Multispecific antibodies targeting BCMA, GPRC5D, and T cells and their applications

Abstract

This application relates to multi-specific binding molecules targeting BCMA, GPRC5D, and T cell receptors. Specifically, this application discloses a BCMA-GPRC5D-CD3 multi-specific antibody, which can bind to tumor surface antigens, activate T cells, and further promote specific killing of tumor cells by T cells, especially multiple myeloma positive for BCMA or GPRC5D. At the same time, this application further provides a method for manufacturing the multi-specific binding molecule and its applications.

Description

Technical Field

[0001] <Cross - reference to related applications> This application claims the priority of a Chinese patent application with application number 202210631397.9, filed on June 6, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0002] <Technical Field> The present disclosure relates to the field of biopharmaceuticals, and specifically, to multispecific antibodies targeting BCMA, GPRC5D, and T cells, and their applications.

Background Art

[0003] Multiple myeloma (MM) is a plasma cell malignancy, characterized by unrestricted proliferation of plasma cells in the bone marrow like tumor cells, accompanied by the secretion of monoclonal immunoglobulins, which causes a series of clinical symptoms such as multiple osteolytic lesions, hypercalcemia, anemia, kidney damage, and recurrent infections. The incidence of multiple myeloma accounts for 1% of all tumors and 10 - 15% of hematological malignancies. The male - to - female ratio is 1.6:1, and the age of the majority of patients is >40 years. The treatment of multiple myeloma includes chemotherapy and hematopoietic stem cell transplantation. Here, immunomodulatory agents represented by lenalidomide and protease inhibitors represented by bortezomib show good medicinal effects in the form of single - agent or combination use, and have become common treatment means for multiple myeloma patients. However, multiple myeloma is still considered an incurable disease. Current treatment means only relieve the symptoms of multiple myeloma and none of them can completely remove the tumor, and most patients will eventually relapse. Therefore, there is an urgent need for new treatment methods.

[0004] In recent years, CAR - T therapy or ADC targeted chemotherapy has been used in multiple myeloma and achieved remarkable progress. In particular, CAR T targeting B - cell maturation antigen (BCMA) and antibody - ADC drugs targeting BCMA have shown positive clinical effects in the treatment of multiple myeloma, and the sales of various drugs have been approved.

[0005] B cell maturation antigen (BCMA), also known as CD269 or TNFRSF17, is a member of the tumor necrosis factor receptor superfamily and was first discovered in the early 1990s. It is mainly expressed on the surface of mature B lymphocytes and plasma cells and is a marker protein for B lymphocyte maturation. Structurally, BCMA consists of three main domains: an extracellular segment (aa1 - 54), a transmembrane region (aa55 - 77), and an intracellular segment (aa78 - 184). B cell activating factor (BAFF) and a proliferation - inducing ligand (APRIL) are the main ligands of BCMA. Through interaction with BCMA, they transmit cell - stimulating signals, activate the TRAF - dependent NF - κB and JNK pathways, and increase the proliferation and survival rate of B cells. BCMA is mainly expressed on the surface of mature B lymphocytes and plasma cells and is scarcely expressed in other tissue cells. And since BCMA is highly expressed in the tumor cells of the majority of multiple myeloma patients, it is an ideal therapeutic target for multiple myeloma.

[0006] However, for multiple myeloma patients with BCMA - negative or low - expressing BCMA, and patients who relapse after BCMA - targeted therapy, the emergence of more effective treatment means is urgently needed. Therefore, for the treatment of multiple myeloma, it is necessary to find other more potential new targets.

Summary of the Invention

Means for Solving the Problems

[0007] To solve the above - mentioned technical problems, the present application provides the following technical solutions. In a first aspect, the present disclosure provides a multispecific binding molecule, and the multispecific binding molecule (a) a first antigen - binding molecule (ABM1) that specifically binds to a first tumor - associated antigen (TAA1), (b) a second antigen - binding molecule (ABM2) that specifically binds to a second tumor - associated antigen (TAA2), (c) and a third antigen - binding molecule (ABM3) that specifically binds to an antigen expressed on human immune cells.

[0008] In a second aspect, the disclosure provides an isolated nucleic acid that encodes the aforementioned multispecific binding molecule.

[0009] In a third aspect, the disclosure provides a recombinant vector that contains the aforementioned isolated nucleic acid.

[0010] In a fourth aspect, the disclosure provides a host cell that contains the aforementioned isolated nucleic acid or the aforementioned recombinant vector.

[0011] In a fifth aspect, the disclosure provides a method for producing a multispecific binding molecule that includes culturing the aforementioned host cell under suitable conditions and isolating the multispecific binding molecule.

[0012] In a sixth aspect, the disclosure provides a pharmaceutical composition that includes the aforementioned multispecific binding molecule, the aforementioned isolated nucleic acid, the aforementioned recombinant vector, the aforementioned host cell, or a product produced according to the aforementioned method, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0013] In a seventh aspect, the disclosure provides a method for activating T cells that includes administering to a subject a therapeutically effective amount of the aforementioned multispecific binding molecule or pharmaceutical composition.

[0014] In an eighth aspect, the disclosure provides the use of the aforementioned multispecific binding molecule, the aforementioned isolated nucleic acid, the aforementioned host cell, a product produced by the aforementioned method, or the aforementioned pharmaceutical composition for manufacturing a drug for treating and / or preventing cancer, or the use for treating and / or preventing cancer.

[0015] In a ninth aspect, the disclosure provides a method for treating and / or preventing cancer that includes administering to a subject a therapeutically effective amount of the aforementioned multispecific binding molecule, the aforementioned isolated nucleic acid, the aforementioned host cell, a product produced by the aforementioned method, or the aforementioned pharmaceutical composition.

[0016] The beneficial effects are as follows. The development of bispecific multi-specific antibodies and related bioproducts targeting GPRC5D and BCMA can cover more tumor populations, bring about a more thorough tumor clearance effect, have the advantage of delaying tumor recurrence, and there is a huge potential clinical need.

Brief Description of the Drawings

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[0018] Definition and Explanation of Terms Unless otherwise specifically defined in the present disclosure, scientific and technical terms related to the present disclosure have the meanings understood by those skilled in the art.

[0019] Also, unless otherwise specifically explained in this specification, singular terms in this specification should include the plural form, and plural terms should include the singular form. More specifically, unless otherwise clearly indicated, the singular forms "one" and "such" used in this specification and the appended claims include multiple referents.

[0020] The terms "comprising", "including", and "having" used in this specification are used synonymously and are intended to indicate the inclusiveness of the scheme, meaning that other elements may exist in addition to the listed elements in the said scheme. At the same time, it should be understood that the descriptions "comprising", "including", and "having" used in this specification also provide a scheme of "consisting of...".

[0021] The term "and / or", when used in this specification, includes the meanings of "and", "or", and "all or any other combination of elements linked by the terms belonging to".

[0022] As used herein, the term "BCMA" is an abbreviation for B cell maturation antigen and belongs to the tumor necrosis factor receptor family. BCMA is mainly expressed on the surface of late B cells, short-lived proliferating plasmablasts and long-lived plasma cells, and is not expressed on early B cells, CD34-positive hematopoietic stem cells and other normal tissue cells, but is highly expressed in MM cells. By mediating downstream signaling pathways, BCMA plays an important role in the survival, proliferation, metastasis and drug resistance of MM cells. Therefore, BCMA is an ideal antigen target for MM treatment. An exemplary human BCMA sequence can be found from GenBank Protein Accession No: NP_001183.2.

[0023] As used herein, the term "GPRC5D" refers to G protein-coupled receptor class C group 5 member D, which belongs to the orphan receptor and is a seven-transmembrane protein. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, but its expression in normal tissues is limited to the hair follicle region. Studies have shown that 65% of multiple myeloma patients have GPRC5D with an expression threshold exceeding 50%. Due to this characteristic, GPRC5D has become a potential target for treating MM. An exemplary human GPRC5D sequence can be found from GenBank Protein Accession No: NP_061124.1.

[0024] As used herein, the term "CD3" refers to an antigen that exists as part of the multi-molecular T cell receptor (TCR) in T cells, which consists of a homodimer or heterodimer formed by two of the four receptor chains, namely CD3ε, CD3δ, CD3ζ and CD3γ. Human CD3-ε (hCD3ε) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P07766.2. Human CD3-δ (hCD3δ) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P04234.1. Therefore, unless explicitly indicated as being derived from non-human species, such as "mouse CD3", "monkey CD3", etc., the term "CD3" refers to human CD3.

[0025] As used herein, the term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed on the surface of cancerous B cells, either fully or as a fragment (e.g., MHC / peptide), and which may be used to preferentially target cancerous B cells with a pharmacological agent. As used herein, the term "cancerous B cell" refers to a B cell that is experiencing or has experienced uncontrolled proliferation. In some examples, the TAA is a marker expressed on both normal and cancer cells, such as a germline marker, such as CD19 in B cells. In some examples, the TAA is a B cell surface molecule that is overexpressed on cancerous B cells compared to normal B cells, such as 1-fold, 2-fold, 3-fold, or more overexpression compared to normal B cells, such as BCMA and GPRC5D, which are expressed much higher in cancerous B cells than in normal tissue. In some examples, the TAA is a cell surface molecule that is inappropriately synthesized in cancerous B cells, such as a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal B cells. In some examples, the TAA is expressed only fully or as a fragment (e.g., MHC / peptide) on the cell surface of cancerous cells and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses B cell antigens that are specific to cancer cells and may also be referred to in the art as "tumor-specific antigens" (TSAs).

[0026] As used herein, the term "specific binding" refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically reflected by the equilibrium dissociation constant (KD), where a low KD represents high affinity. Taking an antibody as an example, high affinity is typically about 10 -6 M or less, 10 -7 M or less, about 10 -8 M or less, about 10 -9Refers to having a KD of M or less. The calculation method of KD is as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured using methods well-known in the art, such as surface plasmon resonance methods (e.g., Biacore) or equilibrium dialysis assays.

[0027] As used herein, the term "antigen-binding molecule" is used in the broadest sense and refers to a molecule that specifically binds to an antigen. Exemplarily, antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics. An "antibody mimetic" refers to an organic compound or binding domain that can specifically bind to an antigen but is unrelated to the antibody structure. Exemplarily, antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamers, or Kunitz-type domain peptides.

[0028] As used herein, the term "antibody" is used in the broadest sense and refers to a polypeptide or combination of polypeptides that includes sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region such that it can specifically bind to an antigen. As used herein, "antibody" encompasses various forms and various structures so long as it exhibits the desired antigen-binding activity. As used herein, "antibody" includes alternative protein scaffolds or artificial scaffolds having grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include scaffolds derived from antibodies (which may include mutations introduced, for example, to stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds, for example, including biocompatible polymers. See, for example, Korndorfer, I.P., Beste, G. & Skerra, A. (2003). Proteins, 53, 121-129., Roque, A.C.A., Lowe, C.R. & Taipa, M.A. Antibodies and genetically engineered related molecules: production and purification. Biotechnol. Prog. 20, 639-654 (2004). Such scaffolds may further include scaffolds not derived from antibodies, for example, scaffold proteins known in the art that can be used for CDR grafting, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0029] The term "antibody" includes whole antibodies and any antigen-binding fragments (i.e., "antigen-binding portions") or single chains thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked to each other via disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy-chain variable region (abbreviated herein as VH) and a heavy-chain constant region. The heavy-chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light-chain variable region (abbreviated herein as VL) and a light-chain constant region. The light-chain constant region consists of one domain CL. The VH and VL regions may be further divided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed among more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, and are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains capable of interacting with an antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Immunoglobulin heavy-chain constant regions differ in amino acid composition and sequence order, and thus also in their antigenicity. Accordingly, the "immunoglobulins" herein can be divided into five classes, or may be referred to as isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be divided into various subclasses due to differences in the amino acid composition of its hinge region and the number and position of the heavy-chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into κ chains or λ chains by the constant region. Each class of Ig among the five classes of Ig can have either a κ chain or a λ chain.

[0030] As used herein, the term "antibody" further includes antibodies without light chains, such as heavy-chain antibodies (HCAbs) produced from camelid animals such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe), and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (IgNARs) found in cartilaginous fish such as sharks.

[0031] The term "antibody" as used herein may be derived from any animal, including but not limited to humans and non-human animals, and said non-human animals may be selected from primates, mammals, rodents, and vertebrates, such as camelid animals, llamas, guanacos, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0032] The term "heavy-chain antibody" as used herein refers to an antibody lacking the light chain of a conventional antibody. Specifically, the term includes, but is not limited to, homodimeric antibodies comprising a VH antigen-binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0033] The term "nanobody" as used herein refers to the fact that in animals such as camels, there are natural heavy-chain antibodies lacking light chains, and if the variable region thereof is cloned, a single-domain antibody consisting only of the heavy-chain variable region can be obtained, which is also called VHH (Variable domain of heavy chain of heavy chain antibody) and is the smallest functional antigen-binding fragment.

[0034] In this specification, the terms "VHH domain", "nanobody", and "single domain antibody" (sdAb) have the same meaning and are used synonymously, referring to the cloning of the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function. Usually, after obtaining a heavy chain antibody that is naturally lacking in the light chain and the heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0035] For further explanations regarding "heavy chain antibody", "single domain antibody", "VHH domain", and "nanobody", please refer to the following: Hamers-Casterman et al., Nature. 363, 446-8 (1993). Naturally occurring antibodies devoid of light chains., the review article by Muyldermans (J Biotechnol. 2001 Jun; 74(4): 277-302. Single domain camel antibodies: current status), as well as the following patent applications mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103, WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193, WO97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527, WO 03 / 050531, WO 01 / 90190, WO03 / 025020, WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787, WO 06 / 122825 and other prior art mentioned in these applications.

[0036] As used herein, the term "multispecificity" refers to the ability of an antibody or an antigen-binding fragment thereof to bind, for example, to at least two different epitopes on different antigens or the same antigen. Thus, terms such as "bispecificity", "trispecificity", "tetraspecificity" refer to the number of different epitopes to which an antibody can bind. For example, a common monospecific IgG-type antibody has two identical antigen-binding sites (complementary sites) and can therefore bind only to the same epitope (rather than to different epitopes). In contrast, a multispecific antibody has at least two different types of complementary sites / binding sites and can therefore bind to at least two different epitopes. As described herein, "complementary determining regions" refer to the antigen-binding sites of an antibody. Also, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions in a single antibody (the actual number of complementary determining regions / binding sites in a single antibody molecule is referred to as the "valence"). For example, a single native IgG antibody has two identical complementary sites and is therefore monospecific and bivalent. Accordingly, a multispecific antibody includes at least two (different) complementary determining regions / binding sites. Thus, the term "multispecific antibody" refers to an antibody that has more than one complementary site and has the ability to bind to two or more different epitopes. The term "multispecific antibody" specifically includes bispecific antibodies as defined above, but usually further includes antibodies that specifically bind to three or more different epitopes, scaffolds, i.e., antibodies that have three or more complementary sites / binding sites.

[0037] As used herein, the term "valence" indicates the presence of a given number of antigen-binding domains in an antibody / antigen-binding molecule. Thus, the terms "trivalent", "tetravalent", "pentavalent", and "hexavalent" refer to the presence of three, four, five, and six antigen-binding domains, respectively, in an antibody / antigen-binding molecule.

[0038] As used herein, the term "trivalent" refers to an antigen-binding molecule having three antigen-binding domains. The multispecific antigen-binding molecules of the present disclosure are trispecific and specifically bind to TAA1, TAA2, and CD3. Therefore, the trivalent multispecific binding molecules of the present disclosure have at least three antigen-binding domains that bind to different antigens. Examples of the trivalent multispecific binding molecules of the present disclosure are illustratively shown in FIGS. 1N or 1O.

[0039] As used herein, the term "tetravalent" refers to an antigen-binding molecule having four antigen-binding domains. The multispecific antigen-binding molecules of the present disclosure are trispecific and specifically bind to TAA1, TAA2, and CD3. Therefore, the tetravalent multispecific binding molecules of the present disclosure typically have two antigen-binding domains that bind to the same antigen (e.g., TAA1 or TAA2), and two antigen-binding domains that bind to separate antigens (e.g., CD3, and TAA1 or TAA2). Examples of the tetravalent multispecific binding molecules of the present disclosure are illustratively shown in FIGS. 1H-1J.

[0040] As used herein, the term "pentavalent" refers to an antigen-binding molecule having five antigen-binding domains. The multispecific binding molecules of the present disclosure are trispecific and specifically bind to TAA1, TAA2, and CD3. Therefore, the pentavalent multispecific binding molecules of the present disclosure typically have two pairs of antigen-binding domains that bind to the same antigen, respectively, and a single antigen-binding domain that binds to a third antigen (e.g., CD3). Examples of the pentavalent multispecific binding molecules of the present disclosure are illustratively shown in FIGS. 1K-1M.

[0041] As used herein, the term "hexavalent" refers to an antigen-binding molecule having six antigen-binding domains. The multispecific binding molecules of the present disclosure are trispecific and specifically bind to TAA1, TAA2, and CD3. Different structures (e.g., three antigen-binding domains that bind to TAA1, two antigen-binding domains that bind to TAA2, and one antigen-binding domain that binds to CD3, or three antigen-binding domains that bind to TAA1, two antigen-binding domains that bind to CD3, and one antigen-binding domain that binds to TAA2) are within the scope of the present disclosure, but the hexavalent multispecific binding molecules of the present disclosure typically have three pairs of two antigen-binding domains that each bind to the same antigen. Examples of the hexavalent multispecific binding molecules of the present disclosure are illustratively shown in FIGS. 1A-1G.

[0042] As used herein, the terms "full-length antibody", "intact antibody", and "whole antibody" are used synonymously herein and refer to having a structure substantially similar to the structure of a natural antibody.

[0043] As used herein, the terms "antigen-binding fragment" and "antibody fragment" are used synonymously herein, which do not have all the structures of a whole antibody and only include local or local variants of the whole antibody, and the local or local variants have the ability to bind to an antigen. Illustratively, the "antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, F(ab’)2, Fab’, Fab’-SH, Fd, Fv, scFv, diabody, and single-domain antibodies.

[0044] As used herein, the terms "linker", "linker", and "linker" are used synonymously herein and refer to a linking polypeptide sequence for linking protein domains, which typically has a certain degree of flexibility and the original function of the protein domain is not lost by the use of the linker.

[0045] As used herein, the term "chimeric antibody" refers to an antibody having a variable sequence of an immunoglobulin derived from one source organism (e.g., rat, mouse, rabbit or alpaca) and a constant region of an immunoglobulin derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-1207. Transfectomas Provide Novel Chimeric Antibodies, Gillies et al., J Immunol Methods. 1989 Dec 20;125(1-2):191-202, which are hereby incorporated by reference in their entirety. See also Patent US5807715A.

[0046] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to improve its homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and / or constant region) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies usually retain or partially retain the desired properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance the activity of immune cells or the ability to enhance the immune response.

[0047] As used herein, "fully human antibody" refers to an antibody having a variable region in which both the FR and CDR are derived from human germline immunoglobulin sequences. Also, when the antibody includes a constant region, the constant region is also derived from a human germline immunoglobulin sequence. A "fully human antibody" as used herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or somatic mutations in vivo). However, a "fully human antibody" as used herein does not include an antibody in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been transplanted into a human framework sequence.

[0048] As used herein, the term "variable region" refers to the region in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen, and "heavy chain variable region" is used synonymously with "VH" and "HCVR", and "light chain variable region" is used synonymously with "VL" and "LCVR". The variable regions of the heavy and light chains of a native antibody generally have a similar structure, and each region contains four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL is sufficient to confer antigen-binding specificity.

[0049] As used herein, "complementary determining region" is used synonymously with "CDR" and generally refers to the hypervariable regions (HVRs) found in both the variable domains of the light and heavy chains. The more conserved portions of the variable domains are called the framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on context and the various definitions known in the art. Some positions within the variable domain may be considered to be within the hypervariable region under one set of standards (e.g., IMGT or Kabat), while they may be considered to be outside the hypervariable region under a different set of standards (e.g., Kabat or IMGT), and thus may be considered heterozygous hypervariable positions. One or more of these positions may also be found in an extended hypervariable region. The present disclosure includes antibodies in which modifications are included at these heterozygous hypervariable positions. The heavy chain variable region CDR may be abbreviated as HCDR, and the light chain variable region may be abbreviated as LCDR. The variable domains of the native heavy and light chains each contain four framework regions that mainly use sheet structures, which are connected via three CDRs (CDR1, CDR2, and CDR3). These three CDRs form loops that connect the sheet structures and, in some cases, form part of the sheet structure. The CDRs in each chain are held tightly via the FR regions in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and contribute to the formation of the antigen-binding site of the antibody with the CDRs from the other antibody chain.

[0050] "CDR" in this specification may be labeled and defined in a manner well-known in the art, including, but not limited to, the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. Tools used include, but are not limited to, the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDR in this specification includes overlaps and subsets of amino acid residues of different definition methods.

[0051] The term "Kabat numbering system" in this specification generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0052] The term "Chothia numbering system" in this specification generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of the CDR region based on the positions of the loop regions of the structure (see, for example, Chothia, C., & Lesk, A. M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. Journal of molecular biology, 196(4), 901-917.).

[0053] As used herein, the term "IMGT numbering system" generally refers to a numbering system based on the international ImMunoGeneTics information system (IMGT) proposed by Lefranc et al., see Lefranc, M.P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L.,... & Lefranc, G. (2003). IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental & Comparative Immunology, 27(1), 55-77.

[0054] As used herein, the term "heavy chain constant region" refers to the carboxyl-terminal portion of an antibody heavy chain, which does not directly participate in the binding of the antibody to the antigen, but represents effector functions such as interaction with Fc receptors, and has a more conserved amino acid sequence relative to the variable domain of the antibody. The "heavy chain constant region" can be selected from the CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes "full-length heavy chain constant region" and "heavy chain constant region fragment", the former having a structure substantially similar to the constant region of a natural antibody, while the latter contains only "a part of the full-length heavy chain constant region". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of CH1 domain - hinge region - CH2 domain - CH3 domain. When the antibody is IgE, it further includes a CH4 domain, and when the antibody is a heavy chain antibody, it does not include a CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from Fc or CH3 domain.

[0055] As used herein, the term "light chain constant region" refers to the carboxyl-terminal portion of an antibody light chain, which is not directly involved in the binding of the antibody to an antigen, and the light chain constant region may be selected from a constant κ domain or a constant λ domain.

[0056] As used herein, the term "Fc region" is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes both native sequence Fc regions and variant Fc regions. Exemplarily, the human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational cleavage and remove one or more, particularly one or two amino acids from the C-terminus of the heavy chain. Thus, expression of a particular nucleic acid molecule encoding the full-length heavy chain can result in antibodies produced by host cells that either contain the full-length heavy chain or contain a cleavage variant of the full-length heavy chain. This can apply when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) of the Fc region, or the C-terminal glycine (Gly446) and lysine (Lys447), may or may not be present. Typically, the IgG Fc region includes the IgG CH2 and IgG CH3 domains, and optionally may further include, in addition to this, all or part of the hinge region, but does not include the CH1 domain. The "CH2 domain" of the human IgG Fc region typically extends from the amino acid residue at approximately position 231 to the amino acid residue at approximately position 340. In one embodiment, the carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" includes the residues at the C-terminus of the CH2 domain in the Fc region (i.e., from the amino acid residue at approximately position 341 of IgG to the amino acid residue at approximately position 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having a "bump", "knob", introduced into one of its strands and a corresponding "cavity", "hole", introduced into another strand, see U.S. Patent No. 5,821,333, which is hereby expressly incorporated by reference herein).As described herein, such variant CH3 domains may be used to promote heterodimerization of two different antibody heavy chains.

[0057] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0058] As used herein, the term "Fc variant" refers to a change in Fc structure or function caused by one or more amino acid substitution, insertion or deletion mutations present at appropriate sites in the Fc. "Interaction between Fc variants" refers to the formation of steric filling effects, electrostatic induction, hydrogen bonding, and hydrophobic interactions between Fc variants designed by mutation. The interaction between Fc variants serves to form stable heterodimeric proteins. A preferred mutation design is the "Knob-into-hole (KIH)" form of mutation design.

[0059] The mutation design technology of Fc variants has been widely applied in the art for the production of bispecific antibodies or Fc fusion protein forms of heterodimers. Representative examples include the "Knob-into-Hole" form proposed by Carter et al. (Ridgway, J.B., Presta, L.G., & Carter, P. (1996). ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering, Design and Selection, 9(7), 617-621.), the Fc-containing heterodimer form formed by Amgen engineers using electrostatic steering (US 20100286374 A1), the heterodimer form (SEEDbodies) formed by IgG / IgA chain exchange proposed by Jonathan H. Davis et al. (Davis, J.H., Aperlo, C., Li, Y., Kurosawa, E., Lan, Y., Lo, K.M., & Huston, J.S. (2010). SEEDbodies: fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Engineering, Design & Selection, 23(4), 195-202.), and DuoBody of Genmab (Gramer, M.J., van den Bremer, E.T., van Kampen, M.D., Kundu, A., Kopfmann, P., Etter, E.,... & Parren, P.W. (2013, November).Production of stable bispecific IgG1 by controlled Fab-arm exchange: scalability from bench to large-scale manufacturing by application of standard approaches. In MAbs (Vol.5, No.6, pp.962-973). Taylor & Francis.) Bispecific molecules formed by platform technology, integrated by Xencor engineers with structural calculations and Fc amino acid mutations, and heterodimeric protein forms formed by integrating different modes of action (Moore, G.L., Bautista, C., Pong, E., Nguyen, D.H.T., Jacinto, J., Eivazi, A.,... & Lazar, G.A. (2011, November). A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. In MAbs (Vol.3, No.6, pp.546-557). Taylor & Francis.), by Suzhou Conning Jerry, a method for Fc modification based on a charge network (CN201110459100.Other genetic engineering methods for achieving the formation of heterodimeric functional proteins can be mentioned based on the heterodimeric protein forms obtained in (7) and Fc amino acid changes or functional modification means. The Knob / Hole structure in the Fc mutant fragment described in the present disclosure refers to the fact that two Fc fragments are each mutated and can bind in the form of "Knob-into-Hole" after mutation. It is preferable to perform site-directed mutagenesis modification in the Fc region using the "knob-into-hole" model of Cater et al. so that the obtained first Fc mutant and second Fc mutant can bind together in the "knob-into-hole" form to form a heterodimer. Selecting a specific immunoglobulin Fc region from a specific immunoglobulin class and subclass is within the scope of understanding of those skilled in the art. Preferably, it is the Fc region of human antibodies IgG1, IgG2, IgG3, IgG4, and more preferably, the Fc region of human antibody IgG1. Randomly and arbitrarily select from the first Fc mutant or the second Fc mutant, and perform a knob mutation on one of them and a hole mutation on the other.

[0060] The term "effector function" as used herein refers to the activity of an antibody molecule, which is mediated by binding through domains of the antibody rather than the antigen-binding domain and is typically mediated by binding of an effector molecule. Effector function includes complement-mediated effector function, which is mediated, for example, by the binding of the C1 component of said complement to said antibody. Activation of complement is important for conditioning and lysis of cellular pathogens. Activation of complement can further stimulate an inflammatory response and be involved in autoimmune hypersensitivity reactions. Effector function further includes Fc receptor (FcR)-mediated effector function, which can be induced by the binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of an antibody to an Fc receptor on the cell surface induces a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, removal of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), inflammatory mediator release, placental transfer, and regulation of immunoglobulin production. The effector function of an antibody can be altered by changing the affinity of the antibody for an effector molecule such as an Fc receptor or complement component, for example, by enhancing or decreasing it. Binding affinity is usually changed by modifying the effector molecule binding site, and in this case, it is appropriate to localize the site of interest and modify at least some of the sites in a suitable manner. Changing the binding site in an antibody for an effector molecule is not necessarily required to significantly alter the overall binding affinity, but it is also envisioned to change the geometric shape of the interaction, as in the case of non-productive binding, to abrogate the effector mechanism. It is further envisioned that effector function can be altered by modifying sites that are not directly involved in the binding of an effector molecule but are involved in the performance of effector function in other ways.

[0061] As used herein, the term "conservative amino acid" generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side-chain size, hydrophobicity, hydrophilicity, main-chain conformation, and rigidity). Exemplarily, the amino acids in each of the following groups belong to each other's conservative amino acid residues, and substitutions of amino acid residues within a group belong to conservative amino acid substitutions: 1) Alanine (A), Serine (S), Threonine (T), 2) Aspartic acid (D), Glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), Histidine (H); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0062] As used herein, the term "identity" can be obtained by calculating in the following manner: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison). Thereafter, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecule is the same at this position. Considering the number of gaps that need to be introduced to optimally align these two sequences and the length of each gap, the percentage of identity between the two sequences varies according to the same-position changes common to the sequences.

[0063] Array comparison and calculation of the identity ratio between two arrays can be realized using mathematical algorithms. For example, the Needlema and Wunsch algorithms incorporated in the GAP program of the GCG software package (available from www.gcg.com), the Blossum 62 matrix or the PAM250 matrix, and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 are used to determine the identity ratio between two amino acid sequences. Further, for example, the GAP program in the GCG software package (available from www.gcg.com), the NWSgapdna.CMP matrix, and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6 are used to determine the identity ratio between two nucleotide sequences. A particularly preferred parameter set (and the parameter set to be used unless otherwise specified) is the Blossum62 score matrix using a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Further, the identity ratio between two amino acid sequences or nucleotide sequences can be determined using the E. Meyers and W. Miller algorithms incorporated in the ALIGN program (version 2.0), using the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0064] Additionally or alternatively, the nucleic acid and protein sequences described in the present disclosure may be further used as "query sequences" to perform searches against common databases to identify, for example, other family member sequences or related sequences. For example, such searches can be performed by the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10. To obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure, a BLAST nucleotide search may be performed using the NBLAST program with a score = 100 and word length = 12. To obtain amino acid sequences homologous to the protein molecules of the present disclosure, a BLAST protein search may be performed using the XBLAST program with a score = 50 and word length = 3. To obtain gapped alignment results for comparison purposes, gapped BLAST may be used as described in Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research, 25(17), 3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0065] As used herein, the term "nucleic acid" includes any compound and / or substance that is a polymer containing nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Usually, a nucleic acid molecule is described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually represented as 5' to 3'. As used herein, the term "nucleic acid molecule" includes, for example, deoxyribonucleic acid (DNA) including complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers including mixtures of two or more of these molecules. The nucleic acid molecule may be linear or circular. Also, the term "nucleic acid molecule" includes both sense and antisense strands, and single-stranded and double-stranded forms. And the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues. The nucleic acid molecule further includes DNA and RNA molecules and is suitable for direct expression of the antibodies of the present disclosure as vectors in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified.For example, the mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, whereby the mRNA can be injected into a subject to produce antibodies in vivo (see, for example, Stadler, C.R., Bahr-Mahmud, H., Celik, L., Hebich, B., Roth, A.S., Roth, R.P.,... & Sahin, U. (2017). Elimination of large tumors in mice by mRNA-encoded bispecific antibodies. Nature medicine, 23(7), 815-817. or EP2101823B1).

[0066] As used herein, "isolated" nucleic acid refers to a nucleic acid molecule separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in the following cells, which normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0067] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked thereto. The term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which the vector has been introduced. Some vectors can direct the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors".

[0068] As used herein, the term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include, without regard to the number of generations, the original transformed cells and their progeny. Progeny may not be identical to the parental cell with respect to the nucleic acid material and may include mutations. As used herein, mutant progeny are included that have the same function or biological activity as those initially screened or selected from the transformed cells.

[0069] As used herein, the term "pharmaceutical composition" refers to a formulation that exists in a form that permits the biological activity of the active ingredient(s) contained therein to be effective and that contains no other components that have unacceptable toxicity for the subject to whom the pharmaceutical composition is administered.

[0070] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial and antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, adhesives, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, dyes, and the like, and combinations thereof, which are known to those of ordinary skill in the art (see, e.g., Remington’s Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pages 1289-1329). The application of any conventional carrier in a therapeutic or pharmaceutical composition is contemplated, except in cases where incompatibility with the active ingredient(s) would occur.

[0071] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in a subject to be treated, such as cancer and tumors. Beneficial or desirable clinical results include, but are not limited to, alleviation of symptoms, reduction in the degree of disease, stabilization of a disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of a disease state, and remission (whether partial or complete remission). Subjects in need of treatment include subjects suffering from a disorder or disease, subjects susceptible to a disorder or disease, or subjects in which it is desired to prevent a disorder or disease. When terms such as slow down, reduce, decrease, alleviate, remission, etc. are mentioned, their meanings also include cases such as removal, disappearance, non-occurrence, etc.

[0072] As used herein, the term "subject" refers to a living organism that is undergoing treatment for a particular disease or disorder described herein. Exemplarily, "subject" includes mammals undergoing treatment for a disease or disorder, such as humans, primates (e.g., monkeys) or primate mammals.

[0073] As used herein, the term "effective amount" refers to a dosage of a therapeutic agent that, when administered to a cell, tissue or subject alone or in combination with another therapeutic agent, can effectively prevent or alleviate a disease disorder or the progression of the disease. "Effective amount" further refers to a dosage of a compound sufficient to alleviate symptoms, e.g., treat, cure, prevent or alleviate a related medical disorder, or increase the rate at which these disorders are treated, cured, prevented or alleviated. When the active ingredient is administered to an individual alone, the therapeutically effective dosage refers to that ingredient only. When applying a combination, the therapeutically effective dosage refers to the combined dosage of the active ingredients that leads to a therapeutic effect, whether by combination, sequential administration or simultaneous administration.

[0074] As used herein, the term "cancer" refers to or describes a physiological state typically characterized by disordered cell growth in mammals. This definition includes both benign and malignant cancers. As used herein, "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, whether malignant or benign. The terms "cancer" and "tumor" are not mutually exclusive as used herein.

[0075] As used herein, the term "EC50" refers to the half-maximal effective concentration, which includes the antibody concentration that induces a midpoint response between baseline and maximum after a specific exposure time. EC50 essentially represents 50% of the antibody concentration at which its maximal effect is observed and can be measured by methods known in the art. **DETAILED DESCRIPTION OF THE INVENTION**

[0076] GPRC5D is a G protein-coupled receptor class C group 5 member D, belonging to the orphan receptor, and is a seven-transmembrane protein. GPRC5D is a new specific target for multiple myeloma following BCMA. Tissue expression profiling studies have found that GPRC5D is specifically highly expressed in multiple myeloma plasma cells, but is lowly expressed in normal tissues, limited to the immunologically tolerant hair follicle region, and the expression of GPRC5D does not overlap with the expression of BCMA. In a tumor recurrence model in which BCMA is lost, GPRC5D CAR-T still has a therapeutic effect and overcomes tumor avoidance. The CD3 molecule is an important marker of T cells, is part of the T cell receptor (TCR), can induce the activation of T cells, and is related to tumor immunity.

[0077] The present disclosure provides a new multispecific antigen-binding molecule that simultaneously targets the tumor antigens BCMA, GPRC5D, and T cells (CD3), binds to two different targets of multiple myeloma, induces T cells to participate in tumor killing, and provides a new tumor treatment means by enhancing the tumor killing effect.

[0078] At the same time, the present disclosure provides a nucleic acid fragment, a vector, a host cell, an immune effector cell, a manufacturing method, a pharmaceutical composition, a manufacturing use, and a treatment method for tumors or cancers (such as B cell lymphoma or multiple myeloma) that encode a multispecific antibody or antigen-binding fragment that specifically binds to B cell maturation antigen (BCMA), GPRC5D, and T cells.

[0079] In a first aspect, the present disclosure provides a multispecific binding molecule, wherein the multispecific binding molecule comprises (a) a first antigen-binding molecule (ABM1) that specifically binds to a first tumor-associated antigen (TAA1), (b) a second antigen-binding molecule (ABM2) that specifically binds to a second tumor-associated antigen (TAA2), and (c) a third antigen-binding molecule (ABM3) that specifically binds to an antigen expressed on human immune cells.

[0080] In some embodiments, the TAA1 and / or the TAA2 are expressed on cancerous B cells.

[0081] In some embodiments, the cancerous B cells are derived from plasmablasts or B cells.

[0082] In some embodiments, the immune cells are selected from T cells, NK cells, or macrophages.

[0083] In some embodiments, the TAA1 and TAA2 are each independently selected from BCMA and GPRC5D, and / or the antigen expressed on the immune cells is CD3.

[0084] In some embodiments, the ABM1, ABM2, and ABM3 may each independently be selected from an antibody, an antibody fragment, F(ab’)2, Fab’, Fab, Fv, scFv, a nanobody, or VHH.

[0085] In some embodiments, the multispecific binding molecule comprises four polypeptide chains: a first heavy chain, a first light chain, a second heavy chain, and a second light chain.

[0086] In some specific embodiments, (1) the first heavy chain and the second heavy chain are the same, (a) VH a -CH1-L1-VHH b -Fc-L2-VH CD3 -L3-VL CD3 , (b) VH a -CH1-L1-VH CD3 -L2-VL CD3 -Fc-L3-VHH b , (c) VHH b -L1-VH a -CH1-L2-VH CD3 -L3-VL CD3 -Fc, (d) VH a -CH1-L1-VHHb -L2-VH CD3 -L3-VL CD3 -Fc, or (e)VH a -CH1-L1-VH b -L2-VL b -Fc-L3-VH CD3 -L4-VL CD3 has the structure shown in and / or, (2) the first light chain and the second light chain are the same and have the structure of VL a -CL where VH a and VL a are respectively the heavy chain variable region and the light chain variable region that specifically bind to GPRC5D, VHH b is a nanobody that specifically binds to BCMA, VH b and VL b are respectively the heavy chain variable region and the light chain variable region that specifically bind to BCMA, VH CD3 and VL CD3 are respectively the heavy chain variable region and the light chain variable region that specifically bind to CD3, Fc is the Fc region of any one antibody, and L1, L2, L3 and L4 are respectively the same or different linkers.

[0087] In some specific embodiments, (1) the first heavy chain and the second heavy chain are different, (a) the first heavy chain VH a -CH1-L1-VHH b -Fc1 and the second heavy chain VH a -CH1-L2-VH CD3 -L3-VL CD3 -Fc2, (b) the first heavy chain VHH b -L1-VH a -CH1-Fc1 and the second heavy chain VH a -CH1-L2-VH CD3 -L3-VL CD3 -Fc2, (c) the first heavy chain VH a -CH1-L1-VH b -L2-VL b-Fc1 and the second heavy chain VH a -CH1-L3-VH b -L4-VL b -Fc2-L5-VH CD3 -L6-VL CD3 、 (d) The first heavy chain VH a -CH1-L1-VHH b -Fc1 and the second heavy chain VH a -CH1-L2-VHH b -Fc2-L3-VH CD3 -L4-VL CD3 、or (e) The first heavy chain VHH b -L1-VH a -CH1-Fc1 and the second heavy chain VHH b -L2-VH a -CH1-Fc2-L3-VH CD3 -L4-VL CD3 has the structure shown in, Optionally, a Flag tag is further attached to the carboxy group terminus of the first heavy chain in (a) to (e) above, and / or a His tag is further attached to the carboxy group terminus of the second heavy chain, (2) The first light chain and the second light chain are the same and have the structure of VL a -CL Here, VH a and VL a are the heavy chain variable region and the light chain variable region that specifically bind to GPRC5D, respectively, VHH b is a nanobody that specifically binds to BCMA, VH b and VL b are the heavy chain variable region and the light chain variable region that specifically bind to BCMA, respectively, VH CD3 and VL CD3 are the heavy chain variable region and the light chain variable region that specifically bind to CD3, respectively, Fc1 and Fc2 are the Fc regions of one of the antibodies, and L1, L2, L3, L4, L5 and L6 are the same or different linkers, respectively.

[0088] In some embodiments, the multispecific binding molecule comprises three polypeptide chains, a first heavy chain, a second heavy chain, and a light chain, wherein (1) The first heavy chain and the second heavy chain are (a) the first heavy chain VHH b -L1-VHH b -Fc1 and the second heavy chain VH a -CH1-L2-VH CD3 -L3-VL CD3 -Fc2, (b) the first heavy chain VHH b -L1-VH CD3 -L2-VL CD3 -Fc1 and the second heavy chain VH a -CH1-Fc2, or (c) the first heavy chain VHH b -Fc1 and the second heavy chain VH a -CH1-L1-VH CD3 -L2-VL CD3 -Fc2, having the structure shown, Optionally, a Flag tag is further attached to the carboxy-terminal of the first heavy chain in (a)-(c) above, and / or a His tag is further attached to the carboxy-terminal of the second heavy chain. (2) The light chain has the structure VL a -CL, wherein VH a and VL a are respectively the heavy chain variable region and the light chain variable region that specifically bind to GPRC5D, VHH b is a nanobody that specifically binds to BCMA, VH b and VL b are respectively the heavy chain variable region and the light chain variable region that specifically bind to BCMA, VH CD3 and VL CD3 are respectively the heavy chain variable region and the light chain variable region that specifically bind to CD3, and L1, L2, and L3 are each the same or different linkers.

[0089] In some embodiments, Fc1 and Fc2 of the multispecific binding molecule are different, Fc1 is knob-Fc, Fc2 is hole-Fc, or Fc1 is hole-Fc and Fc2 is knob-Fc. Preferably, Fc1 and Fc2 are the Fc regions of IgG1 or IgG4.

[0090] In some specific embodiments, the knob-Fc contains the T366W mutation, and / or the hole-Fc contains the T366S, L368A, and / or Y407V mutations.

[0091] In some embodiments, ABM3 in the multispecific binding molecule is a CD3 antibody or an antigen-binding fragment thereof, which comprises a light chain variable region and a heavy chain variable region. (a) The light chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs. 54 to 56, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 54 to 56. And (b) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs. 51 to 53, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 51 to 53.

[0092] In some specific embodiments, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99%, or more identity compared to SEQ ID NO: 10, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99%, or more identity compared to SEQ ID NO: 9.

[0093] In some specific embodiments, the ABM3 is scFv.

[0094] In some embodiments, TAA1 and TAA2 in the multispecific binding molecule are selected from the group consisting of (a) TAA1 is BCMA and TAA2 is GPRC5D, or (b) TAA1 is GPRC5D and TAA2 is BCMA.

[0095] In some specific embodiments, the antigen-binding molecule that specifically binds to GPRC5D in the multispecific binding molecule comprises a light-chain variable region and a heavy-chain variable region of an antibody, wherein (a) the light-chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs. 42 to 44, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 42 to 44, the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs. 39 to 41, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 39 to 41, or (b) the light-chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs. 48 to 50, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 48 to 50, the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs. 45 to 47, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 45 to 47.

[0096] In some specific embodiments, (a) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO:6, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO:5, or, (b) The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO:8, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO:7.

[0097] In some specific embodiments, the antigen-binding molecule that specifically binds to GPRC5D in the multispecific binding molecule is a Fab.

[0098] In some specific embodiments, the antigen-binding molecule that specifically binds to BCMA in the multispecific binding molecule comprises the heavy chain variable region of an antibody, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3. The amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO.27 - 29, or as shown in SEQ ID NO.30 - 32, or have 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to SEQ ID NO.27 - 29 or SEQ ID NO.30 - 32.

[0099] In some specific embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO.1 or SEQ ID NO.2.

[0100] In some specific embodiments, the antigen-binding molecule that specifically binds to BCMA is a VHH.

[0101] In some specific embodiments, the antigen-binding molecule that specifically binds to BCMA in the multispecific binding molecule comprises a light-chain variable region and a heavy-chain variable region of an antibody, and (a) the light-chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs. 36 to 38, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 36 to 38, and (b) the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs. 33 to 35, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 33 to 35.

[0102] In some specific embodiments, the light-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99%, or more identity compared to SEQ ID NO: 4, and / or the heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99%, or more identity compared to SEQ ID NO: 3.

[0103] In some specific embodiments, the antigen-binding molecule that specifically binds to BCMA in the multispecific binding molecule is a scFv.

[0104] In some embodiments, the ABM1, ABM2, and / or ABM3 are chimeric, humanized, or fully human-derived.

[0105] In some embodiments, the multispecific binding molecule further comprises an Fc fragment of human IgG1, IgG2, IgG3 or IgG4, preferably comprising an Fc fragment of human IgG1 or human IgG4.

[0106] In some specific embodiments, the Fc fragment of the multispecific binding molecule has an amino acid mutation that changes the effector function, preferably, the amino acid mutation that changes the effector function includes S228P, F234A, L234A and / or L235A mutations, and more preferably, the amino acid mutation that changes the effector function includes (a) L234A and / or L235A mutations of IgG1 Fc, and / or (b) S228P, F234A and / or L235A mutations of IgG4 Fc.

[0107] In some embodiments, the Fc fragment of the multispecific binding molecule comprises the same or different Fc1 and Fc2.

[0108] In some specific embodiments, the different Fc1 and Fc2 in the multispecific binding molecule have amino acid mutations that promote heterodimerization, preferably, the amino acid mutations that promote heterodimerization include Knob-into-hole (KIH) mutations, and more preferably, the Knob-into-hole mutations include (a) any one Fc region comprising a T366W mutation, and (b) another Fc region comprising T366S, L368A and / or Y407V mutations.

[0109] In some specific embodiments, the Fc1 further comprises a His tag, and the Fc2 further comprises a Flag tag, or the Fc1 further comprises a Flag tag, and the Fc2 further comprises a His tag.

[0110] In some embodiments, the multispecific binding molecule is trivalent, tetravalent, pentavalent or hexavalent.

[0111] In a second aspect, the present disclosure provides an isolated nucleic acid that encodes the aforementioned multispecific binding molecule.

[0112] In a third aspect, the present disclosure provides a recombinant vector that contains the aforementioned isolated nucleic acid.

[0113] In a fourth aspect, the present disclosure provides a host cell that contains the aforementioned isolated nucleic acid or the aforementioned recombinant vector. Preferably, the host cell is a eukaryotic cell or a prokaryotic cell. More preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli, and / or Bacillus subtilis. Even more preferably, the host cell is Expi293 cells.

[0114] In a fifth aspect, the present disclosure provides a method for producing a multispecific binding molecule that includes culturing the aforementioned host cell under appropriate conditions and isolating the multispecific binding molecule.

[0115] In a sixth aspect, the present disclosure provides a pharmaceutical composition that includes the aforementioned multispecific binding molecule, the aforementioned isolated nucleic acid, the aforementioned recombinant vector, the aforementioned host cell, or a product produced according to the aforementioned method, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. Preferably, the pharmaceutical composition further includes an additional cancer therapeutic agent.

[0116] In a seventh aspect, the present disclosure provides a method for activating T cells that includes administering a therapeutically effective amount of the aforementioned multispecific binding molecule or pharmaceutical composition to a subject.

[0117] In an eighth aspect, the present disclosure provides the use of the aforementioned multispecific binding molecule, the aforementioned isolated nucleic acid, the aforementioned host cell, a product produced by the aforementioned method, or the aforementioned pharmaceutical composition for manufacturing a drug for treating and / or preventing cancer, or the use for treating and / or preventing cancer.

[0118] In a preferred embodiment, the cancer is B-cell lymphoma, and more preferably, the B-cell lymphoma is multiple myeloma.

[0119] In a ninth aspect, the present disclosure provides a method for treating and / or preventing cancer, which comprises administering to a subject a therapeutically effective amount of the aforementioned multispecific binding molecule, the aforementioned isolated nucleic acid, the aforementioned host cell, the product manufactured by the aforementioned method, or the aforementioned pharmaceutical composition.

[0120] In a preferred embodiment, the cancer is B-cell lymphoma, and more preferably, the B-cell lymphoma is multiple myeloma.

[0121] Examples Hereinafter, the present disclosure will be further described with reference to specific examples, and the advantages and features of the present disclosure will become apparent from the description. When specific conditions are not specified in the examples, they are carried out according to conventional conditions or the conditions proposed by the manufacturer. When the reagents or equipment used are not specified by the manufacturer, they are all common products that can be purchased commercially.

[0122] The examples of the present disclosure are merely illustrative and do not limit the scope of the present disclosure. Those skilled in the art can make modifications or substitutions to the details and forms of the technical solutions of the present disclosure without departing from the spirit and scope of the present disclosure, and it will be understood that all these modifications and substitutions are included within the protection scope of the present disclosure.

[0123] Example 1 Construction of anti-BCMA-GPRC5D-CD3 trispecific antibody molecule Through a series of experiments such as animal immunization, sequencing, and humanization, suitable humanized anti-BCMA specific antibodies (BCMA-VHH01, BCMA-VHH02, and BCMA-Hab01), humanized anti-GPRC5D specific antibodies (GPRC5D-Hab01 and GPRC5D-Hab02), and humanized anti-CD3 specific antibodies (CD3-Hab01) were obtained. Subsequently, these specific antibodies were assembled as a trispecific antibody BCMA-GPRC5D-CD3, and the assembly methods could be 2:2:2 (BCMA:GPRC5D:CD3) (exemplarily as shown in A - G of Figure 1), or 1:2:1 (BCMA:GPRC5D:CD3 or GPRC5D:BCMA:CD3) (exemplarily as shown in H - J of Figure 1), or 1:1:1 (BCMA:GPRC5D:CD3) (exemplarily as shown in N or O of Figure 1), or 2:2:1 (BCMA:GPRC5D:CD3) (exemplarily as shown in K - M of Figure 1). The antibody fragments could be scFv, Fab, or VHH, the subtype of the heavy chain constant region was IgG1 or IgG4. When the molecule had an asymmetric structure, "Knob in Hole" was used to reduce mismatching. Finally, each fragment was linked with a Linker and constructed into a PTT5 expression vector to obtain the target plasmid. The schematic structural diagram of the trispecific antibody molecule is as shown in Figure 1, the molecular composition of each trispecific antibody is as shown in Table 1, the amino acid sequence of the antibody molecule is as shown in Table 2, and the CDRs determined by the Kabat numbering system of the main antibody variable regions are as shown in Table 3. The positive control antibodies used in this example were the bispecific antibody REGN5459 (WO 2020018820A1, targeting the BCMA antigen, the CD3 receptor expressed on the T cell surface) from Regeneron and the bispecific antibody JNJ7564 (WO 2019220368A1, targeting the GPRC5D antigen, the CD3 receptor expressed on the T cell surface) from Johnson & Johnson. The negative control antibody was antiFITC×CD3-Hab01 (prepared in-house). The structures of the positive and negative control antibodies are as shown in P - R of Figure 1, where the Fc regions of REGN5459 and JNJ7564 both contain mutations that prevent homodimerization.

[0124]

Table 1

[0125]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0126]

Table 3

[0127] Example 2 Expression and Purification of BCMA-GPRC5D-CD3 Tertiary Antibody Molecule and Control Antibody 2.1 Expression and Purification of Tertiary Antibody Molecule The plasmid and the transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021) and mixed uniformly, then left standing for 15 min, added to Expi293F cells (Thermofisher, catalog number: A14527), and cultured in a shaker at 5% CO2, 120 rpm, and 37 °C. On the day after transfection, OPM-293 ProFeed (Shanghai OPMI, catalog number: F081918-001) and 6 g / L glucose (Sigma, catalog number: G7528) were added. On the 6th day after transfection, the cell supernatant was collected. After collecting the culture supernatant, Protein A affinity and molecular sieve purification were performed on the protein using AKTA Pure. Quantitative and qualitative analyses were performed on the obtained antibody by SDS-PAGE, SEC-HPLC, and CE-SDS. Depending on the structure of the antibody, purification may be performed by any of the following several methods: (A) Purification method for symmetric tertiary antibodies The tertiary antibodies Tri-17, Tri-29, Tri-32, Tri-35, Tri-55, Tri-59, and Tri-60 are suitable for this method. The specific purification method is as follows.

[0128] 1. Coarse purification was performed using a Protein A column (Mabselect SuRe TM , purchased from Cytiva). First, the Protein A column was equilibrated with an equilibration buffer (PBS buffer, pH 7.4) that was 3 to 5 times the column volume, and the clarified culture supernatant was loaded at a flow rate of 8 mL / min. After loading was completed, 3 to 5 column volumes were rinsed with a high-salt rinse solution (20 mM phosphate buffer, 1 M NaCl, pH 7.4). An eluent (20 mM citrate buffer, pH 3.5) was used to elute the protein bound to the Protein A column, and A 280 the elution of the protein was monitored by the ultraviolet absorption peak. The eluted protein was collected and neutralized to pH 5 - 6 by adding 1 M pH 8.0 Tris-HCl.

[0129] 2. Perform precise purification using a molecular sieve (Ezload 16 / 60 Chromdex 200 pg, purchased from Boster Biological Technology or Superdex 200 Increase 10 / 300GL, purchased from Cytiva), collect the target sample, concentrate it, dialyze it, exchange it with Buffer 559 (10 mM acetic acid, 9% sucrose, pH 5.5), perform sterile filtration with a 0.22 μm filter, and store it aseptically to obtain the purified corresponding antibody.

[0130] (B) Purification method for type I asymmetric tertiary antibodies The tertiary antibodies Tri-38, Tri-41, Tri-46, Tri-49, Tri-52, Tri-56, and Tri-62 are suitable for this method. The specific purification method is as follows.

[0131] 1. Perform rough purification using a Protein A column. First, equilibrate the Protein A column with an equilibration buffer (PBS buffer, pH 7.4) that is 3 to 5 times the column volume, and then load the clarified culture supernatant at a flow rate of 8 mL / min. After loading is complete, rinse with a high-salt rinse solution for 3 to 5 times the column volume. Remove excess hapten with Eluent A (20 mM citrate buffer, pH 3.85), and elute the target protein with Eluent B (20 mM citrate buffer, pH 3.5). 280 Monitor the elution of the protein by the ultraviolet absorption peak. Collect the eluted protein, add 1 M pH 8.0 Tris-HCl to neutralize the pH to 5 - 6.

[0132] 2. Perform precise purification using a molecular sieve, collect the target sample, concentrate it, dialyze it, exchange it with Buffer 559, perform sterile filtration with a 0.22 μm filter, and store it aseptically to obtain the purified corresponding antibody.

[0133] (C) Purification method for type II asymmetric tertiary antibodies The tertiary antibodies Tri-63, Tri-64, Tri-65, and Tri-66 are suitable for this method. The specific purification method is as follows.

[0134] 1. Crude purification was performed using a Protein A column. First, the Protein A column was equilibrated with an equilibration buffer (PBS buffer, pH 7.4) that was 3 to 5 times the column volume, and the clarified culture supernatant was loaded at a flow rate of 8 mL / min. After loading was completed, 3 to 5 times the column volume was rinsed with a high-salt rinse solution. Using an elution solution (20 mM citrate buffer, pH 3.5), the protein bound to the Protein A column was eluted, and the elution of the protein was monitored by the A280 ultraviolet absorption peak. The eluted protein was collected and neutralized to pH 5 - 6 by adding 1M pH 8.0 Tris-HCl.

[0135] 2. Precision purification was performed using HiTrap KappaSelect (purchased from Cytiva). First, it was equilibrated with an equilibration buffer (PBS buffer, pH 7.4) that was 3 to 5 times the column volume, and the crudely purified protein solution was loaded at a flow rate of 5 mL / min. After loading was completed, 3 to 5 times the column volume was rinsed with PBS, and then 10 times the column volume was rinsed with 10 mM phosphate buffer (pH 7.4). Using elution solution C (50 mM glycine, pH 3.6), the protein bound to the KappaSelect column was eluted, and using elution solution D (50 mM glycine, pH 3.0), the impurity proteins were eluted, and the elution of the protein was monitored by the A280 ultraviolet absorption peak. The eluted protein was collected and neutralized to pH 5 - 6 by adding 1M pH 8.0 Tris-HCl.

[0136] 3. Precision purification was performed using a molecular sieve. The target sample was collected, concentrated, then dialyzed, exchanged into 559 buffer, and sterile filtered through a 0.22 μm filter and stored aseptically to obtain the purified corresponding antibody.

[0137] Tertiary antibodies purified by different methods were each analyzed by SEC-HPLC to characterize the molecular size homogeneity and purity of the protein sample. The HPLC used was an Agilent 1260, the chromatography column TSKgel G3000SWXL was purchased from Tosoh Bioscience, the mobile phase was 200 mM phosphate buffer, pH 7.0 / isopropyl alcohol (v / v 9:1), the detection temperature was 25 °C, the flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. The SEC-HPLC data were analyzed by manual integration of the chromatogram, the purity of the protein was calculated according to the area normalization method, the main peak was considered to be the monomer, the chromatographic peak before the main peak was called the aggregate, and the chromatographic peak after the main peak was called the fragment. The results are shown in Table 4.

[0138]

Table 4

[0139] 2.2 Expression and purification of control antibodies Anti-hel-hIgG1 (which is often abbreviated as hIgG1 in this application) is an antibody targeting hen egg lysozyme (purchased from Beyotime, catalog number: B117901) and is commonly used as a negative control antibody in the antibody screening and identification process. The expression methods of REGN5459, JNJ7564, and antiFITC×CD3-Hab01 are the same as in 2.1, and the expression and purification methods of the tertiary antibody molecules are as follows.

[0140] (A) Purification methods of REGN5459 and antiFITC×CD3-Hab01 1. Crude purification was performed using a Protein A column. First, the Protein A column was equilibrated with an equilibration buffer (PBS buffer, pH 7.4) that was 3 to 5 times the column volume, and the clarified culture supernatant was loaded at a flow rate of 8 mL / min. After loading was completed, 3 to 5 column volumes were rinsed with a high-salt rinse solution (20 mM phosphate buffer, 1 M NaCl, pH 7.4). The target protein was eluted with a linear gradient using elution solution A (20 mM citrate buffer, pH 3.85) and elution solution B (20 mM citrate buffer, pH 3.5), and the elution of the protein was monitored by the A280 ultraviolet absorption peak. The eluted protein was collected, and 1 M pH 8.0 Tris-HCl was added to neutralize the pH to 5 - 6.

[0141] 2. Precision purification was performed using a molecular sieve, the target sample was collected, and finally stored in PBS buffer, sterilization filtration was performed with a 0.22 μm filter, and after sterilization preservation, the purified REGN5459 and antiFITC×CD3-Hab01 antibodies were obtained.

[0142] (B) JNJ7564 purification method 1. Using a Protein A column, the corresponding monospecific antibodies JNJ7564-anti-GPRC5D and JNJ7564-anti-CD3 were each purified. First, the Protein A column was equilibrated with an equilibration buffer (PBS buffer, pH 7.4) that was 3 to 5 times the column volume, and the clarified culture supernatant was loaded at a flow rate of 8 mL / min. After loading was completed, 3 to 5 column volumes were rinsed with a high-salt rinse solution (20 mM phosphate buffer, 1 M NaCl, pH 7.4). The protein bound to the Protein A column was eluted using an elution solution (20 mM citrate buffer, pH 3.5), and the elution of the protein was monitored by the A280 ultraviolet absorption peak. The eluted protein was collected, 1 M pH 8.0 Tris-HCl was added to neutralize the pH to 5 - 6, and dialysis was performed to exchange it with PBS buffer.

[0143] 2. Bispecific antibodies were generated by in vitro reduction and reoxidation. Anti-GPRC5D / anti-CD3 antibodies at 1 - 20 mg / ml were mixed at a molar ratio of 1.08:1, 2-mercaptoethanol (2-MEA) with a final concentration of 75 mM was added, and the mixture was incubated at 25 - 37 °C for 2 - 6 hours. 2-Mercaptoethanol (2-MEA) was removed by dialysis or ultrafiltration or desalting and buffer exchange. Bispecific antibodies were generated by oxidation and reassembly at 20 °C.

[0144] Tertiary antibodies purified by different methods were all analyzed by SEC-HPLC to characterize the molecular size homogeneity and purity of the protein samples. The method was the same as above. The results are as shown in Table 5.

[0145]

Table 5

[0146] Example 3 Construction and Identification of Engineered Cell Lines 3.1 Production of CHOK1-human GPRC5D cells and CHOK1-monkey GPRC5D cells The nucleotide sequences encoding the human GPRC5D amino acid sequence (NCBI Gene ID: 55507) and the monkey GPRC5D amino acid sequence (NCBI Gene ID: 102122120) were cloned into the pLVX lentiviral vector respectively, and virus particles were produced in HEK293T cells. After infecting the CHOK1 cell line with lentivirus, it was selectively cultured for one week in Advanced DMEM / F12 Medium (purchased from Gibco, catalog number 12634028) containing 10% (w / w) fetal bovine serum (purchased from ExCell Bio, catalog number FND500) and 10 μg / ml puromycin (purchased from Gibco, catalog number A1113803). JNJ7564 was used as the primary antibody, and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088) was used as the secondary antibody, and detected by a flow cytometer FACS CantoII (purchased from BD Biosciences). Cells with high expression levels and a single peak shape were amplified, and the amplified cells were re-detected by flow cytometry analysis. A positive cell population with good growth, high fluorescence intensity, and high uniformity was selected and continuously expanded in culture, and then cryopreserved in liquid nitrogen.

[0147] Previously produced JNJ7564-anti-GPRC5D-hIgG1 (commissioned by General Biosystems (Anhui) Co., Ltd., the GPRC5D-VH and VL sequences in JNJ7564 were cloned into the expression vectors PTT5-huIgG1 and PTT5-huIgGLC (Kappa) containing the signal peptide MGWSWILLFLLSVTAGVHS (SEQ ID NO.57) and the human-derived heavy / light chain constant regions respectively to construct a chimeric monoclonal antibody) was used for identification, and the antibody has cross-reactivity with human and monkey GPRC5D. The FACS results are as shown in Figures 2A - 2B, the results show a single peak, and it can be used for monitoring the binding activity between the multi-specific binding molecule and human and monkey GPRC5D.

[0148] 3.2 Production of the Flp-in CHO-human BCMA cell line, a cell line with overexpression of the target antigen BCMA The nucleotide sequence encoding the human BCMA amino acid sequence (NCBI Gene ID: 608) was cloned into the PcDNA5 / FRT vector (purchased from Clontech). After transfecting the Flp-in CHO cell line, it was selectively cultured for two weeks in F12K Medium (purchased from Gibco, catalog number 21127030) containing 10% (w / w) fetal bovine serum (purchased from ExCell Bio, catalog number FND500) and 600 μg / ml hygromycin (purchased from ThermoFisher, catalog number 10687010). Using the REGN5459 antibody as the primary antibody and the goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088) as the secondary antibody, it was detected with a flow cytometer FACS CantoII (purchased from BD Biosciences). Cells with high expression levels and a single peak shape were amplified, and the amplified cells were redetected by flow cytometry analysis. A positive cell population with good growth, high fluorescence intensity, and high uniformity was selected and continuously expanded in culture, and then cryopreserved in liquid nitrogen.

[0149] Previously prepared REGN5459-anti-BCMA-hIgG1 (commissioned from General Biosystems (Anhui) Co., Ltd., the BCMA-VH and VL sequences in REGN5459 were cloned into the expression vectors PTT5-huIgG1 and PTT5-huIgGLC (Kappa) containing the signal peptide MGWSWILLFLLSVTAGVHS and the human-derived heavy / light chain constant regions respectively to construct a chimeric monoclonal antibody) was used for identification, and the antibody has cross-reactivity with human and monkey BCMA. The FACS results are as shown in Figure 3, the results show a single peak, and it can be used to monitor the binding activity between the multispecific binding molecule and human BCMA.

[0150] 3.3 Production of a stable transfection cell line of the luciferase reporter gene Lentivirus packaging was performed on the pLvx-luciferase-P2A-GFP-RES-hygro vector system. Next, virus infection was carried out on Molp-8 cells, the medium was changed at 24 h, 0.125 mg / ml hygro was added for pressure screening, and pressure screening was performed on the cell pool with hygro for 2 weeks. The GFP expression peak was detected by FACS to be about 40-fold, indicating successful pool infection. Using the limiting dilution method, the cells were seeded at 1 cell / well into a total of 3 96-well cell plates and cultured at 37 °C for 2 - 3 weeks. A total of about 30 monoclonal cell lines grew. These monoclonal cells were transferred to 24-well plates and cultured for 3 - 4 days. Based on the cell growth status and GFP intensity, a total of 3 monoclonal cell lines were selected for expansion culture, and the luciferase expression in the cells was further detected. Finally, monoclonal Molp-8-Luc cells with strong fluorescence were obtained. Both H929-Luc pool and RPMI8226-Luc pool were purchased from Nanjing Kebai, and the method for producing monoclonal cells was the same as that for Molp-8-Luc.

[0151] Example 4 Identification of the Binding of the BCMA-GPRC5D-CD3 Tertiary Antibody Molecule to the Target Antigen and CD3 (A) Detection of the Binding of the BCMA-GPRC5D-CD3 Tertiary Antibody to Human BCMA Protein and Cross-Binding to Rhesus BCMA Protein by Enzyme-Linked Immunosorbent Assay (ELISA) The specific method is as follows: Human BCMA protein (purchased from Acro, catalog number: BCA-H522y) and monkey BCMA protein (purchased from Acro, catalog number: BCA-C52H7) were each diluted with PBS to a final concentration of 1 μg / mL, and then added to a 96-well ELISA plate at 50 μl / well. The plate was sealed with a plastic film and incubated overnight at 4°C. The next day, the plate was washed twice with PBS, and the blocking solution [PBS + 2% (w / v) BSA] was added and blocked at room temperature for 2 hours. The blocking solution was discarded, and the plate was washed three times with PBS. Serial diluted 100 nM BCMA-GPRC5D-CD3 antibody or control antibody was added at 50 μl / well. After incubation at 37°C for 2 hours, the plate was washed three times with PBS. Goat anti-human secondary antibody labeled with HRP (horseradish peroxidase) (purchased from Merck, catalog number: AP113P) was added, and after incubation at 37°C for 1 hour, the plate was washed five times with PBS. TMB substrate was added at 50 μl / well, and after incubation at room temperature for 10 minutes, stop solution (1.0 M HCl) was added at 50 μl / well. The OD450nm value was read using an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The results are as shown in Figures 4A and 4B, and all tertiary antibody molecules can bind to human BCMA and have cross-binding activity with monkey BCMA. The IgG subtype control is human IgG1 (anti-HEL-IgG1).

[0152] (B) Detection by cell-based ELISA of the binding of BCMA-GPRC5D-CD3 tertiary antibody to CHOK1-human GPRC5D cells and to CHOK1-monkey GPRC5D cells The specific method is as follows: Expand the required cells in a T-75 cell culture flask until the logarithmic growth phase, aspirate and remove the culture medium, wash twice with PBS buffer, digest the cells with pancreatin, and then terminate the digestion with complete medium. Pipette the cells until a single-cell suspension is formed. After counting the cells, centrifuge them and resuspend the cell pellet with complete medium to 4×10 5 cells / ml, add to a 96-well flat-bottom cell culture plate at 100 μl / well, and culture overnight in an incubator at 37°C and 5% carbon dioxide. The next day, discard the medium in the 96-well plate, wash the cells once with PBS, and then fix the cells with a fixing solution for immunostaining (purchased from Beyotime, catalog number: P0098-500 ml) at 50 μl / well and fix at room temperature for 0.5 hour. Add the blocking solution [PBS + 5% (w / v) skim milk] and block at room temperature for 2 hours. Discard the blocking solution, wash the plate three times with PBS, and add the gradient-diluted 100 nM BCMA-GPRC5D-CD3 antibody or control antibody at 50 μl / well. After incubating at 37°C for 2 hours, wash the plate three times with PBS. Add a goat anti-human secondary antibody labeled with HRP (horseradish peroxidase), incubate at 37°C for 1 hour, and then wash the plate five times with PBS. Add the TMB substrate at 50 μl / well, incubate at room temperature for 10 minutes, and then add the stop solution (1.0 M HCl) at 50 μl / well. Read the OD450nm value with an ELISA plate reader. The results are as shown in Figures 5A and 5B, and all the tertiary antibody molecules can bind to CHOK1-human GPRC5D cells and have cross-binding activity with CHOK1-monkey GPRC5D cells.

[0153] (C) Detection of the binding of BCMA-GPRC5D-CD3 tertiary antibody to human CD3e / d protein and human CD3e protein by enzyme-linked immunosorbent assay (ELISA) The specific method is the same as that in Example 4(A). The proteins coated on the 96-well ELISA plate are human CD3e / d protein (purchased from Sino Biological, catalog number: CT032-H2508) and human CD3e protein (purchased from Sino Biological, catalog number: 10977-H08H), respectively. The results are as shown in Figures 6A and 6B. The trispecific antibody molecule binds to both human CD3e / d protein and human CD3e protein to a certain extent.

[0154] (D) Detection of the binding of BCMA-GPRC5D-CD3 trispecific antibody to the target antigen BCMA overexpressing cell line and the target antigen GPRC5D overexpressing cell line by flow cytometry (FACS) Flp-in CHO-human BCMA cells and CHO K1-human GPRC5D cells were each expanded in a T-75 cell culture flask until the logarithmic growth phase, and the cells were pipetted until they became a single cell suspension. After counting the cells, they were centrifuged, and the cell pellet was resuspended in FACS buffer (PBS + 2% fetal bovine serum) at 2×10 6Resuspend to cells / milliliter, add to a 96-well FACS reaction plate at 50 μl / well, add BCMA-GPRC5D-CD3 antibody or control antibody, start from 100 nM and dilute 5-fold with an 8% gradient, 50 μl per well, and incubate at 4 °C for 1 hour. Centrifuge and wash three times with PBS buffer, add secondary antibody anti-hIgG(Fc) Alexa 647 (purchased from Jackson, catalog number: 109-605-098) at 50 μl / well, and incubate on ice for 1 hour. Centrifuge and wash three times with PBS buffer, take 100 μl, detect by FACS (FACS CantoTM, purchased from BD), and analyze the results. Analyze the data with software (FlowJo) to obtain the mean fluorescence intensity (MFI) of the cells. Next, analyze with software (GraphPad Prism8), perform data fitting, and calculate the EC50. The results are as shown in Table 6 and Figure 7 and Table 7 and Figure 8. All trispecific antibody molecules bound well to the target antigens BCMA and GPRC5D in the overexpressing cells respectively.

[0155]

Table 6

[0156]

Table 7

[0157] (E) Detection by FACS of the binding of BCMA-GPRC5D-CD3 trispecific antibody to endogenous tumor cells co-expressing target antigens BCMA and GPRC5D The specific method is the same as in Example 4(D). In this experiment, the binding ability of the trispecific antibody molecule was detected using multiple myeloma cell line NCI-H929 (all purchased from ATCC) as endogenous cells. NCI-H929 is a cell line with high expression of both GPRC5D and BCMA. The results are as shown in Table 8 and Figure 9. The trispecific antibody molecule can strongly bind to the endogenous tumor cell NCI-H929 expressing antigens BCMA and GPRC5D.

[0158]

Table 8

[0159] (F) Detection by FACS of the binding of the (F) BCMA-GPRC5D-CD3 trispecific antibody to endogenous tumor cells expressing CD3 and activated human and monkey PBMC cells expressing CD3 The endogenous cells expressing CD3 are Jurkat (purchased from ATCC). The production processes of human PBMC and monkey PBMC expressing CD3 are as follows: PBMC isolated from human blood were activated according to the methods of the human T cell activation kit (purchased from Miltenyi Biotec, catalog number: 130-091-441) and the monkey T cell activation kit (purchased from Miltenyi Biotec, catalog number: 130-092-919), respectively, and cultured for 3 days. Then, the activated T cells were cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum containing 100 units of human IL2 protein (purchased from Acro, catalog number: IL2-H4113) for 2 weeks. The FACS detection method is the same as that in Example 4 (D). The results are as shown in Table 9, Figure 10, and Tables 10, Figures 11A and 11B. The trispecific antibody molecule binds to some extent to all of Jurkat cells, human PBMC, and monkey PBMC cells.

[0160]

Table 9

[0161]

Table 10

[0162] (G) Detection of the activation activity of the BCMA-GPRC5D-CD3 trispecific antibody against Jurkat-NFAT-NanoLuc cells Preparation of Jurkat-NFAT-NanoLuc cells: Jurkat cells (purchased from ATCC) were seeded at 1E6 cells / well into 6-well culture dishes (Costar: 3516) containing 2mL of complete medium (RPM1640, 10% FBS, penicillin-streptomycin) and continued to be cultured at 37°C under 5% CO2 for 24 hours. The next day, 3μg of pNL[NLucP / NFAT-RE / Hygro] vector and 3μl of PLUS reagent (Invitrogen: 11514-015) were diluted in 150μL of Opti-MEM (Gibco: 11058021) and incubated at room temperature for 5 minutes. The pNL[NLucP / NFAT-RE / Hygro] vector contains NFAT, which drives the luciferase reporter gene NanoLuc to transcribe in response to TCR activation. After incubation, the diluted DNA solution was pre-incubated with 1:1 Lipofectamine LTX solution (Invitrogen:15338-100) (6 μl Lipofectamine LTX + 144 μl Opti-MEN) and incubated at room temperature for 15 minutes to form DNA-Lipofectamine LTX complexes. After incubation, 300 μl of DNA-Lipofectamine complexes were added directly to the cell wells. Jurkat cells were transfected at 37°C under 5% CO2 for 24 hours. After incubation, the cells were washed with 3 mL of PBS and selectively cultured in 1640 (purchased from Gibco, Cat. No.: 31870082) medium containing 10% (w / w) fetal bovine serum containing 125 μg / ml of hygromycin (Invitrogen: 10687010) for 2 weeks to obtain the required Jurkat-NFAT-NanoLuc cell line.

[0163] The BCMA-GPRC5D-CD3 trispecific antibody and the control antibody were serially diluted 5-fold starting from 100 nM (final concentration) or serially diluted 3-fold starting from 100 nM (final concentration). The diluent was RPMI 1640 medium containing 5% fetal bovine serum. The diluted antibodies were added to a 96-well flat-bottom black clear-bottom cell culture plate at 50 μl / well. H929 cells or negative cells K562 and Jurkat-NFAT-NanoLuc cells were each expanded in a T-75 cell culture flask until the logarithmic growth phase and pipetted until they became single cell suspensions. After counting the cells, they were centrifuged, and the cell pellet was resuspended in 1640 medium containing 5% fetal bovine serum to 2×10 6 cells / ml. The three types of cells were each added to the above 96-well cell culture plate at 25 μl / well. After mixing evenly, they were placed in a 37°C, 5% (v / v) CO2 incubator and incubated for 5 - 6 hours. After incubation, 50 μl of Nano-Glo luciferase reporter gene detection system (purchased from Promega, catalog number: N1110) was added to each well, placed on a shaker and shaken for 10 minutes of incubation. Finally, the fluorescence values were read by a plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The results are as shown in Table 11 and Figures 12A and 12B. The trispecific antibody molecule can well activate Jurkat-NFAT-NanoLuc cells mediated by H929 cells expressing the target antigens BCMA and GPRC5D, but under the mediation of negative cells K562 that do not express BCMA and GPRC5D, it either does not activate Jurkat-NFAT-NanoLuc cells or causes extremely weak non-specific activation.

[0164] [Table 11]

[0165] Example 5 Detection of the killing activity and cytokine production of BCMA-GPRC5D-CD3 trispecific antibody-mediated T cells against tumor cells in vitro In this experiment, three types of human multiple myeloma cell lines were selected as target cells for the T cell in vitro killing experiment. Here, NCI-H929 is a cell line with high expression of both GPRC5D and BCMA, RPMI8226 is a cell line with moderate to low expression of GPRC5D and medium expression of BCMA, and Molp-8 is a cell line with high expression of GPRC5D and low expression of BCMA. To facilitate subsequent experiments and detections, stable transfection strains of the above three cell lines that stably express luciferase were constructed, and hereinafter, they are abbreviated as H929-Luc, RPMI8226-Luc, and Molp-8-Luc (refer to 3.3 of Example 3 for the construction method).

[0166] On the first day of the experiment, healthy donor PBMCs (human peripheral monocytes) cryopreserved in liquid nitrogen were taken out and resuscitated, then resuspended with RPMI-1640 medium and cultured overnight in an incubator at 5% CO2 and 37 °C in a culture flask for standby. The next day, single cell suspensions were prepared by taking H929-Luc, RPMI8226-Luc, and Molp-8-Luc cells respectively. The density of tumor cells was adjusted to 1.33×10 5 cells / ml with complete medium, and the PBMC density was adjusted to 1.33×10 6 cells / ml. 75 μl of the adjusted H929-Luc, RPMI8226-Luc, or Molp-8-Luc and 75 μl of PBMC were each taken and uniformly mixed in equal volume (the number of tumor cells and PBMCs per well was 1×10 4 cells and 1×10 5(in number), add to each well of a 96-well plate with a U-bottom using a micropipette. The volume of the cell mixture is 150 μl / well. The antibodies were serially diluted in complete medium (the starting concentration of the antibody is 100 nM, diluted 7-fold, with 8 gradients), and 100 μl of antibody dilutions with different concentrations were added to each well to make the final volume of each well 250 μl. After uniformly mixing the cells, they were placed in an incubator at 5% CO2 and 37 °C. After culturing for 48 hours, 100 μl of the supernatant was taken and used for detecting cytokines IFNγ and TNFα in the cell culture supernatant. The remaining cells were continuously cultured in an incubator at 5% CO2 and 37 °C. After continuously culturing for 24 hours, the cell viability was detected using the Bright-Glo Luciferase kit (purchased from Promega, catalog number E2620, refer to the instruction manual for usage). The well with only PBMC was defined as a positive control with 100% killing, and the one with an antibody concentration of 0 was defined as a blank negative control. The cell survival rate = (reading value of the sample well - reading value of the positive well) / (reading value of the blank well - reading value of the positive well) × 100%, and the results were calculated and plotted using GraphPad Prism 9.0 software.

[0167] The target cells used in the in vitro killing of some tertiary antibodies are Molp-8 cells that do not express luciferase, and the method of mixed incubation of PBMC, tumor cells, and antibodies is the same as above. After co-culturing for 24 hours, the cell culture plate was centrifuged at a rotation speed of 300 g, 100 μl of the supernatant was transferred and used for detecting the released cytokines in the cell culture supernatant. The remaining 100 μl of the supernatant was transferred to a flat-bottom 96-well plate, and the LDH value of cell release per well in the remaining supernatant medium was detected using an LDH detection kit (purchased from DO JINDO, catalog number CK12, refer to the instruction manual for usage), and the killing percentage of the target cells was calculated according to the method in the kit's instruction manual.

[0168] There are two methods for detecting cytokines. One is to use the Cytometric Bead Array human Th1 / Th2 / Th17 cytokine kit (purchased from BD, catalog number 560484, refer to the kit's instruction manual for usage) to detect the cytokine release amount in the medium with a flow cytometer. The other is to detect cytokines IFNγ and TNFα in the cell culture supernatant by the ELISA method (the kits are purchased from BD, catalog numbers are 555142 and 55521 respectively, refer to the kit's instruction manual for usage).

[0169] The results of tumor cell killing by tertiary antibody-mediated T cells are as shown in Figures 13, 15, 17 and Table 12 respectively. According to the results, for different tumor cells, the tertiary antibody can cause a more excellent or equivalent killing effect compared to the positive control. The T cells mediated by the tertiary antibodies Tri-38 and Tri-41 had a weaker killing effect on Molp-8 than the positive control. The cytokines TNFα and IFNγ secreted when killing tumor cells in vitro are as shown in Figures 14A - 14B, 16A - 16B and 18A - 18B respectively. According to the results, the T cells mediated by the tertiary antibodies Tri-29, Tri-35, Tri-46 and Tri-55 can secrete a certain amount of cytokines when killing tumor cells.

[0170]

Table 12

Claims

1. A multispecific binding molecule, wherein the multispecific binding molecule comprises: (a) a first antigen-binding molecule (ABM1) that specifically binds to a first tumor-associated antigen (TAA1); (b) a second antigen-binding molecule (ABM2) that specifically binds to a second tumor-associated antigen (TAA2); (c) a third antigen-binding molecule (ABM3) that specifically binds to an antigen expressed on human immune cells, and optionally, the TAA1 and / or the TAA2 is expressed on cancerous B cells, the multispecific binding molecule.

2. The cancerous B cells are derived from plasma cells or B cells, and optionally, the immune cells are selected from T cells, NK cells or macrophages, the multispecific binding molecule according to claim 1.

3. The TAA1 and TAA2 are each independently selected from BCMA and GPRC5D, and / or the antigen expressed on the human immune cells is CD3, the multispecific binding molecule according to claim 1 or 2.

4. The ABM1, ABM2, and ABM3 are each independently selected from an antibody, an antibody fragment, F(ab'), ２ , Fab', Fab, Fv, scFv, a nanobody, or VHH, and is the multispecific binding molecule according to any one of claims 1 to 3.

5. The multispecific binding molecule comprises four polypeptide chains, namely a first heavy chain, a first light chain, a second heavy chain and a second light chain, the multispecific binding molecule according to any one of claims 1 to 4.

6. (1) The first heavy chain and the second heavy chain are the same; (a) VH ａ -CH1-L1-VHH ｂ -Fc-L2-VH ＣＤ３ -L3-VL ＣＤ３ , (b) VH ａ -CH1-L1-VH ＣＤ３ -L2-VL ＣＤ３ -Fc-L3-VHH ｂ , (c) VHH ｂ -L1-VH ａ -CH1-L2-VH ＣＤ３ -L3-VL ＣＤ３ -Fc, (d) VH ａ -CH1-L1-VHH ｂ -L2-VH ＣＤ３ -L3-VL ＣＤ３ -Fc, or, (e) VH ａ -CH1-L1-VH ｂ -L2-VL ｂ -Fc-L3-VH ＣＤ３ -L4-VL ＣＤ３ has the structure shown in, and / or, (2) The first light chain and the second light chain are the same and have a structure of VL ａ -CL, Here, VH ａ and VL ａ are respectively the heavy-chain variable region and the light-chain variable region that specifically bind to GPRC5D, and VHH ｂ is a nanobody that specifically binds to BCMA, VH ｂ and VL ｂ are respectively the heavy-chain variable region and the light-chain variable region that specifically bind to BCMA, VH ＣＤ３ and VL ＣＤ３ are respectively the heavy-chain variable region and the light-chain variable region that specifically bind to CD3, Fc is the Fc region of any one of the antibodies, and L1, L2, L3, and L4 are respectively the same or different linkers. The multispecific binding molecule according to claim 5.

7. (1) The first heavy chain and the second heavy chain are different; (a) First heavy chain VH ａ -CH1-L1-VHH ｂ -Fc1 and second heavy chain VH ａ -CH1-L2-VH ＣＤ３ -L3-VL ＣＤ３ -Fc2, (b) The first heavy chain VHH ｂ -L1-VH ａ -CH1-Fc1 and the second heavy chain VH ａ -CH1-L2-VH ＣＤ３ -L3-VL ＣＤ３ -Fc2, (c) First heavy chain VH ａ -CH1-L1-VH ｂ -L2-VL ｂ -Fc1 and second heavy chain VH ａ -CH1-L3-VH ｂ -L4-VL ｂ -Fc2-L5-VH ＣＤ３ -L6-VL ＣＤ３ , (d) First heavy chain VH ａ -CH1-L1-VHH ｂ -Fc1 and second heavy chain VH ａ -CH1-L2-VHH ｂ -Fc2-L3-VH ＣＤ３ -L4-VL ＣＤ３ or, (e) The first heavy chain VHH ｂ -L1-VH ａ -CH1-Fc1 and the second heavy chain VHH ｂ -L2-VH ａ -CH1-Fc2-L3-VH ＣＤ３ -L4-VL ＣＤ３ has the structure shown, Optionally, a Flag tag is further attached to the carboxy-terminal of the first heavy chain in (a) to (e) above, and / or a His tag is further attached to the carboxy-terminal of the second heavy chain. (2) The first light chain and the second light chain are the same and have a structure of VL ａ -CL, Here, VH ａ and VL ａ are respectively a heavy chain variable region and a light chain variable region that specifically bind to GPRC5D, and VHH ｂ is a nanobody that specifically binds to BCMA, VH ｂ and VL ｂ are respectively a heavy chain variable region and a light chain variable region that specifically bind to BCMA, VH ＣＤ３ and VL ＣＤ３ are respectively a heavy chain variable region and a light chain variable region that specifically bind to CD3, and L1, L2, L3, L4, L5 and L6 are respectively the same or different linkers, the multispecific molecule according to claim 5.

8. The multispecific binding molecule comprises three polypeptide chains, namely a first heavy chain, a second heavy chain and a light chain, wherein (1) The first heavy chain and the second heavy chain are (a) First heavy chain VHH ｂ -L1-VHH ｂ -Fc1 and second heavy chain VH ａ -CH1-L2-VH ＣＤ３ -L3-VL ＣＤ３ -Fc2, (b) First heavy chain VHH ｂ -L1-VH ＣＤ３ -L2-VL ＣＤ３ -Fc1 and second heavy chain VH ａ -CH1-Fc2, or, (c) First heavy chain VHH ｂ -Fc1 and second heavy chain VH ａ -CH1-L1-VH ＣＤ３ -L2-VL ＣＤ３ - having the structure shown in Fc2, Optionally, a Flag tag is further attached to the carboxy-terminal of the first heavy chain in (a) to (c) above, and / or a His tag is further attached to the carboxy-terminal of the second heavy chain. (2) The light chain has a structure of VL ａ - CL, and Here, VH ａ and VL ａ are respectively the heavy chain variable region and the light chain variable region that specifically bind to GPRC5D, VHH ｂ is a nanobody that specifically binds to BCMA, VH ｂ and VL ｂ are respectively the heavy chain variable region and the light chain variable region that specifically bind to BCMA, VH ＣＤ３ and VL ＣＤ３ are respectively the heavy chain variable region and the light chain variable region that specifically bind to CD3, Fc1 and Fc2 are respectively the Fc regions of any one antibody, and L1, L2 and L3 are respectively the same or different linkers. The multispecific binding molecule according to any one of claims 1 to 4.

9. Fc1 and Fc2 of the multispecific binding molecule are different, Fc1 is a knob-Fc, Fc2 is a hole-Fc, or Fc1 is a hole-Fc and Fc2 is a knob-Fc. Preferably, the knob-Fc contains a T366W mutation, and / or the hole-Fc contains a T366S, L368A and / or Y407V mutation. Preferably, Fc1 and Fc2 are Fc regions of IgG1 or IgG4. The multispecific binding molecule according to any one of claims 6 to 8.

10. Fc1 and Fc2 of the multispecific molecule are the same or different and have amino acid mutations that alter effector function. Preferably, the mutations that alter effector function include S228P, F234A, L234A and / or L235A. More preferably, the mutations that alter effector function include (a) the L234A and / or L235A mutations of IgG1 Fc, and / or (b) the S228P, F234A and / or L235A mutations of IgG4 Fc. The multispecific binding molecule according to any one of claims 6 to 8.

11. ABM3 is a CD3 antibody or an antigen-binding fragment thereof, which includes a light chain variable region and a heavy chain variable region. (a) The light chain variable region includes LCDR1, LCDR2 and LCDR3, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO. 54 to 56, or have 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to SEQ ID NO. 54 to 56. And (b) the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO. 51 to 53, or have 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to SEQ ID NO. 51 to 53. The multispecific binding molecule according to any one of claims 1 to 3.

12. The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO: 10, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO: 9, the multispecific binding molecule according to claim 11.

13. The multispecific binding molecule according to claim 12, wherein the ABM3 is a scFv.

14. The multispecific binding molecule according to any one of claims 1 to 3, wherein the TAA1 and the TAA2 are selected from the group consisting of (a) TAA1 is BCMA and TAA2 is GPRC5D, or (b) TAA1 is GPRC5D and TAA2 is BCMA.

15. In the multi-specific binding molecule, the antigen-binding molecule that specifically binds to GPRC5D comprises a light chain variable region and a heavy chain variable region of an antibody, (a) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NOs. 42 to 44, or have 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to SEQ ID NOs. 42 to 44, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NOs. 39 to 41, or have 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to SEQ ID NOs. 39 to 41, or (b) the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NOs. 48 to 50, or have 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to SEQ ID NOs. 48 to 50, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NOs. 45 to 47, or have 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to SEQ ID NOs. 45 to 47. The multi-specific binding molecule according to claim 14.

16. (a) the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO: 6, and / or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO: 5, or, (b) The light chain variable region includes the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO: 8, and / or the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO:

7. The multispecific binding molecule according to claim 15.

17. The multispecific binding molecule according to claim 16, wherein the antigen-binding molecule that specifically binds to GPRC5D in the multispecific binding molecule is a Fab.

18. In the multispecific binding molecule, the antigen-binding molecule that specifically binds to BCMA includes the heavy chain variable region of an antibody. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs. 27 to 29, or as shown in SEQ ID NOs. 30 to 32, or have 1, 2, 3 or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 27 to 29 or SEQ ID NOs. 30 to 32. The multispecific binding molecule according to claim 14.

19. The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99% or more identity compared to SEQ ID NO. 1 or SEQ ID NO.

2. The multispecific binding molecule according to claim 18.

20. The multispecific binding molecule according to claim 19, wherein the antigen-binding molecule that specifically binds to BCMA is a VHH.

21. In the multispecific binding molecule, the antigen-binding molecule that specifically binds to BCMA comprises a light-chain variable region and a heavy-chain variable region of an antibody, and (a) the light-chain variable region comprises LCDR1, LCDR2, and LCDR3, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NOs. 36 to 38, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 36 to 38, and (b) the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3, and the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NOs. 33 to 35, or have 1, 2, 3, or more amino acid insertions, deletions, and / or substitutions compared to SEQ ID NOs. 33 to 35. The multispecific binding molecule according to claim 14.

22. The light-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99%, or more identity compared to SEQ ID NO: 4, and / or the heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having 90%, 95%, 96%, 97%, 98%, 99%, or more identity compared to SEQ ID NO:

3. The multispecific binding molecule according to claim 21.

23. The antigen-binding molecule that specifically binds to BCMA is scFv. The multispecific binding molecule according to claim 22.

24. The ABM1, ABM2, and / or ABM3 are chimeric, humanized, or fully human-derived. The multispecific binding molecule according to any one of claims 1 to 23.

25. Further comprising an Fc fragment of human IgG1, IgG2, IgG3, or IgG4, preferably comprising an Fc fragment of human IgG1 or human IgG4. The multispecific binding molecule according to claim 24.

26. The Fc fragment comprises the same or different Fc1 and Fc2. The multispecific binding molecule according to claim 25.

27. The Fc fragment has amino acid mutations that alter effector function, preferably, the amino acid mutations that alter effector function include S228P, F234A, L234A and / or L235A mutations, more preferably, the amino acid mutations that alter effector function are (a) the L234A and / or L235A mutations of IgG1 Fc, and / or (b) the S228P, F234A and / or L235A mutations of IgG4 Fc, the multispecific binding molecule according to claim 25.

28. The different Fc1 and Fc2 have amino acid mutations that promote heterodimerization, preferably, the amino acid mutations that promote heterodimerization include Knob-into-hole (KIH) mutations, more preferably, the Knob-into-hole mutations are (a) any one of the Fc regions contains the T366W mutation, and (b) the other Fc region contains the T366S, L368A and / or Y407V mutations, the multispecific binding molecule according to claim 27.

29. The Fc1 further comprises a His tag (His-tag), and the Fc2 further comprises a Flag tag (Flag-tag), or Fc1 further comprises a Flag-tag, and Fc2 further comprises a His-tag, the multispecific binding molecule according to claim 28.

30. The multispecific binding molecule is trivalent, tetravalent, pentavalent or hexavalent, the multispecific binding molecule according to any one of claims 11 to 29.

31. An isolated nucleic acid, wherein the nucleic acid encodes the multispecific binding molecule according to any one of claims 1 to 30, the isolated nucleic acid.

32. A recombinant vector comprising the isolated nucleic acid according to claim 31.

33. A host cell, wherein the host cell comprises the isolated nucleic acid according to claim 31 or the recombinant vector according to claim 32, preferably, the host cell is a eukaryotic cell or a prokaryotic cell, more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and / or Bacillus subtilis, more preferably, the host cell is Expi293 cells, the host cell.

34. A method for producing a multispecific binding molecule, the method comprising culturing the host cell according to claim 33 under appropriate conditions and isolating the multispecific binding molecule.

35. A pharmaceutical composition, the pharmaceutical composition comprising the multispecific binding molecule according to any one of claims 1 to 30, the isolated nucleic acid according to claim 31, the recombinant vector according to claim 32, the host cell according to claim 33, or a product produced according to the method according to claim 34, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients, preferably, the pharmaceutical composition further comprises an additional cancer therapeutic agent, preferably, the pharmaceutical composition is used for the treatment and / or prevention of cancer, preferably, the cancer is B-cell lymphoma, more preferably, the B-cell lymphoma is multiple myeloma, pharmaceutical composition.

36. A method for activating T cells, the method comprising administering to a subject a therapeutically effective amount of the multispecific binding molecule according to any one of claims 1 to 30 or the pharmaceutical composition according to claim 35.

37. Use of the multispecific binding molecule according to any one of claims 1 to 30, the isolated nucleic acid according to claim 31, the host cell according to claim 33, the product produced by the method according to claim 34 or the pharmaceutical composition according to claim 35 for the manufacture of a medicament for treating and / or preventing cancer, or for the use for treating and / or preventing cancer, preferably, the cancer is B-cell lymphoma, more preferably, the B-cell lymphoma is multiple myeloma, use.

38. A method for treating and / or preventing cancer, the method comprising administering to a subject a therapeutically effective amount of the multispecific binding molecule according to any one of claims 1 to 30, the isolated nucleic acid according to claim 31, the host cell according to claim 33, the product produced by the method according to claim 34 or the pharmaceutical composition according to claim 35, preferably, the cancer is B-cell lymphoma, more preferably, the B-cell lymphoma is multiple myeloma, method.

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