Bispecific antigen binding molecules and uses thereof

Abstract

The present disclosure provides forms of bispecific antigen binding molecules, the invention also provides a bispecific antigen binding molecule aiming at GPRC5D and CD3 constructed based on the form, a specific antigen binding molecule aiming at GPRC5D or an antigen binding fragment thereof, a pharmaceutical composition containing the specific antigen binding molecule or the antigen binding fragment thereof, and related application of the pharmaceutical composition in tumor treatment.

Description

Bispecific antigen-binding molecules and their applications

[0001] Cross-reference to related applications

[0002] This application claims the priority benefit of the Chinese invention patent application with application date of May 16, 2023, application number 202310552022.8, and invention name “Bispecific antigen binding molecules and their applications”, and the contents of the application are incorporated by reference. Technical Field

[0003] The present disclosure belongs to the field of immunology and relates to a bispecific antigen-binding molecule. The present disclosure also relates to related encoding nucleic acids, vectors, host cells, drugs, and related applications in treating cancer. Background Art

[0004] Multiple myeloma (MM) is a malignant tumor of the blood system and the second most common hematological malignancy after non-Hodgkin's lymphoma. It is common in middle-aged and elderly people. Multiple myeloma can damage the bones, immune system, kidneys, and red blood cell count, often leading to extensive bone destruction, accompanied by osteolytic lesions, osteopenia, and pathological fractures. According to statistics, the number of multiple myeloma patients in my country increased from 69,800 in 2016 to 101,900 in 2019. With the acceleration of aging, it is expected to increase to 167,200 in 2024 and further to 266,300 in 2030. With advances in diagnosis and treatment, the disease is also showing a trend of younger patients. Multiple myeloma is very prone to relapse. The median survival time of patients in my country is 24-36 months, and the five-year survival rate is only about 24%.

[0005] Like most cancer treatments, the standard treatment for multiple myeloma is chemotherapy, with melphalan being the most commonly used medication. These drugs often lead to bone marrow suppression and are less than ideal in efficacy, with only approximately 5% achieving complete remission. These treatments are primarily intended to alleviate the disease. In recent years, advances in medicine have revolutionized the treatment of multiple myeloma with the development of targeted drugs, such as proteasome inhibitors, immunomodulators, and antibodies. Compared to existing treatment options, the survival rate of multiple myeloma patients has significantly increased, significantly improving patient prognosis. However, multiple myeloma remains incurable, and most patients relapse after multiple lines of treatment. Consequently, more researchers and clinicians are seeking new targets for relapsed / refractory multiple myeloma.

[0006] GPRC5D is a G protein-coupled receptor C5 family subtype D, an orphan receptor and a seven-transmembrane protein. GPRC5D is specifically and highly expressed in plasma cells of multiple myeloma and is found only in hair follicles in normal tissues. Antibodies and CAR-T cell therapies targeting GPRC5D have shown promising preclinical results, with no hair loss observed in experimental animals. Furthermore, GPRC5D expression is not restricted by BCMA expression. In models of tumor recurrence caused by BCMA antigen loss, GPRC5D-targeted CAR-T therapies can overcome tumor escape. These findings suggest that GPRC5D is an ideal clinical target.

[0007] T cell-based therapies have demonstrated significant anti-tumor effects in numerous animal models, and many T cell therapies have recently made significant progress in treating cancer indications. Therefore, leveraging the potential of T cells to develop novel, highly effective, and low-toxic T cell bispecific antibodies is crucial. Bispecific antibodies targeting the CD3 antigen can bring T cells and tumor cells closer together, leveraging T cells to specifically kill tumor cells. These antibodies are known as T cell engagers (TCE bispecific antibodies). Nearly half of clinical-stage bispecific antibody candidates target the CD3 antigen, and the safety and efficacy of TCE bispecific antibodies are being investigated. While relevant research has made some progress, many challenges remain, such as balancing anti-tumor activity and safety. Developing novel, highly effective, and low-toxic T cell bispecific antibodies is crucial.

[0008] Therefore, there is a need to develop new forms of bispecific antibodies and to utilize them to further develop highly effective and low-toxic bispecific antibodies. The present disclosure provides a bispecific antibody against GPRC5D and CD3. This molecule can recruit T cells to tumor sites through the CD3 target and specifically kill tumor cells that overexpress GPRC5D, making it possible to target the cytotoxic effects of T cells to cancer cells.

[0009] SUMMARY OF THE INVENTION

[0010] The first object of the present disclosure is to provide a novel bispecific antigen-binding molecule, in which the binding portion that specifically binds to the second antigen is encapsulated between the binding portion that specifically binds to the first antigen and the Fc structure, thereby reducing the exposure of the second antigen-binding portion and thereby reducing the risk of non-specific cytokine release.

[0011] The bispecific antigen-binding molecules of the present disclosure comprise the following polypeptides:

[0012] A first polypeptide comprising: (i) an antigen-binding fragment Fab heavy chain domain capable of specifically binding to a first antigen, (ii) a single-chain antibody (scFv) domain capable of specifically binding to a second antigen, and (iii) a first Fc domain;

[0013] A second polypeptide comprises: an antigen-binding fragment Fab light chain domain capable of specifically binding to the first antigen;

[0014] The third polypeptide comprises: a second Fc domain.

[0015] The antigen-binding fragment Fab heavy chain domain of the first polypeptide and the antigen-binding fragment Fab light chain domain of the second polypeptide form a first binding site for the first antigen, the single-chain antibody (scFv) domain forms a second binding site for the second antigen, and the first Fc domain and the second Fc domain are associated with each other.

[0016] Optionally, the bispecific binding molecule further comprises a fourth polypeptide, comprising an antigen-binding fragment Fab light chain domain that is identical to the antigen-binding fragment Fab light chain domain of the second polypeptide and that is capable of specifically binding to the first antigen, and the third polypeptide further comprises an antigen-binding fragment Fab heavy chain domain that is capable of specifically binding to the first antigen, the antigen-binding fragment Fab heavy chain domain being identical to the antigen-binding fragment Fab heavy chain domain of the first polypeptide, and its C-terminus is linked to the N-terminus of the second Fc domain, the antigen-binding fragment Fab heavy chain domain of the third polypeptide and the antigen-binding fragment Fab light chain domain of the fourth polypeptide forming a third binding site for the first antigen.

[0017] In one embodiment, the single-chain antibody (scFv) domain comprises a heavy chain variable region and a light chain variable region;

[0018] Preferably, the heavy chain variable region of the single-chain antibody (scFv) domain and the light chain variable region of the single-chain antibody (scFv) domain are connected via a first linker, wherein the C-terminus of the heavy chain variable region of the single-chain antibody (scFv) domain is fused to the N-terminus of the first linker, and the C-terminus of the first linker is fused to the N-terminus of the light chain variable region of the single-chain antibody (scFv) domain;

[0019] More preferably, the first linker comprises the amino acid sequence (G4S) n , n is any integer from 1 to 10.

[0020] In one embodiment, the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, and the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain; the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, and the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain.

[0021] Preferably, the first CH3 domain comprises a "knob" structure, and the second CH3 domain comprises a "hole" structure; more preferably, the "knob" structure comprises amino acid substitutions S354C and T366W, and the "hole" structure comprises amino acid substitutions Y349C, T366S, L368A and Y407V.

[0022] Preferably, the Fc domain is derived from IgG1.

[0023] Preferably, the single-chain antibody (scFv) domain is connected to the first Fc domain via a second linker, wherein the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the second linker, and the C-terminus of the second linker is fused to the N-terminus of the first Fc domain.

[0024] More preferably, the second linker comprises the amino acid sequence EPKSS.

[0025] In one embodiment, the antigen-binding fragment Fab heavy chain domain comprises the heavy chain variable region and CH1 domain of an immunoglobulin, and the C-terminus of the Fab heavy chain variable region is fused to the N-terminus of the CH1 domain; the antigen-binding fragment Fab light chain domain comprises the light chain variable region and light chain constant region of an immunoglobulin, and the C-terminus of the Fab light chain variable region is fused to the N-terminus of the light chain constant region.

[0026] Preferably, the antigen-binding fragment Fab heavy chain domain of the first polypeptide is connected to the single-chain antibody (scFv) domain via a third linker, wherein the C-terminus of the antigen-binding fragment Fab heavy chain domain is fused to the N-terminus of the third linker, and the C-terminus of the third linker is fused to the N-terminus of the single-chain antibody (scFv) domain.

[0027] More preferably, the third linker comprises the amino acid sequence (G4S) n , n is any integer from 1 to 10.

[0028] Optionally, the C-terminus of the antigen-binding fragment Fab heavy chain domain of the third polypeptide is connected to the N-terminus of the second Fc domain via a fourth linker.

[0029] In one embodiment, the first polypeptide comprises the following structure: Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain, preferably comprises the following structure: Fab heavy chain variable region-Fab CH1-third linker-scFv heavy chain variable region-first linker-scFv light chain variable region-second linker-first CH2-first CH3; the second polypeptide comprises the following structure: Fab light chain variable region-light chain constant region; and the third polypeptide comprises the following structure: second CH2-second CH3.

[0030] In an optional embodiment, the third polypeptide comprises the following structure: Fab heavy chain domain-second Fc domain, preferably comprises the following structure: Fab heavy chain variable region-Fab CH1-second CH2-second CH3, and the fourth polypeptide comprises the following structure: Fab light chain variable region-light chain constant region.

[0031] In one embodiment, the Fc domain of the first polypeptide and / or the third polypeptide comprises the following amino acid substitutions: L234A and / or L235A.

[0032] In one embodiment, the Fc domain of the third polypeptide comprises the following amino acid substitution: H435R.

[0033] In one embodiment, the second antigen is CD3, preferably CD3ε; preferably, the single-chain antibody (scFv) domain comprises a HCDR1 as shown in SEQ ID NO: 19, a HCDR2 as shown in SEQ ID NO: 20, a HCDR3 as shown in SEQ ID NO: 21, a LCDR1 as shown in SEQ ID NO: 22, a LCDR2 as shown in SEQ ID NO: 23, and a LCDR3 as shown in SEQ ID NO: 24.

[0034] More preferably, the single-chain antibody (scFv) domain comprises a heavy chain variable region having a sequence as shown in SEQ ID NO: 17 and a light chain variable region having a sequence as shown in SEQ ID NO: 18;

[0035] More preferably, the single-chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:8.

[0036] In one embodiment, the first Fc domain comprises the amino acid sequence shown in SEQ ID NO: 26; and the second Fc domain comprises the amino acid sequence shown in SEQ ID NO: 4.

[0037] In one embodiment, the first antigen is GPRC5D; preferably, the antigen-binding fragment Fab heavy chain domain comprises a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 12, and a HCDR3 as shown in SEQ ID NO: 13, and / or a LCDR1 as shown in SEQ ID NO: 14, a LCDR2 as shown in SEQ ID NO: 15, and a LCDR3 as shown in SEQ ID NO: 16;

[0038] More preferably, the antigen-binding fragment Fab heavy chain domain comprises a heavy chain variable region having a sequence as shown in SEQ ID NO: 9, and / or the antigen-binding fragment Fab light chain domain comprises a light chain variable region having a sequence as shown in SEQ ID NO: 10;

[0039] More preferably, the antigen-binding fragment Fab heavy chain domain comprises the amino acid sequence shown in SEQ ID NO: 25; and the antigen-binding fragment Fab light chain domain comprises the amino acid sequence shown in SEQ ID NO: 3.

[0040] In one embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3; and the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4. In an optional embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, the second polypeptide and / or the fourth polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3, and the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0041] The second object of the present disclosure is to provide a bispecific antigen-binding molecule against GPRC5D (preferably against GPRC5D and CD3), which can recruit T cells to the tumor site by targeting immune cell surface antigens (preferably CD3 antigens), specifically killing tumor cells that highly express GPRC5D, while the level of cytokine release in vitro is low and the safety is good.

[0042] Based on this, the present disclosure provides a bispecific antigen-binding molecule that binds to a specific epitope, wherein the epitope that the bispecific antigen-binding molecule is capable of binding to is:

[0043] The epitope is the same as or overlaps with that of an antibody comprising HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 12, HCDR3 as shown in SEQ ID NO: 13, LCDR1 as shown in SEQ ID NO: 14, LCDR2 as shown in SEQ ID NO: 15, and LCDR3 as shown in SEQ ID NO: 16.

[0044] In one embodiment, the bispecific antigen binding molecule is capable of binding to an epitope that:

[0045] The epitope is the same as or overlaps with the epitope directed by the antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 9 and the light chain variable region amino acid sequence shown in SEQ ID NO: 10.

[0046] In one embodiment, the bispecific antigen binding molecule is capable of binding to CD3, preferably CD3ε.

[0047] The present disclosure also provides a bispecific antigen-binding molecule comprising:

[0048] (A) a first binding moiety capable of specifically binding to GPRC5D; and

[0049] (B) a second binding moiety that specifically binds to a different antigen or epitope than the first binding moiety;

[0050] The first binding portion comprises:

[0051] The HCDR1 sequence is shown in SEQ ID NO: 11, the HCDR2 sequence is shown in SEQ ID NO: 12, the HCDR3 sequence is shown in SEQ ID NO: 13, the LCDR1 sequence is shown in SEQ ID NO: 14, the LCDR2 sequence is shown in SEQ ID NO: 15 and the LCDR3 sequence is shown in SEQ ID NO: 16.

[0052] In one embodiment, the first binding moiety comprises:

[0053] The sequence of the heavy chain variable region is shown in SEQ ID NO: 9 and the sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0054] In one embodiment, the second binding moiety is capable of binding to CD3, preferably CD3ε.

[0055] Another object of the present disclosure is to provide a specific antigen-binding molecule or an antigen-binding fragment thereof for GPRC5D, wherein the binding molecule or the antigen-binding fragment thereof can specifically bind to GPRC5D without non-specific binding to the same family protein GPRC5A.

[0056] The GPRC5D binding molecule or antigen-binding fragment thereof comprises: a HCDR sequence of a heavy chain variable region as shown in SEQ ID NO: 9 and a LCDR sequence of a light chain variable region as shown in SEQ ID NO: 10; preferably, the GPRC5D binding molecule or antigen-binding fragment thereof comprises a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 12, and a HCDR3 as shown in SEQ ID NO: 13, and / or a LCDR1 as shown in SEQ ID NO: 14, a LCDR2 as shown in SEQ ID NO: 15, and a LCDR3 as shown in SEQ ID NO: 16; further preferably, the GPRC5D binding molecule or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 9 and a light chain variable region amino acid sequence as shown in SEQ ID NO: 10.

[0057] In one embodiment, the GPRC5D binding molecule or antigen-binding fragment thereof comprises a heavy chain constant region and a light chain constant region; preferably, it comprises an IgG1 heavy chain constant region and a kappa light chain constant region.

[0058] The present disclosure also provides a nucleic acid molecule encoding the bispecific antigen-binding molecule or the GPRC5D-binding molecule or an antigen-binding fragment thereof.

[0059] The present disclosure also provides an expression vector comprising the nucleic acid molecule.

[0060] The present disclosure also provides a host cell comprising the nucleic acid molecule or expression vector; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, Expi293 or HEK293 cell.

[0061] The present disclosure also provides a method for preparing a bispecific antigen-binding molecule or the GPRC5D-binding molecule or an antigen-binding fragment thereof, the method comprising: culturing the aforementioned host cell under suitable conditions.

[0062] The present disclosure also provides an antibody-drug conjugate, which is formed by coupling the aforementioned bispecific antigen-binding molecule or the GPRC5D-binding molecule or its antigen-binding fragment to other biologically active molecules; preferably, the other biologically active molecule is a small molecule drug; preferably, the bispecific antigen-binding molecule and the other biologically active molecule are connected via a linker.

[0063] The present disclosure also provides a pharmaceutical composition comprising the aforementioned bispecific antigen-binding molecule, the GPRC5D-binding molecule or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a host cell, and / or an antibody-drug conjugate.

[0064] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0065] In one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0066] The present disclosure also provides the use of the aforementioned bispecific antigen-binding molecules, nucleic acid molecules, expression vectors, host cells, antibody-drug conjugates, and / or pharmaceutical compositions in the preparation of a medicament for treating, alleviating, and / or preventing tumors. Preferably, the tumor is a GPRC5D-positive tumor.

[0067] The present disclosure also provides a method for inducing cell death expressing GPRC5D in vivo or in vitro, the method comprising contacting the cell with the aforementioned bispecific antigen-binding molecule, the GPRC5D-binding molecule or antigen-binding fragment thereof, nucleic acid molecule, expression vector, host cell, antibody-drug conjugate and / or pharmaceutical composition. Preferably, the cell expressing GPRC5D3 is a tumor cell.

[0068] The present disclosure also provides a method for treating a disease associated with GPRC5D expression in a subject, the method comprising administering to a subject in need thereof a bispecific antigen-binding molecule, a GPRC5D-binding molecule or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a host cell, an antibody-drug conjugate, and / or a pharmaceutical composition. Preferably, the disease is a tumor. Preferably, the subject has relapsed or refractory disease to prior treatment with an anti-cancer therapeutic agent.

[0069] Preferably, the tumor / tumor cells are selected from the group consisting of lymphomas such as multiple myeloma, and metastases of the aforementioned tumors.

[0070] In one embodiment, the method further comprises administering to the subject an additional therapeutic agent.

[0071] The present disclosure also provides the aforementioned bispecific antigen-binding molecules, the GPRC5D-binding molecules or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, host cells, antibody-drug conjugates and / or pharmaceutical compositions for use in treatment.

[0072] The substitutions referred to in this disclosure are preferably expressed using the EU numbering system.

[0073] The technical solution of the present invention has the following beneficial effects: while maintaining a strong in vitro tumor cell killing ability, the level of in vitro non-specific cytokine release is weak or absent, and the safety is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The accompanying drawings further illustrate the novel features disclosed in this specification. The features and advantages disclosed in this specification can be better understood with reference to these drawings, but it should be understood that these drawings are only used to illustrate specific implementations of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0075] FIG1 shows the structures of two bispecific antibodies constructed in the examples, namely, bispecific antibody 1 and bispecific antibody 2.

[0076] FIG2A shows the binding of double antibody 1 and double antibody 2 to cells expressing hGPRC5D (NCI-H929 cells).

[0077] FIG2B shows the binding of double antibody 1 and double antibody 2 to cells expressing hGPRC5D (RPMI8226 cells).

[0078] FIG2C shows the binding of double antibody 1 and double antibody 2 to cells expressing hCD3 (Jurkat cells).

[0079] FIG3A shows the results of T cell-mediated cytotoxicity assay (TDCC), with the target cells being NCI-H929.

[0080] FIG3B shows the results of T cell-mediated cytotoxicity assay (TDCC), with RPMI8226 cells as the target cells.

[0081] Figure 3C shows the release of cytokine IFNγ under the conditions of co-incubation of PBMC and NCI-H929 tumor cells with dual antibody molecules.

[0082] Figure 3D shows the release of cytokine IFNγ when the bispecific antibody molecules were incubated with PBMCs only.

[0083] Figure 3E shows the release of cytokine IL6 under the conditions of co-incubation of PBMC and NCI-H929 tumor cells plus bispecific antibody molecules.

[0084] Figure 3F shows the release of cytokine IL6 when the bispecific antibody molecules were incubated with PBMCs only.

[0085] Figure 4 shows the efficacy of the bispecific antibody molecule in a mouse model reconstructed with human immune cells (tumor cells are NCI-H929).

[0086] Figure 5 shows the efficacy of the bispecific antibody molecule in a mouse model reconstructed with human immune cells (the tumor cells are RPMI8226).

[0087] FIG6A and FIG6B show the binding of the mouse hybridoma-derived anti-GPRC5D chimeric antibody of the present disclosure to GPRC5D-expressing cells (human GPRC5D-CHOK1 cell line).

[0088] FIG6C and FIG6D show the binding of the mouse hybridoma-derived anti-GPRC5D chimeric antibody of the present disclosure to GPRC5D-expressing cells (cynomolgus monkey GPRC5D-CHOK1 cell line).

[0089] FIG7 shows that the mouse hybridoma-derived anti-GPRC5D chimeric antibody disclosed herein does not non-specifically bind to the same family member GPRC5A.

[0090] FIG8A and FIG8B show the binding of the mouse hybridoma-derived anti-GPRC5D humanized antibody of the present disclosure to GPRC5D-expressing cells (human GPRC5D-CHOK1 cell line).

[0091] FIG8C and FIG8D show the binding of the mouse hybridoma-derived anti-GPRC5D humanized antibody disclosed herein to cells expressing GPRC5D (NCI-H929 tumor cells).

[0092] Detailed Description of the Invention

[0093] the term

[0094] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0095] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0096] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0097] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0098] As used herein, the three-letter and one-letter codes for amino acids are as described in J. Biol. Chem, 243, p3558 (1968).

[0099] The term "antibody" herein may include intact antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragments thereof (i.e., antigen-binding portions) or single chains thereof, and may also include products having antigen-specific binding ability formed by modifications (e.g., linking other peptide segments, rearrangement of functional units, etc.) based on intact antibodies or their antigen-binding fragments or single chains.

[0100] In one embodiment, an antibody typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain comprises a variable domain (VH) and a constant region.

[0101] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM. The corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0102] In the case of IgG, IgA, and IgD antibodies, the constant region comprises three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a segment of variable length rich in proline and cysteine. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0103] The term "variable region" or "variable domain" shows significant changes in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light chain / heavy chain pair forms an antibody binding site, so that a complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVRs), or more generally, referred to as complementary determining regions (CDRs). VH and VL each have four framework regions, FRs, represented by FR1, FR2, FR3, and FR4, respectively. Therefore, CDR and FR sequences typically appear in the following sequence of a heavy chain variable domain (or light chain variable domain): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0104] The term "antibody fragment" comprises at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or its immunogenicity-determining portion, or a fusion protein product further derived from this fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific antibodies or multispecific antibodies formed by antibody fragments.

[0105] The term "Fab" or "Fab fragment" refers to a monovalent antibody fragment consisting of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain. The term "F(ab')2" or "F(ab')2 fragment" comprises two Fab fragments and a hinge region and is a bivalent antibody fragment.

[0106] The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0107] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. As used herein, "amino acid substitution" or "substitution" or "replacement" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution S32A refers to the replacement of serine at position 32 with alanine.

[0108] When antibodies, binding molecules, bispecific binding molecules, or multispecific binding molecules are prepared using the variable regions of the present disclosure, the constant regions are not particularly limited, and constant regions known to those skilled in the art or obtained independently can be used. Amino acid mutations (e.g., mutations that increase or decrease binding to Fc receptors or FcRn) can also be introduced into the constant region portion.

[0109] The term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, e.g., an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index.

[0110] The bispecific antigen binding molecules of the present disclosure may also include substitutions or modifications of the constant region (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which produce compounds with the following preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered Fc ligand binding to Fc receptors (FcRs), enhanced or reduced ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity. In certain aspects, the antibody variants include Fc regions with one or more amino acid substitutions that weaken FcγR binding (e.g., substitutions at positions 234 and 235 of the Fc region). In one aspect, the substitutions are L234A and L235A.

[0111] The term "knob-into-hole" refers to a modification for promoting the association of the two polypeptide chains of Fc, which comprises a "knob" modification in one of the two polypeptide chains of Fc and a "hole" modification in the other of the two polypeptide chains of Fc. This technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng, 9, 617-621 (1996) and Carter, J Immunol Meth, 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of the first polypeptide chain and introducing a corresponding cavity ("hole") in the interface of the second polypeptide chain, so that the protrusion can be placed in the cavity to promote heterodimer formation and hinder homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide chain with larger side chains (e.g., tyrosine or tryptophan). A complementary cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide chain by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0112] Thus, in a specific embodiment, in the CH3 domain of the first polypeptide chain of the Fc domain of the bispecific antigen-binding molecule of the present disclosure, one amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance in the CH3 domain of the first polypeptide chain, which can be accommodated in the cavity in the CH3 domain of the second polypeptide chain, and in the CH3 domain of the second polypeptide chain of the Fc domain, one amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second polypeptide chain, in which the protuberance in the CH3 domain of the first polypeptide chain can be accommodated. Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0113] The term "linker" refers to any tool used to connect two different functional units (e.g., antigen binding fragments). The types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of the polypeptide linker is not limited. The polypeptide linker is preferably non-immunogenic and flexible, such as those comprising serine and glycine sequences. Depending on the specific construct, the linker can be long or short.

[0114] According to the present disclosure, the linker connecting the different functional units preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises the amino acid sequence (G4S) n or (G4S) n A, wherein n is any integer selected from 1 to 10, preferably comprises the amino acid sequence (G4S)3 or (G4S)3A. The linker connecting the VH and VL domains to form the scFv domain of VH-VL or VL-VH preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises the amino acid sequence (G4S) n or (G4S) n A, wherein n is any integer selected from 1 to 10, preferably comprises the amino acid sequence (G4S)3 or (G4S).

[0115] As used herein, "antibody" may be used in the broadest sense and may include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

[0116] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced by a single cell clone that is directed against a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or a combination of these techniques.

[0117] It should be noted that the CDR and FR divisions of the variable regions of the antibodies and bispecific antigen-binding molecules disclosed herein are determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies containing one or more CDRs derived from any naming system based on the antibody sequences disclosed herein are clearly within the scope of this disclosure.

[0118] The term "humanized antibody" refers to an antibody in which all or part of the amino acids outside the CDRs of a non-human antibody (such as a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigenic specificity similar to that of the original antibody.

[0119] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0120] The terms "homologous" and "heterogeneous" used in this article are relative concepts, which can refer to the fact that different elements in a construct have the same or different origins. They can also refer to the fact that after the construction of the construct is completed, some elements that originally had the same origin, i.e., "homologous", have been transformed and changed compared to other original elements that have not been transformed, thus becoming "heterogeneous".

[0121] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, a complete extracellular domain (ECD) or a protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that triggers an immune response. Any form of antigen or a cell or preparation containing the antigen can be used to generate an antibody specific for an antigenic determinant.

[0122] The terms "epitope" and "antigenic determinant" refer to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope can be formed by adjacent amino acids or by non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope typically exists in a unique spatial conformation and comprises at least 3-15 amino acids.

[0123] The term "bispecific" refers to an antigen binding molecule that is capable of specifically binding to two different antigenic determinants. The term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and their derivatives, such as fragments. The term "bispecific antigen binding molecule" or "bispecific binding molecule" refers to a binding molecule (e.g., an antibody or a molecule comprising an antibody fragment) that is specific for two different antigens (or epitopes), particularly a bispecific antibody.

[0124] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.

[0125] When antibodies, binding molecules, bispecific binding molecules, or multispecific binding molecules are prepared using the variable regions of the present disclosure, the constant regions are not particularly limited, and constant regions known to those skilled in the art or obtained independently can be used. Amino acid mutations (e.g., mutations that increase or decrease binding to Fc receptors or FcRn) can also be introduced into the constant region.

[0126] The method for obtaining the binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules disclosed herein is not particularly limited and may be obtained by any method. The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules of the invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid containing the secreted antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0127] The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0128] The term "stable transfection" or "stable transfection" refers to the introduction and integration of exogenous nucleic acid, DNA or RNA, into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated the foreign DNA into its genomic DNA.

[0129] The term "antibody drug conjugate" (ADC) refers to an antibody to which a therapeutically active substance or active pharmaceutical ingredient (API) has been covalently coupled so that the therapeutically active substance or active pharmaceutical ingredient (API) can be targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient can be a cytotoxin that can kill cells targeted by the ADC, preferably malignant or cancerous cells. Covalent attachment of the therapeutically active substance, active pharmaceutical ingredient or cytotoxin can be performed in a non-site-specific manner using standard chemical linkers that couple payloads to lysine or cysteine ​​residues, or preferably, conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC.

[0130] The term "amino acid substitution" or "substitution" or "replacement" means replacing the amino acid at a specific position in the parent polypeptide sequence with another amino acid.

[0131] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). d " refers to the dissociation constant for a specific antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, bio-layer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0132] The term "biological activity" refers to the ability of an antibody to bind to an antigen and result in a measurable biological response, which can be measured in vitro or in vivo.

[0133] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. When a "pharmaceutical composition" is present as a combination of separate formulations containing two or more different active ingredients, they can be administered simultaneously, sequentially, separately, or at intervals, with the goal of exerting the biological activities of the multiple active ingredients for combined treatment of a disease.

[0134] The binding molecules or antigen-binding fragments disclosed herein can be used in combination with other drugs, and the active ingredients can be mixed together to form a single administration unit, or can be independently formed into administration units and used separately.

[0135] The term "effective amount" refers to a dosage of a pharmaceutical formulation of an antibody or fragment of the present disclosure that produces the desired effect in a treated patient after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as ethnic differences; weight, age, and health status; the specific disease involved; the severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0136] As used herein, the term "individual" or "subject" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0137] As used herein, the terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, the term "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance defects (e.g., transplant rejection).

[0138] As used herein, the term "tumor" refers to a disease characterized by pathological proliferation of cells or tissues, and their subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation.

[0139] As used herein, the term "treatment" refers to clinical intervention aimed at altering the course of a disease in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, ameliorating or relieving the condition, and alleviating or improving the prognosis.

[0140] The terms "G protein coupled receptor C5 family subtype D" and "GPRC5D" specifically include human GPRC5D protein, and include variants, subtypes, homologous species and analogs of human GPRC5D that have at least one common epitope with GPRC5D (e.g., human GPRC5D). Exemplary human GPRC5D sequences can be found in GenBank Accession No. BC069341, NCBI Reference Sequence: NP_061124.1 and those described in UniProtKB / Swiss-Prot Accession No. Q9NZD1 (see also Brauner-Osborne, H et al., 2001, Biochim. Biophys. Acta 1518, 237-248). DETAILED DESCRIPTION

[0141] The present disclosure is further described below with reference to specific examples. It should be understood that these examples are intended only to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Experimental methods in the following examples, where specific conditions are not specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0142] Example 1. Design and sequence of anti-CD3-GPRC5D bispecific antibody

[0143] The bispecific antigen-binding molecule (hereinafter referred to as the bispecific antibody) constructed in the embodiment is formed by connecting the heavy chain of the anti-GPRC5D full-length antibody Fab segment to the binding domain of the human T cell receptor subunit CD3ε through a flexible linker. Among them, the anti-GPRC5D full-length antibody is GPRC5D mAb, whose heavy chain sequence is shown in SEQ ID NO: 5, and the light chain sequence is shown in SEQ ID NO: 3. The CD3ε binding domain is from the full-length antibody CD3 mAb, whose heavy chain sequence is shown in SEQ ID NO: 6, and the light chain sequence is shown in SEQ ID NO: 7. In order to reduce the ADCC activity of the antibody, the Fc segment of the finally constructed bispecific antibody has been subjected to amino acid substitutions of L234A and L235A.

[0144] The heavy and light chain variable regions of the CD3 mAb were connected via a flexible linker to form a single-chain antibody (scFv) with the structure: VH-(G4S)3-VL, with the sequence shown in SEQ ID NO: 8. The scFv was then fused to the C-terminus of the heavy chain of the GPRC5D full-length antibody Fab fragment via a flexible linker.

[0145] The scFv was fused to the C-terminus of the heavy chain of the Fab segment of the full-length GPRC5D antibody, and also contained another complete GPRC5D binding domain and an Fc portion with a "knob" and "hole" structure. The resulting bispecific antibody was named Bis-Antibody 1. The sequences of the fused chains are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and a schematic diagram is shown in Figure 1.

[0146] The scFv was fused to the C-terminus of the heavy chain of the Fab segment of the full-length GPRC5D antibody, and also contained an Fc portion with a "knob" and "hole" structure. The resulting bispecific antibody was named Bis-Anti 2. The sequences of the fused chains are shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, and a schematic diagram is shown in Figure 1.

[0147] Of the two heterologous heavy chains, the one containing the scFv was designed as a "knob" structure, including amino acid substitutions at S354C and T366W. Of the two heterologous heavy chains, the one not containing the scFv was designed as a "hole" structure, including amino acid substitutions at Y349C, T366S, L368A, and Y407V. Furthermore, to facilitate purification of the bispecific antibody, the heavy chain in the "hole" structure also underwent an H435R substitution.

[0148] The structures of bispecific antibody 1 and bispecific antibody 2 and their related molecular sequences are summarized in Tables 1 and 2, respectively.

[0149] Table 1 Structure of bispecific antibodies

[0150] Table 2 Amino acid sequences of bispecific antibodies

[0151] Example 2. Construction of anti-CD3-GPRC5D bispecific antibody and its transient transfection expression in eukaryotic cells

[0152] The gene fragments encoding the aforementioned bispecific antibody molecules were cloned into the pTT5 expression vector to prepare transfection-grade expression plasmids.

[0153] Cultivation of Expi293F in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, it was further purified using a gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, collect the monomer peak, and exchange the solution into PBS for aliquoting. The final purified antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0154] Example 3. Affinity detection experiment of anti-CD3-GPRC5D bispecific antibody

[0155] A. Affinity Detection of Anti-CD3-GPRC5D Bispecific Antibody to Cells Expressing hGPRC5D and hCD3

[0156] FACS was used to detect the binding of the anti-CD3-GPRC5D bispecific antibody to NCI-H929 cells and RPMI8226 cells expressing hGPRC5D, and T lymphocytes (Jurkat) naturally expressing hCD3.

[0157] NCI-H929 cells (ATCC, CRL-9068), RPMI8226 cells (ATCC, CCL-155), and Jurkat cells (ATCC, TIB-152) were cultured in RPMI1640 + 10% FBS + 0.05 mM mercaptoethanol for NCI-H929 cells and RPMI1640 + 10% FBS for RPMI8226 and Jurkat cells. The cells were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. When ready for use, NCI-H929, RPMI8226, and Jurkat cells were placed directly into 50 mL centrifuge tubes without digestion.

[0158] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA (in PBS). The cells were counted and adjusted to a cell density of 1E6 / mL. The cells were plated into a 96-well round-bottom culture plate (Corning, 3799) and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the cells were resuspended in 200 μL of 1% BSA (in PBS). The cells were centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate was stored at 4°C until use. The test antibody and negative control IgG1 AA (purchased from Bio-Bio, catalog number B109802) were diluted in 1% BSA (in PBS) at a starting concentration of 100 nM and then diluted 10-fold to seven concentrations. The cells were resuspended in the diluted antibody at 100 μL / well and incubated at 4°C for 1 hour. The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant discarded. Resuspend and wash with 160 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Dilute the secondary antibody (goat anti human IgG Fc PE) at 1:400 with 1% BSA (in PBS) according to the instructions, resuspend the cells with the diluted secondary antibody, 100 μL / well, and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes, and discard the supernatant. Resuspend the cells with 100 μL of 1% BSA (in PBS), filter the cells through 300 mesh gauze, and detect the mean fluorescence intensity of the PE channel by flow cytometry.

[0159] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50 ) and the highest mean fluorescence intensity (Top MFI), as shown in Table 3, Figure 2A (NCI-H929 cells), Figure 2B (RPMI8226 cells), and Figure 2C (Jurkat cells). The binding ability of the two bispecific antibodies to the Jurkat cell line was weaker than that to the NCI-H929 cell line. Literature reports that the expression level of GPRC5D in RPMI8226 cells is lower than that in NCI-H929 cells. Therefore, the binding ability of the two bispecific antibodies to the RPMI8226 cell line is weaker than that to the NCI-H929 cell line.

[0160] Table 3 Affinity of bispecific antibodies to hGPRC5D (NCI-H929 cell line and RPMI8226 cell line) and hCD3 (Jurkat cell line)

[0161] B. In vitro recombinant protein binding affinity and kinetics of the anti-CD3-GPRC5D bispecific antibody

[0162] The affinity and kinetic properties of the anti-CD3-GPRC5D bispecific antibody to human / cynomolgus monkey CD3 were analyzed using a Biacore 8K instrument.

[0163] To determine the affinity and kinetic properties of human CD3 (purchased from Acro, Catalog No. CDD-H52W1) and cynomolgus monkey CD3 (purchased from Acro, Catalog No. CDD-C52W4), a CM5 chip was used to directly immobilize human / cynomolgus monkey CD3 molecules. The CM5 chip was first activated with EDC and NHS. Human / cynomolgus monkey CD3 molecules were diluted to 1 μg / mL in acetate solution, pH 5, and immobilized at a flow rate of 10 μL / min for 60 s. The chip was then blocked with ethanolamine. The anti-CD3-GPRC5D bispecific antibody was diluted two-fold in HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer to a concentration series (100 nM to 0.39 nM). Binding was allowed to proceed for 90 s and dissociation for 360 s at a flow rate of 50 μL / min.

[0164] After each experimental round, the chip was rinsed with 3M MgCl₂ solution at a flow rate of 30 μL / min for 30 seconds to remove the anti-CD3-GPRC5D bispecific antibody molecules, completing chip regeneration. Raw data were analyzed using Biacore Insight Evaluation Software (3.0.12.15655) and fitted with a (1:1) Langmuir model. The resulting bispecific antibody affinity and kinetics data are shown in Table 4.

[0165] Table 4 Binding affinity and kinetics of anti-CD3-GPRC5D bispecific antibodies to human / cynomolgus monkey CD3 proteins

[0166] The experimental results showed that the two bispecific antibody molecules bound to both human and cynomolgus monkey CD3 with comparable affinity.

[0167] Example 4. In vitro functional experiments of anti-CD3-GPRC5D bispecific antibodies

[0168] AT cell-mediated cytotoxicity assay (TDCC), target cells are NCI-H929

[0169] Culture NCI-H929 cells (ATCC, CRL-9068) in RPMI 1640 medium with 10% FBS and 0.05 mM mercaptoethanol in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When ready to use, place NCI-H929 cells directly into a 50 mL centrifuge tube without digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI 1640 medium with 2% FBS, count the cells, and adjust the cell density to 1.5E5 / mL.

[0170] Target cells were plated into a flat-bottom 96-well plate (Corning, 3599) at 100 μL / well and incubated overnight at 37°C, 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and adjusted to a cell density of 3E6 / mL in RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C, 5% CO2.

[0171] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 400 nM and dilute 10-fold downward. Add the diluted antibody to the cell culture plate at 50 μL / well, for a starting and ending concentration of 100 nM. Incubate at 37°C, 5% CO2 for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0172] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0173] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0174] Import cell killing data into GraphPad Prism, draw cell killing / concentration curves, and calculate EC 50 The results are shown in Table 5 and Figure 3A. The maximum killing capacity of the two bispecific antibodies against NCI-H929 cells can reach about 90%, and the half-effective concentration of bispecific antibody 1 molecule is lower than that of bispecific antibody 2 molecules.

[0175] BT cell-mediated cytotoxicity assay (TDCC), target cells are RPMI8226

[0176] Culture RPMI8226 cells (ATCC, CCL-155) in RPMI1640 + 10% FBS in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When ready to use, place the RPMI8226 cells directly into a 50 mL centrifuge tube without digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI1640 + 2% FBS, count the cells, and adjust the cell density to 1.5E5 / mL.

[0177] Target cells were plated into a flat-bottom 96-well plate (Corning, 3599) at 100 μL / well and incubated overnight at 37°C, 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and adjusted to a cell density of 3E6 / mL in RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C, 5% CO2.

[0178] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 400 nM and dilute 10-fold downward. Add the diluted antibody to the cell culture plate at 50 μL / well, for a starting and ending concentration of 100 nM. Incubate at 37°C, 5% CO2 for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0179] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0180] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0181] Import cell killing data into GraphPad Prism, draw cell killing / concentration curves, and calculate EC 50 The results are shown in Table 5 and Figure 3B. Literature reports indicate that GPRC5D expression in RPMI8226 cells is lower than that in NCI-H929 cells. Therefore, the maximum killing ability of the two bispecific antibodies against RPMI8226 cells is much weaker than that against NCI-H929 cells. The half-effective concentration of the two bispecific antibodies against RPMI8226 cells is higher than that against NCI-H929 cells; bispecific antibody 1 has a stronger killing effect than bispecific antibody 2.

[0182] Table 5 T cell-mediated cytotoxicity assay (TDCC) results of anti-CD3-GPRC5D bispecific antibodies

[0183] C. Cytokine release assay

[0184] NCI-H929 cells (ATCC, CRL-9068) were cultured in RPMI1640 + 10% FBS + 0.05mM mercaptoethanol in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When ready to use, NCI-H929 cells were placed directly into a 50mL centrifuge tube without digestion. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend in RPMI1640 + 2% FBS medium, count the cells, and adjust the cell density to 1.5E5 / mL. NCI-H929 cells were plated into a 96-well plate (corning 3799) at 50μL / well and incubated at 37°C in 5% CO2.

[0185] Purchase fresh PBMCs, count the cells, and adjust the cell density to 6E6 / mL using RPMI1640 + 2% FBS. Plate the effector cells into a 96-well plate at 50 μL / well and incubate at 37°C in 5% CO2.

[0186] Dilute the antibody in RPMI1640 + 2% FBS medium to a starting concentration of 400 nM and dilute 10-fold downward. Add the diluted antibody to the cell culture plate at 50 μL / well, for a starting and ending concentration of 100 nM. Incubate at 37°C, 5% CO2 for 24 hours.

[0187] The culture plate was centrifuged at 400 g for 10 minutes, and 100 μL of the supernatant was taken and frozen at -80°C for later use.

[0188] Dilute the standard sample according to the instructions for the cytokine assay kits (Biolegend, 430104 (Human IFN-γ ELISA MAX Deluxe); 430504 (ELISA MAX Deluxe Set Human IL-6)) in two-fold dilutions, with a maximum concentration of 8000 pg / mL and a minimum concentration of 0 pg / mL, for a total of 12 concentration points. Add 100 μL / well of the prepared standard sample or thawed sample to a 96-well plate (samples require additional dilution) and read the OD450 value on an Envision plate according to the kit instructions.

[0189] The results are shown in Figures 3C-3F. When PBMCs were co-incubated with tumor cells, the levels of IFNγ and IL6 cytokine release induced by the bispecific antibody 2 molecule were weaker than those induced by the bispecific antibody 1 molecule. Cytokine release by both bispecific antibodies was also weak in the presence of PBMCs alone, indicating an appropriate safety window for co-incubation with PBMCs and NCI-H929 tumor cells.

[0190] Example 5. Physical stability test of anti-CD3-GPRC5D bispecific antibody

[0191] NanoDSF (differential fluorescence scanning technology) was used to detect the thermal stability of different antibodies in pH 7.4 PBS buffer. The sample concentration was around 1 mg / mL, and Prometheus NT.Plex (nano DSF) was used for detection. Before detection, each sample was centrifuged at 10,000 g for 10 minutes. 40 μL of sample was added to each well of the sample plate (the instrument loading volume was 10 μL, and each sample had one replicate well). The scanning temperature started at 30°C and ended at 95°C, with a scanning rate of 0.5°C / min. The experimental results are shown in Table 6. Both bispecific antibodies exhibited good thermal stability.

[0192] Table 6 NanoDSF detection results of anti-CD3-GPRC5D bispecific antibodies

[0193] Example 6. Pharmacokinetics of anti-CD3-GPRC5D bispecific antibody

[0194] Two naive cynomolgus monkeys were used in the experiment and were given free access to water. The bispecific antibody was administered at a dose of 1 mg / kg, and the intravenous infusion was completed over 30 minutes. Blood was collected at the following time points: Pre-dose, 30 minutes, 2 hours, 4 hours, 6 hours, 24 hours (1 day), 48 hours (2 days), 72 hours (3 days), 120 hours (5 days), 168 hours (7 days), 240 hours (10 days), and 336 hours (14 days). Whole blood samples were collected in polyethylene tubes without anticoagulants, placed at room temperature for about 1 hour, centrifuged at 6000g at 25°C, and immediately divided into two portions (PK samples and cytokine detection samples), immediately placed on dry ice, and transferred to a -80°C refrigerator for long-term storage.

[0195] The concentration of CD3 in serum was determined by ELISA. A 96-well plate was coated with human CD3 protein at a concentration of 1 μg / mL, with 100 μL per well added and incubated at 4°C overnight. The plate was washed three times with 200 μL of PBST per well, followed by the addition of 300 μL of blocking reagent (5% milk powder) and incubation at 37°C for 1 hour. The plate was then washed three times with 200 μL of PBST per well, followed by the addition of 100 μL of the test sample and incubation at 37°C for 1 hour. The plate was then washed six times with 300 μL of PBST per well, followed by the addition of anti-human IgG (H&L) HRP (1:2000) and TMB. After incubation in the dark for 10 minutes, the color reaction was stopped by the addition of 100 μL of stop solution. The CD3 concentration was quantified based on the color reaction.

[0196] The absorbance at a wavelength of 450 nm was detected using an M5 plate reader from MD, and the data were processed using softmax software.

[0197] Phoenix Winnolin 8.2 software was used to calculate the monkey serum concentration of the bispecific antibody and obtain the pharmacokinetic parameters of the bispecific antibody.

[0198] The results showed that the pharmacokinetic properties of bispecific antibodies 1 and 2 were good.

[0199] Example 7. Pharmacokinetics of anti-CD3-GPRC5D bispecific antibody accompanied by cytokine detection

[0200] The assay kit is a Multi-Analyte Flow assay kit (Biolegend, Cat. No. 740391). Before use, add 250 μL of buffer to the lyophilized NHP Th cytokine, mix thoroughly, and let it stand at room temperature for 10 minutes. Dilute the standard sample 1:4 with the assay buffer provided in the kit, and dilute the PK serum sample 1:4 with the assay buffer provided in the kit. Add 10 mL of LEGENDplex Assay Buffer to lyophilized Matrix B and dissolve at room temperature for 15 minutes before use.

[0201] Add 25 μL of Matrix B and 25 μL of standard to the standard wells. Add 25 μL of assay buffer and 25 μL of serum sample to the sample wells. Vortex the detection beads, then mix the various detection beads in a 1:1 ratio and dilute with assay buffer to the working concentration, adding 25 μL to each well. Seal the plate with parafilm and incubate at 800 rpm in the dark for 2 hours. Centrifuge at 250 g for 5 minutes, discard the supernatant, and wash with 200 μL / well of wash buffer. Add 25 μL of detection antibody to the plate, seal the plate with parafilm, and incubate at 600 rpm in the dark for 1 hour at room temperature. Add 25 μL of SA-PE, seal the plate with parafilm, and incubate at 600 rpm in the dark for 0.5 hour at room temperature. Centrifuge at 250 g for 5 minutes at room temperature, discard the supernatant, and wash with 200 μL / well of wash buffer. The sample was resuspended in 1% BSA (PBS solution), filtered through 300-mesh gauze, and detected by flow cytometry.

[0202] The FCS files were exported and the cytokine release of the samples was analyzed using LENEGDplex 8.0 software. The results are shown in Table 7.

[0203] Table 7 Pharmacokinetics of anti-CD3-GPRC5D bispecific antibodies accompanied by cytokine detection results

[0204] The experimental results showed that the pharmacokinetic detection of various cytokines by the two bispecific antibodies in crab-eating monkeys showed that the overall release level of various cytokines was very low, with a slightly higher release of IL6, which returned to the initial level after 72 hours and had good safety.

[0205] Example 8. In vivo efficacy experiment of anti-CD3-GPRC5D bispecific antibody

[0206] A. Mouse model reconstructed with human immune cells, using NCI-H929 tumor cells

[0207] All experimental animals were housed in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 20.0-26.0℃, the humidity was 40-70%, and the light-dark cycle was 12h / 12h.

[0208] NCI-H929 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.05 mM β-ME. NCI-H929 cells were collected in the exponential growth phase, resuspended in 0.1 mL (1:1) PBS and Matrigel suspension, and 5×10 cells were inoculated on the right side of the back of the experimental mice. 6 NCI-H929 cells were used to observe tumor growth regularly. 3 The mice were randomly divided into groups according to tumor size and body weight.

[0209] Donor PBMCs were purchased from AllCells, LLC. 1×10 7 PBMC / 0.1 mL PBS was inoculated intraperitoneally into each mouse on the day of NCI H929 cell inoculation to establish a mouse model reconstructed with human immune cells.

[0210] Before the start of administration, all animals were weighed and the tumor volume was measured with a vernier caliper. In view of the fact that tumor volume affects the effectiveness of treatment, the randomized group design method was used to group the mice according to their tumor volume to ensure that the tumor volumes between different groups were similar. Grouping was performed using StudyDirector™ (version 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA). The day of mouse grouping was defined as day 0, and intraperitoneal injection was started on day D0, once every three days, for 6 doses. The hIgG1AA negative control group (purchased from Baiying Bio, catalog number B109802) and the test bispecific antibody experimental grouping and dosing regimen are shown in Table 8.

[0211] Table 8 Experimental groups and dosing regimens

[0212] After administration, the animals' daily behavior was monitored for 16 days. During the entire experiment, the length and width of the tumor were measured every 2 days using a vernier caliper, and the tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ) was calculated. Relative tumor inhibition rate TGI (%): TGI% = (1-T / C) × 100%. T / C% is the relative tumor growth rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the hIgG1 AA control group at a certain time point. T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the hIgG1 AA control group at a specific time point, respectively. The experimental results of tumor volume, mouse body weight, tumor weight, etc. of each group of animals are expressed as mean ± standard error (Mean ± SEM). The independent sample T test was used to compare whether there were significant differences between different treatment groups and the control group. The data were analyzed using SPSS. P < 0.05 was considered to be significantly different. The experimental results are shown in Table 9 and Figure 4.

[0213] Different dose treatment groups of Dual Antibody 1 and Dual Antibody 2 showed very significant tumor inhibition effects in the MiXeno model of human myeloma NCI-H929 subcutaneously transplanted NPG female mice (p values ​​were all less than 0.001). The tumor inhibition rates of Dual Antibody 1 and Dual Antibody 2 both reached 100%, and the mice tolerated the test drugs well, with no obvious weight loss or toxicity.

[0214] Table 9 Tumor volume and tumor inhibition rate of each drug group

[0215] B. Reconstruction of mouse model with human immune cells, and RPMI8226 tumor cells

[0216] All experimental animals were housed in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 20.0-26.0℃, the humidity was 30-70%, and the light-dark cycle was 12h / 12h.

[0217] RPMI8226 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. RPMI8226 cells were collected in the exponential growth phase and resuspended in 0.1 mL (1:1) PBS and Matrigel suspension. 5×10 6 RPMI8226 cells were used to observe the tumor growth regularly. 3 The mice were randomly divided into groups according to tumor size and body weight.

[0218] Donor PBMCs were purchased from AllCells, LLC. 1×10 7 PBMC / 0.1 mL PBS was inoculated intraperitoneally into each mouse on the day of RPMI8226 cell inoculation to establish a mouse model reconstructed with human immune cells.

[0219] Before the start of drug administration, all animals were weighed and the tumor volume was measured with a vernier caliper. In view of the fact that tumor volume affects the effectiveness of treatment, the randomized group design method was used to group the mice according to their tumor volume to ensure that the tumor volumes between different groups were similar. Grouping was performed using StudyDirector™ (version 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA). The day of mouse grouping was defined as day 0, and intraperitoneal injection was started on day D0, once every three days, for 6 doses. The hIgG1AA negative control group (purchased from Baiying Bio, item number B109802) and the test bispecific antibody experimental grouping and dosing regimen are shown in Table 10.

[0220] Table 10 Experimental groups and dosing regimens

[0221] After administration, the animals' daily behavior was monitored for 18 days. During the entire experiment, the length and width of the tumors and the tumor volume (mm) were measured twice a week using a vernier caliper. 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2) was calculated. Relative tumor inhibition rate TGI (%): TGI% = (1-T / C) × 100%. T / C% is the relative tumor growth rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the hIgG1 AA control group at a certain time point. T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the hIgG1 AA control group at a specific time point, respectively. The experimental results of tumor volume, mouse body weight, tumor weight, etc. of each group of animals are expressed as mean ± standard error (Mean ± SEM). The independent sample T test was used to compare whether there were significant differences between different treatment groups and the control group. The data were analyzed using SPSS. P < 0.05 was considered to be significantly different. The experimental results are shown in Table 11 and Figure 5.

[0222] Different dose treatment groups of dual antibody 1 and dual antibody 2 showed very significant tumor inhibition effects in the MiXeno model of human myeloma RPMI8226 subcutaneously transplanted NPG female mice (p values ​​were all less than 0.005), and the mice tolerated the test drugs well, with no obvious weight loss or toxicity.

[0223] Table 11 Tumor volume and tumor inhibition rate of each drug group

[0224] Example 9. Obtaining anti-GPRC5D antibodies from mouse hybridomas

[0225] Construction of cell lines expressing human GPRC5D and GPRC5A, and cynomolgus monkey GPRC5D.

[0226] The nucleotide sequence encoding human GPRC5D (Uniprot ID: Q9NZD1) was cloned into the pCMV3 vector (SinoBiological, Catalog No. CV011) to generate a vector for constructing a human GPRC5D cell line. This vector was transfected into CHOK1 cells (ATCC, Catalog No. CCL-61) to generate a CHOK1 cell line expressing human GPRC5D (abbreviated as: human GPRC5D-CHOK1 cell line).

[0227] The nucleotide sequence encoding cynomolgus macaque GPRC5D (Uniprot ID: A0A2K5W6I2) was cloned into the pCMV3 vector to generate a vector for constructing the cynomolgus macaque GPRC5D cell line. This vector was then transfected into CHOK1 cells to generate the CHOK1 cell line expressing cynomolgus macaque GPRC5D (abbreviated as the cynomolgus macaque GPRC5D-CHOK1 cell line).

[0228] The nucleotide sequence encoding human GPRC5A (Uniprot ID: Q8NFJ5) was cloned into the pCMV3 vector to obtain a vector for constructing a human GPRC5A cell line. The obtained vector was transfected into CHOK1 cells to obtain a CHOK1 cell pool expressing human GPRC5A (abbreviated as: human GPRC5A-CHOK1 cell line).

[0229] Anti-GPRC5D monoclonal antibodies were generated by immunizing mice.

[0230] The experiment used Swiss Webster white mice, female, 6 weeks old (Charles River Company). Housing environment: SPF grade. After purchase, the mice were kept in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. The immunogen was the full-length human GPRC5D plasmid, and 4 μg of plasmid was used for each immunization on days 0, 14, 28, and 42. CHO-K1 cells that highly express human GPRC5D were used for booster immunization 4 days before spleen cell fusion. During this period, the mouse serum was detected by FACS to determine the antibody titer in the mouse serum. After the booster immunization, mice with high antibody titers in the serum and titers approaching a plateau were selected for spleen cell fusion, and the spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) were fused to obtain hybridoma cells.

[0231] After 7-14 days of culture of the fused hybridoma cells, the culture supernatant was taken and the hybridoma supernatant was screened for antibodies using CHO-K1 cells that highly expressed human GPRC5D by FACS. The positive antibody strains were further screened using CHO-K1 cells that highly expressed cynomolgus monkey GPRC5D and blank CHO-K1 cells to exclude non-specific binding antibody hybridoma strains. The strains were screened using flow cytometry and further excluded using CHO-K1 cells that highly expressed human GPRC5A. Thus, hybridomas that specifically bound to human / cynomolgus monkey GPRC5D but not to human GPRC5A were selected. Hybridoma cells in the logarithmic growth phase were collected, RNA was extracted using Trizol (Invitrogen, 15596-018), and reverse transcribed (PrimeScript TMReverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using Mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced to obtain 11 monoclonal antibodies: 18CH-1, 18CH-2C, 18CH-3, 18CH-4, 18CH-5, 18CH-7A, 18CH-9, 18CH-10A, 18CH-15, 18CH-16, and 18CH-18. The amino acid sequence of the variable region of 18CH-16 is shown in Table 12.

[0232] Table 12 Amino acid sequences of the variable regions of mouse hybridoma-derived monoclonal antibodies against GPRC5D

[0233] Based on the above amino acid sequence, the CDRs and FRs of the antibody variable region were divided using the Kabat numbering convention. The sequence composition of the six CDRs of 18CH-16 is shown in Table 13 below.

[0234] Table 13 CDR sequences of anti-GPRC5D monoclonal antibodies derived from mouse hybridomas

[0235] Example 10. Construction of anti-GPRC5D chimeric antibody derived from mouse hybridoma and its transient transfection expression in eukaryotic cells

[0236] The sequenced nucleic acid sequences encoding the heavy and light chain variable regions of the monoclonal antibody disclosed herein were spliced ​​with the nucleic acid sequences encoding the IgG1 heavy chain constant region and the κ light chain constant region, respectively, to generate target gene fragments, which were cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0237] Cultivation of Expi293F in serum-free medium TMCells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified chimeric antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0238] Example 11. Binding of mouse hybridoma-derived anti-GPRC5D chimeric antibodies to GPRC5D-expressing cells

[0239] Human GPRC5D-CHOK1 cells and cynomolgus macaque GPRC5D-CHOK1 cells were cultured in a medium containing F12K + 10% FBS + 400 μg / mL hygromycin in T75 cell culture flasks at 37°C in a 5% CO2 incubator. Before use, cells were washed twice with sterile DPBS and digested with 0.25% trypsin-EDTA for approximately 5 minutes before rinsing with complete culture medium.

[0240] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. Discard the supernatant and resuspend the cells in 200 μL of 1% BSA in PBS. Centrifuge again at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and store at 4°C until needed. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant. Wash the cells with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. Discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells with the diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash the cells with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0241] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The obtained MFI was imported into Graphpad to analyze the half-binding concentration (EC) of the antibody to the cells. 50 ) and the highest mean fluorescence intensity (Top MFI), the results are shown in Table 14 and Figures 6A-6D. The screened anti-GPRC5D chimeric antibodies had good binding to human GPRC5D and cynomolgus monkey GPRC5D at the cellular level.

[0242] Table 14 Binding of anti-GPRC5D chimeric antibodies derived from mouse hybridoma to cells expressing GPRC5D

[0243] Example 12. Binding of anti-GPRC5D chimeric antibodies derived from mouse hybridomas to GPRC5A, a member of the same family

[0244] The culture medium for human GPRC5A-CHOK1 cells is F12K + 10% FBS + 400 μg / mL Hygromycin. The cells are cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. When ready for use, the cells are washed twice with sterile DPBS, digested with 0.25% trypsin EDTA for approximately 5 minutes, and then terminated with complete culture medium.

[0245] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. Discard the supernatant and resuspend the cells in 200 μL of 1% BSA in PBS. Centrifuge again at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and store at 4°C until needed. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant. Wash the cells with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. Discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells with the diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash the cells with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes. Discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0246] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The obtained MFI was imported into Graphpad to analyze the half-binding concentration (EC) of the antibody to the cells. 50 ) and the highest mean fluorescence intensity (Top MFI), and the results are shown in FIG7 , indicating that the tested antibodies had no non-specific binding to the same family member GPRC5A.

[0247] Example 13. Humanized design of anti-GPRC5D antibody derived from mouse hybridoma

[0248] The 11 chimeric antibodies were subjected to expression purification tests and cell-level binding tests, and two clones were further selected for humanized design.

[0249] Humanization of the murine anti-human GPRC5D monoclonal antibody was performed using methods well-documented in the field. Briefly, human constant domains were substituted for the parental (murine antibody) constant domains, and human antibody sequences were selected based on homology between murine and human antibodies. Based on the representative murine antibody VH / VL CDR structures, the heavy and light chain variable region sequences were compared with a human antibody germline database to obtain human germline templates with high homology.

[0250] The CDR regions of the murine antibody were transplanted onto the selected humanized template, replacing the humanized variable regions. These regions were then recombined with the IgG constant regions (preferably IgG1 heavy chain and κ light chain). Next, based on the three-dimensional structure of the murine antibody, backmutations were performed on buried residues, residues directly interacting with the CDR regions, and residues critically influencing VL and VH conformations. Multiple antibodies were designed, combining humanized light and heavy chain variable region sequences, including 18CH16H2L1, as shown in Table 15.

[0251] Table 15 Amino acid sequences of the variable regions of the humanized antibody 18CH16H2L1 derived from mouse hybridoma

[0252] Example 14. Preparation of humanized anti-GPRC5D antibodies derived from mouse hybridomas

[0253] Referring to Example 10, the target gene fragments generated by splicing the nucleic acid sequences encoding the heavy chain variable region and light chain variable region of the humanized antibody with the nucleic acid sequences encoding the IgG1 heavy chain constant region and the κ light chain constant region were cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0254] Cultivation of Expi293F in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified humanized antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0255] Example 15. Binding of mouse hybridoma-derived anti-GPRC5D humanized antibodies to GPRC5D-expressing cells

[0256] Human GPRC5D-CHOK1 cells were cultured in F12K + 10% FBS + 400 μg / mL Hygromycin, while NCI-H929 cells were cultured in RPMI 1640 + 10% FBS + 0.05 mM mercaptoethanol. T75 cell culture flasks were cultured in a 37°C, 5% CO2 incubator. Human GPRC5D-CHOK1 cells were washed twice with sterile DPBS and digested with 0.25% trypsin-EDTA for approximately 5 minutes before quenching with complete culture medium. NCI-H929 cells were transferred directly to a 50 mL centrifuge tube using a pipette.

[0257] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. Discard the supernatant and resuspend the cells in 200 μL of 1% BSA in PBS. Centrifuge again at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and store at 4°C until needed. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant. Wash the cells with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. Discard the supernatant. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 with 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells with the diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash the cells with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0258] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The obtained MFI was imported into Graphpad to analyze the half-binding concentration (EC) of the antibody to the cells. 50) and the highest mean fluorescence intensity (Top MFI), the results are shown in Table 16 and Figures 8A-8D. The binding of clone 18CH16 humanized antibody to human GPRC5D-CHOK1 cells and NCI-H929 tumor cells was basically equivalent to that of the parent antibody.

[0259] Table 16 Binding of mouse hybridoma-derived anti-GPRC5D humanized antibody 18CH16H2L1 to cells expressing GPRC5D

[0260] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

1. A bispecific antigen-binding molecule comprising: A first polypeptide comprises: (i) an antigen-binding fragment Fab heavy chain domain capable of specifically binding to a first antigen, (ii) a single-chain antibody (scFv) domain capable of specifically binding to a second antigen, and (iii) a first Fc domain, A second polypeptide comprises: an antigen binding fragment Fab light chain domain capable of specifically binding to the first antigen, and A third polypeptide comprises: a second Fc domain; The antigen-binding fragment Fab heavy chain domain of the first polypeptide and the antigen-binding fragment Fab light chain domain of the second polypeptide form a first binding site for the first antigen, the single-chain antibody (scFv) domain forms a second binding site for the second antigen, and the first Fc domain and the second Fc domain are associated with each other; Optionally, the bispecific binding molecule further comprises a fourth polypeptide, the fourth polypeptide comprising an antigen-binding fragment Fab light chain domain that is identical to the antigen-binding fragment Fab light chain domain of the second polypeptide and is capable of specifically binding to the first antigen, and the third polypeptide further comprises an antigen-binding fragment Fab heavy chain domain that is capable of specifically binding to the first antigen, the antigen-binding fragment Fab heavy chain domain is identical to the antigen-binding fragment Fab heavy chain domain of the first polypeptide, and its C-terminus is connected to the N-terminus of the second Fc domain, and the antigen-binding fragment Fab heavy chain domain of the third polypeptide and the antigen-binding fragment Fab light chain domain of the fourth polypeptide form a third binding site for the first antigen.

2. The bispecific antigen binding molecule of claim 1, wherein the single-chain antibody (scFv) domain comprises a heavy chain variable region and a light chain variable region; Preferably, the heavy chain variable region of the single-chain antibody (scFv) domain is connected to the light chain variable region of the single-chain antibody (scFv) domain via a first linker, wherein the C-terminus of the heavy chain variable region of the single-chain antibody (scFv) domain is fused to the N-terminus of the first linker, and the C-terminus of the first linker is fused to the N-terminus of the light chain variable region of the single-chain antibody (scFv) domain; More preferably, the first linker comprises the amino acid sequence (G4S) n , n is any integer from 1 to 10.

3. The bispecific antigen binding molecule of claim 1 or 2, wherein the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, and the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain; the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, and the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain; Preferably, the first CH3 domain comprises a "knob" structure, and the second CH3 domain comprises a "hole" structure; more preferably, the "knob" structure comprises amino acid substitutions S354C and T366W, and the "hole" structure comprises amino acid substitutions Y349C, T366S, L368A and Y407V; Preferably, the Fc domain is derived from IgG1; Preferably, the single-chain antibody (scFv) domain is connected to the first Fc domain via a second linker, wherein the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the second linker, and the C-terminus of the second linker is fused to the N-terminus of the first Fc domain; More preferably, the second linker comprises the amino acid sequence EPKSS.

4. The bispecific antigen-binding molecule according to any one of claims 1 to 3, wherein the Fab heavy chain domain of the antigen-binding fragment comprises a heavy chain variable region and a CH1 domain of an immunoglobulin, and the C-terminus of the heavy chain variable region is fused to the N-terminus of the CH1 domain; the Fab light chain domain of the antigen-binding fragment comprises a light chain variable region and a light chain constant region of an immunoglobulin, and the C-terminus of the light chain variable region is fused to the N-terminus of the light chain constant region; Preferably, the antigen-binding fragment Fab heavy chain domain of the first polypeptide is connected to the single-chain antibody (scFv) domain via a third linker, wherein the C-terminus of the antigen-binding fragment Fab heavy chain domain is fused to the N-terminus of the third linker, and the C-terminus of the third linker is fused to the N-terminus of the single-chain antibody (scFv) domain; More preferably, the third linker comprises the amino acid sequence (G4S) n , n is any integer between 1 and 10; Optionally, the C-terminus of the antigen-binding fragment Fab heavy chain domain of the third polypeptide is connected to the N-terminus of the second Fc domain via a fourth linker.

5. The bispecific antigen binding molecule according to any one of claims 1 to 4, wherein the first polypeptide comprises the following structure: Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain, preferably the first polypeptide comprises the following structure: Fab heavy chain variable region-Fab CH1-third linker-scFv heavy chain variable region-first linker-scFv light chain variable region-second linker-first CH2-first CH3; the second polypeptide comprises the following structure: Fab light chain variable region-light chain constant region; the third polypeptide comprises the following structure: second CH2-second CH3; Optionally, the third polypeptide comprises the following structure: Fab heavy chain domain-second Fc domain, preferably comprises the following structure: Fab heavy chain variable region-Fab CH1-second CH2-second CH3, and the fourth polypeptide comprises the following structure: Fab light chain variable region-light chain constant region.

6. The bispecific antigen binding molecule of any one of claims 1 to 5, comprising one or more amino acid substitutions selected from the following group: L234A, L235A and H435R; preferably, the L234A and L235A are substitutions on the first polypeptide and / or the third polypeptide; preferably, the H435R substitution is a substitution on the third polypeptide.

7. The bispecific antigen binding molecule according to any one of claims 1 to 6, wherein the second antigen is CD3, preferably CD3ε; Preferably, the single-chain antibody (scFv) domain comprises a HCDR1 having a sequence as shown in SEQ ID NO: 19, a HCDR2 having a sequence as shown in SEQ ID NO: 20, a HCDR3 having a sequence as shown in SEQ ID NO: 21, a LCDR1 having a sequence as shown in SEQ ID NO: 22, a LCDR2 having a sequence as shown in SEQ ID NO: 23, and a LCDR3 having a sequence as shown in SEQ ID NO: 24; More preferably, the single-chain antibody (scFv) domain comprises a heavy chain variable region having a sequence as shown in SEQ ID NO: 17 and a light chain variable region having a sequence as shown in SEQ ID NO: 18; More preferably, the single-chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:

8.

8. The bispecific antigen binding molecule of any one of claims 1 to 7, wherein the first Fc domain comprises the amino acid sequence shown in SEQ ID NO: 26; and the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:

4.

9. The bispecific antigen binding molecule of any one of claims 1 to 8, wherein the first antigen is GPRC5D; Preferably, the antigen-binding fragment Fab heavy chain domain comprises HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 12 and HCDR3 as shown in SEQ ID NO: 13; the antigen-binding fragment Fab light chain domain comprises LCDR1 as shown in SEQ ID NO: 14, LCDR2 as shown in SEQ ID NO: 15 and LCDR3 as shown in SEQ ID NO: 16; More preferably, the antigen-binding fragment Fab heavy chain domain comprises a heavy chain variable region with a sequence as shown in SEQ ID NO: 9; the antigen-binding fragment Fab light chain domain comprises a light chain variable region with a sequence as shown in SEQ ID NO: 10; More preferably, the antigen-binding fragment Fab heavy chain domain comprises an amino acid sequence as shown in SEQ ID NO: 25; and the antigen-binding fragment Fab light chain domain comprises an amino acid sequence as shown in SEQ ID NO:

3.

10. The bispecific antigen-binding molecule of any one of claims 1 to 9, wherein the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3, and / or the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; optionally, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, the second polypeptide and / or the fourth polypeptide comprises the amino acid sequence shown in SEQ ID NO: 3, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:

2.

11. A bispecific antigen-binding molecule, which can bind to an epitope: The epitope is the same as or overlaps with that of an antibody comprising HCDR1 as shown in SEQ ID NO: 11, HCDR2 as shown in SEQ ID NO: 12, HCDR3 as shown in SEQ ID NO: 13, LCDR1 as shown in SEQ ID NO: 14, LCDR2 as shown in SEQ ID NO: 15, and LCDR3 as shown in SEQ ID NO: 16; Preferably, the epitope it is capable of binding to: The epitope is the same as or overlaps with that of the antibody comprising the heavy chain variable region amino acid sequence shown in SEQ ID NO: 9 and the light chain variable region amino acid sequence shown in SEQ ID NO:

10.

12. The bispecific antigen binding molecule of claim 11, which is capable of binding to CD3, preferably to CD3ε.

13. A bispecific antigen-binding molecule comprising: (A) a first binding moiety capable of specifically binding to GPRC5D; and (B) a second binding moiety that specifically binds to a different antigen or epitope than the first binding moiety; The first binding portion comprises: HCDR1 with the sequence shown in SEQ ID NO: 11, HCDR2 with the sequence shown in SEQ ID NO: 12, HCDR3 with the sequence shown in SEQ ID NO: 13, LCDR1 with the sequence shown in SEQ ID NO: 14, LCDR2 with the sequence shown in SEQ ID NO: 15, and LCDR3 with the sequence shown in SEQ ID NO: 16; Preferably, the first binding moiety comprises: The heavy chain variable region sequence is shown in SEQ ID NO:9 and the light chain variable region sequence is shown in SEQ ID NO:

10.

14. The bispecific antigen binding molecule of claim 13, wherein the second binding moiety is capable of binding to CD3, preferably to CD3ε.

15. A GPRC5D binding molecule or an antigen-binding fragment thereof, comprising: a HCDR sequence of a heavy chain variable region as shown in SEQ ID NO: 9, and / or a LCDR sequence of a light chain variable region as shown in SEQ ID NO: 10; preferably, the GPRC5D binding molecule or an antigen-binding fragment thereof comprises a HCDR1 as shown in SEQ ID NO: 11, a HCDR2 as shown in SEQ ID NO: 12, and a HCDR3 as shown in SEQ ID NO: 13, and / or a LCDR1 as shown in SEQ ID NO: 14, a LCDR2 as shown in SEQ ID NO: 15, and a LCDR3 as shown in SEQ ID NO: 16; More preferably, it comprises a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 9 and a light chain variable region amino acid sequence as shown in SEQ ID NO: 10; Further preferably, it comprises a heavy chain constant region and a light chain constant region; preferably, it comprises an IgG1 heavy chain constant region and a kappa light chain constant region.

16. A nucleic acid molecule encoding the bispecific antigen binding molecule of any one of claims 1 to 14 or the GPRC5D binding molecule of claim 15 or an antigen binding fragment thereof.

17. An expression vector comprising the nucleic acid molecule of claim 16.

18. A host cell comprising the nucleic acid molecule of claim 16 or the expression vector of claim 17; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, Expi293 or HEK293 cell.

19. A method for preparing the bispecific antigen binding molecule of any one of claims 1 to 14 or the GPRC5D binding molecule or antigen binding fragment thereof of claim 15, the method comprising: The host cell of claim 18 is cultured under suitable conditions.

20. An antibody-drug conjugate, which is formed by coupling the bispecific antigen binding molecule according to any one of claims 1 to 14 or the GPRC5D binding molecule or antigen binding fragment thereof according to claim 15 with other biologically active molecules; preferably, the other biologically active molecules are small molecule drugs; preferably, the bispecific antigen binding molecule and the other biologically active molecules are connected via a linker.

21. A pharmaceutical composition comprising the bispecific antigen binding molecule of any one of claims 1 to 14, the GPRC5D binding molecule or antigen binding fragment thereof of claim 15, the nucleic acid molecule of claim 16, the expression vector of claim 17, the host cell of claim 18 and / or the antibody drug conjugate of claim 20; preferably, it further comprises a pharmaceutically acceptable carrier and / or one or more additional therapeutic agents.

22. Use of the bispecific antigen binding molecule of any one of claims 1 to 14, the GPRC5D binding molecule or antigen binding fragment thereof of claim 15, the nucleic acid molecule of claim 16, the expression vector of claim 17, the host cell of claim 18, the antibody-drug conjugate of claim 20 and / or the pharmaceutical composition of claim 21 in the preparation of a drug for treating, alleviating and / or preventing tumors; Preferably, the tumor is a GPRC5D-positive tumor; Preferably, the tumor is selected from the group consisting of lymphomas such as multiple myeloma, and metastases of the above tumors.

23. A method for inducing cell death expressing GPRC5D in vivo or in vitro, the method comprising contacting the cell with the bispecific antigen binding molecule of any one of claims 1 to 14, the GPRC5D binding molecule or antigen binding fragment thereof of claim 15, the nucleic acid molecule of claim 16, the expression vector of claim 17, the host cell of claim 18, the antibody drug conjugate of claim 20 and / or the pharmaceutical composition of claim 21, preferably, the cell expressing GPRC5D is a tumor cell; Preferably, the tumor cell is a cell selected from the group consisting of lymphomas such as multiple myeloma, and metastases of the above tumors.

24. A method for treating a disease associated with expression of GPRC5D in a subject, the method comprising administering to a subject in need thereof the bispecific antigen binding molecule of any one of claims 1 to 14, the GPRC5D binding molecule or antigen binding fragment thereof of claim 15, the nucleic acid molecule of claim 16, the expression vector of claim 17, the host cell of claim 18, the antibody drug conjugate of claim 20, and / or the pharmaceutical composition of claim 21; Preferably, the disease is a tumor; Preferably, the tumor is a lymphoma such as multiple myeloma, and metastatic cancer of the above tumors; Preferably, it further comprises administering to said subject an additional therapeutic agent.

25. The bispecific antigen binding molecule of any one of claims 1 to 14, the GPRC5D binding molecule or antigen binding fragment thereof of claim 15, the nucleic acid molecule of claim 16, the expression vector of claim 17, the host cell of claim 18, the antibody drug conjugate of claim 20 and / or the pharmaceutical composition of claim 21 for use in treatment.

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