the natural cysteine ​​with a non-cysteine. In some embodiments, the CH1 in the antigen binding region has a substitution of a cysteine ​​amino acid by a non-cysteine ​​amino acid at position 220 (EU numbering). In some embodiments, the light chain CL in the antigen binding region has a substitution of a cysteine ​​amino acid to a non-cysteine ​​amino acid at position 214 (EU numbering). In some embodiments, the disulfide bond remodeling mutation comprises C220S / A / V in CH1 in the antigen binding region (IgG1 subtype, or C131S / A / V at the corresponding position in IgG2, IgG3, or IgG4), and C214S / A / V in CL (EU numbering). In some embodiments, the Fab comprises CH1 and CL, the CH1 comprises an F126C mutation, and the CL comprises a Q124C mutation (EU numbering). In some embodiments, the antigen binding region comprises CH1 and CL, the CH1 comprises an F126C and C220S mutation (or corresponding C131S), and the CL comprises Q124C and C214S mutations (EU numbering).

[0160] In some embodiments, the Fab fragment comprising a disulfide bond remodeling mutation comprises a CH1, wherein the CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 85 and comprises F126C. In some embodiments, the CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 86 and comprises F126C and C220S. In some embodiments, the CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 86.

[0161] In some embodiments, the Fab fragment comprising a disulfide bond remodeling mutation comprises a CL, wherein the CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 97 and comprises Q124C. In some embodiments, the CL comprises or consists of the amino acid sequence of SEQ ID NO: 97. In some embodiments, the CL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 98 and comprises Q124C and C124S. In some embodiments, the CL comprises or consists of the amino acid sequence of SEQ ID NO: 98.

[0162] Charge mutation:

[0163] In some embodiments, the charge mutation is a mutation of an amino acid pair on the contact surface of the heavy chain and light chain of at least one antigen binding region, wherein the two amino acids in the amino acid pair are mutated to opposite charges. In some embodiments, the charge mutation comprises mutating two amino acids to amino acids with opposite charges at the amino acid pair at the following positions: position 39 of the heavy chain variable region and position 38 of the light chain variable region (Kabat numbering). In some embodiments, the charge mutation comprises mutating two amino acids to amino acids with opposite charges at the amino acid pair at the following positions: Q39 of the heavy chain variable region and Q38 of the light chain variable region (Kabat numbering).

[0164] 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.

[0165] As used herein, "two antigen-binding regions contain opposite charge mutations" means that the two antigen-binding regions contain charge mutations at one or more identical amino acid pairs in the first and second antigen-binding regions, but the charge mutations at the amino acid pairs in one antigen-binding region are different from the charge mutations at the amino acid pairs in the other antigen-binding region, for example, at the same positions, 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 mutated to a positively charged amino acid, and the light chain amino acid at position X2 is mutated to a negatively charged amino acid, while in the second antigen-binding region, the heavy chain amino acid at position X1 is mutated to a negatively charged amino acid, and the light chain amino acid at position X2 is mutated to a positively charged amino acid, wherein the positively charged amino acid in the first antigen-binding region and the positively charged amino acid in the second antigen-binding region can be the same or different, for example, the same; and the negatively charged amino acid in the first antigen-binding region and the negatively charged amino acid in the second antigen-binding region can be the same or different, for example, the same.

[0166] 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.

[0167] In one embodiment, the trispecific antibody of the invention comprises two antigen binding regions, wherein

[0168] 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. Meanwhile, in the other 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.

[0169] Antigen binding region

[0170] In some embodiments, the trispecific antibody of the present invention comprises an antigen binding region that specifically binds BCMA, an antigen binding region that specifically binds CD3, and an antigen binding region that specifically binds GPRC5D.

[0171] Antigen binding region that specifically binds to BCMA

[0172] In some embodiments, the antigen binding region that specifically binds to BCMA comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds to CD3 comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen binding region that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0173] In some aspects, the antigen binding region that specifically binds to BCMA comprises a heavy chain variable region (VH). In some aspects, the antigen binding region that specifically binds to CD3 comprises a light chain variable region (VL). In some aspects, the antigen binding region that specifically binds to BCMA comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR and LCDR are determined by the Kabat scheme (Kabat numbering system described in Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0174] In some embodiments, the heavy chain variable region VH that specifically binds to the antigen binding region of BCMA

[0175] (i) comprises 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 amino acid sequence of SEQ ID NO: 56; or

[0176] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 56; or

[0177] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 56, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0178] In some embodiments, the light chain variable region VL that specifically binds to the antigen binding region of BCMA

[0179] (i) comprises 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 amino acid sequence of SEQ ID NO: 60; or

[0180] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 60; or

[0181] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 60, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0182] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, of the antigen binding region that specifically binds to BCMA are the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 56.

[0183] In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, of the antigen binding region that specifically binds to BCMA are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in any one of SEQ ID NO:60.

[0184] In some embodiments, in the antigen binding region of the present invention that specifically binds to BCMA,

[0185] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 59; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 61; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 62; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 63.

[0186] In some embodiments of the present invention, the antigen binding region that specifically binds to BCMA comprises VH and VL, wherein

[0187] The VH comprises, or consists of, the amino acid sequence of SEQ ID NO:56, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:60, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0188] In some embodiments of the present invention, the antigen-binding region that specifically binds to BCMA comprises the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO:56, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO:60.

[0189] In some embodiments of the present invention, the antigen binding region that specifically binds to BCMA comprises: HCDR1 as set forth in SEQ ID NO:57, HCDR2 as set forth in SEQ ID NO:58, HCDR3 as set forth in SEQ ID NO:59; LCDR1 as set forth in SEQ ID NO:61, LCDR2 as set forth in SEQ ID NO:62, and LCDR3 as set forth in SEQ ID NO:63.

[0190] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to BCMA comprises VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 56, and the VL comprises the amino acid sequence of SEQ ID NO: 60. In some specific embodiments of the present invention, the antigen-binding region that specifically binds to BCMA comprises VH and VL, wherein the VH consists of the amino acid sequence of SEQ ID NO: 56, and the VL consists of the amino acid sequence of SEQ ID NO: 60.

[0191] In one embodiment, the antigen-binding region that specifically binds to BCMA is an antigen-binding fragment of an anti-BCMA antibody, which is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), or linear antibody. Preferably, the antigen-binding region that specifically binds to BCMA is Fab.

[0192] In some embodiments, the first antigen-binding region contained in the trispecific 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 contained in the trispecific antibody of the present invention is derived from an anti-BCMA antibody.

[0193] In some embodiments, the BCMA-specific Fab fragment of the trispecific antibody suitable for use in the present invention comprises disulfide bond remodeling mutations, for example, it comprises F126C, or F126C and C220S in CH1, and it comprises Q124C, or Q124C and C214S in CL.

[0194] In some embodiments, the BCMA-specific binding Fab fragment of the trispecific antibody suitable for use in the present invention comprises a charge mutation, for example, it comprises Q39K in the heavy chain variable region and Q38D in the light chain variable region; or it comprises Q38K in the light chain variable region and Q39D in the heavy chain variable region.

[0195] The BCMA-specific binding Fab fragment of the trispecific antibody suitable for the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, Q39K in the heavy chain variable region and F126C in CH1, or F126C and C220S; and Q38D in the light chain variable region and Q124C in CL, or Q124C and C214S.

[0196] The BCMA-specific binding Fab fragment of the trispecific antibody suitable for the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, Q39D in the heavy chain variable region and F126C in CH1, or F126C and C220S; and Q38K in the light chain variable region and Q124C in CL, or Q124C and C214S.

[0197] In some embodiments, the Fab that specifically binds to BCMA suitable for the trispecific antibody of the present invention comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a heavy chain variable region, and the Fab light chain comprises a light chain variable region.

[0198] wherein the heavy chain variable region of the Fab heavy chain

[0199] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 76, or

[0200] (ii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 76, and comprises the Q39K mutation; and / or

[0201] The light chain variable region of the Fab light chain

[0202] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, or

[0203] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 75, and comprises the Q38D mutation.

[0204] Antigen binding region that specifically binds to GPRC5D

[0205] In some embodiments, the antigen binding region that specifically binds to GPRC5D comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds to CD3 comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen binding region that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0206] In some aspects, the antigen binding region that specifically binds to GPRC5D comprises a heavy chain variable region (VH). In some aspects, the antigen binding region that specifically binds to GPRC5D comprises a light chain variable region (VH). In some aspects, the antigen binding region that specifically binds to GPRC5D comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, HCDRs and LCDRs are determined by the Kabat scheme (Kabat numbering system described in Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0207] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to GPRC5D, such as an antibody that specifically binds to GPRC5D disclosed in PCT / CN2022 / 076832. In some embodiments, the antigen-binding region comprises one, two, three, four, five, or six CDRs of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region comprises one, two, and three heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region comprises one, two, and three light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region comprises both a heavy chain variable region and a light chain variable region of a known antibody that specifically binds to GPRC5D.

[0208] In some embodiments, the heavy chain variable region VH that specifically binds to the antigen binding region of GPRC5D

[0209] (i) comprises 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 amino acid sequence of SEQ ID NO: 40 or 48; or

[0210] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 40 or 48; or

[0211] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 40 or 48, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0212] In some embodiments, the light chain variable region VL specifically binds to the antigen binding region of GPRC5D

[0213] (i) comprises 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 amino acid sequence of SEQ ID NO: 44 or 52; or

[0214] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 44 or 52; or

[0215] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 44 or 52, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions.

[0216] In some embodiments, the three complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, of the antigen binding region that specifically binds to GPRC5D are the three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in any one of SEQ ID NOs: 40 or 48.

[0217] In some embodiments, the three complementary determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, of the antigen binding region that specifically binds to GPRC5D are the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in any one of SEQ ID NOs: 44 or 52.

[0218] In some embodiments, in the antigen binding region of the present invention that specifically binds to GPRC5D,

[0219] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:41; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:42; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:43; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:45; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:46; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; or

[0220] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:49; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:50; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:51; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:53; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:54; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:55.

[0221] In some embodiments of the present invention, the antigen binding region that specifically binds to GPRC5D comprises VH and VL, wherein

[0222] the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:44, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0223] The VH comprises the amino acid sequence of SEQ ID NO:48, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO:52, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0224] In some embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 40, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 44. In some embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 48, and the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 52.

[0225] In some embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises: HCDR1 as set forth in SEQ ID NO:41, HCDR2 as set forth in SEQ ID NO:42, HCDR3 as set forth in SEQ ID NO:43; LCDR1 as set forth in SEQ ID NO:45, LCDR2 as set forth in SEQ ID NO:46, and LCDR3 as set forth in SEQ ID NO:47. In some embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises: HCDR1 as set forth in SEQ ID NO:49, HCDR2 as set forth in SEQ ID NO:50, HCDR3 as set forth in SEQ ID NO:51; LCDR1 as set forth in SEQ ID NO:53, LCDR2 as set forth in SEQ ID NO:54, and LCDR3 as set forth in SEQ ID NO:55.

[0226] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 40, and the VL comprises the amino acid sequence shown in SEQ ID NO: 44. In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO: 40, and the VL consists of the amino acid sequence shown in SEQ ID NO: 44.

[0227] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 48, and the VL comprises the amino acid sequence shown in SEQ ID NO: 52. In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO: 48, and the VL consists of the amino acid sequence shown in SEQ ID NO: 52.

[0228] In one embodiment, the antigen-binding region that specifically binds to GPRC5D is an antigen-binding fragment of an anti-GPRC5D antibody, which is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody. Preferably, the antigen-binding region that specifically binds to GPRC5D is Fab.

[0229] In some embodiments, the third antigen-binding region comprised in the trispecific 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 comprised in the trispecific antibody of the present invention is derived from an anti-GPRC5D antibody.

[0230] In some embodiments, the Fab fragment that specifically binds to GPRC5D of the trispecific antibody suitable for use in the present invention comprises a disulfide bond remodeling mutation, for example, it comprises F126C, or F126C and C220S in CH1, and it comprises Q124C, or Q124C and C214S in CL.

[0231] In some embodiments, the Fab fragment that specifically binds to GPRC5D of the trispecific antibody suitable for use in the present invention comprises a charge mutation, for example, it comprises Q39K in the heavy chain variable region and Q38D in the light chain variable region; or it comprises Q38K in the light chain variable region and Q39D in the heavy chain variable region.

[0232] The Fab fragment that specifically binds to GPRC5D and is suitable for use in the trispecific antibody of the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, Q39K in the heavy chain variable region and F126C, or F126C and C220S, in CH1; and Q38D in the light chain variable region and Q124C, or Q124C and C214S in CL.

[0233] The Fab fragment that specifically binds to GPRC5D of the trispecific antibody suitable for the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, Q39D in the heavy chain variable region and F126C in CH1, or F126C and C220S; and Q38K in the light chain variable region and Q124C in CL, or Q124C and C214S.

[0234] In some embodiments, the Fab that specifically binds to GPRC5D suitable for the trispecific antibody of the present invention comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a heavy chain variable region, and the Fab light chain comprises a light chain variable region.

[0235] wherein the heavy chain variable region of the Fab heavy chain

[0236] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, or

[0237] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 80, and comprises the Q39K mutation; and / or

[0238] The light chain variable region of the Fab light chain

[0239] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 79, or

[0240] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 79, and comprises the Q38D mutation.

[0241] Antigen binding region that specifically binds to CD3

[0242] In some embodiments, the antigen binding region that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds to CD3 comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen binding region that specifically binds to CD3 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0243] In some aspects, the antigen binding region that specifically binds to CD3 comprises a heavy chain variable region (VH). In some aspects, the antigen binding region that specifically binds to CD3 comprises a light chain variable region (VH). In some aspects, the antigen binding region that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises 3 complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, HCDR and LCDR are determined by the Kabat scheme (Kabat numbering system described in Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0244] In some embodiments, the antigen binding region is derived from an antibody that specifically binds to CD3, such as the CD3 antibodies disclosed in WO2022068809, such as sp34.24 or sp34.87 therein.

[0245] In some embodiments, the antigen binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD3, such as the CD3 antibodies disclosed in WO2022068809, such as sp34.24 or sp34.87 therein.

[0246] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809, such as 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of sp34.24 or sp34.87 therein.

[0247] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809, such as 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of sp34.24 or sp34.87 therein.

[0248] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809, such as three heavy chain variable region CDRs and three light chain variable region CDRs of sp34.24 or sp34.87 therein.

[0249] In some embodiments, the antigen binding region comprises a known antibody that specifically binds to CD3, such as the CD3 antibodies disclosed in WO2022068809, such as the heavy chain variable region and light chain variable region of sp34.24 or sp34.87 therein, and the mutations described herein.

[0250] In some embodiments, the heavy chain variable region VH that specifically binds to the antigen binding region of CD3

[0251] (i) comprises 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 amino acid sequence of SEQ ID NO: 64 or 72; or

[0252] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 64 or 72; or

[0253] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 64 or 72, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions.

[0254] In some embodiments, the light chain variable region VL specifically binds to the antigen binding region of CD3

[0255] (i) comprises 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 amino acid sequence of SEQ ID NO: 68; or

[0256] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 68; or

[0257] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 68, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0258] In some embodiments, the three complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, of the antigen binding region that specifically binds to CD3 are the three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in any one of SEQ ID NOs: 64 or 72.

[0259] In some embodiments, the three complementary determining regions (LCDRs) from the light chain variable region of the antigen binding region that specifically binds to CD3, LCDR1, LCDR2 and LCDR3, are the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in any one of SEQ ID NO:68.

[0260] In some embodiments, in the antigen binding region of the present invention that specifically binds to CD3,

[0261] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 65; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 66; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 67 or 73; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 70; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 71.

[0262] In some embodiments of the present invention, the antigen binding region that specifically binds to CD3 comprises VH and VL, wherein

[0263] The VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 64 or 72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0264] In some embodiments of the present invention, the antigen binding region that specifically binds to CD3 comprises the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 64 or 72, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 68.

[0265] In some specific embodiments of the present invention, the antigen binding region that specifically binds to CD3 comprises: HCDR1 as shown in SEQ ID NO:65, HCDR2 as shown in SEQ ID NO:66, HCDR3 as shown in SEQ ID NO:67 or 73; LCDR1 as shown in SEQ ID NO:69, LCDR2 as shown in SEQ ID NO:70 and LCDR3 as shown in SEQ ID NO:71.

[0266] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 comprises VH and VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 64 or 72, and the VL comprises the amino acid sequence shown in SEQ ID NO: 68. In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO: 64 or 72, and the VL consists of the amino acid sequence shown in SEQ ID NO: 68.

[0267] In one embodiment, the antigen-binding region that specifically binds to CD3 is an antigen-binding fragment of an anti-CD3 antibody, which is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), or linear antibody. Preferably, the antigen-binding region that specifically binds to CD3 is Fab.

[0268] In some embodiments, the second antigen-binding region contained in the trispecific 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 contained in the trispecific antibody of the present invention is derived from an anti-CD3 antibody.

[0269] In some embodiments, the CD3-specific Fab fragment of the trispecific antibody suitable for use in the present invention comprises a disulfide bond remodeling mutation, for example, it comprises F126C, or F126C and C220S in CH1, and it comprises Q124C, or Q124C and C214S in CL.

[0270] In some embodiments, the CD3-specific Fab fragment of the trispecific antibody suitable for use in the present invention comprises a charge mutation, for example, it comprises Q39K in the heavy chain variable region and Q38D in the light chain variable region; or it comprises Q38K in the light chain variable region and Q39D in the heavy chain variable region.

[0271] The CD3-specific Fab fragment suitable for the trispecific antibody of the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, Q39K in the heavy chain variable region and F126C in CH1, or F126C and C220S; and Q38D in the light chain variable region and Q124C in CL, or Q124C and C214S.

[0272] The CD3-specific Fab fragment suitable for the trispecific antibody of the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, Q39D in the heavy chain variable region and F126C in CH1, or F126C and C220S; and Q38K in the light chain variable region and Q124C in CL, or Q124C and C214S.

[0273] In some embodiments, a Fab specifically binding to CD3 suitable for use in the trispecific antibody of the present invention comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises a heavy chain variable region, and the Fab light chain comprises a light chain variable region.

[0274] wherein the heavy chain variable region of the Fab heavy chain

[0275] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 77 or 78, or

[0276] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 77 or 78, and comprises the Q39D mutation; and / or

[0277] The light chain variable region of the Fab light chain

[0278] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 74, or

[0279] (ii) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%,

[0280] An amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical to a polypeptide having a p-terminal domain and comprising the Q38K mutation.

[0281] Fc dimers suitable for the trispecific antibodies of the present invention

[0282] In one embodiment, the two Fc regions in the trispecific antibody of the present invention dimerize to form a dimeric Fc. Preferably, the two Fc regions heterodimerize to form a heterodimeric Fc.

[0283] In some embodiments, the first and second Fc regions are different and are capable of dimerizing to form a heterodimeric Fc scaffold.

[0284] In some embodiments, the Fc region encompasses native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520). In some embodiments, the Fc region is human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of an amino acid sequence of SEQ ID NO: 87 or 88 or an amino acid sequence having at least 90% identity thereto, for example 95%, 96%, 97%, 99% or higher identity thereto.

[0285] In some embodiments, the Fc region of the present invention comprises antibody CH2 and CH3. In some embodiments, the antibody Fc region may also have an IgG hinge region or a portion of an IgG hinge region at the N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region. The hinge region may contain a mutation.

[0286] As will be appreciated by those skilled in the art, to promote the formation of heterodimers of the multispecific antibodies of the present invention, the Fc regions comprised by the multispecific antibodies of the present invention may include mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions.

[0287] Methods for promoting heterodimerization of the Fc region are known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner so that each CH3 region (or a heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the other CH3 region of the complementary engineering transformation (such that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). Preferably, based on the Knob-in-Hole technology, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region.

[0288] In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is substituted with a tryptophan residue (T366W) (knob mutation); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is substituted with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is substituted with a serine residue (T366S), and / or the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the EU index).

[0289] In a further embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is substituted with a tryptophan residue (T366W) and the serine residue at position 354 is substituted with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is substituted with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is substituted with a cysteine ​​residue); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is substituted with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the EU index), optionally the tyrosine residue at position 349 is substituted with a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0290] In a specific embodiment, one Fc region comprises the amino acid substitution T366W and the other Fc region comprises the amino acid substitutions T366S, L368A, and Y407V (numbering is according to the EU index).

[0291] In a specific embodiment, one Fc region comprises amino acid substitutions S354C and T366W, and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering is according to the EU index).

[0292] Thus, in a specific embodiment, the trispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein 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.

[0293] Thus, in a specific embodiment, the trispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 91, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 92.

[0294] Thus, in a specific embodiment, the trispecific antibody of the invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 92 and comprises the mutation T366W, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 91, comprises the mutations T366S, L368A, and Y407V.

[0295] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0296] The Fc region of the binding molecules of the invention, such as antibodies, can also be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0297] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function; or performing Fc region modification.

[0298] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector function, eg, comprises a mutation that achieves the above function.

[0299] As will be appreciated by those skilled in the art, depending on the intended use of the binding molecules of the present invention, such as antibody molecules, the binding molecules of the present invention, such as antibody molecules, may also include modifications in the Fc domain that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces 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 another preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn.

[0300] In some embodiments, the Fc region comprising a mutation that reduces binding to an Fcγ receptor comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 89 or 90, or an amino acid sequence that is at least 90% identical thereto, e.g., 95%, 96%, 97%, 99% or more identical thereto.

[0301] In some embodiments, the Fc region comprises an amino acid sequence that is at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical to the amino acid sequence shown in SEQ ID NO: 89 or 90 and comprises an L234A / L235A mutation.

[0302] In a preferred embodiment, the heterodimeric Fc of the trispecific antibody suitable for use in the present invention comprises two Fc regions, wherein

[0303] One Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 93, and the other Fc region comprises

[0304] The amino acid sequence shown in ID NO: 94 or consisting thereof; or

[0305] One Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 94 and comprises the mutations L234A / L235A and T366W, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 93 and comprises the mutations L234A / L235A, T366S, L368A, and Y407V.

[0306] Trispecific antibody structures and exemplary trispecific antibodies of the present invention

[0307] In some embodiments, the present invention provides a trispecific antibody comprising a first Fab that specifically binds to BCMA, a Fab that specifically binds to CD3, and a Fab that specifically binds to GPRC5D, and an Fc heterodimer.

[0308] In some embodiments, the present invention provides a trispecific antibody comprising a first Fab that specifically binds to BCMA, a Fab that specifically binds to CD3, and a Fab that specifically binds to GPRC5D, and an Fc heterodimer.

[0309] In some preferred embodiments, the trispecific 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, and a third Fab that specifically binds to a third antigen, and an Fc heterodimer, wherein the first antigen is selected from one of BCMA, CD3, and GPRC5D, the second antigen is selected from one of BCMA, CD3, and GPRC5D, and the third antigen is selected from one of BCMA, CD3, and GPRC5D, and the first antigen, the second antigen, and the third antigen are different, wherein

[0310] (1) a first Fab that specifically binds to a 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 of an Fc heterodimer (the first Fc region, e.g., an Fc region comprising a knob mutation or an Fc region comprising a hole mutation);

[0311] A second Fab that specifically binds to a second antigen is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of another Fc region (a second Fc region, such as an Fc region comprising a hole mutation or an Fc region comprising a knob) of the Fc heterodimer, and a third Fab that specifically binds to a third antigen is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of the VH of the Fab heavy chain of the second Fab; or

[0312] A third Fab that specifically binds to a 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 of the Fc heterodimer (the first Fc region, e.g., the Fc region comprising a knob mutation or the Fc region comprising a hole mutation);

[0313] 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 (the second Fc region, e.g., an Fc region comprising a hole mutation or an Fc region comprising a knob) of the Fc heterodimer, and 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 N-terminus of the VH of the Fab heavy chain of the second Fab;

[0314] For example, the structure shown in FIG1A ;

[0315] (2) a first Fab that specifically binds to a 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 of the Fc heterodimer (the first Fc region, e.g., an Fc region comprising a knob mutation or an Fc region comprising a hole);

[0316] A second Fab that specifically binds to a second antigen is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of another Fc region (a second Fc region, e.g., an Fc region comprising a hole mutation or an Fc region comprising a knob) of the Fc heterodimer, and a third Fab that specifically binds to a third antigen is fused at the N-terminus of the VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab; or

[0317] A third Fab that specifically binds to a 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 of the Fc heterodimer (the first Fc region, e.g., an Fc region comprising a knob mutation or an Fc region comprising a hole);

[0318] 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 another Fc region (the second Fc region, e.g., an Fc region comprising a hole mutation or an Fc region comprising a knob) of the Fc heterodimer, and the first Fab that specifically binds to the first antigen is fused at the N-terminus of the VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab;

[0319] For example, the structure shown in FIG. 1B .

[0320] In some embodiments, the second antigen is CD3. In some embodiments, the first antigen and the third antigen are GPRC5D or BCMA, respectively, or the third antigen and the first antigen are GPRC5D or BCMA, respectively.

[0321] 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 or the third antigen is BCMA.

[0322] In some embodiments, the fusion comprises direct fusion or fusion through a linker. In some embodiments, the linker is (GGGGS)n, wherein n=1, 2, 3, or 4.

[0323] In some embodiments, the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation. In some embodiments, the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation. In some embodiments, one or both of the first and second Fc regions comprise L234A and L235A mutations. In some embodiments, the first Fc region comprises a knob mutation and L234A and L235A mutations, and the second Fc region comprises a hole mutation and L234A and L235A mutations. In some embodiments, the first Fc region comprises a hole mutation and L234A and L235A mutations, and the second Fc region comprises a knob mutation and L234A and L235A mutations.

[0324] In some embodiments, wherein the second Fab comprises a charge mutation. In some embodiments, wherein one or both of the first Fab or the third Fab comprises a charge mutation and a disulfide bond remodeling mutation.

[0325] In some embodiments, wherein the second Fab comprises a charge mutation, and one of the first or third Fab comprises a charge mutation and a disulfide bond remodeling mutation. In some embodiments, the two Fabs comprising charge mutations respectively comprise opposite charge mutations.

[0326] In some embodiments, the second Fab comprises a charge mutation comprising a Q38K mutation in the Fab light chain and a Q39D mutation in the Fab heavy chain. In some embodiments, the first or third Fab comprises a charge mutation and a disulfide bond remodeling mutation comprising a Q39K and F126C mutation in the Fab heavy chain and a Q38D and Q124C mutation in the Fab light chain. In some embodiments, the first or third Fab comprises a charge mutation and a disulfide bond remodeling mutation comprising a Q39K, F126C, and C220S mutation in the Fab heavy chain and a Q38D, Q124C, and C214S mutation in the Fab light chain.

[0327] In some embodiments, the second Fab comprises a charge mutation comprising a Q38D mutation in the Fab light chain and a Q39K mutation in the Fab heavy chain. In some embodiments, the first or third Fab comprises a charge mutation and a disulfide bond remodeling mutation comprising a Q39D and F126C mutation in the Fab heavy chain and a Q38K and Q124C mutation in the Fab light chain. In some embodiments, the first or third Fab comprises a charge mutation and a disulfide bond remodeling mutation comprising a Q39D, F126C, and C220S mutation in the Fab heavy chain and a Q38K, Q124C, and C214S mutation in the Fab light chain.

[0328] In some embodiments, the first or third Fab comprising a charge mutation and a disulfide bond remodeling mutation is fused to the second Fab, or is fused to the second Fab via the Fc region.

[0329] In some specific embodiments, the trispecific antibody of the present invention is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which are composed of the following peptide chains:

[0330] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0331] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0332] A peptide chain 3# that contains a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0333] A peptide chain 4# comprising a Fab light chain that specifically binds to BCMA, and

[0334] A peptide chain 5# that contains a Fab light chain that specifically binds to CD3,

[0335] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired to form the third 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 first and second Fab, as shown in Figure 1A.

[0336] In some specific embodiments, peptide chain 1# comprises the amino acid sequence shown in SEQ ID NO:5, or comprises 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:5, or consists of the amino acid sequence;

[0337] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:4, or comprises 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 of SEQ ID NO:4;

[0338] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO: 3 or 6, or comprises 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 of SEQ ID NO: 3 or 6, or consists of the amino acid sequence;

[0339] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises 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: 2, or consists of the amino acid sequence; and / or

[0340] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0341] In some specific embodiments, peptide chain 1# comprises the amino acid sequence of SEQ ID NO: 15, or comprises 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 of SEQ ID NO: 15, or consists of the amino acid sequence;

[0342] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:14, or comprises 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 of SEQ ID NO:14;

[0343] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO: 3 or 6, or comprises 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 of SEQ ID NO: 3 or 6, or consists of the amino acid sequence;

[0344] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises 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: 2, or consists of the amino acid sequence; and / or

[0345] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0346] In some specific embodiments, the trispecific antibody of the present invention is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which are composed of the following peptide chains:

[0347] A peptide chain 1# that contains a Fab heavy chain and an Fc domain that specifically binds to BCMA.

[0348] A peptide chain 2# containing a Fab light chain that specifically binds to BCMA,

[0349] A peptide chain 3# that contains a Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0350] A peptide chain 4# comprising a Fab light chain that specifically binds to GPRC5D, and

[0351] A peptide chain 5# containing a Fab light chain that specifically binds to CD3,

[0352] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired 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 and second Fab, as shown in Figure 1A.

[0353] In some specific embodiments, peptide chain 1# comprises the amino acid sequence of SEQ ID NO: 11, or comprises 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 of SEQ ID NO: 11, or consists of the amino acid sequence;

[0354] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:10, or comprises 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 of SEQ ID NO:10;

[0355] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO:9, or comprises 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 of SEQ ID NO:9;

[0356] Peptide chain 4# comprises the amino acid sequence of SEQ ID NO:8, or comprises 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 of SEQ ID NO:8; and

[0357] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0358] In some specific embodiments, the trispecific antibody of the present invention is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which are composed of the following peptide chains:

[0359] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0360] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0361] A peptide chain 3# that sequentially contains a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain that specifically binds to the antigen binding region of BCMA.

[0362] A peptide chain 4# comprising a Fab light chain that specifically binds to CD3, and

[0363] A peptide chain 5# of a Fab light chain containing an antigen-binding region that specifically binds to BCMA,

[0364] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain 2# are paired to form the third 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 second and first Fab, as shown in Figure 1B.

[0365] In some specific embodiments, peptide chain 1# comprises the amino acid sequence shown in SEQ ID NO:5, or comprises 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:5, or consists of the amino acid sequence;

[0366] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:4, or comprises 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 of SEQ ID NO:4;

[0367] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO:7, or comprises 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 of SEQ ID NO:7;

[0368] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises 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: 1, or consists of the amino acid sequence; and / or

[0369] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:2, or comprises 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:2, or consists of the amino acid sequence.

[0370] II. Multispecific Antibody Production and Purification

[0371] In one embodiment, the present invention provides a method for preparing an antibody molecule of the present invention, or a fragment thereof, or a chain thereof, wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding an antibody molecule of the present invention, or a fragment thereof, or a chain thereof, and optionally isolating the antibody, or fragment thereof, or a chain thereof. In a certain embodiment, the method further comprises recovering the antibody molecule of the present invention, or a fragment thereof, or a chain thereof from the host cell.

[0372] 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 a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising 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. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0373] The antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and such factors, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0374] III immunoconjugates

[0375] In some embodiments, the present invention provides immunoconjugates comprising any of the antibodies provided herein and other substances, such as therapeutic agents or labels. In some embodiments, the therapeutic agent may be a therapeutic agent suitable for forming an immunoconjugate with an antibody. In some embodiments, the therapeutic agent is selected from any of the following categories (i)-(iii): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs with tumor suppressive effects. In some embodiments, the therapeutic agent is a chemotherapeutic agent.

[0376] In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC).

[0377] IV Nucleic Acids and Host Cells

[0378] The present invention provides nucleic acids encoding any chain, monomer, domain, or antigen-binding region of a multispecific antibody, such as a trispecific antibody, of the present invention. Polynucleotide sequences encoding each chain can be generated using methods well known in the art. In addition, the polynucleotides and nucleic acids of the present invention may include a segment encoding a secretory signal peptide, which can be operably linked to an antibody or protein encoding a multispecific antibody, such as a trispecific antibody, of the present invention, thereby directing secretory expression of the multispecific antibody, such as a trispecific antibody, and each chain thereof.

[0379] The present invention also provides vectors comprising the nucleic acids of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is a pCDNA vector, such as the pCDNA3.1 expression vector.

[0380] The present invention also provides host cells containing the nucleic acid or the vector. Host cells suitable for replicating and supporting the expression of the multispecific antibodies, such as trispecific antibodies, of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient quantities of multispecific antibodies, such as trispecific antibodies, for clinical use.

[0381] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells, such as Expi293 cells or HEK293 cells). Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (as described, for example, in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC5 cells, and FS4 cells. Other useful 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, NSO, P3X63 and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphocyte (e.g., Y0, NSO, Sp20 cell).

[0382] V. Pharmaceutical Compositions, Pharmaceutical Combinations, and Kits

[0383] In one aspect, the invention provides a composition or a medicament or a formulation, e.g., a pharmaceutical composition, comprising a trispecific antibody molecule of the invention and, optionally, a pharmaceutical excipient. In some embodiments, the pharmaceutical excipient is a pharmaceutically acceptable carrier.

[0384] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Common preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral injection or infusion. As used herein, the phrases "parenteral administration" and "parenteral administration" mean modes of administration other than enteral administration and topical administration, typically administered by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intradermal, peritoneal, transtracheal, subcutaneous injection and infusion.

[0385] In a preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

[0386] In some embodiments, the antibody molecules of the invention are the sole active ingredient in a pharmaceutical composition. In other embodiments, a pharmaceutical composition may comprise an antibody molecule described herein and one or more other therapeutic agents.

[0387] The pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody molecule of the present invention.

[0388] Kits comprising the antibody molecules described herein are also within the scope of the present invention. The kits may comprise one or more other elements, such as instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0389] In another aspect, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the antibody molecule described herein and one or more other therapeutic agents.

[0390] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0391] - a first container containing a pharmaceutical composition comprising the trispecific antibody of the invention;

[0392] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0393] The other therapeutic agent suitable for use in the pharmaceutical compositions and drug combinations of the present invention can be a therapeutic agent selected from any of the following categories (i)-(iii): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor-suppressing effects. In some embodiments, the other therapeutic agent is a chemotherapeutic agent.

[0394] VI. Uses and Methods of the Molecules of the Invention

[0395] In one aspect, the present invention provides in vivo and in vitro uses and methods of using the antibody molecules of the present invention.

[0396] In some embodiments, the uses and methods of the present invention involve the use of the antibody molecules of the present invention in vivo and / or in vitro for:

[0397] - Binds to GPRC5D antigen with high affinity, including GPRC5D antigen expressed on the surface of cells;

[0398] - targeting T cells (e.g., CD4+ and / or CD8+ T cells) to cells expressing GPRC5D on their surface, particularly GPRC5D-positive tumor cells;

[0399] - Targeting T cells (e.g., CD4+ and / or CD8+ T cells) to cells expressing BCMA on their surface, particularly BCMA-positive tumor cells;

[0400] -Activate CD3 downstream signaling pathways in T cells;

[0401] -Mediate T cell killing of GPRC5D-positive tumor cells and / or BCMA-positive tumor cells;

[0402] - Induce T cells to release cytokines such as TNF-α, IFN-γ, and IL-2;

[0403] - Inhibit or kill GPRC5D-positive and / or BCMA-positive tumor cells, or

[0404] - treating GPRC5D-positive and / or BCMA-positive tumors, such as multiple myeloma, for example, treating a BCMA-positive patient population, a GPRC5D-positive patient population, or a GPRC5D-positive patient population that has lost BCMA after binding of an anti-BCMA molecule;

[0405] -For the prevention of tumor relapse mediated by BCMA escape, such as relapse of multiple myeloma.

[0406] In some embodiments, the present invention provides a method for preventing or treating a GPRC5D and / or BCMA-related disease in a subject, comprising administering to the subject a trispecific antibody of the present invention, or an immunoconjugate, composition, or drug or formulation comprising the same. In some embodiments, the present invention provides a method for the above-mentioned use in a subject, comprising administering to the subject a trispecific antibody of the present invention, or an immunoconjugate, composition, or drug or formulation comprising the same.

[0407] In some embodiments, the disease is, for example, a tumor, such as cancer. The cancer may be in the early, middle, or late stages or may be a metastatic cancer. In some embodiments, the cancer is a GPRC5D single-positive cancer, a BCMA single-positive cancer, a GPRC5D and BCMA double-positive cancer, or a BCMA and GPRC5D double-low expression cancer, such as a GPRC5D low expression and / or BCMA low expression cancer (e.g., a cancer that recurs after anti-BCMA treatment or anti-GPRC5D treatment). In some embodiments, the cancer may be a solid tumor or a hematologic tumor. In some embodiments, the cancer is multiple myeloma.

[0408] Due to the antigen binding specificity for BCMA and GRPC5D, the trispecific antibody molecules of the present invention can have a wider population of cancer patients for treatment than bispecific antibodies targeting BCMA and CD3 and bispecific antibodies targeting GPRC5D and CD3. In some embodiments, the present invention provides the use of the trispecific antibody molecules of the present invention to treat GPRC5D single-positive cancer. In some embodiments, the present invention provides the use of the trispecific antibody molecules of the present invention to treat BCMA single-positive cancer. In some embodiments, the present invention provides the use of the trispecific antibody molecules of the present invention to treat GPRC5D and BCMA double-positive cancer. In some embodiments, the present invention also provides the use of the trispecific antibody molecules of the present invention to treat BCMA and / or GPCR5D low-expressing cancer methods, such as GRPC5D low-expression and / or BCMA low-expressing cancer or BCMA and GPRC5D double-low-expressing cancer (e.g., cancer that relapsed after anti-BCMA treatment and / or anti-GPRC5D treatment).

[0409] The term "GPRC5D single-positive cancer" as used herein refers to cancer cells that only express GPRC5D, or GPRC5D expression is elevated (i.e., overexpressed) in cancer cells compared to healthy cells (e.g., cells of the same tissue of a healthy subject or healthy cells adjacent to cancer cells). The term "GPRC5D low-expressing cancer" as used herein refers to cancer cells that express GPRC5D at a low level, for example, at a slightly higher level than in healthy cells of normal tissue.

[0410] The term "BCMA-single-positive cancer" as used herein refers to cancer cells that express only BCMA, or BCMA expression is elevated (i.e., overexpressed) in cancer cells compared to healthy cells (e.g., cells of the same tissue of a healthy subject or healthy cells adjacent to cancer cells). The term "BCMA-low-expressing cancer" as used herein refers to cancer cells that express low levels of BCMA, for example, slightly higher than the expression level in healthy cells of normal tissue.

[0411] The "GPRC5D and BCMA double-positive cancer" of the present invention refers to cancer cells that have GPRC5D and BCMA expression, or GPRC5D and BCMA expression in cancer cells is increased compared to healthy cells (e.g., the same tissue cells of a healthy subject or healthy cells adjacent to cancer cells). "GPRC5D and BCMA double-positive cancer" also encompasses cancers that have BCMA expression and GPRC5D expression but both or one of them are expressed at a low level, such as BCMA and / or GPCR5D low-expressing cancers, such as GRPC5D low-expressing and / or BCMA low-expressing cancers or BCMA and / or GPRC5D double-low-expressing cancers (e.g., cancers that relapse after anti-BCMA treatment and / or anti-GPRC5D treatment).

[0412] When "BCMA-positive" is mentioned herein, it covers 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 lead to resistance of individuals to immunotherapy targeting BCMA. Therefore, the "BCMA-positive cancer" of the present invention also covers cancers in which BCMA variant expression is increased (i.e., overexpressed) in cancer cells compared to healthy cells (e.g., cells of the same tissue of a healthy subject or healthy cells adjacent to the cancer cells).

[0413] In some embodiments, the BCMA variant comprises one or more or all of the following mutations:

[0414] (i) Proline P deletion at position 34 (P34del);

[0415] (ii) deletion of Serine S at position 30 (S30del);

[0416] (iii) amino acid substitution at position 39, e.g., substitution of arginine R to alanine A (R39A);

[0417] (iv) amino acid substitution at position 27, for example, substitution of arginine R to proline P (R27P).

[0418] In some embodiments, the present invention provides methods for inhibiting or killing GPRC5D-positive and / or BCMA-positive cells.

[0419] In some embodiments, the GPRC5D-positive cells are cells having GPRC5D expression or overexpression in the cells. In some embodiments, the BCMA-positive cells are cells having BCMA expression or overexpression in the cells.

[0420] In some embodiments, the BCMA variant in the BCMA-positive cells comprises one or more or all of the following mutations:

[0421] (i) Proline P deletion at position 34 (P34del);

[0422] (ii) deletion of Serine S at position 30 (S30del);

[0423] (iii) amino acid substitution at position 39, e.g., substitution of arginine R to alanine A (R39A);

[0424] (iv) amino acid substitution at position 27, for example, substitution of arginine R to proline P (R27P).

[0425] In some embodiments, the BCMA-positive cells of the present invention having BCMA variant expression or overexpression are resistant to immunotherapy targeting BCMA (e.g., Elranatamab or Teclistamab treatment). In some embodiments, the BCMA-positive cells of the present invention having BCMA variant expression or overexpression are resistant to immunotherapy targeting BCMA and GPRC5D.

[0426] In some embodiments, in the BCMA-positive cells expressing or overexpressing BCMA variants of the present invention, BCMA has a P34del, S30del, R39A, or R27P mutation. In some embodiments, in the BCMA-positive cells expressing or overexpressing BCMA variants of the present invention, BCMA having a P34del, S30del, R39A, or R27P mutation is expressed, or its expression is elevated (i.e., overexpressed) compared to healthy cells (e.g., cells of the same tissue of a healthy subject or healthy cells adjacent to cancer cells).

[0427] In some embodiments, the BCMA-positive cells expressing or overexpressing a BCMA variant of the present invention express or overexpress a BCMA variant containing a P34del or R27P mutation and are resistant to elranatamab.

[0428] In some embodiments, the BCMA-positive cells having expression or overexpression of a BCMA variant of the present invention have expression or overexpression of a BCMA variant containing a P34del, S30del, R39A, or R27P mutation and are resistant to Teclistamab.

[0429] In some embodiments, the antibody molecule of the present invention or a pharmaceutical composition comprising the antibody molecule of the present invention is used as a drug for treating and / or preventing GRPC5D and / or BCMA-related diseases or for killing or inhibiting GPRC5D-positive and / or BCMA-positive cells in an individual, or as a diagnostic tool for GRPC5D and / or BCMA-related diseases, preferably, the individual is a mammal, more preferably a human.

[0430] In other aspects, the present invention provides use of the trispecific antibodies of the present invention, or immunoconjugates or compositions or combination products comprising the same, in the production or preparation of medicaments for the uses described herein, e.g., for preventing or treating the relevant diseases or disorders mentioned herein, or for killing or inhibiting GPRC5D-positive and / or BCMA-positive cells.

[0431] In some embodiments, the trispecific antibodies of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or disorders mentioned herein or for killing or inhibiting GPRC5D-positive and / or BCMA-positive cells.

[0432] In some embodiments, the treatment modality is, for example, surgery or radiation therapy.

[0433] In one aspect, the invention provides a diagnostic method for detecting the presence of a relevant antigen in a biological sample, such as serum, semen or urine or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion) in vitro or in vivo. The diagnostic method comprises: (i) contacting the sample (and optionally a control sample) with an antibody molecule as described herein or administering the antibody molecule to a subject under conditions that allow interaction to occur and (ii) detecting the formation of a complex between the antibody molecule and the sample (and optionally a control sample). The formation of a complex indicates the presence of a relevant antigen and can indicate the suitability or need for treatment and / or prevention as described herein.

[0434] In some embodiments, before treatment, for example, before initial treatment or before a certain treatment after the treatment interval, related antigens, such as GRPC5D and / or BCMA, are detected. Useful detection methods include immunohistochemistry, immunocytochemistry, FACS, ELISA determinations, PCR technology (for example, RT-PCR) or in vivo imaging technology. Generally, the antibody molecules used in vivo and in vitro detection methods are directly or indirectly labeled with detectable substances to facilitate detection of bound or unbound conjugates. Suitable detectable substances include a variety of biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, paramagnetic (for example, nuclear magnetic resonance activity) substances and radioactive substances.

[0435] In some embodiments, the level and / or distribution of an antigen of interest, such as GRPC5D and / or BCMA, is determined in vivo, e.g., non-invasively (e.g., by detecting an antibody molecule of the invention that is detectably labeled using a suitable imaging technique (e.g., positron emission tomography (PET) scanning). In one embodiment, the level and / or distribution of an antigen of interest is determined in vivo, e.g., by detecting an antibody molecule of the invention that is detectably labeled with a PET agent (e.g., 18F-fluorodeoxyglucose (FDG)).

[0436] In one embodiment, the invention provides a diagnostic kit comprising an antibody molecule described herein and instructions for use.

[0437] VII. Specific Implementation Plan

[0438] In one aspect of the present invention, the present invention relates to the following specific embodiments:

[0439] 1. A trispecific 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.

[0440] 2. The trispecific antibody of embodiment 1, wherein the second antigen-binding region specifically binds CD3, and / or the third antigen-binding region specifically binds GPRC5D.

[0441] 3. The trispecific antibody of embodiment 1 or 2, wherein the first antigen-binding region, the second antigen-binding region and the third antigen-binding region are a first Fab, a second Fab and a third Fab, respectively.

[0442] 4. The trispecific antibody of embodiment 3, wherein

[0443] (1) The first Fab 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 of the Fc heterodimer; the second Fab 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, and the third Fab is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of the VH of the Fab heavy chain of the second Fab fragment;

[0444] or

[0445] The third Fab 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 of the Fc heterodimer; the second Fab 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, and the first Fab is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of the VH of the Fab heavy chain of the second Fab fragment;

[0446] or

[0447] (2) the first Fab 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 of the Fc heterodimer; the second Fab 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, and the third Fab is fused at the N-terminus of VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab; or

[0448] The third Fab 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 of the Fc heterodimer; the second Fab 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, and the first Fab is fused at the N-terminus of VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab.

[0449] 5. The trispecific antibody of embodiment 4, wherein the fusion comprises direct fusion or fusion via a linker.

[0450] 6. The trispecific antibody of embodiment 5, wherein the linker is (GGGGS)n, wherein n=1, 2, 3 or 4.

[0451] 7. The trispecific antibody of any one of embodiments 3-6, 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.

[0452] 8. The trispecific antibody of embodiment 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, e.g.

[0453] The second Fab comprises VH and VL, wherein the VH comprises 39D, and the VL comprises 38K, and

[0454] The first or third Fab fused to the second Fab via the Fc region comprises VH and VL, wherein the VH comprises 39K and the VL comprises 38D; or

[0455] The second Fab comprises VH and VL, wherein the VH comprises 39K, and the VL comprises 38D, and

[0456] The first or third Fab fused to the second Fab via the Fc region comprises VH and VL, wherein the VH comprises 39D and the VL comprises 38K.

[0457] 9. The trispecific antibody of embodiment 7 or 8, wherein the disulfide bond remodeling mutation comprises F126C in CH1 of Fab and Q124C in CL of Fab.

[0458] 10. The trispecific antibody of embodiment 9, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1, wherein the CH1

[0459] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 85, and comprises F126C; or

[0460] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 85.

[0461] 11. The trispecific antibody of embodiment 9 or 10, wherein the Fab comprising a disulfide bond remodeling mutation comprises CL, wherein the CL

[0462] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 97 and comprises Q124C; or

[0463] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 97.

[0464] 12. The trispecific antibody of embodiment 9, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1 and CL, wherein the CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85, and the CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 97.

[0465] 13. The trispecific antibody of embodiment 7 or 8, wherein the disulfide bond remodeling mutations comprise F126C and C220S in CH1 of Fab and Q124C and C214S in CL of Fab.

[0466] 14. The trispecific antibody of embodiment 13, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1, wherein the CH1

[0467] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 86, and comprises F126C and C220S; or

[0468] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 86.

[0469] 15. The trispecific antibody of embodiment 13 or 14, wherein the Fab comprising a disulfide bond remodeling mutation comprises CL, wherein the CL

[0470] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 98, and comprises Q124C and C214S; or

[0471] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 98.

[0472] 16. The trispecific antibody of embodiment 13, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1 and CL, wherein the CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 86, and the CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 98.

[0473] 17. The trispecific antibody of any one of embodiments 1-16, wherein the trispecific 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.

[0474] 18. The trispecific antibody of embodiment 17, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 87 or 88, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or higher identity thereto.

[0475] 19. The trispecific antibody of embodiment 18, wherein the first and / or second Fc region comprises L234A / L235A mutations.

[0476] 20. The trispecific antibody of embodiment 19, wherein the first and / or second Fc region comprises the amino acid sequence of SEQ ID NO: 89 or 90; or an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or higher identity, to the amino acid sequence of SEQ ID NO: 89 or 90 and comprising the amino acid substitutions L234A / L235A.

[0477] 21. The trispecific antibody of any one of embodiments 17-20, wherein one of the first and second Fc regions comprises a Knob mutation and the other comprises a Hole mutation.

[0478] 22. The trispecific antibody of embodiment 21, wherein one Fc region comprises the amino acid substitution T366W and the other Fc region comprises the amino acid substitutions T366S, L368A and Y407V (numbering according to the EU index).

[0479] 23. The trispecific antibody of embodiment 22, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 91, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 92.

[0480] 24. The trispecific antibody of embodiment 23, wherein 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.

[0481] 25. The trispecific antibody of embodiment 17, wherein one Fc region comprises amino acid substitutions L234A / L235A and T366W, and the other Fc region comprises amino acid substitutions L234A / L235A and T366S, L368A and Y407V (numbering according to the EU index).

[0482] 26. The trispecific antibody of embodiment 25, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 94, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 93.

[0483] 27. The trispecific antibody of embodiment 26, wherein one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 94, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 93.

[0484] 28. The trispecific antibody of any one of embodiments 1-27, 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

[0485] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 59; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 61; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 62; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 63.

[0486] 29. The trispecific antibody of embodiment 28, wherein the VH of the first antigen-binding region that specifically binds to BCMA comprises or consists of the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0487] 30. The trispecific antibody of embodiment 28 or 29, wherein the VL of the first antigen-binding region that specifically binds to BCMA comprises or consists of the amino acid sequence shown in SEQ ID NO:60, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0488] 31. The trispecific antibody of embodiment 30, wherein the first antigen-binding region that specifically binds to BCMA comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:56, and the VL of the first antigen-binding region that specifically binds to BCMA consists of the amino acid sequence shown in SEQ ID NO:60.

[0489] 32. The trispecific antibody of embodiment 28, wherein the first antigen-binding region that specifically binds to BCMA comprises VH and VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K

[0490] For example,

[0491] The VH of the first antigen-binding region

[0492] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 76, or

[0493] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 76, and comprises a Q39K mutation;

[0494] and / or the VL of the first antigen binding region

[0495] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, or

[0496] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 75, and comprises the Q38D mutation.

[0497] 33. The trispecific antibody of any one of embodiments 1-32, 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, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein

[0498] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:41; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:42; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:43; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:45; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:46; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; or

[0499] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:49; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:50; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:51; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:53; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:54; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:55.

[0500] 34. The trispecific antibody of embodiment 33, wherein the VH that specifically binds to the third antigen binding region of GPRC5D contains

[0501] (i) the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence; or

[0502] (ii) the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence.

[0503] 35. The trispecific antibody of embodiment 33 or 34, wherein the VL that specifically binds to the third antigen binding region of GPRC5D comprises

[0504] (i) the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence; or

[0505] (ii) the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0506] 36. The trispecific antibody of embodiment 35, wherein the third antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:48, and the VL consists of the amino acid sequence shown in SEQ ID NO:52; or the VH consists of the amino acid sequence shown in SEQ ID NO:40, and the VL consists of the amino acid sequence shown in SEQ ID NO:44.

[0507] 37. The trispecific antibody of any one of embodiments 33-36, wherein the third antigen-binding region that specifically binds to GPRC5D comprises a VH and a VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K, for example

[0508] The VH that specifically binds to the third antigen binding region of GPRC5D

[0509] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, or

[0510] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 80, and comprises a Q39K mutation;

[0511] And the VL that specifically binds to the third antigen binding region of GPRC5D

[0512] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 79, or

[0513] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 79, and comprises the Q38D mutation.

[0514] 38. The trispecific antibody of any one of embodiments 1-37, wherein the second antigen-binding region 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

[0515] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 65; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 66; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 67 or 73; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 70; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 71.

[0516] 39. The trispecific antibody of embodiment 38, wherein the VH that specifically binds to the second antigen-binding region of CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 64 or 72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0517] 40. The trispecific antibody of embodiment 38 or 39, wherein the VL of the second antigen-binding region that specifically binds to CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO:68, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0518] 41. The trispecific antibody of embodiment 40, wherein the second antigen-binding region that specifically binds to CD3 comprises VH and VL, wherein 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.

[0519] 42. The trispecific antibody of any one of embodiments 38-41, wherein the second antigen-binding region that specifically binds to CD3 comprises a VH and a VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K.

[0520] 43. The trispecific antibody of embodiment 42, wherein the VH that specifically binds to the second antigen-binding region of CD3

[0521] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 77 or 78, or

[0522] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 77 or 78, and comprises the Q39D mutation;

[0523] And the VL that specifically binds to the second antigen binding region of CD3

[0524] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 74, or

[0525] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 74, and comprises the Q38K mutation.

[0526] 44. The trispecific antibody according to any one of embodiments 3-43, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, wherein the trispecific antibody is composed of the following peptide chains:

[0527] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0528] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0529] A peptide chain 3# that contains a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0530] A peptide chain 4# comprising a Fab light chain that specifically binds to BCMA, and

[0531] A peptide chain 5# that contains a Fab light chain that specifically binds to CD3,

[0532] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain 2# are paired to form the third 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 first and second Fab.

[0533] 45. The trispecific antibody of embodiment 44, wherein

[0534] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:5, or comprises 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 of SEQ ID NO:5, or consists of the amino acid sequence;

[0535] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:4, or comprises 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 of SEQ ID NO:4;

[0536] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO: 3 or 6, or comprises 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 of SEQ ID NO: 3 or 6, or consists of the amino acid sequence;

[0537] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises 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: 2, or consists of the amino acid sequence; and / or

[0538] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0539] 46. ​​The trispecific antibody of embodiment 44, wherein

[0540] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:15, or comprises 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 of SEQ ID NO:15;

[0541] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:14, or comprises 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 of SEQ ID NO:14;

[0542] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO: 3 or 6, or comprises 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 of SEQ ID NO: 3 or 6, or consists of the amino acid sequence;

[0543] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises 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: 2, or consists of the amino acid sequence; and / or

[0544] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0545] 47. The trispecific antibody according to any one of embodiments 1-43, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which is composed of the following peptide chains:

[0546] A peptide chain 1# that contains a Fab heavy chain and an Fc domain that specifically binds to BCMA.

[0547] A peptide chain 2# containing a Fab light chain that specifically binds to BCMA,

[0548] A peptide chain 3# that contains a Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0549] A peptide chain 4# comprising a Fab light chain that specifically binds to GPRC5D, and

[0550] A peptide chain 5# containing a Fab light chain that specifically binds to CD3,

[0551] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired 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 and second Fab.

[0552] 48. The trispecific antibody of embodiment 47, wherein

[0553] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:11, or comprises 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 of SEQ ID NO:11, or consists of the amino acid sequence;

[0554] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:10, or comprises 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 of SEQ ID NO:10;

[0555] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO:9, or comprises 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 of SEQ ID NO:9;

[0556] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO:8, or comprises 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:8, or consists of the amino acid sequence; and / or

[0557] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0558] 49. The trispecific antibody according to any one of embodiments 1-43, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which is composed of the following peptide chains:

[0559] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0560] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0561] A peptide chain 3# that sequentially contains a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain that specifically binds to the antigen binding region of BCMA.

[0562] A peptide chain 4# comprising a Fab light chain that specifically binds to CD3, and

[0563] A peptide chain 5# of a Fab light chain containing an antigen-binding region that specifically binds to BCMA,

[0564] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain 2# are paired to form the third 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 second and first Fab.

[0565] 50. The trispecific antibody of embodiment 49, wherein

[0566] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:5, or comprises 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 of SEQ ID NO:5, or consists of the amino acid sequence;

[0567] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:4, or comprises 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 of SEQ ID NO:4;

[0568] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO:7, or comprises 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 of SEQ ID NO:7;

[0569] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises 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: 1, or consists of the amino acid sequence; and / or

[0570] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:2, or comprises 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:2, or consists of the amino acid sequence.

[0571] 51. A nucleic acid molecule encoding any one chain of the trispecific antibody according to any one of embodiments 1 to 50, or consisting of said nucleic acid sequence.

[0572] 52. An expression vector comprising the nucleic acid molecule of embodiment 51, for example, the expression vector is a pCNDA vector, such as a pCDNA3.1 expression vector.

[0573] 53. A host cell comprising the nucleic acid molecule of embodiment 51 or the expression vector of embodiment 52. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as HEK293 cells.

[0574] 54. A method for preparing the trispecific antibody of any one of embodiments 1-50, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 51 or the expression vector of embodiment 52 under conditions suitable for expression of the antibody chains, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0575] 55. An immunoconjugate comprising the trispecific antibody of any one of embodiments 1-50.

[0576] 56. A pharmaceutical composition, medicament or formulation comprising the trispecific antibody according to any one of embodiments 1 to 50, or the immunoconjugate according to embodiment 55, and optionally a pharmaceutically acceptable excipient.

[0577] 57. A pharmaceutical combination comprising the trispecific antibody of any one of embodiments 1-50, or the immunoconjugate of embodiment 55, and one or more other therapeutic agents, such as chemotherapeutic agents.

[0578] 58. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the trispecific antibody of any one of embodiments 1-50, or the immunoconjugate of embodiment 55, or the pharmaceutical composition or formulation of embodiment 56; or the pharmaceutical combination of embodiment 57.

[0579] 59. The method of embodiment 58, wherein the cancer is a GPRC5D single-positive cancer, a BCMA single-positive cancer, a GPRC5D and BCMA double-positive cancer, or a BCMA and GPCR5D double-low-expressing cancer, such as a GRPC5D low-expressing and / or BCMA low-expressing cancer (e.g., a cancer that relapsed after anti-BCMA treatment or anti-GPRC5D treatment).

[0580] 60. The method of embodiment 58 or 59, wherein the cancer is a solid tumor or a hematological tumor, for example, the cancer is multiple myeloma.

[0581] 61. The method of any one of embodiments 58-60, wherein the method further comprises administering in combination with other therapies, such as treatment modalities (e.g., surgery or radiation therapy) and / or other therapeutic agents (e.g., chemotherapeutic agents).

[0582] In one aspect of the present invention, the present invention also relates to the following specific embodiments:

[0583] 1. A trispecific antibody that specifically binds to GPRC5D, comprising a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region that specifically binds to BCMA, wherein the first antigen-binding region 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

[0584] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 57; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 58; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 59; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 61; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 62; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 63;

[0585] Preferably, the second antigen binding region specifically binds to CD3 and / or the third antigen binding region specifically binds to GPRC5D.

[0586] 2. The trispecific antibody of embodiment 1, wherein the VH first antigen-binding region that specifically binds to BCMA comprises, or consists of, the amino acid sequence of SEQ ID NO: 56, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0587] 3. The trispecific antibody of embodiment 1 or 2, wherein the VL of the first antigen-binding region that specifically binds to BCMA comprises or consists of the amino acid sequence of SEQ ID NO: 60, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0588] 4. The trispecific antibody of embodiment 1, wherein the first antigen-binding region that specifically binds to BCMA comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO: 56, and the VL of the first antigen-binding region consists of the amino acid sequence shown in SEQ ID NO: 60.

[0589] 5. The trispecific antibody according to any one of embodiments 1-4, wherein the first antigen-binding region that specifically binds to BCMA comprises a VH and a VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K.

[0590] 6. The trispecific antibody of embodiment 5, wherein the VH that specifically binds to the first antigen-binding region of BCMA

[0591] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 76, or

[0592] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 76, and comprises a Q39K mutation;

[0593] And the VL that specifically binds to the first antigen binding region of BCMA

[0594] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, or

[0595] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 75, and comprises the Q38D mutation.

[0596] 7. The trispecific antibody according to any one of embodiments 1-6, wherein the first antigen-binding region that specifically binds to BCMA is a Fab.

[0597] 8. The trispecific antibody of any one of embodiments 1-7, wherein the third antigen-binding region that specifically binds to GPRC5D 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

[0598] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:41; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:42; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:43; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:45; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:46; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; or

[0599] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:49; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:50; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:51; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:53; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:54; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:55.

[0600] 9. The trispecific antibody of embodiment 8, wherein the VH that specifically binds to the third antigen-binding region of GPRC5D comprises

[0601] (i) the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence; or

[0602] (ii) the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence.

[0603] 10. The trispecific antibody of embodiment 8 or 9, wherein the VL that specifically binds to the third antigen-binding region of GPRC5D contains

[0604] (i) the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence; or

[0605] (ii) the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0606] 11. The trispecific antibody of embodiment 8, wherein the third antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:48, and the VL consists of the amino acid sequence shown in SEQ ID NO:52; or the VH consists of the amino acid sequence shown in SEQ ID NO:40, and the VL consists of the amino acid sequence shown in SEQ ID NO:44.

[0607] 12. The trispecific antibody of any one of embodiments 1-11, wherein the third antigen-binding region that specifically binds to GPRC5D comprises VH and VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K.

[0608] 13. The trispecific antibody of embodiment 12, wherein the VH that specifically binds to the third antigen binding region of GPRC5D

[0609] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, or

[0610] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 80, and comprises a Q39K mutation;

[0611] And the VL of the third antigen binding region

[0612] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 79, or

[0613] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 79, and comprises the Q38D mutation.

[0614] 14. The trispecific antibody according to any one of embodiments 1 to 13, wherein the third antigen-binding region that specifically binds to GPRC5D is a Fab.

[0615] 15. The trispecific antibody of any one of embodiments 1-14, wherein the second antigen-binding region that specifically binds to CD3 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

[0616] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 65; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 66; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 67 or 73; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 69; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 70; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 71.

[0617] 16. The trispecific antibody of embodiment 15, wherein the VH of the second antigen-binding region that specifically binds to CD3 comprises or consists of the amino acid sequence of SEQ ID NO: 64 or 72, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0618] 17. The trispecific antibody of embodiment 15 or 16, wherein the VL of the second antigen-binding region that specifically binds to CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO:68, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0619] 18. The trispecific antibody of embodiment 15, wherein the second antigen-binding region that specifically binds to CD3 comprises VH and VL, wherein 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.

[0620] 19. The trispecific antibody of any one of embodiments 15-18, wherein the second antigen-binding region that specifically binds to CD3 comprises VH and VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K.

[0621] 20. The trispecific antibody of embodiment 19, wherein the VH that specifically binds to the second antigen-binding region of CD3

[0622] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 77 or 78, or

[0623] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 77 or 78, and comprises the Q39D mutation;

[0624] And the VL that specifically binds to the second antigen binding region of CD3

[0625] (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 74, or

[0626] (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 74, and comprises the Q38K mutation.

[0627] 21. The trispecific antibody of any one of embodiments 1-20, wherein the second antigen binding region that specifically binds to CD3 is a Fab.

[0628] 22. The trispecific antibody according to any one of embodiments 1-21, wherein the first antigen-binding region, the second antigen-binding region and the third antigen-binding region are a first Fab, a second Fab and a third Fab, respectively.

[0629] 23. The trispecific antibody of embodiment 22, wherein the first Fab or the third Fab further comprises a disulfide bond remodeling mutation.

[0630] 24. The trispecific antibody of embodiment 23, wherein the disulfide bond remodeling mutation comprises F126C in CH1 of Fab and Q124C in CL of Fab.

[0631] 25. The trispecific antibody of embodiment 24, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1, wherein the CH1

[0632] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 85, and comprises F126C; or

[0633] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 85.

[0634] 26. The trispecific antibody of embodiment 24 or 25, wherein the Fab comprising a disulfide bond remodeling mutation comprises CL, wherein the CL

[0635] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 97 and comprises Q124C; or

[0636] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 97.

[0637] 27. The trispecific antibody of embodiment 24, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1 and CL, wherein the CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85, and the CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 97.

[0638] 28. The trispecific antibody of embodiment 24, wherein the disulfide bond remodeling mutations comprise F126C and C220S in CH1 of Fab and Q124C and C214S in CL of Fab.

[0639] 29. The trispecific antibody of embodiment 28, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1, wherein the CH1

[0640] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 86, and comprises F126C and C220S; or

[0641] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 86.

[0642] 30. The trispecific antibody of embodiment 28 or 29, wherein the Fab comprising a disulfide bond remodeling mutation comprises CL, wherein the CL

[0643] (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 98, and comprises Q124C; or

[0644] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 98.

[0645] 31. The trispecific antibody of embodiment 28, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1 and CL, wherein the CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 86, and the CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 98.

[0646] 32. The trispecific antibody of any one of embodiments 1-31, wherein the trispecific 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.

[0647] 33. The trispecific antibody of embodiment 32, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 87 or 88 or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or higher identity thereto.

[0648] 34. The trispecific antibody of embodiment 33, wherein the first and / or second Fc region comprises L234A / L235A mutations.

[0649] 35. The trispecific antibody of embodiment 34, wherein the first and / or second Fc region comprises the amino acid sequence of SEQ ID NO: 89 or 90; or comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or higher identity, to the amino acid sequence of SEQ ID NO: 89 or 90 and comprises the amino acid substitution L234A / L235A.

[0650] 36. The trispecific antibody of any one of embodiments 32-35, wherein one of the first and second Fc regions comprises a Knob mutation and the other comprises a Hole mutation.

[0651] 37. The trispecific antibody of embodiment 36, wherein one Fc region comprises the amino acid substitution T366W and the other Fc region comprises the amino acid substitutions T366S, L368A and Y407V (numbering according to the EU index).

[0652] 38. The trispecific antibody of embodiment 37, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:91, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:92.

[0653] 39. The trispecific antibody of 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 and T366S, L368A and Y407V (numbering according to the EU index).

[0654] 40. The trispecific antibody of embodiment 39, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:94, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO:93.

[0655] 41. The trispecific antibody of any one of embodiments 1-40, wherein the first antigen-binding region, the second antigen-binding region and the third antigen-binding region are a first Fab, a second Fab and a third Fab, respectively, and wherein

[0656] (1) The first Fab 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 of the Fc heterodimer; the second Fab 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, and the third Fab is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of the VH of the Fab heavy chain of the second Fab fragment;

[0657] or

[0658] The third Fab 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 of the Fc heterodimer; the second Fab 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, and the first Fab is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of the VH of the Fab heavy chain of the second Fab fragment;

[0659] or

[0660] (2) the first Fab 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 of the Fc heterodimer; the second Fab 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, and the third Fab is fused at the N-terminus of VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab; or

[0661] The third Fab 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 of the Fc heterodimer; the second Fab 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, and the first Fab is fused at the N-terminus of VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab.

[0662] 42. The trispecific antibody of embodiment 41, wherein the fusion comprises direct fusion or fusion through a linker.

[0663] 43. The trispecific antibody of embodiment 42, wherein the linker is (GGGGS)n, wherein n=1, 2, 3 or 4.

[0664] 44. The trispecific antibody of any one of embodiments 41-43, wherein the second Fab comprises a charge mutation comprising a Q38K or Q38D mutation in the Fab light chain and a Q39D or Q39K mutation in the Fab heavy chain.

[0665] 45. The trispecific antibody of any one of embodiments 41-44, wherein in the first or third Fab fused to the second Fab, or fused to the second Fab via the Fc region, the Fab heavy chain comprises Q39K or Q39D and F126C mutations, and the Fab light chain comprises Q38D or Q38K and Q124C mutations; or

[0666] The first or third Fab is fused to the second Fab, or is fused to the second Fab via the Fc region, wherein the Fab heavy chain comprises Q39K or Q39D and F126C mutations and C220S, and the Fab light chain comprises Q38D or Q38K and Q124C mutations and C214S.

[0667] 46. ​​The trispecific antibody of any one of embodiments 41-43, wherein

[0668] the second Fab comprises a charge mutation comprising a Q38K mutation in the Fab light chain and a Q39D mutation in the Fab heavy chain; and is fused to the second Fab, or to the first or third Fab fused to the second Fab via the Fc region, wherein the Fab heavy chain comprises Q39K and F126C mutations, and the Fab light chain comprises Q38D and Q124C mutations; or

[0669] the second Fab comprises a charge mutation comprising a Q38K mutation in the Fab light chain and a Q39D mutation in the Fab heavy chain; and is fused to the second Fab, or to the first or third Fab fused to the second Fab via the Fc region, wherein the Fab heavy chain comprises Q39K and F126C mutations and C220S, and the Fab light chain comprises Q38D and Q124C mutations and C214S; or

[0670] the second Fab comprises a charge mutation comprising a Q38D mutation in the Fab light chain and a Q39K mutation in the Fab heavy chain; and is fused to the second Fab, or to the first or third Fab fused to the second Fab via the Fc region, wherein the Fab heavy chain comprises Q39D and F126C mutations, and the Fab light chain comprises Q38K and Q124C mutations; or

[0671] The second Fab comprises a charge mutation comprising a Q38D mutation in the Fab light chain and a Q39K mutation in the Fab heavy chain; and is fused to the second Fab, or to the first or third Fab fused to the second Fab via the Fc region, wherein the Fab heavy chain comprises Q39D and F126C mutations and C220S, and the Fab light chain comprises Q38K and Q124C mutations and C214S.

[0672] 47. The trispecific antibody according to any one of embodiments 1-46, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, wherein the trispecific antibody is composed of the following peptide chains:

[0673] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0674] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0675] A peptide chain 3# that contains a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0676] A peptide chain 4# comprising a Fab light chain that specifically binds to BCMA, and

[0677] A peptide chain 5# that contains a Fab light chain that specifically binds to CD3,

[0678] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain 2# are paired to form the third 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 first and second Fab.

[0679] 48. The trispecific antibody of embodiment 47, wherein

[0680] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:5, or comprises 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 of SEQ ID NO:5, or consists of the amino acid sequence;

[0681] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:4, or comprises 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 of SEQ ID NO:4;

[0682] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO: 3 or 6, or comprises 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 of SEQ ID NO: 3 or 6, or consists of the amino acid sequence;

[0683] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises 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: 2, or consists of the amino acid sequence; and / or

[0684] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0685] 49. The trispecific antibody of embodiment 47, wherein

[0686] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:15, or comprises 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 of SEQ ID NO:15;

[0687] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:14, or comprises 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 of SEQ ID NO:14;

[0688] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO: 3 or 6, or comprises 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 of SEQ ID NO: 3 or 6, or consists of the amino acid sequence;

[0689] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 2, or comprises 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: 2, or consists of the amino acid sequence; and / or

[0690] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0691] 50. The trispecific antibody according to any one of embodiments 1-46, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which is composed of the following peptide chains:

[0692] A peptide chain 1# that contains a Fab heavy chain and an Fc domain that specifically binds to BCMA.

[0693] A peptide chain 2# containing a Fab light chain that specifically binds to BCMA,

[0694] A peptide chain 3# that contains a Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0695] A peptide chain 4# comprising a Fab light chain that specifically binds to GPRC5D, and

[0696] A peptide chain 5# containing a Fab light chain that specifically binds to CD3,

[0697] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired 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 and second Fab.

[0698] 51. The trispecific antibody of embodiment 50, wherein

[0699] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:11, or comprises 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 of SEQ ID NO:11, or consists of the amino acid sequence;

[0700] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:10, or comprises 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 of SEQ ID NO:10;

[0701] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO:9, or comprises 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 of SEQ ID NO:9;

[0702] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO:8, or comprises 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:8, or consists of the amino acid sequence; and / or

[0703] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

[0704] 52. The trispecific antibody according to any one of embodiments 1-46, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which is composed of the following peptide chains:

[0705] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0706] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0707] A peptide chain 3# that sequentially contains a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain that specifically binds to the antigen binding region of BCMA.

[0708] A peptide chain 4# comprising a Fab light chain that specifically binds to CD3, and

[0709] A peptide chain 5# of a Fab light chain containing an antigen-binding region that specifically binds to BCMA,

[0710] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain 2# are paired to form the third 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 second and first Fab.

[0711] 53. The trispecific antibody of embodiment 52, wherein

[0712] Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:5, or comprises 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 of SEQ ID NO:5, or consists of the amino acid sequence;

[0713] Peptide chain 2# comprises the amino acid sequence of SEQ ID NO:4, or comprises 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 of SEQ ID NO:4;

[0714] Peptide chain 3# comprises the amino acid sequence of SEQ ID NO:7, or comprises 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 of SEQ ID NO:7;

[0715] Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO: 1, or comprises 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: 1, or consists of the amino acid sequence; and / or

[0716] Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:2, or comprises 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:2, or consists of the amino acid sequence.

[0717] 54. A nucleic acid molecule encoding any one chain of the trispecific antibody according to any one of embodiments 1 to 53, or consisting of said nucleic acid sequence.

[0718] 55. An expression vector comprising the nucleic acid molecule of embodiment 54, for example, the expression vector is a pCNDA vector, such as a pCDNA3.1 expression vector.

[0719] 56. A host cell comprising the nucleic acid molecule of embodiment 54 or the expression vector of embodiment 52. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as HEK293 cells.

[0720] 57. A method for preparing the trispecific antibody of any one of embodiments 1-53, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 54 or the expression vector of embodiment 55 under conditions suitable for expression of the antibody chains, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0721] 58. An immunoconjugate comprising the trispecific antibody of any one of embodiments 1-53.

[0722] 59. A pharmaceutical composition, medicament or formulation comprising the trispecific antibody of any one of embodiments 1-53, or the immunoconjugate of embodiment 58, and optionally a pharmaceutically acceptable excipient.

[0723] 60. A pharmaceutical combination comprising the trispecific antibody of any one of embodiments 1-53, or the immunoconjugate of embodiment 55, and one or more other therapeutic agents, such as chemotherapeutic agents.

[0724] 61. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the trispecific antibody of any one of embodiments 1-53, or the immunoconjugate of embodiment 58, or the pharmaceutical composition or formulation of embodiment 59; or the pharmaceutical combination of embodiment 57.

[0725] 62. The method of embodiment 61, wherein the cancer is a GPRC5D single-positive cancer, a BCMA single-positive cancer, a GPRC5D and BCMA double-positive cancer, or a BCMA and GPCR5D double-low-expressing cancer, such as a GRPC5D low-expressing and / or BCMA low-expressing cancer (e.g., a cancer that relapsed after anti-BCMA treatment or anti-GPRC5D treatment).

[0726] 63. The method of embodiment 61 or 62, wherein the cancer is a solid tumor or a hematological tumor, for example, the cancer is multiple myeloma.

[0727] 64. The method of any one of embodiments 58-60, wherein the method further comprises administering in combination with other therapies, such as treatment modalities (e.g., surgery or radiation therapy) and / or other therapeutic agents (e.g., chemotherapeutic agents).

[0728] It should be understood that the present invention also covers any combination of any technical features or any combination of any technical solutions described herein. Example

[0729] Example 1. Molecular structure design and construction of trispecific antibody (Ts)

[0730] The example antibody is a multispecific T-cell engager antibody that simultaneously targets GPRC5D, BCMA, and CD3. It can simultaneously bind to two tumor-associated antigens (GPRC5D and BCMA) on the surface of multiple myeloma cells (MM) and the CD3 receptor on the surface of T cells. Based on two anti-GPRC5D antibodies with different affinities (hz7F5.3, hz5E12.1.P1), one anti-BCMA antibody (ADI-38497), and two anti-CD3 antibodies with different affinities (hzsp34.24, hzsp34.87), multispecific antibodies that simultaneously target GPRC5D, BCMA, and CD3 and contain disulfide bond remodeling mutations and charge mutations were designed. The sequence numbers and specific sequences of the variable region and CDR sequences of the above three antibodies are shown in the sequence information.

[0731] Specifically, the present invention designed six example antibodies with a 1+1+1 structure (B3, B3b, B5) as described in Figure 1 and Table 1, and achieved better efficacy than bispecific antibodies. Furthermore, the example antibody design utilizes specific mutation technology (disulfide bond remodeling mutation and charge mutation, see the sequence information table for specific mutation information) to resolve the light chain mispairing of non-BCMA and CD3 Fab, and the Fc portion uses "knob-in-hole" technology to resolve the heavy chain mispairing of asymmetric IgG-like bispecific antibodies, wherein the Fc is the heavy chain constant region of IgG1 and introduces L234A, L235A ("EU" numbering) amino acid mutations that weaken effector function.

[0732] The designed structures of the above 6 exemplary antibodies are shown in Figure 1 and Table 1.

[0733] Table 1. Fragment numbers and descriptions of example antibodies

[0734] The structures of the six exemplary antibodies mentioned above are in the B3, B3b and B5 forms.

[0735] The B3 form (for details such as chain #, see Figure 1A) consists of five polypeptide chains forming a bilaterally asymmetric IgG-like pentamer, which is composed of the following peptide chains:

[0736] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0737] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0738] A peptide chain 3# that contains a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0739] A peptide chain 4# comprising a Fab light chain that specifically binds to BCMA, and

[0740] A peptide chain 5# that contains a Fab light chain that specifically binds to CD3,

[0741] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired to form the third 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 first and second Fab, as shown in Figure 1A-Format B3.

[0742] The B3b form (for details such as chain #, see Figure 1A) consists of five polypeptide chains forming a bilaterally asymmetric IgG-like pentamer, which is composed of the following peptide chains:

[0743] A peptide chain 1# that contains a Fab heavy chain and an Fc domain that specifically binds to BCMA.

[0744] A peptide chain 2# containing a Fab light chain that specifically binds to BCMA,

[0745] A peptide chain 3# that contains a Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0746] A peptide chain 4# comprising a Fab light chain that specifically binds to GPRC5D, and

[0747] A peptide chain 5# containing a Fab light chain that specifically binds to CD3,

[0748] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired 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 and second Fab, as shown in Figure 1A-Format B3b.

[0749] The B5 form (for details such as chain #, see Figure 1B) consists of five polypeptide chains forming a bilaterally asymmetric IgG-like pentamer, which is composed of the following peptide chains:

[0750] A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D,

[0751] A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D,

[0752] A peptide chain 3# that sequentially contains a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain that specifically binds to the antigen binding region of BCMA.

[0753] A peptide chain 4# comprising a Fab light chain that specifically binds to CD3, and

[0754] A peptide chain 5# of a Fab light chain containing an antigen-binding region that specifically binds to BCMA,

[0755] The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain 2# are paired to form the third 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 second and first Fab, as shown in Figure 1B.

[0756] At the same time, this patent expresses and purifies the following bispecific or trispecific antibodies:

[0757] Inno-B (control Hel / BCMA / CD3, Hel is an egg lysozyme antibody that does not bind to any human protein and is used as a control), Inno-G (control Hel / GPRC5D / CD3), F2(24) (GPRC5D / BCMA / CD3, the structure is derived from WO2022174813A1 (BCMA antigen binding region is scFv)), F2(87) (GPRC5D / BCMA / CD3, the structure is derived from WO2022174813A1 (BCMA antigen binding region is scFv)), JNJ-BCMA / CD3 (BCMA / CD3, from Derived from patent WO2017031104A1, also known as Teclistamab), Roche-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 WO 2019 / 154890A1), Elranatamab (Pfizer BCMAxCD3, derived from patent WO2016166629A1).

[0758] The following negative control antibody was also expressed and purified: anti-Hel antibody (labeled as IgG or hIgG1 in the figures), whose heavy chain sequence is SEQ ID NO: 101 and light chain sequence is SEQ ID NO: 100.

[0759] Example 2. Preparation and purification of trispecific antibodies

[0760] Plasmid preparation

[0761] The nucleic acids encoding each chain of each antibody of the present invention were synthesized by GeneWeiZi and constructed into the pCDNA3.1 template plasmid respectively. A certain amount of plasmid was prepared and filtered through a 0.22 μm filter for transient expression in cells.

[0762] Transfection and expression

[0763] Expi293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 3 × 10 6 cells / ml. On the day of transfection, the cell density was adjusted to 3×10 6cells / ml. Each molecule was expressed in two vials, A and B. To vial A, the plasmid containing peptide chains 1# and 2# was added at a 1:1 ratio. To vial B, the plasmid containing peptide chains 3#, 4#, and 5# was added at a 1:1:1 ratio. Opti-MEM medium (Gibco catalog number: 31985-070) was used as transfection buffer to dilute the plasmid and mix thoroughly. The appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid from the previous step (at a plasmid to PEI mass ratio of 1:3). After mixing thoroughly, the mixture was incubated at room temperature for 10 minutes to obtain a DNA / PEI mixture. The DNA / PEI mixture was gently poured into HEK293 cells and mixed thoroughly. After incubation at 37°C, 8% CO2 for 24 h, VPA (Sigma, Catalog No.: P4543-100G) was added to a final concentration of 2 mM and 2% (v / v) Feed (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone) was added. Culture was continued for 6 days.

[0764] purification

[0765] The culture was collected and centrifuged at 4000 rpm for 30 minutes. The cell supernatant was filtered with a 0.45 μM filter membrane and purified by Protein A affinity chromatography. It was further purified by KappaSelect affinity chromatography and LambdaFabSelect affinity chromatography. Finally, the complete molecule was purified by in vitro recombination and ion exchange chromatography.

[0766] The specific Protein A affinity chromatography purification procedure is as follows: the supernatant is purified using a prepacked column, Hitrap Mabselect Sure (GE, 11-0034-95). The procedure is as follows: Before purification, the column is equilibrated with 5 column volumes of equilibration buffer (20 mM Tris, 150 mM NaCl, pH 7.2); the collected supernatant is passed through the column, and the column is then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins; the column is rinsed with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluate is collected. 2 M Tris is added to neutralize the pH to 6.5. The purified antibody is then quantified using liquid chromatography-mass spectrometry (LC-MS) to determine the correct binding ratio.

[0767] The specific KappaSelect affinity chromatography purification procedure is as follows: the supernatant is purified using a prepacked HiTrap KappaSelect column (GE, 17-5458-11). The procedure is as follows: Before purification, the column is equilibrated with 5 column volumes of equilibration buffer (PBS, pH 7.4); the collected supernatant is passed through the column, and the column is then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins; the column is rinsed with 5 column volumes of elution buffer (0.1 M glycine buffer, pH 2.5), and the eluate is collected. 2 M Tris is added to neutralize the pH to 6.5. The purified antibody is then quantified using liquid chromatography-mass spectrometry (LC-MS) to determine the correct binding ratio.

[0768] The specific LambdaFabSelect affinity chromatography purification procedure is as follows: the supernatant is purified using a prepacked HiTrap LambdaFabSelect column (GE, 17-5482-11). The procedure is as follows: Before purification, the column is equilibrated with 5 column volumes of equilibration buffer (PBS, pH 7.4); the collected supernatant is passed through the column, and the column is then washed with 10 column volumes of equilibration buffer to remove nonspecifically bound proteins; the column is rinsed with 5 column volumes of elution buffer (0.1 M acetate buffer, pH 3.5), and the eluate is collected. 2 M Tris is added to neutralize the pH to 6.5. The purified antibody is then quantified using liquid chromatography-mass spectrometry (LC-MS) to determine the correct binding ratio.

[0769] In vitro reconstitution

[0770] The specific in vitro reconstitution method is as follows: molecule A obtained in bottle A and molecule B obtained in bottle B are mixed at a molar ratio of 1:1, GSH (Sigma Aldrich, G4251) with a final concentration of 5 mM and arginine (Sigma Aldrich, A5006) with a final concentration of 50 mM are added, and the mixture is incubated at 37°C for 6 hours.

[0771] Fine purification

[0772] The specific ion exchange chromatography purification procedure is as follows: the in vitro recombinant product is exchanged into pH 6.0 low-salt PB buffer (10 mM phosphate, pH 6.0) using ultrafiltration concentrators (MILLIPORE, Catalog No.: UFC901096), and fine purification is performed using Capto HiRes S10 / 100 (GE, Catalog No. 29275879). Before purification, the column is equilibrated with 5 column volumes of pH 6.0 low-salt PB buffer; the sample after the exchange is passed through the column, and the column is then washed with 10 column volumes of pH 6.0 low-salt PB buffer to remove non-specifically bound proteins; during elution, the concentration of pH 6.0 high-salt PB buffer (10 mM phosphate, pH 6.0, 1 M NaCl) is linearly increased (linear gradient: pH 6.0 high-salt PB buffer increases from 1% to 100% over 30 column volumes), and the main eluted peak is collected. The solution was exchanged into PBS buffer (Gibco, catalog number: 70011-044) using an ultrafiltration concentration tube (MILLIPORE, catalog number: UFC901096).

[0773] FIG7 shows the difference in aggregation between the B3(24) molecule and the control molecule (F2(24)).

[0774] Compared with the trispecific antibody (F2(24) in the WO2022174813A1 patent application, the B3(24) molecule of the present invention has obvious improvements in protein properties. Specifically, the proportion of aggregates produced during the cation exchange purification process after in vitro reconstitution is greatly reduced. After in vitro reconstitution of the trispecific antibody F2(24) in the WO2022174813A1 patent application, cation exchange chromatography showed approximately 35% aggregates (Figure 7A), while the aggregates produced by the B3(24) molecule after in vitro reconstitution were very few or basically invisible (Figure 7B). Since aggregates are also one of the impurities that need to be removed during the purification process, a significant reduction in aggregates can greatly improve the recovery rate during the purification process.

[0775] Example 3. Determination of the affinity of trispecific antibodies for cells expressing antigens

[0776] 3.1 Determination of the affinity of trispecific antibodies for antigen-expressing cells

[0777] In the exemplary trispecific antibodies, the affinity to human (h-) and cynomolgus macaque (cyno-) BCMA was tested on GS-CHO-hBCMA and GS-CHO-cynoBCMA cell lines; the affinity to human (h-) and cynomolgus macaque (cyno-) GPRC5D was tested on GS-CHO-hGPRC5D and GS-CHO-cynoGPRC5D cell lines; and the affinity to human CD3 was tested on T cells in the Jurkat cell line (Jiman Biotech, GM-C01459) and human cryopreserved PBMCs (Saily, SLB-HP050B).

[0778] Among them, the method for obtaining GS-CHO cell lines overexpressing GPRC5D (GS-CHO-hGPRC5D and GS-CHO-cynoGPRC5D) is as follows: the coding nucleic acids of the full-length sequences of human and monkey GPRC5D (human GPRC5D (Q9NZD1, uniprot database); monkey GPRC5D (A0A2K5W6I7, uniprot database)) were respectively inserted into the PEE17.4 plasmid (Lonza, GS Xceed Expression System), and the stable cell lines were constructed through pressure screening;

[0779] The method for obtaining GS-CHO cell lines overexpressing BCMA (GS-CHO-hBCMA and GS-CHO-cynoBCMA) is as follows: the coding nucleic acids of the full-length sequences of human and monkey BCMA (human BCMA (Q02223, uniprot database); monkey BCMA (A0A2K5UD97, uniprot database)) are respectively inserted into the T2A-EGFP plasmid (GenWeichi), and stable cell lines are constructed through pressure screening.

[0780] Specifically, cultured cells were obtained or T cells were isolated from human PBMC according to the relevant instructions, and the cells were seeded in a 96-well V-bottom plate at a cell number of 1E5 / well. Then, the test and control molecules were serially diluted (see Figure 2 for details) and incubated with cells expressing the corresponding antigen at 4 degrees Celsius for 40 minutes. Then, fluorescently labeled secondary antibodies were added (diluted at 1:200; mixed with the cell death and viability stain DCM, diluted at 1:2000; CD4 and CD8 antibodies were added to the isolated human T cells to mark and distinguish the corresponding T cells, and the antibodies were diluted at 1:200). Finally, the cell fluorescence intensity was detected by flow cytometry (BD), and the curve was fitted using GraphPad Prism 8.0 to calculate the EC 50 Figures 2A-H and Tables 2-9 show the affinity of the antibodies of the present invention for binding to the corresponding antigens.

[0781] The specific results are shown in Figure 2 and Table 2-10.

[0782] Table 2. Affinity of exemplary antibodies to human BCMA-1

[0783] ND: not detected

[0784] Table 3. Affinity of exemplary antibodies to human BCMA-2

[0785] Table 4. Affinity of example antibodies to cynomolgus monkey BCMA-1

[0786] Table 5. Affinity of exemplary antibodies to human GPRC5D-1

[0787] Table 6. Affinity of exemplary antibodies to human GPRC5D-2

[0788] Table 7. Affinity of example antibodies to cynomolgus monkey GPRC5D-1

[0789] Table 8. Affinity of exemplary antibodies to human CD3-1

[0790] Table 9. Affinity of exemplary antibodies for human T cells

[0791] 3.2 Affinity determination of trispecific antibodies for cells expressing mutant antigens

[0792] 293T cell lines overexpressing BCMA mutants (293T-hBCMA-P34del, 293T-hBCMA-S30del, 293T-hBCMA-R39A, 293T-hBCMA-R27P) and GS CHO cell lines overexpressing BCMA mutants (GS CHO-hBCMA-P34del, GS CHO-hBCMA-S30del, GS CHO-hBCMA-R39A, GS The method for obtaining CHO-hBCMA-R27P is as follows: the full-length human BCMA sequence (human BCMA (Q02223, Uniprot database) is subjected to corresponding point mutations or deletion mutations, the mutated encoding nucleic acids are inserted into the T2A-EGFP plasmid (GenWeizhi), and a stable cell line is constructed through pressure screening. The amino acid sequence and encoding nucleic acid sequence of the full-length human BCMA sequence, hBCMA-P34del, hBCMA-S30del, hBCMA-R39A, and -hBCMA-R27P can be found in the sequence information.

[0793] 1Specific construction method:

[0794] After 2-3 passages, the 293T cells were revived and the culture medium was discarded. The cells were washed with 10 ml PBS and digested with 2 ml trypsin. After terminating with culture medium, the cells were centrifuged at 400 x g for 3 min and the supernatant was discarded. The cells were resuspended in culture medium and 20 μl of cell suspension was transferred to a 1.5 ml EP tube. The cells were counted and the number of cells was 1.5 x 10 7 Cells were seeded into a T175 flask, shaken evenly, and placed horizontally in a 37°C incubator for overnight culture. Remove the 293T cells that were plated in advance, discard the culture medium, and add 20 ml of DMEM culture medium containing 10% FBS and 1% PS to each flask to prepare transfection mixture 1:

[0795] Mixture 1 was slowly added to mixture 2 at once, mixed well and allowed to stand at room temperature for 15 minutes;

[0796] The transfection mixture was added to the culture medium of 293T cells, marked, and the culture flask was shaken to evenly distribute the transfection mixture in the culture medium. 4-6 hours after transfection, the culture medium was discarded and 20 mL of fresh DMEM containing 2% FBS was added. 48 and 72 hours after transfection, the cell culture supernatant was collected, centrifuged at 500 × g for 10 minutes at 4°C, and filtered through a 0.45 μm low protein binding membrane. After removing the supernatant, the cells were trypsinized with 2 mL of culture medium, centrifuged at 400 × g for 3 minutes, and the supernatant was discarded. The cells were resuspended in culture medium and placed in a culture flask to obtain 293T cell lines overexpressing BCMA mutants (293T-hBCMA-P34del, 293T-hBCMA-S30del, 293T-hBCMA-R39A, and 293T-hBCMA-R27P). The cells were placed in an incubator and continued to culture.

[0797] Mix the virus supernatant and Takara virus concentrate in a ratio of 3:1 (v / v), seal with parafilm, and incubate at 4°C overnight. Centrifuge the mixture at 1500 × g for 45 min at 4°C and discard the supernatant. Resuspend the lentiviral pellet in 1 mL of CD CHO medium, incubate at 4°C until completely dissolved, and store in aliquots at -80°C.

[0798] Construction of GS CHO cell line overexpressing BCMA mutants: GS CHO cells were collected by centrifugation at 400×g for 4 min, the supernatant was discarded, and 3 mL of complete medium was added to resuspend the cells. The cells were counted and 1×10 6 Cells were seeded in 6-well plates, and each well was filled with culture medium to 1 mL. 4 h after infection, the culture medium was added to 2 mL. 24 h after infection, the cells were placed in a centrifuge tube and centrifuged at 400 × g for 4 min to collect the cells. The virus-containing culture medium was removed, and the cells were resuspended in fresh CD CHO culture medium containing 10% FBS and 1% PS. The cells were seeded in a T25 flask and placed upright in a CO2 incubator for incubation. After 48 h of cell culture, the culture medium was added to 10 mL and the cells were placed in a CO2 incubator for further incubation. The CHO cells cultured at 37°C were removed, centrifuged at 400 × g for 4 min, and the supernatant was discarded. The cells were inoculated into a 125 mL Erlenmeyer flask and cultured in a 37°C shaker. GS CHO cell lines overexpressing BCMA mutants (GS CHO-hBCMA-P34del, GS CHO-hBCMA-S30del, GS CHO-hBCMA-R39A, and GS CHO-hBCMA-R27P) were obtained.

[0799] 2. Detection method:

[0800] The cultured 293T cell lines overexpressing BCMA mutants (293T-hBCMA-P34del, 293T-hBCMA-S30del, 293T-hBCMA-R39A, 293T-hBCMA-R27P) were removed, the culture medium was discarded, the cells were washed with 10ml PBS, digested with 2ml trypsin, and centrifuged at 400xg for 5min after termination with culture medium. The supernatant was discarded and the culture medium was resuspended. 20ul of cell suspension was transferred to a 1.5ml EP tube, counted, and plated in a 96-well U-bottom plate at a cell number of 1E5 / well. Then, the antibody to be tested and the control molecule were serially diluted. The antibody was serially diluted 3-fold with PBS, with a starting concentration of 1500nM and a total of 12 dilutions. 50ul of serially diluted drugs were incubated with the plated cells at 4 degrees Celsius for 30 minutes, and then fluorescently labeled secondary antibodies (Anti-human Fc-PE, Abcam, ab98596, diluted 1:200) was incubated at 4°C for 30 minutes. The cell fluorescence intensity was detected by flow cytometry (BD), and the curve was fitted and the EC was calculated using GraphPad Prism 8.0. 50 value.

[0801] The 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 the culture, centrifuged at 400×g for 5 minutes, and the supernatant was discarded. The cells were resuspended in the culture medium, and 20 μl of the cell suspension was transferred to a 1.5 ml EP tube. The cells were counted and seeded into a 96-well U-bottom plate at a cell number of 1E5 / well. The test and control molecules were then serially diluted. The antibody was serially diluted 3-fold with PBS, with a starting concentration of 800 nM and a total of 12 dilutions. 50 μl of the serially diluted drug was incubated with the plated cells at 4°C for 30 minutes. Fluorescently labeled secondary antibodies (diluted at 1:200) were then added and incubated at 4°C for 30 minutes. Finally, the cell fluorescence intensity was detected by flow cytometry (BD), and the curve was fitted using GraphPad Prism 8.0 to calculate the EC 50 value.

[0802] 3 Experimental results:

[0803] Figures 2I and 2J show the following results:

[0804] B3(24) can bind to cells overexpressing wild-type BCMA, cells overexpressing BCMA-P34del, cells overexpressing BCMA-P30del, cells overexpressing BCMA-R39A, and cells overexpressing BCMA-R27P;

[0805] Alnuctamab (Roche-BCMAxCD3) can bind to cells overexpressing wild-type BCMA, cells overexpressing BCMA-P34del, cells overexpressing BCMA-P30del, cells overexpressing BCMA-R39A, and cells overexpressing BCMA-R27P;

[0806] Elranatamab (Pfizer BCMAxCD3) can bind to cells overexpressing wild-type BCMA and cells overexpressing BCMA-P34del, and weakly binds to cells overexpressing BCMA-R39A;

[0807] Teclistamab / JNJ-BCMA / CD3 (J&J BCMAxCD3) can weakly bind to cells overexpressing wild-type BCMA and has extremely weak binding to cells overexpressing BCMA-R39A.

[0808] Example 4. Determination of trispecific antibody affinity by BLI

[0809] In the trispecific example antibody of the present invention, the affinity of the anti-BCMA end and the anti-CD3 end is detected by biofilm optical interferometry (BLI). The present invention is based on the BLI of the fiber optic biosensor to measure the binding kinetics of the example antibody with human BCMA, monkey BCMA, human CD3E&G and monkey CD3E&G. When the biomolecules bind to the sensor surface, a layer of biofilm is formed. The biofilm causes interference with the waveform of the light passing through the sensor. The interference phenomenon is detected in the form of phase shift, so that the change in the number of molecules bound to the sensor can be detected; the kinetic curve is fitted according to the change in the real-time response value, and k is calculated. on 、k off , K D .

[0810] Specifically, probe sensors conjugated with streptavidin protein (SA) or anti-human Fc (AHC) were pre-wetted in 200 μl of SD buffer (1x PBS, 0.1% BSA, 0.05% tween-20). Sample antibodies and biotinylated or Fc-tagged BCMA and CD3E&G heterodimer antigens were diluted in SD buffer. Then, 200 μl of SD buffer and each diluted sample (100 nM) were added to a 96-well black plate. The probes and samples were placed in an Octet (Fortebio, Red96e). Open Data Acquisition 10.0, select "New Kinetic Experiment," arrange the plate according to sample positions, select sensor positions, and set the run steps and durations: Baseline 60 s, Loading 250 s, Baseline 100 s, Association 600 s, and Dissociation 600 s. The experimental speed was 1000 rpm and the temperature was 30°C.

[0811] The results were analyzed in "Data Analysis 10.0" software. The buffer reference channel was deducted, and 1:1 binding was selected to fit the data. The kon, koff, and KD values ​​of the example antibody were calculated.

[0812] Antigen information

[0813] Reagent and material information

[0814] In the experiment performed as described in the above assay, the results are shown in Table 10. The results show that the affinities of the four exemplary antibodies of B3 to BCMA and CD3 are close to those of the F2 antibody.

[0815] Table 10 Affinity of exemplary antibodies detected by BLI

[0816] ND: not detected

[0817] Example 5. Killing experiment of human multiple myeloma cells by trispecific antibodies and cytokine release

[0818] When polyclonal antibodies bind to GPRC5D and / or BCMA on the surface of MM cells and CD3E on the surface of primary T cells, they can cross-link with T cells in a tumor-associated antigen (TAA, i.e., GPRC5D and / or BCMA)-dependent manner, stimulating T cell activation and mediating tumor cell killing. Because there is strong heterogeneity in the expression of BCMA and GPRC5D in tumor cells in actual multiple myeloma patients, this specificity is manifested in the expression of one or more antigens by different subtypes of tumor cells, or in individual differences in the levels of antigen expression by tumor cells.

[0819] KO cell line preparation and flow cytometry

[0820] H929 cells expressing BCMA and GPRC5D were prepared by RNP transfection. Using the gRNA design website (broadinstitute.org), gRNAs with high MIT specificity scores (Genescript) were selected. Three chemically synthesized gRNAs per target protein were mixed with Cas9 protein (Genscript SC1841 NA) and transfected into the cells by electroporation. After stable culture, primers were designed near the gRNA cleavage site for PCR. The amplified products were sequenced, and the cleavage efficiency was calculated using Sanger sequencing (Genescript) followed by CRISPR analysis to determine the target gene cleavage rate. The knockout cell pools were then labeled for BCMA and GPRC5D using a flow cytometer (BD), and target protein-negative cells were isolated. Results: The final selected gRNA sequences and the flow cytometric analysis results of the resulting cell lines are shown in the table below and Figure 6.

[0821] Table 11. gRNA sequences selected for knockout of H929 cell lines:

[0822] Killing assay and cytokine release

[0823] To further test the killing effect of the example antibodies in multiple myeloma, 1) H929 cells expressing a single TAA, namely BCMA-KO and GPRC5D-KO (prepared as described above); 2) H929 cells expressing both TAAs (MM cells, Nanjing Kebai, CBP60243) or L363 cells expressing both TAAs (MM cells, Nanjing Kebai, CBP60240) were co-incubated with PBMCs (Saily, SLB-HP050B) at a 10:1 effector cell to tumor cell ratio. Example antibodies and control antibodies were added, and cell death was detected 16 hours later using the lactate dehydrogenase (LDH) release assay. Calu-6 cells (NSCLC cells, Chinese Academy of Sciences Cell Bank, TCHu144), non-target cells expressing neither antigen, were co-incubated with the corresponding antibodies and PBMCs as a control for nonspecific killing. 3) H929 KO GPRC5D, H929 KO BCMA expressing a single TAA, and parental H929 cells expressing dual antigens were mixed at a 1:1:1 ratio and incubated with PBMCs. The example antibody and control antibody were added, and the survival rate of the target tumor cells was analyzed by flow cytometry after 16 hours. Reagents and Materials:

[0824] Experimental procedures: PBMC cells (Saily, SLB-HP050B) were removed from liquid nitrogen, thawed rapidly in a 37°C water bath, added to 9 ml of serum-free medium, centrifuged at 300 g for 5 min, discarded the supernatant, and resuspended in 10% FBS-free phenol red 1640 medium to adjust the cell density to 2 × 10 6 Collect logarithmic growth tumor cells and wash them twice with PBS to remove excess serum. If flow cytometry is required to detect cell death, add CTV staining solution (1 μM, CellTrace TM Violet DMSO stock solution: PBS = 1:50,000; 2E6 cells / mL CTV staining solution) incubate at 37°C in the dark for 15 minutes. If using LDH to detect tumor cell death, skip this step and centrifuge directly. Centrifuge at 300 × g for 5 minutes, discard the supernatant and retain the cell pellet. Resuspend the cells in phenol red-free medium and adjust the cell density to 4 × 10 5Cell / mL. Prepare eight 10-fold serial dilutions of the antibody in complete medium, as needed for different cell types. (For H929 cells (including KO cells), the starting concentration is 200 nM, with a maximum final concentration of 50 nM. For L363 and Calu-6 cells, the starting concentration is 400 nM, with a maximum final concentration of 100 nM.) Add the final concentration to a 96-well cell culture plate (U-bottom, COSTAR, Cat. 7007) containing 50 μL of tumor cells, 50 μL of serially diluted antibody, and 100 μL of PBMC cells for co-incubation. If LDH detection of tumor cell death is desired, establish a well for spontaneous LDH release from target cells containing only 50 μL of tumor cells, as well as a well for maximum LDH release from target cells containing 50 μL of tumor cells in Lysis buffer (provided in the LDH kit, diluted 1:10). Bring the volume to 200 μL / well with complete medium. The mixed cells were placed in a CO2 incubator at 37 degrees Celsius for 16 hours.

[0825] After incubation, the cell culture plate was centrifuged at 400g for 5 minutes. 160 μL of supernatant was aspirated from each well and 50 μL of supernatant was transferred to a new 96-well flat-bottom plate (Biyuntian, Cat. FPT019) for LDH assay. The remaining supernatant was frozen (-80°C) for subsequent cytokine assays. The cell pellet was used for flow cytometry analysis of cell death.

[0826] LDH detection of tumor cell death: Add 50 μL of cell supernatant to each well of a 96-well white-bottom plate, followed by 50 μL of detection solution. Incubate at room temperature in the dark for 10-30 minutes (depending on the cell type and reaction color), then add 50 μL of stop solution. Read the absorbance at 492 / 650 nm on a multifunctional microplate reader (SPARK) within 1 hour after adding the stop solution. The wells containing zero concentration of antibody plus tumor cells and immune cells serve as the background, the wells containing only tumor cells serve as the minimum value, and the wells containing tumor cells treated with lysis solution serve as the maximum value. Killing is calculated as (sample value - background value) / (maximum value - minimum value).

[0827] Flow cytometry for tumor cell death: 200 μL FACS buffer (1XPBS, 2% FBS, 2mM EDTA) was added to each well of the above cell culture plate, washed once, centrifuged and the liquid was decanted, 45 μL DCM staining solution (1:2000 prepared with FACS buffer) was added to each well, incubated at 4°C in the dark for 20 minutes, washed once with 200 μL FACS buffer, dried, resuspended in 150 μL FACS buffer, and analyzed on a flow cytometer (BD). Killing was calculated as DCM + CTV + Cell count / CTV+ Total cell count.

[0828] Cisbio cytokine detection: Dissolve the standard in H2O to create the Standard Stock solution; dilute it 3-fold with Diluent to create Standard 7 (Std 7); then perform a 2-fold serial dilution, with a total of 6 wells (Std 1-6), and Std 0 as the Diluent blank control. Add 16 μL of the supernatant to be tested to each well of a 96-well plate (#66PL96025); similarly, add 16 μL of the standard to each well. Both Eu Cryptate and d2antibody are diluted 1:20 in detection buffer. The two dilutions are mixed 1:1, and 4 μL of the mixture is added to each well. Seal the plate with sealing film and incubate at room temperature for 2 hours. Emission signals are detected using a multifunctional microplate reader (SPARK), and the 665 nm / 620 nm ratio represents the relative value of cytokine content.

[0829] Experimental results: The exemplary antibodies can activate and mediate the killing effect of PBMC on MM cells in a dose-dependent manner ( Figures 3A-3O , Tables 12-24 ).

[0830] 1. The killing results of the corresponding antibodies in H929 ko GPRC5D and H929 ko BCMA expressing a single TAA are shown in Figures 3A-3F and Tables 12-16. The specific experimental results are as follows:

[0831] The example antibodies, like the corresponding antibody control molecules, can activate T cells in PBMC to kill them in a dose-dependent manner in cells expressing TAAs. In contrast, no killing effect was observed in non-target cells that do not express TAAs (Figure 3P). This indicates that the killing is a specific killing that depends on binding to the corresponding TAA. The higher the anti-CD3 end affinity of the example antibody, the better its ability to kill tumor cells, that is, the molecule whose antigen binding region specifically binds to CD3 contains the heavy chain variable region from sp34.24 (SEQ ID NO: 77) (i.e., marked with (24) in the figure) is stronger than the molecule whose antigen binding region specifically binds to CD3 contains the heavy chain variable region from sp34.87 (SEQ ID NO: 78) (i.e., marked with (87) in the figure).

[0832] In the cytotoxicity assay against H929-KO GPRC5D cells, the B3, B3b, and B5 versions of the exemplary antibodies showed superior tumor cell cytotoxicity compared to the F2 version. This suggests that the scFv-free design of B3, B3b, and B5 enhances BCMA function.

[0833] In terms of mediating PBMC killing of H929 ko BCMA, the ability of the exemplary antibodies in the form of B3 and B5 to mediate tumor cell killing was close to that of the molecule in the form of F2 and was better than that of the molecule in the form of B3b.

[0834] The above results indicate that the anti-GPRC5D and anti-BCMA ends of the exemplary antibodies in the form of B3, B5, and B3b can successfully serve as TAA antibodies for TCE and play a role in mediating T cell killing of tumor cells.

[0835] 2. In cells H929 and L363 expressing both antigens (Figures 3G-3M, Tables 18-24): BCMA and GPRC5D levels on the L363 cell surface were relatively low; BCMA and GPRC5D levels on the H929 cell surface were relatively high (compared to the antibodies disclosed in WO2022174813). The specific results are shown below:

[0836] In H929 cells, as observed above, molecules whose antigen-binding regions specifically bind to CD3 and whose heavy chain variable regions are derived from sp34.24 (SEQ ID NO: 77) are more potent than molecules whose antigen-binding regions specifically bind to CD3 and whose heavy chain variable regions are derived from sp34.87 (SEQ ID NO: 78). Furthermore, the killing activity of the trispecific exemplary antibodies B3 (24) and B3-7F5.3 (24) was superior to that of JNJ's bispecific control molecules, i.e., JNJ-BCMA / CD3 and JNJ-GPRC5D / CD3, and was slightly weaker or comparable to that of Roche's bispecific control molecules, i.e., Roche-GPRC5D and Roche-BCMA / CD3.

[0837] At the same time, in the comparison of cytokine release induced during H929 cell killing, there was no significant difference in cytokine release induced by the trispecific exemplary antibodies and the control bispecific antibodies, namely JNJ-BCMA / CD3, JNJ-GPRC5D / CD3 and REGN-BCMA / CD3 ( Figures 3Q-3R ).

[0838] In the absence of target cells, the trispecific example antibodies, like the control bispecific antibodies, namely JNJ-BCMA / CD3, JNJ-GPRC5D / CD3, Roche-GPRC5D / CD3, Roche-BCMA / CD3, and REGN-BCMA / CD3, did not induce nonspecific cytokine release, that is, the level was comparable to that of the hIgG control ( Figures 3S-3T ).

[0839] In L363 cells, the exemplary trispecific antibodies B3 (24) and B3-7F5.3 (24) showed stronger killing effects than JNJ's bispecific control molecules (JNJ-BCMA / CD3, JNJ-GPRC5D / CD3), and comparable to Roche's control bispecific molecules (Roche-GPRC5D / CD3, Roche-BCMA / CD3). When used together with control molecules (Inno-B, Inno-G or Inno-G-7F5.3) for killing, both the anti-GPRC5D end and the anti-BCMA end of the exemplary trispecific antibodies of the present invention can play a role; when the control BCMA / CD3 and GPRC5D / CD3 bispecific molecules of JNJ and Roche were mixed at a ratio of 1:1 and compared with the exemplary trispecific antibodies of the present invention at the same concentration, the killing of L363 cells mediated by the exemplary trispecific antibodies of the present invention was superior to the combination of the control BCMA / CD3 and GPRC5D / CD3 bispecific molecules. The above results indicate that the anti-GPRC5D end and anti-BCMA end of the trispecific antibody have a mutual synergistic effect on killing tumor cells, which is better than the combination of two bispecific antibodies.

[0840] 3. After a 1:1:1 mixture of single TAA-expressing H929 ko GPRC5D and H929 ko BCMA cells and dual-antigen-expressing parental H929 cells, the killing curve of the exemplary trispecific antibody of the present invention was higher than that of the control molecule, i.e., its maximum killing ratio was higher than that of the control molecule ( Figures 3N , 3O ).

[0841] Table 12 illustrates the antibody-mediated killing effect of PBMC on H929 ko GPRC5D cells. NA means not analyzed.

[0842] Table 13 shows the killing effect of PBMC on H929 ko GPRC5D cells mediated by antibodies-2

[0843] Table 14 shows the killing effect of PBMC on H929 ko GPRC5D cells mediated by antibodies-3

[0844] Table 15 illustrates the antibody-mediated killing of H929 ko BCMA cells by PBMC-1. NA indicates not analyzed.

[0845] Table 16 illustrates the antibody-mediated killing of H929 ko BCMA cells by PBMC-2. NA indicates not analyzed.

[0846] Table 17 illustrates the antibody-mediated killing of H929 ko BCMA cells by PBMC-3. NA indicates not analyzed.

[0847] Table 18 illustrates the antibody-mediated killing of H929 cells by PBMC-1.

[0848] Table 19 illustrates antibody-mediated killing of H929 cells by PBMC-2.

[0849] Table 20 shows the killing effect of PBMC on H929 cells mediated by antibodies-3

[0850] Table 21 illustrates antibody-mediated killing of L363 cells by PBMC-1.

[0851] Table 22 illustrates antibody-mediated killing of L363 cells by PBMC-2.

[0852] Table 23 illustrates antibody-mediated killing of L363 cells by PBMC-3.

[0853] Table 24 illustrates antibody-mediated killing of L363 cells by PBMC-4.

[0854] Killing assay against cells expressing mutant antigens

[0855] Experimental procedures: PBMC cells (Saily, SLB-HP100B) were removed from liquid nitrogen, thawed rapidly in a 37°C water bath, added to 9 ml of serum-free medium, centrifuged at 300 g for 5 min, discarded the supernatant, and resuspended in 10% FBS-free phenol red 1640 medium to adjust the cell density to 2 × 10 6 cell / ml. Collect logarithmically growing tumor cells and wash them twice with PBS to remove excess serum. Centrifuge the GS CHO cell line overexpressing the BCMA mutant constructed in Example 1 at 300×g for 5 minutes, discard the supernatant and retain the cell pellet. Resuspend the cells in phenol red-free medium and adjust the cell density to 4×10 5cell / mL. The antibody was diluted 10-fold in a gradient using complete culture medium for a total of 8 concentrations (the starting concentration during preparation was 200nM and the maximum final concentration was 50nM), with the last concentration being 0. 50μL of tumor cells, 50μL of gradiently diluted antibodies, and 100μL of PBMC cells were added to a 96-well cell culture plate (U-bottom, COSTAR, Cat.7007) for co-incubation. A spontaneous LDH release well for target cells with only 50μL of tumor cells and a maximum LDH release well for target cells with 50μL of tumor cells added to Lysis buffer (provided with the LDH kit, diluted 1:10) were set up, and the volume was made up to 200μL / well with complete culture medium. The mixed cells were cultured in a 37°C CO2 incubator for 16 hours.

[0856] After co-incubation, the cell culture plate was centrifuged at 400 g for 5 min, 160 μL of supernatant was aspirated from each well, and 50 μL of supernatant was transferred from each well to a new 96-well flat-bottom plate (Biyuntian, Cat. FPT019) for LDH detection.

[0857] LDH detection of tumor cell death: Add 50 μL of cell supernatant to each well of a 96-well white-bottom plate, followed by 50 μL of detection solution. Incubate at room temperature in the dark for 10-30 minutes (depending on the cell type and reaction color), then add 50 μL of stop solution. Read the absorbance at 492 / 650 nm on a multifunctional microplate reader (SPARK) within 1 hour after adding the stop solution. The wells containing zero concentration of antibody plus tumor cells and immune cells serve as the background, the wells containing only tumor cells serve as the minimum value, and the wells containing tumor cells treated with lysis solution serve as the maximum value. Killing is calculated as (sample value - background value) / (maximum value - minimum value).

[0858] Experimental results: B3(24) can activate PBMC in a dose-dependent manner to kill GS CHO overexpressing BCMA mutants (GS CHO-hBCMA-P34del, GS CHO-hBCMA-S30del, GS CHO-hBCMA-R39A, GS CHO-hBCMA-R27P); Teclistamab has no in vitro killing function on BCMA mutants R27P and R39A (Figure 3U).

[0859] Example 6. Trispecific Antibodies Overcome the Effects of sBCMA

[0860] Soluble BCMA (sBCMA) is shed from the plasma cell surface by γ-secretase cleavage. It is elevated in MM patients and correlates with adverse clinical outcomes (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5395119 / ). sBCMA consists of an extracellular domain and a portion of the BCMA transmembrane domain. It not only reduces the density of target antigens but also provides a soluble decoy that can limit the effectiveness of BCMA / CD3 molecules. Because it contains the GPRC5D end, which is not restricted by sBCMA, the GPRC5D / BCMA / CD3 molecule is theoretically less affected by sBCMA.

[0861] Experimental procedures: Human cryopreserved PBMC cells (SAILY) were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, added to 9 ml of serum-free medium, centrifuged at 300 g for 4 min, discarded the supernatant, and resuspended in 1% FBS-free phenol red-free 1640 medium (complete medium) to adjust the cell density to 4 × 10 6 Collect the cell culture medium into a 50ml sterile centrifuge tube, centrifuge at 300×g for 5 minutes, discard the supernatant and retain the cell pellet. Use complete culture medium to adjust the cell density to 4×10 5 cell / mL. The antibody was diluted to 200nM with complete medium and diluted 10-fold in a gradient. At the same time, human recombinant BCMA (hBCMA; the stock solution was 0.5mg / mL, prepared by PBS) was diluted to 800ng / mL using complete medium to obtain sBCMA solution. 50μL of tumor cells (H929), 50μL of gradient diluted antibodies, 50μL of PBMC cells and 50μL of sBCMA solution were added to a 96-well cell culture plate (U-shaped, Costar). At this time, there was 200ng / mL of sBCMA in the co-incubation system. Place in a 37-degree Celsius CO2 incubator and culture for 16h. After co-incubation, the cell culture plate was centrifuged at 400g for 5min, and 50μL of supernatant was transferred from each well to a new 96-well flat-bottom plate (Biyuntian, Cat.FPT019) for LDH detection. The cell pellet was detected by flow cytometry (BD) to determine the percentage of CD69-positive cells in T cells. For information on the cells used, see Example 5.

[0862] LDH detection of tumor cell death: Add 50 μL of cell supernatant to each well of a 96-well white-bottom plate, followed by 50 μL of detection solution. Incubate at room temperature in the dark for 10-30 minutes (depending on the cell type and reaction color), then add 50 μL of stop solution. Read the absorbance at 492 / 650 nm on a multifunctional microplate reader (SPARK) within 1 hour after adding the stop solution. The wells containing zero concentration of antibody plus tumor cells and immune cells serve as the background, the wells containing only tumor cells serve as the minimum value, and the wells containing tumor cells treated with lysis solution serve as the maximum value. Killing is calculated as (sample value - background value) / (maximum value - minimum value).

[0863] Flow cytometry for CD8 and CD4 T cell activation: Wash each well of the cell culture plate with 200 μL of FACS buffer (1X PBS, 2% FBS, 2 mM EDTA). Centrifuge and discard the liquid. Add 45 μL of staining solution (prepared with FACS buffer) to each well. Incubate at 4°C in the dark for 20 min. Wash once with 200 μL of FACS buffer, drain dry, and resuspend in 150 μL of FACS buffer before analysis. Calculate the percentage of CD69-positive cells among CD8 and CD4 T cells.

[0864] Reagents and materials:

[0865] The experimental results are shown in Figures 4A, 4B, and 4C, and Tables 25 and 26. These results indicate that trispecific antibody-mediated T cell activation and H929 cell killing are less affected by sBCMA.

[0866] Table 25 illustrates antibody-mediated activation of T cells in PBMC (w / o: without, w: with)

[0867] Table 26 illustrates antibody-mediated activation of T cells in PBMC (w / o: without, w: with)

[0868] Example 7. Antitumor efficacy of trispecific antibodies in a tumor-bearing humanized mouse model

[0869] Female NOG mice (35-48 days old) were purchased from Beijing Weitongda Laboratory Animal Technology Co., Ltd. They were SPF grade. Upon arrival, the mice were acclimated and quarantined for 5-7 days before the study began.

[0870] Tumor cells were routinely subcultured for subsequent in vivo experiments, and cells were collected by centrifugation and dispersed with PBS. NOG mice were intravenously inoculated with PBMC cells at 4x10 6On the third day after PBMC inoculation, the right dorsal and abdominal areas of the mice were shaved and tumor cells were subcutaneously inoculated at a volume of 200 ul per mouse (inoculation density: H929 ko BCMA: 4x10 6 pcs / pcs,H929 ko GPRC5D:4x10 6 MM.1S (MM cells, Nanjing Kebai, CBP60239): 3x10 6 pcs / pcs,L363:1x10 6 / mouse. On the 7th day after tumor cell inoculation, mice were divided into groups according to tumor volume (6-7 mice per group) and intraperitoneally administered (see Figure 5 and the accompanying figure for details on the frequency and dosage of administration). The tumor volume and body weight of mice were monitored twice a week. Tumor volume measurement: A vernier caliper was used to measure the maximum long axis (L) and maximum wide axis (W) of the tumor, and the tumor volume was calculated according to the following formula: V = L × W2 / 2. Body weight was measured using an electronic balance. During the entire study, mice were euthanized when the tumor reached the endpoint or when the mice had a weight loss of >20%. Tumor size was counted.

[0871] Experimental results:

[0872] H929 ko GPRC5D tumor (Figure 5A): B3(24) molecule can effectively inhibit the growth of H929 ko GPRC5D cells, and its anti-tumor ability is consistent with that of JNJ-BCMA / CD3 control molecule. It is stronger than B3(87) molecule, REGN-BCMA / CD3 molecule and F2(87) molecule.

[0873] H929 ko BCMA tumor (Figure 5B): B3(24) molecules can effectively inhibit the growth of H929 ko BCMA cells, and its anti-tumor ability is consistent with that of the JNJ-GPRC5D / CD3 control molecule; its anti-tumor ability is stronger than that of all BCMA / CD3 and (JNJ-GPRC5D / CD3+JNJ-BCMA / CD3) combinations; it is slightly weaker than that of Roche-GPRC5D / CD3 and (Roche-BCMA / CD3+Roche-GPRC5D / CD3) combinations.

[0874] MM1S tumor (Figure 5C): Both the example antibodies and the control molecules showed good anti-tumor effects.

[0875] L363 tumor (Figure 5D): B3 (24) molecules can effectively inhibit the growth of L363 cells. Its anti-tumor ability is stronger than that of the combination of JNJ-GPRC5D / CD3, JNJ-BCMA / CD3 and (JNJ-BCMA / CD3+JNJ-GPRC5D / CD3); and is closer to that of the combination of Roche-GPRC5D / CD3, Roche-BCMA / CD3 and (Roche-GPRC5D / CD3+Roche-GPRC5D / CD3).

[0876] The B3(24) molecule showed no difference in effect on mouse body weight compared with other control molecules in all models.

[0877] 2. Antibody sequence from which the antigen-binding region is derived

[0878] 3. Other sequences

[0879] 4. Specific sequence information

Claims

1. A trispecific 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 trispecific antibody of claim 1, wherein the second antigen binding region specifically binds CD3, and / or the third antigen binding region specifically binds GPRC5D.

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

4. The trispecific antibody of claim 3, wherein (1) The first Fab 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 of the Fc heterodimer; the second Fab 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, and the third Fab is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of the VH of the Fab heavy chain of the second Fab fragment; or The third Fab 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 of the Fc heterodimer; the second Fab 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, and the first Fab is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of the VH of the Fab heavy chain of the second Fab fragment; or (2) the first Fab 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 of the Fc heterodimer; the second Fab 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, and the third Fab is fused at the N-terminus of the VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab; or The third Fab 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 of the Fc heterodimer; the second Fab 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, and the first Fab is fused at the N-terminus of VH of the Fab heavy chain to the C-terminus of the Fc region fused to the second Fab. The trispecific antibody of claim 4 , wherein the fusion comprises direct fusion or fusion through a linker. The trispecific antibody of claim 5 , wherein the linker is (GGGGS)n, wherein n=1, 2, 3 or 4.

7. The trispecific antibody of any one of claims 3-6, 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. The trispecific antibody of 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 and VL, and the VH comprises 39D, and the VL comprises 38K, and The first or third Fab fused to the second Fab via the Fc region comprises VH and VL, wherein the VH comprises 39K, and the VL comprises 38D; or The second Fab comprises VH and VL, and the VH comprises 39K, and the VL comprises 38D, and The first or third Fab fused to the second Fab via the Fc region comprises VH and VL, wherein the VH comprises 39D and the VL comprises 38K.

9. The trispecific antibody of claim 7 or 8, wherein the disulfide bond remodeling mutations comprise F126C in CH1 of Fab and Q124C in CL of Fab.

10. The trispecific antibody of claim 9, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1, wherein the CH1 (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 85, and comprises F126C; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:

85.

11. The trispecific antibody of claim 9 or 10, wherein the Fab comprising disulfide bond remodeling mutations comprises CL, wherein the CL (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:97, and comprises Q124C; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:

97.

12. The trispecific antibody of claim 9, wherein the Fab comprising disulfide bond remodeling mutations comprises CH1 and CL, wherein the CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85, and the CL comprises or consists of the amino acid sequence shown in SEQ ID NO:

97.

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

14. The trispecific antibody of claim 13, wherein the Fab comprising a disulfide bond remodeling mutation comprises CH1, wherein the CH1 (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:86, and comprises F126C and C220S; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:

86.

15. The trispecific antibody of claim 13 or 14, wherein the Fab comprising disulfide bond remodeling mutations comprises CL, wherein the CL (i) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:98, and comprises Q124C and C214S; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:

98.

16. The trispecific antibody of claim 13, wherein the Fab comprising disulfide bond remodeling mutations comprises CH1 and CL, wherein the CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 86, and the CL comprises or consists of the amino acid sequence shown in SEQ ID NO:

98.

17. The trispecific antibody of any one of claims 1-16, wherein the trispecific 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.

18. The trispecific antibody of claim 17, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, e.g., comprising or consisting of the amino acid sequence SEQ ID NO: 87 or 88 or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto.

19. The trispecific antibody of claim 18, wherein the first and / or second Fc region comprises a L234A / L235A mutation.

20. The trispecific antibody of claim 19, wherein the first and / or second Fc region comprises the amino acid sequence of SEQ ID NO: 89 or 90; or comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity, to the amino acid sequence shown in SEQ ID NO: 89 or 90 and comprises the amino acid substitution L234A / L235A.

21. The trispecific antibody of any one of claims 17-20, wherein one of the first and second Fc regions comprises a Knob mutation and the other comprises a Hole mutation.

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

23. The trispecific antibody of claim 22, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:91, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:

92.

24. The trispecific antibody of claim 23, wherein 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.

25. The trispecific antibody of 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 and T366S, L368A and Y407V (numbering according to EU index).

26. The trispecific antibody of claim 25, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:94, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence shown in SEQ ID NO:

93.

27. The trispecific antibody of claim 26, wherein one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:94, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:

93.

28. The trispecific antibody of any one of claims 1-27, 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 HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:57; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:58; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:59; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:61; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:62; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

63.

29. The trispecific antibody of claim 28, wherein the VH of the first antigen-binding region that specifically binds to BCMA comprises or consists of the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

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

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

60.

32. The trispecific antibody of claim 28, wherein the first antigen-binding region that specifically binds to BCMA comprises VH and VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K For example, The VH of the first antigen binding region (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 76, or (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:76, and comprises a Q39K mutation; and / or the VL of the first antigen binding region (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, or (ii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:75, and comprises the Q38D mutation.

33. The trispecific antibody of any one of claims 1-32, 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, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:41; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:42; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:43; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:45; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:46; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:47; or HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:49; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:50; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:51; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:53; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:54; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

55.

34. The trispecific antibody of claim 33, wherein the VH that specifically binds to the third antigen binding region of GPRC5D comprises (i) an amino acid sequence as shown in SEQ ID NO:40, or an amino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence; or (ii) the amino acid sequence of SEQ ID NO:48, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

35. The trispecific antibody of claim 33 or 34, wherein the VL that specifically binds to the third antigen binding region of GPRC5D comprises (i) the amino acid sequence of SEQ ID NO:44, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consisting of said amino acid sequence; or (ii) the amino acid sequence of SEQ ID NO:52, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

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

44.

37. The trispecific antibody of any one of claims 33-36, wherein the third antigen-binding region that specifically binds to GPRC5D comprises VH and VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K, e.g. The VH that specifically binds to the third antigen binding region of GPRC5D (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 80, or (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:80, and comprises a Q39K mutation; And the VL that specifically binds to the third antigen binding region of GPRC5D (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 79, or (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:79, and comprises the Q38D mutation.

38. The trispecific antibody of any one of claims 1-37, wherein the second antigen-binding region specifically binds 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 HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:65; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:66; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:67 or 73; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:69; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:70; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

71.

39. The trispecific antibody of claim 38, wherein the VH of the second antigen-binding region 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

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

41. The trispecific antibody of claim 40, wherein the second antigen-binding region that specifically binds to CD3 comprises VH and VL, wherein 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. The trispecific antibody of any one of claims 38-41, wherein the second antigen-binding region that specifically binds to CD3 comprises VH and VL, and the VH comprises 39K and the VL comprises 38D; or the VH comprises 39D and the VL comprises 38K.

43. The trispecific antibody of claim 42, wherein the VH that specifically binds to the second antigen binding region of CD3 (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 77 or 78, or (ii) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 77 or 78, and comprises the Q39D mutation; And the VL that specifically binds to the second antigen binding region of CD3 (i) comprises or consists of the amino acid sequence shown in SEQ ID NO: 74, or (ii) comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:74, and comprises a Q38K mutation.

44. The trispecific antibody according to any one of claims 3 to 43, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, and which is composed of the following peptide chains: A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D, A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D, A peptide chain 3# that contains a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain. A peptide chain 4# comprising a Fab light chain that specifically binds to BCMA, and A peptide chain 5# comprising a Fab light chain that specifically binds to CD3, The Fab heavy chain of peptide chain 1# and the Fab light chain of peptide chain 2# are paired to form the third 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 first and second Fab.

45. The trispecific antibody of claim 44, wherein Peptide chain 1# comprises the amino acid sequence shown in SEQ ID NO:5, or comprises 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. An amino acid sequence, or consisting of the amino acid sequence; Peptide chain 2# comprises the amino acid sequence shown in SEQ ID NO:4, or comprises 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:4, or consists of the amino acid sequence; Peptide chain 3# comprises the amino acid sequence shown in SEQ ID NO:3 or 6, or comprises 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; Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO:2, or comprises 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:2, or consists of the amino acid sequence; and / or Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

46. ​​The trispecific antibody of claim 44, wherein Peptide chain 1# comprises the amino acid sequence shown in SEQ ID NO:15, or comprises 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; Peptide chain 2# comprises the amino acid sequence shown in SEQ ID NO:14, or comprises 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:14, or consists of the amino acid sequence; Peptide chain 3# comprises the amino acid sequence shown in SEQ ID NO:3 or 6, or comprises 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; Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO:2, or comprises 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:2, or consists of the amino acid sequence; and / or Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

47. The trispecific antibody according to any one of claims 1 to 43, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which is composed of the following peptide chains: A peptide chain 1# that contains a Fab heavy chain and an Fc domain that specifically binds to BCMA. A peptide chain 2# containing a Fab light chain that specifically binds to BCMA, A peptide chain 3# comprising a Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and an Fc domain, in sequence, A peptide chain 4# which in turn comprises a Fab light chain that specifically binds to GPRC5D, and A peptide chain 5# comprising a Fab light chain that specifically binds to CD3, The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired 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 to form the third and second Fab.

48. The trispecific antibody of claim 47, wherein Peptide chain 1# comprises the amino acid sequence of SEQ ID NO:11, or comprises 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 of SEQ ID NO:11, or consists of the amino acid sequence; Peptide chain 2# comprises the amino acid sequence shown in SEQ ID NO:10, or comprises 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:10, or consists of the amino acid sequence; Peptide chain 3# comprises the amino acid sequence shown in SEQ ID NO:9, or comprises 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:9, or consists of the amino acid sequence; Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO:8, or comprises 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:8, or consists of the amino acid sequence; and / or Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence.

49. The trispecific antibody according to any one of claims 1 to 43, which is a bilaterally asymmetric IgG-like pentamer composed of five polypeptide chains, which is composed of the following peptide chains: A peptide chain 1# comprising a Fab heavy chain and an Fc domain that specifically bind to GPRC5D, A peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D, A peptide chain 3# comprising a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain that specifically binds to the antigen binding region of BCMA, A peptide chain 4# comprising a Fab light chain that specifically binds to CD3, and A peptide chain 5# of a Fab light chain containing an antigen binding region that specifically binds to BCMA, The Fab heavy chain of peptide chain #1 and the Fab light chain of peptide chain #2 are paired to form the third 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 second and first Fab.

50. The trispecific antibody of claim 49, wherein Peptide chain 1# comprises the amino acid sequence shown in SEQ ID NO:5, or comprises 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, or consists of the amino acid sequence; Peptide chain 2# comprises the amino acid sequence shown in SEQ ID NO:4, or comprises 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:4, or consists of the amino acid sequence; Peptide chain 3# comprises the amino acid sequence shown in SEQ ID NO:7, or comprises 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:7, or consists of the amino acid sequence; Peptide chain 4# comprises the amino acid sequence shown in SEQ ID NO:1, or comprises 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:1, or consists of the amino acid sequence; and / or Peptide chain 5# comprises the amino acid sequence shown in SEQ ID NO:2, or comprises 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:2, or consists of the amino acid sequence.

51. A nucleic acid molecule encoding any one chain of the trispecific antibody according to any one of claims 1 to 50, or consisting of said nucleic acid sequence.

52. An expression vector comprising the nucleic acid molecule of claim 51, for example, the expression vector is a pCNDA vector, such as a pCDNA3.1 expression vector.

53. A host cell comprising the nucleic acid molecule of claim 51 or the expression vector of claim 52, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as HEK293 cells.

54. A method for preparing the trispecific antibody of any one of claims 1-50, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 51 or the expression vector of claim 52 under conditions suitable for expression of the chains of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

55. An immunoconjugate comprising the trispecific antibody of any one of claims 1-50.

56. A pharmaceutical composition or medicament or formulation comprising the trispecific antibody according to any one of claims 1 to 50, or the immunoconjugate according to claim 55, and optionally a pharmaceutically acceptable excipient.

57. A pharmaceutical combination comprising the trispecific antibody of any one of claims 1 to 50, or the immunoconjugate of claim 55, and one or more other therapeutic agents, such as chemotherapeutic agents.

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

59. The method of claim 58, wherein the cancer is a GPRC5D single-positive cancer, a BCMA single-positive cancer, a GPRC5D and BCMA double-positive cancer, or a BCMA and GPCR5D double-low-expressing cancer, such as a GRPC5D low-expressing and / or BCMA low-expressing cancer (e.g., a cancer that relapsed after anti-BCMA treatment or anti-GPRC5D treatment).

60. The method of claim 58 or 59, wherein the cancer is a solid tumor or a hematological tumor, for example the cancer is multiple myeloma.

61. The method of any one of claims 58-60, wherein the method further comprises administration in combination with other therapies, such as treatment modalities (eg, surgery or radiation therapy) and / or other therapeutic agents (eg, chemotherapeutic agents).

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