Trispecific antigen-binding molecules and uses thereof

Abstract

The present invention provides a configuration of a trispecific antigen-binding molecule, and also provides a trispecific antigen-binding molecule constructed based on this configuration against two tumor antigens GPRC5D and BCMA and a T-cell surface antigen CD3, a specific binding molecule for GPRC5D or a fragment thereof, a specific binding molecule for BCMA or a fragment thereof, a pharmaceutical composition comprising the trispecific antigen-binding molecule or the specific binding molecule or a fragment thereof, and their related applications in tumor treatment.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. 2023101003684, filed on February 7, 2023, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to the field of immunology and primarily to trispecific antigen-binding molecules. More specifically, the present invention relates to trispecific antigen-binding molecules that specifically bind to two tumor antigens, GPRC5D and BCMA, and a T cell surface antigen, CD3, as well as pharmaceutical compositions containing the same, preparation methods, and respective uses. [Background technology]

[0003] Multiple myeloma (MM) is a hematologic malignancy and the second most common after non-Hodgkin's lymphoma. It can devastate bone, the immune system, kidneys, and red blood cell count, typically resulting in extensive bone destruction accompanied by osteolytic lesions, osteopenia, and pathological fractures. The disease is common among middle-aged and elderly people, and the number of patients is rapidly increasing due to the accelerated aging population and advances in diagnostic and therapeutic methods.

[0004] Like most cancer treatments, the standard of care for multiple myeloma is chemotherapy, with commonly used drugs such as melphalan. However, these drugs typically cause bone marrow suppression, resulting in less than ideal therapeutic outcomes, with a complete remission rate of only about 5%. In recent years, advances in medicine have led to revolutionary changes in the treatment of multiple myeloma, with the development of targeted drugs such as proteasome inhibitors, immunomodulators, and antibody-based drugs. While survival rates and patient prognoses have improved significantly compared to earlier treatment regimens, multiple myeloma remains incurable, with most patients experiencing relapse after multiple treatments. In China, the median patient survival time is 24-36 months, with a 5-year survival rate of only about 24%, necessitating the urgent need for new therapeutic approaches.

[0005] B cell maturation antigen (BCMA) is a transmembrane glycoprotein receptor (non-tyrosine kinase receptor) and a member of the tumor necrosis factor family. It is primarily expressed on the surface of mature B cells, but also in small amounts on hematopoietic stem cells and other tissue cells. BCMA's ligands are B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Under normal physiological conditions, the BCMA signaling pathway promotes B cell differentiation into plasma cells and maintains bone marrow plasma cell homeostasis throughout its long life cycle. BCMA is overexpressed in bone marrow plasma cells of patients with MM. Overexpressed BCMA can stimulate the proliferation of malignant plasma cells through the intracellular AKT, MAPK, and (NF)-κB signaling pathways, thereby maintaining their activity and promoting disease progression. Blocking the BCMA signaling pathway can inhibit malignant plasma cell proliferation and prevent / ameliorate disease progression, making BCMA an excellent target for clinical treatment of MM. Furthermore, the BCMA protein on the cell membrane can be cleaved by γ-secretase in vivo, and the cleaved BCMA protein is free in serum and called soluble BCMA protein (sBCMA). sBCMA can form conjugates (also called "complexes") with ligands, thereby reducing the concentration of the ligand in serum, inhibiting the binding of the ligand to membrane-bound BCMA, and blocking the BCMA signaling pathway.

[0006] GPRC5D is a G protein-coupled receptor C5 family subtype D, an orphan receptor with seven transmembrane domains. GPRC5D is specifically and highly expressed in plasma cells of multiple myeloma, whereas it is only observed in hair follicles in normal tissues. Antibodies and CAR-T cell therapies targeting GPRC5D have both shown promising preclinical efficacy, but no hair loss was observed in experimental animals. Furthermore, GPRC5D expression is not restricted by BCMA expression, and CAR-T therapy targeting GPRC5D can overcome tumor escape in tumor relapse models due to loss of the BCMA antigen. All these findings suggest that GPRC5D is an ideal target for clinical use.

[0007] Bispecific antibodies targeting tumor-associated antigens (TAA) and CD3 antigens can bring T cells into spatial proximity with tumor cells, forming synapses and achieving specific killing of tumor cells. These antibodies are called T cell-engagers (TCE bispecific antibodies). TCE-based therapeutic strategies depend on the distribution of TAA on the tumor cells being treated. Furthermore, this study also shows that therapeutic approaches targeting a single TAA site may cause disease recurrence due to tumor escape mechanisms, thereby limiting the efficacy of treatment.

[0008] Because the expression of GPRC5D and BCMA in tumors is uncorrelated, designing a trispecific antibody that targets both GPRC5D and BCMA can cover both patients with GPRC5D-positive tumors and patients with BCMA-positive tumors, and can prevent recurrence due to the loss of a single antigen, thereby achieving improved patient coverage and therapeutic efficacy. Summary of the Invention

[0009] To achieve the above objectives, the present invention provides a trispecific antigen-binding molecule targeting GPRC5D / BCMA / CD3. Through careful research into various molecular configurations, multi-target combinations have been achieved, successfully avoiding the disadvantages of multiple drug combinations.

[0010] According to a first aspect of the present invention, there is provided a trispecific antigen-binding molecule, the configuration of which includes: a first polypeptide comprising: (i) a heavy chain domain of an antigen-binding fragment Fab 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 comprising a light chain domain of an antigen-binding fragment Fab capable of specifically binding to the first antigen; (i) a heavy chain single domain antibody (VHH) domain capable of specifically binding to a third antigen, and (ii) a third polypeptide comprising a second Fc domain.

[0011] The heavy chain domain of the antigen-binding fragment Fab of the first polypeptide forms a first binding site for a first antigen together with the light chain domain of the antigen-binding fragment Fab of the second polypeptide; the single-chain antibody (scFv) domain forms a second binding site for a second antigen; the heavy-chain single-domain antibody (VHH) domain forms a third binding site for a third antigen; and the first Fc domain and second Fc domain are associated with each other.

[0012] Optionally, the trispecific antigen-binding molecule further comprises a fourth polypeptide, wherein the fourth polypeptide comprises a light chain domain of an antigen-binding fragment Fab that specifically binds to a first antigen, wherein the light chain domain of the antigen-binding fragment Fab is the same as the light chain domain of the antigen-binding fragment Fab of the second polypeptide; the third polypeptide further comprises a heavy chain domain of an antigen-binding fragment Fab that specifically binds to the first antigen, wherein the heavy chain domain of the antigen-binding fragment Fab of the third polypeptide is the same as the heavy chain domain of the antigen-binding fragment Fab of the first polypeptide and is linked at its C-terminus to the N-terminus of the VHH domain; and the heavy chain domain of the Fab of the third polypeptide forms a fourth binding site for the first antigen with the light chain domain of the Fab of the fourth polypeptide.

[0013] In one embodiment, the scFv domain comprises a heavy chain variable region and a light chain variable region, and preferably the heavy chain variable region of the scFv domain is linked to the light chain variable region by a first linker, wherein the C-terminus of the heavy chain variable region 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. More preferably, the first linker has the amino acid sequence (G4S) n where n is any integer from 1 to 10.

[0014] 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, and 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.

[0015] Preferably, the first CH3 domain comprises a "knob" structure and the second CH3 domain comprises a "hole" structure, more preferably the "knob" structure comprises the amino acid substitutions S354C and T366W and the "hole" structure comprises the amino acid substitutions Y349C, T366S, L368A and Y407V.

[0016] Preferably, the first and / or second Fc domain comprises the amino acid substitutions L234A, L235A and / or G237A to reduce the ADCC activity of the antibody. Preferably, the second Fc domain of said third polypeptide comprises the amino acid substitution H435R. Preferably, the Fc domain is derived from IgG1.

[0017] In one embodiment, the N-terminus of the first Fc domain is fused to the C-terminus of the scFv domain, preferably the N-terminus of the first Fc domain is fused to the C-terminus of the scFv domain via a second linker, more preferably the second linker comprises the amino acid sequence EPKSS.

[0018] In one embodiment, the heavy chain domain of the antigen-binding fragment Fab comprises a heavy chain variable region and a CH1 domain of an immunoglobulin, wherein the C-terminus of the heavy chain variable region is fused to the N-terminus of the CH1 domain, and the light chain domain of the antigen-binding fragment Fab comprises a light chain variable region and a light chain constant region of an immunoglobulin, wherein the C-terminus of the light chain variable region is fused to the N-terminus of the light chain constant region.

[0019] In one embodiment, the heavy chain domain of the antigen-binding fragment Fab of the first polypeptide is linked to the scFv domain via a third linker, wherein the C-terminus of the heavy chain domain of the antigen-binding fragment Fab 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 scFv domain.

[0020] Preferably, the third linker has the amino acid sequence (G4S) n where n is any integer from 1 to 10.

[0021] Optionally, the C-terminus of the heavy chain domain of the antigen-binding fragment Fab of said third polypeptide is linked to the N-terminus of the VHH domain via a fifth linker. Preferably, the fifth linker has the amino acid sequence (G4S) n where n is any integer from 1 to 10.

[0022] Preferably, the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of a second Fc domain, preferably the C-terminus of the VHH domain is fused to the N-terminus of a second Fc domain via a fourth linker, more preferably the fourth linker comprises the amino acid sequence EPKSS.

[0023] In one embodiment, the first polypeptide comprises the following structure: Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain.

[0024] Preferably, the first polypeptide comprises the heavy chain variable region of Fab-Fab CH1-third linker-heavy chain variable region of scFv-first linker-light chain variable region of scFv-second linker-first CH2-first CH3.

[0025] In one embodiment, the second polypeptide comprises the structure of a light chain variable region-light chain constant region of a Fab. In one embodiment, the third polypeptide comprises the structure VHH domain-fourth linker-second Fc domain. Preferably, the third polypeptide comprises the structure VHH-fourth linker-second CH2-second CH3.

[0026] Optionally, the fourth polypeptide comprises a structure of Fab light chain variable region-light chain constant region, and the third polypeptide comprises a structure of Fab heavy chain domain-fifth linker-VHH-fourth linker-second Fc domain, preferably, the third polypeptide comprises a structure of Fab heavy chain variable region-Fab CH1-fifth linker-VHH-fourth linker-second CH2-second CH3.

[0027] In one embodiment, the second antigen is CD3, preferably CD3ε, and preferably the scFv domain comprises an HCDR1 whose sequence is set forth in SEQ ID NO:27, an HCDR2 whose sequence is set forth in SEQ ID NO:28, an HCDR3 whose sequence is set forth in SEQ ID NO:29, an LCDR1 whose sequence is set forth in SEQ ID NO:30, an LCDR2 whose sequence is set forth in SEQ ID NO:31, and an LCDR3 whose sequence is set forth in SEQ ID NO:32.

[0028] More preferably, said scFv domain comprises a heavy chain variable region whose sequence is shown in SEQ ID NO:25 and a light chain variable region whose sequence is shown in SEQ ID NO:26.

[0029] More preferably, the scFv domain comprises the amino acid sequence shown in SEQ ID NO:13.

[0030] In one embodiment, the first Fc domain comprises the amino acid sequence set forth in SEQ ID NO:33 and the second Fc domain comprises the amino acid sequence set forth in SEQ ID NO:34.

[0031] In one embodiment, the first antigen is BCMA, and preferably the antigen-binding fragment Fab comprises an HCDR1 whose sequence is set forth in SEQ ID NO:16, an HCDR2 whose sequence is set forth in SEQ ID NO:17, and an HCDR3 whose sequence is set forth in SEQ ID NO:18, and / or an LCDR1 whose sequence is set forth in SEQ ID NO:19, an LCDR2 whose sequence is set forth in SEQ ID NO:20, and an LCDR3 whose sequence is set forth in SEQ ID NO:21.

[0032] More preferably, the heavy chain domain of said antigen-binding fragment Fab comprises a heavy chain variable region whose sequence is set forth in SEQ ID NO:14, and / or the light chain domain of said antigen-binding fragment Fab comprises a light chain variable region whose sequence is set forth in SEQ ID NO:15.

[0033] More preferably, the heavy chain domain of said antigen-binding fragment Fab comprises the amino acid sequence set forth in SEQ ID NO:35, and / or the light chain domain of said antigen-binding fragment Fab comprises the amino acid sequence set forth in SEQ ID NO:7.

[0034] In one embodiment, the third antigen is GPRC5D, and preferably, the heavy chain single domain antibody (VHH) domain capable of specifically binding to the third antigen comprises an HCDR1 as set forth in SEQ ID NO:22, an HCDR2 as set forth in SEQ ID NO:23, and an HCDR3 as set forth in SEQ ID NO:24, and more preferably, the VHH domain comprises the sequence as set forth in SEQ ID NO:10.

[0035] In one embodiment, the first polypeptide of the trispecific antigen-binding molecule comprises the amino acid sequence set forth in SEQ ID NO:5, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7, and the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6; or, optionally, the first polypeptide of the trispecific antigen-binding molecule comprises the sequence set forth in SEQ ID NO:5, the second polypeptide and / or the fourth polypeptide comprises the sequence set forth in SEQ ID NO:7, and the third polypeptide comprises the sequence set forth in SEQ ID NO:8.

[0036] The present invention further provides nucleic acid molecules encoding the trispecific antigen-binding molecules of the present invention. The present invention further provides a vector comprising the nucleic acid molecule.

[0037] The present invention further provides a host cell comprising the nucleic acid molecule or vector. Preferably, the host cell is a prokaryotic or eukaryotic cell, the prokaryotic cell is preferably Escherichia coli, the eukaryotic cell is preferably a mammalian cell or yeast, and more preferably, the mammalian cell is a CHO cell, Expi293 or HEK293 cell.

[0038] The present invention further provides a method for producing a trispecific antigen-binding molecule, comprising culturing host cells under suitable conditions.

[0039] The present invention further provides an antibody-drug conjugate formed by conjugating the bispecific antigen-binding molecule with another biologically active molecule. Preferably, the other biologically active molecule is a small molecule drug, and the bispecific antigen-binding molecule is preferably linked to the other biologically active molecule via a linker.

[0040] The present invention further provides pharmaceutical compositions comprising the trispecific antigen-binding molecules, nucleic acid molecules, expression vectors, host cells, and / or antibody-drug conjugates.

[0041] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0042] The present invention further provides the use of the trispecific antigen-binding molecule, nucleic acid molecule, vector, host cell, and / or antibody-drug conjugate in the manufacture of a medicament for the treatment, alleviation, and / or prevention of tumors, preferably GPRC5D-positive and / or BCMA-positive tumors.

[0043] The present invention further provides a method for inducing death of a cell expressing GPRC5D3 and / or BCMA, comprising contacting said cell with the trispecific antigen-binding molecule, nucleic acid molecule, vector, host cell and / or pharmaceutical composition, wherein said cell expressing GPRC5D3 and / or BCMA is a tumor cell.

[0044] The present invention further provides a method for treating a disease associated with expression of GPRC5D and / or BCMA in a subject, the method comprising administering the trispecific antigen-binding molecule, nucleic acid molecule, vector, host cell, and / or pharmaceutical composition to a subject in need thereof. Preferably, the disease is a tumor. Preferably, the subject has relapsed or is refractory to treatment with a previous anti-cancer therapeutic agent. In one embodiment, the method further comprises administering an additional therapeutic agent to the subject.

[0045] Preferably, the tumor or tumor cell according to the present invention is selected from the group consisting of lymphomas, such as multiple myeloma, and metastases of said tumors.

[0046] According to another aspect of the present invention, there is provided an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to GPRC5D, wherein the antigen-binding molecule or antigen-binding fragment thereof can specifically bind to GPRC5D but does not specifically bind to GPRC5A, a protein belonging to the same family.

[0047] Preferably, the antigen-binding molecule that specifically binds to GPRC5D (hereinafter also referred to as "GPRC5D antigen-binding molecule") or its antigen-binding fragment comprises a heavy chain single-domain antibody (VHH) domain. Preferably, the GPRC5D antigen-binding molecule or its antigen-binding fragment comprises the HCDR sequence of a heavy chain variable region whose amino acid sequence is set forth in SEQ ID NO: 10. Preferably, the GPRC5D antigen-binding molecule or its antigen-binding fragment comprises an HCDR1 whose sequence is set forth in SEQ ID NO: 22, an HCDR2 whose sequence is set forth in SEQ ID NO: 23, and an HCDR3 whose sequence is set forth in SEQ ID NO: 24. More preferably, the heavy chain single-domain antibody (VHH) comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0048] Preferably, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof further comprises an Fc domain of an immunoglobulin selected from the isotypes IgG1, IgG2, IgG3, or IgG4, preferably, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof further comprises an IgG1 Fc domain, more preferably, the IgG1 Fc domain comprises the amino acid sequence shown in SEQ ID NO:38.

[0049] In one embodiment, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises a heavy chain single-domain antibody (VHH) domain and an IgG1 Fc domain, preferably the C-terminus of the heavy chain single-domain antibody (VHH) domain is fused to the N-terminus of the IgG1 Fc domain, more preferably the C-terminus of the heavy chain single-domain antibody (VHH) domain is fused to the N-terminus of the IgG1 Fc domain via a linker, preferably the linker has the amino acid sequence EPKSS or (G4S) nwherein n is any integer from 1 to 10. In a preferred embodiment, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises, from the N-terminus to the C-terminus, a VHH domain whose sequence is set forth in SEQ ID NO: 10, a G4S linker, and an IgG1 Fc domain whose sequence is set forth in SEQ ID NO: 38.

[0050] The GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present invention may be a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a murine antibody, a humanized antibody, a chimeric antibody, a modified antibody, a fully human antibody, a full-length antibody, a heavy-chain antibody, a nanobody, Fab, Fv, scFv, F(ab')2, a linear antibody, or a heavy-chain single-domain antibody.

[0051] The present invention further provides a conjugate formed by binding the GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present invention to a capture marker or a detectable marker. Preferably, the detectable marker comprises a radionuclide, a luminescent substance, a coloring substance, or an enzyme.

[0052] The present invention further provides an antibody-drug conjugate (ADC) formed by conjugating the GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to the present invention to another biologically active molecule. Preferably, the other biologically active molecule is a small molecule drug, and the GPRC5D antigen-binding molecule or antigen-binding fragment thereof is linked to the other biologically active molecule via a linker.

[0053] The present invention further provides fusion proteins in which one of the fusion moieties comprises a GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to the present invention.

[0054] The present invention further provides a chimeric antigen receptor (CAR), or a cell comprising the chimeric antigen receptor (e.g., a CAR-T cell), comprising the GPRC5D antigen-binding molecule or an antigen-binding fragment thereof according to the present invention.

[0055] The present invention further provides nucleic acids encoding GPRC5D antigen-binding molecules or antigen-binding fragments thereof, recombinant vectors containing the nucleic acids, and host cells containing the nucleic acids or vectors. Preferably, the host cells are prokaryotic cells (preferably E. coli) or eukaryotic cells (preferably mammalian cells or yeast, more preferably mammalian cells CHO cells or HEK293 cells).

[0056] The present invention further provides a method for producing a GPRC5D antigen-binding molecule or an antigen-binding fragment thereof, comprising culturing the host cell under appropriate conditions and purifying the expression product obtained from the cell.

[0057] The present invention further provides the use of a GPRC5D antigen-binding molecule or an antigen-binding fragment thereof in the manufacture of a medicament for the treatment or alleviation of tumors.

[0058] In one embodiment, the agent targets tumor cells in which GPRC5D is aberrantly expressed. In one embodiment, the tumor is selected from the group consisting of lymphomas, such as multiple myeloma, and metastases of said tumors.

[0059] The present invention further provides a method for treating a disease associated with expression of GPRC5D in a subject, the method comprising administering the GPRC5D antigen-binding molecule or an antigen-binding fragment thereof to a subject in need thereof.

[0060] Preferably, said disease is a tumor, and preferably said tumor disease is selected from the group consisting of lymphomas, such as multiple myeloma, and metastatic cancers of said tumors. More preferably, the method further comprises administering an additional therapeutic agent to said subject.

[0061] The present invention further provides use of the GPRC5D antigen-binding molecule or antigen-binding fragment thereof in the manufacture of a detection reagent or diagnostic reagent.

[0062] In one embodiment, the detection reagent is used to detect the expression of GPRC5D, and the diagnostic reagent is used to diagnose a tumor, preferably the tumor is selected from the group consisting of lymphomas such as multiple myeloma, and metastatic cancers of the tumor.

[0063] The present invention provides a method for detecting the expression of GPRC5D in a sample, comprising: (1) contacting a sample with the GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present invention; (2) detecting the formation of a conjugate between the GPRC5D antigen-binding molecule or its antigen-binding fragment and GPRC5D, wherein optionally the GPRC5D antigen-binding molecule or its antigen-binding fragment is detectably labeled.

[0064] The present invention further provides a method for monitoring GPRC5D-expressing cancer in a subject, the method comprising the steps of exposing a sample obtained from or derived from the subject to one or more of the GPRC5D antigen-binding molecules or antigen-binding fragments described herein, measuring the amount of GPRC5D present in the sample that is bound by the antibody or antigen-binding fragment thereof, comparing the amount of GPRC5D present in the sample with the amount of GPRC5D in a known standard or reference sample, or a similar sample previously obtained from the subject, and determining whether the subject's GPRC5D level indicates cancer progression, regression, or stable disease based on the difference in the amount of GPRC5D in the compared samples.

[0065] A sample obtained from or derived from a subject is a biological sample such as urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells, tissue, surgically resected tumor tissue, a biopsy section, a fine needle aspirate, or a histological preparation.

[0066] The present invention further provides a pharmaceutical composition comprising an effective amount of the GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present invention, or an effective amount of the antibody-drug conjugate, fusion protein, or CAR-T cell of the present invention, or an effective amount of the nucleic acid of the present invention, or an effective amount of the recombinant vector of the present invention, or an effective amount of the host cell of the present invention.

[0067] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises one or more additional other therapeutic agents.

[0068] According to another aspect of the present invention, there is further provided an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA, comprising an HCDR sequence of a heavy chain variable region whose sequence is set forth in SEQ ID NO: 14 and / or an LCDR sequence of a light chain variable region whose sequence is set forth in SEQ ID NO: 15. Preferably, the antigen-binding molecule or antigen-binding fragment thereof comprises an HCDR1 whose sequence is set forth in SEQ ID NO: 16, an HCDR2 whose sequence is set forth in SEQ ID NO: 17, and an HCDR3 whose sequence is set forth in SEQ ID NO: 18, and / or an LCDR1 whose sequence is set forth in SEQ ID NO: 19, an LCDR2 whose sequence is set forth in SEQ ID NO: 20, and an LCDR3 whose sequence is set forth in SEQ ID NO: 21.

[0069] Preferably, the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA comprises a heavy chain variable region whose sequence is set forth in SEQ ID NO: 14, and / or the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA comprises a light chain variable region whose sequence is set forth in SEQ ID NO: 15.

[0070] More preferably, the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA comprises the heavy chain amino acid sequence shown in SEQ ID NO:9, and / or the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA comprises the light chain amino acid sequence shown in SEQ ID NO:7.

[0071] The antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA of the present invention further comprises a heavy chain constant region and / or a light chain constant region, and preferably the heavy chain constant region comprises Fc, more preferably the Fc is derived from a mouse or a human, and more preferably the sequence of the Fc is natural or modified.

[0072] The antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA of the present invention may be a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a murine antibody, a humanized antibody, a chimeric antibody, a modified antibody, a fully human antibody, a full-length antibody, a heavy-chain antibody, a nanobody, a Fab, an Fv, an scFv, an F(ab')2, a linear antibody, or a heavy-chain single-domain antibody.

[0073] The antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA according to the present invention may be a full-length antibody.

[0074] The antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA according to the present invention may be of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0075] The present invention further provides a conjugate formed by binding an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA according to the present invention to a capture or detectable marker, preferably the detectable marker comprises a radionuclide, a luminescent substance, a colored substance, or an enzyme.

[0076] The present invention further provides an antibody-drug conjugate (ADC) formed by conjugating the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA according to the present invention to another biologically active molecule, preferably the other biologically active molecule is a small molecule drug, and preferably the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA is linked to the other biologically active molecule via a linker.

[0077] The present invention further provides fusion proteins in which one of the fusion moieties comprises an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA according to the present invention. The present invention further provides chimeric antigen receptors (CARs) comprising the antigen-binding molecules or antigen-binding fragments thereof that specifically bind to BCMA according to the present invention, and cells comprising said chimeric antigen receptors (e.g., CAR-T cells).

[0078] The present invention further provides a nucleic acid encoding an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA, a recombinant vector comprising the nucleic acid, and a host cell comprising the nucleic acid or vector. Preferably, the host cell is a prokaryotic cell (preferably E. coli) or a eukaryotic cell (preferably a mammalian cell or yeast, more preferably a mammalian cell such as a CHO cell or HEK293 cell).

[0079] The present invention further provides a method for producing an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA according to the present invention, the method comprising culturing the host cell under appropriate conditions and purifying the expression product obtained from the cell.

[0080] The present invention further provides the use of an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA in the manufacture of a medicament for the treatment or alleviation of tumors.

[0081] In one embodiment, the drug targets tumor cells that have aberrant expression of BCMA. In one embodiment, the tumor is selected from the group consisting of lymphomas, such as multiple myeloma, and metastases of said tumors.

[0082] The present invention further provides a method for treating a disease associated with expression of BCMA in a subject, the method comprising administering to a subject in need thereof an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA.

[0083] Preferably, said disease is a tumor, and preferably said tumor disease is selected from the group consisting of lymphomas, such as multiple myeloma, and metastatic cancers of said tumors. More preferably, the method further comprises administering an additional therapeutic agent to said subject.

[0084] The present invention further provides the use of an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA in the manufacture of a detection reagent or a diagnostic reagent.

[0085] In one embodiment, the detection reagent is used to detect expression of BCMA and the diagnostic reagent is used to diagnose a tumor, preferably the tumor is selected from the group consisting of lymphomas such as multiple myeloma, and metastases of said tumors.

[0086] The present invention provides a method for detecting BCMA expression in a sample, comprising the steps of: (1) contacting a sample with an antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA according to the present invention; (2) detecting the formation of a conjugate between the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA and BCMA, wherein optionally, the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA is detectably labeled.

[0087] The present invention further provides a method of monitoring a BCMA-expressing cancer in a subject, the method comprising the steps of exposing a sample obtained from or derived from the subject to one or more of the antigen binding molecules or antigen-binding fragments that specifically bind BCMA as described herein; measuring the amount of BCMA present in the sample that is bound by the antigen binding molecule or antigen-binding fragment thereof; comparing the amount of BCMA present in the sample with the amount of BCMA in a known standard or reference sample, or a similar sample previously obtained from the subject; and determining whether the subject's BCMA level is indicative of cancer progression, regression, or stable disease based on the difference in the amount of BCMA in the compared samples.

[0088] A sample obtained from or derived from a subject is a biological sample such as urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells, tissue, surgically resected tumor tissue, a biopsy section, a fine needle aspirate, or a histological preparation.

[0089] The present invention further provides a pharmaceutical composition comprising an effective amount of an antigen-binding molecule or antigen-binding fragment thereof that binds to BCMA according to the present invention, or an effective amount of an antibody-drug conjugate, fusion protein, or CAR-T cell according to the present invention, or an effective amount of a nucleic acid according to the present invention, or an effective amount of a recombinant vector according to the present invention, or an effective amount of a host cell according to the present invention.

[0090] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition further comprises one or more additional other therapeutic agents. The amino acid positions in the substitutions referred to in the present invention are preferably represented by the EU numbering system. [Brief explanation of the drawings]

[0091] The accompanying drawings further illustrate the novel features disclosed herein. A better understanding of the features and advantages disclosed herein can be obtained by reference to these drawings. However, these drawings are not intended to limit the scope of the appended claims, but should be understood merely as illustrating specific embodiments of the principles disclosed herein.

[0092] [Figure 1A] This figure shows the expression of human GPRC5D in CHOK1 cells verified with an anti-GPRC5D positive control antibody detected using flow cytometry. The dotted line in the figure indicates the expression of human GPRC5D in CHOK1 parental cells not transfected with any plasmid, and the solid line in the figure indicates the overexpression of human GPRC5D protein after CHOK1 parental cells were transfected with the human GPRC5D plasmid.

[0093] [Figure 1B] FIG. 1 shows the expression of cynomolgus monkey GPRC5D in CHOK1 cells verified with an anti-GPRC5D positive control antibody detected using flow cytometry. The dotted line in the figure indicates the expression of cynomolgus monkey GPRC5D in CHOK1 parent cells not transfected with any plasmid, and the solid line in the figure indicates the overexpression of cynomolgus monkey GPRC5D protein after transfecting CHOK1 parent cells with the cynomolgus monkey GPRC5D plasmid.

[0094] [Figure 1C] This figure shows the expression of human GPRC5A in CHOK1 cells verified with an anti-GPRC5A positive control antibody detected using flow cytometry. The dotted line in the figure indicates the expression of human GPRC5A in CHOK1 parent cells not transfected with any plasmid, and the solid line in the figure indicates the overexpression of human GPRC5A protein after the CHOK1 parent cells were transfected with the human GPRC5A plasmid.

[0095] [Figure 2A]FIG. 1 shows the expression of human BCMA in CHOK1 cells as detected using flow cytometry and validated with an anti-BCMA positive control antibody. The dotted line in the figure indicates the expression of human BCMA in parental CHOK1 cells not transfected with any plasmid, and the solid line in the figure indicates the overexpression of human BCMA protein after transfection of parental CHOK1 cells with the human BCMA plasmid.

[0096] [Figure 2B] FIG. 1 shows cynomolgus BCMA expression in CHOK1 cells validated with an anti-BCMA positive control antibody detected using flow cytometry. The dotted line in the figure indicates cynomolgus BCMA expression in parental CHOK1 cells not transfected with any plasmid, and the solid line in the figure indicates overexpression of cynomolgus BCMA protein after transfection of parental CHOK1 cells with the cynomolgus BCMA plasmid.

[0097] [Figure 3] FIG. 1 shows the structures of two trispecific antibodies (Trispecific antibody 1 and Trispecific antibody 2, respectively) constructed in the Examples.

[0098] [Figure 4A] FIG. 1 shows binding of Trispecific Antibody 1, Trispecific Antibody 2 and Teclistamab to tumor cells NCI-H929 expressing the dual targets BCMA and GPRC5D. [Figure 4B] FIG. 1 shows binding of Trispecific Antibody 1, Trispecific Antibody 2 and Teclistamab to the dual target BCMA and GPRC5D expressing tumor cell RPMI8226.

[0099] [Figure 4C] FIG. 1 shows the binding of Trispecific Antibody 1, Trispecific Antibody 2, and Teclistamab to a stable transfectant expressing human GPRC5D (human GPRC5D CHOK1 cells). [Figure 4D] FIG. 1 shows binding of Trispecific Antibody 1, Trispecific Antibody 2 and Teclistamab to stable transfectants expressing human BCMA (human BCMA CHOK1 cells).

[0100] [Figure 4E] FIG. 1 shows binding of trispecific antibody 1, BGCB491, and TS-F2-5 to tumor cells NCI-H929 expressing dual targets BCMA and GPRC5D. [Figure 4F] FIG. 1 shows binding of trispecific antibody 1, BGCB491, and TS-F2-5 to tumor cells RPMI8226 expressing the dual targets BCMA and GPRC5D.

[0101] [Figure 4G] FIG. 1 shows the binding of trispecific antibody 1, BGCB491, and TS-F2-5 to a stable transfectant expressing human GPRC5D (human GPRC5D CHOK1 cells). [Figure 4H] FIG. 1 shows binding of Trispecific Antibody 1, BGCB491, TS-F2-5 to stable transfectants expressing human BCMA (human BCMA CHOK1 cells).

[0102] [Figure 5A] FIG. 1 shows the binding of Trispecific Antibody 1, Trispecific Antibody 2, and Teclistamab to human CD3-expressing cells (Jurkat cells). [Figure 5B] FIG. 1 shows binding of Trispecific Antibody 1, Trispecific Antibody 2 and Teclistamab to human PBMC cells. [Figure 5C] FIG. 1 shows the binding of trispecific antibody 1, BGCB491, and TS-F2-5 to human CD3-expressing cells (Jurkat cells).

[0103] [Figure 6A]FIG. 1 shows the binding of Trispecific Antibody 1, Trispecific Antibody 2, and Teclistamab to a stable transfectant expressing Cynomolgus GPRC5D (Cynomolgus GPRC5D CHOK1 cells).

[0104] [Figure 6B] FIG. 1 shows binding of Trispecific Antibody 1, Trispecific Antibody 2 and Teclistamab to stable transfectants expressing Cynomolgus BCMA (Cynomolgus BCMA CHOK1 cells).

[0105] [Figure 7] FIG. 1 shows binding of Trispecific Antibody 1, Trispecific Antibody 2 and Teclistamab to cyno PBMC cells.

[0106] [Figure 8A] FIG. 1 shows a T-cell dependent cytotoxicity (TDCC) experiment in which the target cells were NCI-H929. [Figure 8B] FIG. 1 shows a T-cell dependent cytotoxicity (TDCC) experiment in which the target cells were RPMI8226. [Figure 8C] FIG. 1 shows a T-cell dependent cytotoxicity (TDCC) experiment in which the target cells are human BCMA CHOK1.

[0107] [Figure 8D] FIG. 1 shows a T cell dependent cytotoxicity (TDCC) experiment in which the target cells were human GPRC5D CHOK1. [Figure 8E] FIG. 1 shows a T-cell dependent cytotoxicity (TDCC) experiment in which the target cells are a mixed cell line of human BCMA CHOK1 and human GPRC5D CHOK1.

[0108] [Figure 9]Figure 1 shows a T cell dependent cytotoxicity experiment in the presence of 2500 ng / mL soluble BCMA (dotted line indicates the 2500 ng / mL soluble BCMA condition).

[0109] [Figure 10A] FIG. 1 shows IL6 release for Trispecific Antibody 1, Trispecific Antibody 2, and Teclistamab under co-culture conditions of PBMCs (PBMC donor number: P122080606F) and tumor cells NCI-H929. [Figure 10B] FIG. 1 shows IL6 release for Trispecific Antibody 1, Trispecific Antibody 2, and Teclistamab under co-culture conditions of PBMCs (PBMC donor number: XC11210) and tumor cells NCI-H929.

[0110] [Figure 11] FIG. 1 shows the results of a pharmacodynamic experiment of trispecific antibody 1, trispecific antibody 2, teclistamab, and the combination of teclistamab and talquetamb in a human immune cell-reconstituted mouse model—changes in tumor volume (tumor cells: NCI-H929).

[0111] [Figure 12] FIG. 1 shows the results of pharmacodynamic experiments of trispecific antibody 1, BGCB491, and TS-F2-5 in a human immune cell-reconstituted mouse model—changes in tumor volume (tumor cells: NCI-H929).

[0112] [Figure 13] FIG. 1 shows the results of pharmacodynamic experiments of trispecific antibody 1, BGCB491, and TS-F2-5 in a human immune cell-reconstituted mouse model—changes in tumor volume (tumor cells: NCI-H929).

[0113] [Figure 14A] FIG. 1 shows the binding of the alpaca-derived anti-GPRC5D chimeric antibody according to the present invention to GPRC5D-expressing cells (human GPRC5D-CHOK1 cell line). [Figure 14B] FIG. 1 shows the binding of the alpaca-derived anti-GPRC5D chimeric antibody according to the present invention to GPRC5D-expressing cells (cynomolgus monkey GPRC5D-CHOK1 cell line).

[0114] [Figure 15A] FIG. 1 shows the binding of the alpaca-derived anti-GPRC5D humanized antibody according to the present invention to GPRC5D-expressing cells (NCI-H929 tumor cells). [Figure 15B] FIG. 1 shows the binding of the alpaca-derived anti-GPRC5D humanized antibody according to the present invention to GPRC5D-expressing cells (cynomolgus monkey GPRC5D-CHOK1 cell line).

[0115] [Figure 16] FIG. 1 shows that the alpaca-derived anti-GPRC5D humanized antibody according to the present invention does not bind nonspecifically to GPRC5A, a member of the same family.

[0116] [Figure 17A] FIG. 1 shows the binding of anti-BCMA chimeric antibodies derived from mouse hybridomas according to the present invention to human BCMA-expressing cells (human BCMA-CHOK1 cell line). [Figure 17B] FIG. 1 shows the binding of anti-BCMA chimeric antibodies derived from mouse hybridomas according to the present invention to cynomolgus monkey BCMA-expressing cells (cynomolgus monkey BCMA-CHOK1 cell line).

[0117] [Figure 18A] FIG. 1 shows the binding of anti-BCMA humanized antibodies derived from mouse hybridomas according to the present invention to human BCMA-expressing cells (human BCMA-CHOK1 cell line). [Figure 18B] FIG. 1 shows the binding of anti-BCMA humanized antibodies derived from mouse hybridomas according to the present invention to cynomolgus monkey BCMA-expressing cells (cynomolgus monkey BCMA-CHOK1 cell line).

[0118] [Figure 19A]Figure 1 shows the binding of the murine hybridoma-derived anti-BCMA humanized antibody variants 19CH-16H2L2-NA and 19CH-16H2L2-QT according to the present invention to human BCMA-expressing cells (human BCMA-CHOK1 cell line). [Figure 19B] Figure 1 shows the binding of the murine hybridoma-derived anti-BCMA humanized antibody variants 19CH-16H2L2-NA and 19CH-16H2L2-QT according to the present invention to cynomolgus BCMA-expressing cells (cynomolgus BCMA-CHOK1 cell line).

[0119] [Detailed Description of the Invention] 〔term〕 All publications, patents, and patent applications mentioned in this specification are hereby 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 herein.

[0120] Before describing the present invention in detail as follows, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0121] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present invention can be described in range format. Unless otherwise specified, it should be understood that numerical ranges or range description formats are intended for brevity and convenience only and are not intended to strictly limit the scope of the present invention. Thus, descriptions in range format should be considered to have specifically disclosed all possible subranges and all possible specific numerical points within those ranges, as if those subranges and related numerical points were expressly set forth herein. The above principles apply equally regardless of the numerical range. When describing a range, the range includes the endpoints of the range.

[0122] When referring to a measurable value (e.g., amount, temporal duration, etc.), the term "about" refers to a variation 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 stated value.

[0123] The term "antibody" as used herein may include intact antibodies (e.g., full-length monoclonal antibodies) and antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof, as well as products formed by modification (e.g., by linking to other peptide segments, rearrangement of functional units, etc.) of intact antibodies or antigen-binding fragments or single chains thereof that retain antigen-specific binding ability.

[0124] In one embodiment, an antibody typically refers to a Y-shaped tetrameric protein consisting of two heavy (H) 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 is composed of one variable domain (VL) and one constant domain (CL). Each heavy chain contains one variable domain (VH) and a constant region.

[0125] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, with the corresponding heavy chain constant domains designated 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 classified into one of two clearly distinct types, designated κ and λ, based on the amino acid sequence of their constant domains.

[0126] In IgG, IgA, and IgD antibodies, the constant region contains 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 proline- and cysteine-rich segment of variable length. Each class of antibody further contains inter- and intrachain disulfide bonds formed from paired cysteine ​​residues.

[0127] The terms "variable region" or "variable domain" refer to significant variations in amino acid composition from one antibody to another and are primarily responsible for antigen recognition and binding. The variable regions of each light / heavy chain pair form the antibody binding site, such that an intact IgG antibody has two binding sites (i.e., is bivalent). The heavy chain variable region (VH) and light chain variable region (VL) domains each contain three regions of extreme variability called hypervariable regions (HVRs), or more commonly called complementarity-determining regions (CDRs). Both VH and VL have four framework regions, designated FR1, FR2, FR3, and FR4, respectively. Thus, the CDR and FR sequences typically occur in the following sequence in the heavy chain variable domain (or light chain variable domain): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0128] The term "antibody fragment" includes 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, which refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or an immunogenic determining portion thereof, or a fusion protein product further derived from the fragment, such as a single-chain antibody or an extracellular binding region in a chimeric antigen receptor. 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 or multispecific antibodies formed from antibody fragments.

[0129] 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" is a bivalent antibody fragment containing two Fab fragments and a hinge region.

[0130] The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one variable region antibody fragment comprising a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short, flexible peptide linker) and can be expressed in the form of a single polypeptide chain, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv can have the VL and VH variable regions set forth above in any order (e.g., relative to the N- and C-termini of the polypeptide), and can comprise a VL-linker-VH or a VH linker-VL.

[0131] A "VHH domain" is also known as a heavy-chain single-domain antibody, VHH, VHH antibody fragment, VHH antibody, or nanobody, and is the variable domain of an antigen-binding immunoglobulin called a "heavy-chain antibody" (i.e., an antibody lacking a light chain). The term "VHH domain" is used to distinguish this variable domain from the heavy-chain variable domain (referred to herein as a "VH domain") and light-chain variable domain (referred to herein as a "VL domain") present in conventional tetrapeptide chain antibodies. A VHH domain specifically binds to an epitope without the need for another antigen-binding domain (this is in contrast to the VH or VL domain of a conventional tetrapeptide chain antibody, where the epitope is recognized by both the VL and VH domains). A VHH domain is a small, stable, and efficient antigen-recognition unit formed from a single immunoglobulin domain. The terms "heavy-chain single-domain antibody," "VHH domain," "VHH," "VHH domain," "VHH antibody fragment," "VHH antibody," and "heavy-chain antibody variable region" can be used interchangeably. "VHH domains" include, but are not limited to, natural antibodies produced by camelids, and may be obtained by humanization of antibodies produced by camelids, or by screening phage display technology.

[0132] The term "amino acid modification" (or "modified amino acid") includes an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. As used herein, an "amino acid substitution" or "substitution" or "replacement" refers to the substitution of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. For example, the substitution S32A means that the serine at position 32 has been replaced with an alanine.

[0133] The multispecific antigen-binding molecules of the present invention can further comprise substitutions or alterations in 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 yield, altered Fc ligand binding to Fc receptors (FcRs), enhanced or attenuated ADCC or CDC, altered glycosylation and / or disulfide bonds, and altered binding specificity. According to some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that weaken FcγR binding (e.g., substitutions at positions 234 and 235 of the Fc region). According to one embodiment, the substitutions are L234A and L235A.

[0134] The term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined to extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain; for example, the IgG Fc domain includes 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 performed according to the EU numbering system, also known as the EU index.

[0135] The term "knob-into-hole" refers to modifications used to promote binding of the two polypeptide chains of an Fc, including a "knob" modification on one of the two polypeptide chains and a "hole" modification on the other of the two polypeptide chains of an Fc. Generally, this method involves introducing a protuberance ("knob") at the interface of a first polypeptide chain and a corresponding cavity ("hole") at the interface of a second polypeptide chain, positioning the protuberance in the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide chain with a larger side chain, such as tyrosine or tryptophan. A complementary cavity of the same or similar size as the protuberance is created at the interface of the second polypeptide chain by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0136] Thus, in a specific embodiment, in the CH3 domain of the first polypeptide chain of the Fc domain of the trispecific antigen-binding molecule of the present invention, one amino acid residue is substituted with an amino acid residue having a larger side chain volume, thus creating a protuberance in the CH3 domain of the first polypeptide chain, which can be placed in a 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 substituted with an amino acid residue having a smaller side chain volume, thus creating a cavity in the CH3 domain of the second polypeptide chain, which can be placed in the cavity of the protuberance in the CH3 domain of the first polypeptide chain. Preferably, the amino acid residue having 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 having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0137] The term "linker" refers to any tool used to link two different functional units (e.g., antigen-binding fragments). 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, e.g., a sequence containing serine and glycine. The linker may be long or short depending on the specific construct.

[0138] According to the present invention, 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 has the amino acid sequence (G4S) n or (G4S) n A, where n is any integer from 1 to 10, and preferably comprises the amino acid sequence (G4S)3 or (G4S)3A. The linker connecting the VH and VL domains to form the VH-VL or VL-VH scFv domain 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, where n is any integer from 1 to 10, and preferably contains the amino acid sequence (G4S)3 or (G4S).

[0139] As used herein, the term "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 (recombinantly produced antibodies), intrabodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

[0140] The term "monoclonal antibody" (also known as "monoclonal antibody (mAb)") refers to a substantially homogeneous antibody that is produced by a single cell clone and targets only a specific antigen 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.

[0141] The division of CDRs and FRs into the variable regions of the antibodies and trispecific antigen-binding molecules of the present invention is determined according to the Kabat definition. Other nomenclature and numbering systems, such as Chothia, IMGT, and AHo, are also known to those skilled in the art. Therefore, a humanized antibody comprising one or more CDRs derived from any nomenclature system based on the antibody sequence of the present invention is clearly within the scope of the present invention.

[0142] The term "humanized antibody" refers to an antibody in which all or some of the amino acids other than the CDRs of a non-human antibody (e.g., a mouse antibody) have been replaced with the corresponding amino acids derived from a human immunoglobulin. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted as long as the antibody does not lose its ability to bind to a specific antigen. A "humanized" antibody maintains the same antigen specificity as the original antibody.

[0143] The term "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant region sequences are derived from another species, e.g., the variable regions are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0144] As used herein, "homologous" (also known as "same species") and "heterologous" are relative concepts that refer to whether the different elements of a construct are from the same or different sources, or whether, after construction of the construct is complete, among multiple "homologous" elements from the same original source, some elements have been transformed and altered compared to the other original elements that have not been transformed, making them "heterologous."

[0145] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment. Broadly defined, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, an intact extracellular domain (ECD), or a protein. Peptides, proteins, glycoproteins, polysaccharides, lipids, portions thereof, and combinations thereof can all be configured as antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen, or cells or preparations containing antigens, can be used to generate antibodies specific to antigenic determinants.

[0146] The terms "epitope" and "antigenic determinant" refer to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from nonadjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically exist in a unique spatial conformation and contain at least 3-15 amino acids.

[0147] The term "multispecific" refers to the ability of an antigen-binding molecule to specifically bind to multiple 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 include immunoglobulins and their derivatives, such as fragments. The term "trispecific antigen-binding molecule" or "trispecific binding molecule" refers to a binding molecule (e.g., an antibody, or antibody fragment-containing molecule) specific for three different antigens (or epitopes), particularly a trispecific antibody.

[0148] The term "specific binding" refers to selective binding to an antigen, as distinguished from unwanted or nonspecific 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 well known to those skilled in the art.

[0149] When antibodies, binding molecules, bispecific binding molecules, or multispecific binding molecules are prepared from the variable regions of the present invention, the constant region is not particularly limited; constant regions known to those skilled in the art or obtained by the person skilled in the art may be used, or amino acid mutations (e.g., mutations that increase or decrease Fc binding to receptors or FcRn) may be introduced into the constant region.

[0150] The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules of the present invention can be obtained by any method without particular limitation. The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules of the present invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. A preferred prior art approach is to use mammalian expression systems, which result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones can be obtained by expressing antibodies that specifically bind to human antigens. Positive clones are grown in serum-free medium in a bioreactor to produce antibodies. The culture medium containing secreted antibodies can be purified and recovered using conventional techniques. Antibodies can be filtered and concentrated using conventional methods. Soluble contaminants and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange.

[0151] As used herein, the term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0152] The term "stable transfection" or "to stably transfect" 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 exogenous DNA into its genomic DNA.

[0153] The term "antibody-drug conjugate" (ADC) refers to an antibody covalently linked to a therapeutically active agent or active pharmaceutical ingredient (API), which can target the antibody's binding target and exhibit its pharmacological function. The therapeutically active agent or active pharmaceutical ingredient (API) may be a cytotoxin capable of killing ADC target cells, preferably malignant or cancer cells. Covalent attachment of the therapeutically active agent, active pharmaceutical ingredient, or cytotoxin can be performed non-site-specifically using standard chemical linkers that attach the payload to lysine or cysteine ​​residues, or, preferably, conjugation can be performed site-specifically, allowing full control over the attachment site and the resulting ADC's drug-to-antibody ratio.

[0154] The term "amino acid substitution" or "substitution" or "replacement" refers to the substitution of an amino acid at a particular position in a parent polypeptide sequence with another amino acid.

[0155] The term "affinity" or "binding affinity" refers to the strength of the sum of all noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). d The term "" refers to the dissociation constant of a particular antibody-antigen interaction. Binding affinity can be determined using a variety of techniques known in the art, such as, for example, surface plasmon resonance, biolayer interferometry, dipole interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0156] The term "biological activity" refers to the ability of an antibody to bind to an antigen and elicit a measurable biological response that can be measured in vitro or in vivo.

[0157] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients that allows the active ingredients contained therein to be present in a biologically active, effective form and does not contain additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered. When a "pharmaceutical composition" exists in the form of a combination of separate formulations containing two or more different active ingredients, they can be administered simultaneously, sequentially, separately, or at intervals, with the aim of using them jointly to exert the biological activity of the multiple active ingredients for the treatment of a disease.

[0158] The binding molecules or antigen-binding fragments of the present invention may be used in combination with other drugs, in which case the active ingredients may be mixed to form a single dosage unit or may be used separately as separate, independent dosage units.

[0159] The term "effective amount" refers to a dose of a pharmaceutical formulation of an antibody or fragment thereof of the present invention that, after single or multiple administration to a patient, produces the desired effect in the treated patient. An effective amount can be readily determined by an attending physician skilled in the art by considering various factors, such as ethnicity, body weight, age, and health, the particular disease involved, the severity of the disease, the response of the individual patient, the particular antibody administered, the administration pattern, the bioavailability characteristics of the administered formulation, the selected administration regimen, and the use of concomitant therapies.

[0160] As used herein, the terms "individual" or "subject" refer to any animal, such as a mammal or marsupial. Individuals of the present invention include, but are not limited to, humans, non-human primates (such as cynomolgus monkeys, rhesus monkeys, or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry.

[0161] As used herein, the term "disease" or "condition" or "disorder" refers to a change or imbalance that impairs or interferes with the normal function of a cell, tissue, or organ. For example, the "disease" includes, but is not limited to, a tumor, a pathogen infection, an autoimmune disease, a T-cell dysfunction disease, or an immune tolerance disorder (e.g., transplant rejection).

[0162] As used herein, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, followed by migration or invasion of other tissues or organs. Tumor growth (proliferation) is usually uncontrollable and progressive, and does not induce or inhibit the proliferation of normal cells.

[0163] The term "treatment" as used herein refers to clinical intervention in the process of attempting to address changes in an individual's disease or a cell-induced disease, and may involve prevention or intervention during the clinical pathological process. Therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or alleviation of disease symptoms, alleviation or improvement of prognosis, etc.

[0164] The terms "G protein-coupled receptor C5 family subtype D" and "GPRC5D" specifically include human GPRC5D protein, and include variants, subtypes, homologs, and analogs of human GPRC5D that share at least one common epitope with GPRC5D (e.g., human GPRC5D); exemplary human GPRC5D sequences can be found in GenBank accession number BC069341, NCBI reference sequence: NP_061124.1, and UniProtKB / Swiss-Prot accession number Q9NZD1 (see also Brauner-Osborne, H et al., 2001, Biochim. Biophys. Acta 1518, 237-248).

[0165] The term "BCMA," also known as TNFRSF17, refers to the tumor-associated antigen B-cell maturation antigen; exemplary human BCMA sequences include the human BCMA protein with accession number UniProt Q02223. DETAILED DESCRIPTION OF THE INVENTION

[0166] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. In the following examples, experimental methods for which specific conditions are not specified are carried out according to conventional conditions known in the art or according to the conditions suggested by the manufacturer.

[0167] [Example 1] Antigen information of human GPRC5D, GPRC5A, and cynomolgus monkey GPRC5D The full-length amino acid sequence (SEQ ID NO: 1) (Uniprot ID: Q9NZD1) of human GPRC5D used in the Examples is as follows:

[0168] TIFF2026506565000001.tif31170

[0169] Note: This protein is a seven-transmembrane protein. The double underlined regions represent the extracellular domain (1-27; 85-93; 145-167; 226-239), the underlined regions represent the intracellular domain (49-63; 115-123; 189-204; 261-345), and the italicized regions represent the transmembrane domain (28-48; 64-84; 94-114; 124-144; 168-188; 205-225; 240-260).

[0170] The full-length amino acid sequence (SEQ ID NO: 2) (Uniprot ID: A0A2K5W6I2) of cynomolgus monkey GPRC5D (cyno-GPRC5D) used in the Examples is as follows:

[0171] TIFF2026506565000002.tif32170

[0172] Note: This protein is a seven-transmembrane protein. The double underlined regions are the extracellular domain (1-27; 85-93; 145-167; 226-239), the underlined regions are the intracellular domain (49-63; 115-123; 189-204; 261-344), and the italicized regions are the transmembrane domain (28-48; 64-84; 94-114; 124-144; 168-188; 205-225; 240-260).

[0173] The full-length amino acid sequence of human GPRC5A used in the examples (SEQ ID NO: 36) (Uniprot ID: Q8NFJ5) is as follows:

[0174] TIFF2026506565000003.tif31170

[0175] Note: This protein is a seven-transmembrane protein. The double underlined regions represent the extracellular domain (1-33; 90-97; 151-176; 234-247), the underlined regions represent the intracellular domain (55-68; 119-129; 198-212; 269-357), and the italicized regions represent the transmembrane domain (34-54; 69-89; 98-118; 130-150; 177-197; 213-233; 248-268).

[0176] [Example 2] Preparation of cell lines expressing human GPRC5D, human GPRC5A, and cynomolgus monkey GPRC5D The nucleotide sequence encoding human GPRC5D amino acid sequence shown in SEQ ID NO. 1 was cloned into pCMV3 (Sino Biological, Cat. No. CV011) vector to obtain a plasmid for constructing a human GPRC5D cell line. The resulting plasmid was transfected into CHOK1 cells (ATCC, Cat. No. CCL-61) to obtain a CHOK1 cell line expressing human GPRC5D (abbreviated as human GPRC5D-CHOK1 cell line).

[0177] The nucleotide sequence encoding the cynomolgus monkey GPRC5D amino acid sequence shown in SEQ ID NO. 2 was cloned into the pCMV3 vector to obtain a plasmid for constructing a cynomolgus monkey GPRC5D cell line. The resulting plasmid was transfected into CHOK1 cells to obtain a CHOK1 cell line expressing cynomolgus monkey GPRC5D (abbreviated as cynomolgus monkey GPRC5D-CHOK1 cell line).

[0178] The nucleotide sequence encoding the human GPRC5A amino acid sequence shown in SEQ ID NO. 36 was cloned into the pCMV3 vector to obtain a vector for constructing a human GPRC5A cell line. The resulting vector was transfected into CHOK1 cells to obtain a CHOK1 cell line expressing human GPRC5A (abbreviated as human GPRC5A-CHOK1 cell line).

[0179] The expression of human GPRC5D in the human GPRC5D-CHOK1 cell line obtained above was detected using a FACS assay, and a negative control hIgG1 LALA isotype (Hyakuei Bio, product number B109802) was set in the experiment.

[0180] The expression of cynomolgus monkey GPRC5D in the above-obtained cynomolgus monkey GPRC5D-CHOK1 cell line was detected using FACS assay, and a negative control hIgG1 LALA isotype (Hyakuei Bio, product number B109802) was set in the experiment.

[0181] The expression of human GPRC5A in the human GPRC5A-CHOK1 cell line obtained above was detected using a FACS assay, and a negative control, hIgG1 LALA isotype (Hyakuei Bio, product number B109802), was set in the experiment. The results are shown as follows:

[0182] The expression of human GPRC5D in the human GPRC5D-CHOK1 cell line is shown in Figure 1A. The results indicate that human GPRC5D is well overexpressed in CHOK1 cells, which can be used in subsequent experiments to verify the binding of GPRC5D monoclonal antibodies to human GPRC5D at the cellular level.

[0183] The expression of cynomolgus monkey GPRC5D in the cynomolgus monkey GPRC5D-CHOK1 cell line is shown in Figure 1B. The results show that cynomolgus monkey GPRC5D is well overexpressed in CHOK1 cells. This can be used in subsequent experiments to verify the binding of the GPRC5D monoclonal antibody to cynomolgus monkey GPRC5D at the cellular level.

[0184] The expression of human GPRC5A in the human GPRC5A-CHOK1 cell line is shown in Figure 1C. This result indicates that human GPRC5A is well overexpressed in CHOK1 cells, and can be used in subsequent experiments to verify the absence of nonspecific binding between the GPRC5D monoclonal antibody and human GPRC5A at the cellular level.

[0185] [Example 3] Antigen information of human BCMA and cynomolgus monkey BCMA The full-length amino acid sequence (SEQ ID NO: 3) (Uniprot ID: Q02223) of human BCMA used in the examples is as follows:

[0186] TIFF2026506565000004.tif17170Note: This protein is a single-pass transmembrane protein; the double underlined horizontal line represents the extracellular domain (1-54), the wavy underlined domain represents the transmembrane domain (55-77), and the horizontal underlined domain represents the intracellular domain (78-184).

[0187] The full-length amino acid sequence (SEQ ID NO: 4) (Uniprot ID: G7Q0I4) of cynomolgus monkey BCMA (cyno-BCMA) used in the examples is as follows:

[0188] TIFF2026506565000005.tif17170Note: This protein is a single-pass transmembrane protein; the double underlined horizontal line represents the extracellular domain (1-53), the wavy underlined domain represents the transmembrane domain (54-76), and the horizontal underlined domain represents the intracellular domain (77-183).

[0189] Example 4: Preparation of human BCMA and cynomolgus monkey BCMA cell lines The nucleotide sequence encoding human BCMA amino acid sequence shown in SEQ ID NO:3 was cloned into the pCMV3 (SinoBiological, Catalog No. CV011) vector to obtain a vector for constructing a human BCMA cell line. The resulting vector was transfected into CHOK1 cells (ATCC, Catalog No. CCL-61) to obtain a CHOK1 cell line expressing human BCMA (abbreviated as human BCMA-CHOK1 cell line).

[0190] The nucleotide sequence encoding the cynomolgus BCMA amino acid sequence shown in SEQ ID:4 was cloned into the pCDH-CMV-MCS-EF1-Hygro (Honorgene, product number CD515B-1) vector to obtain a vector for constructing a cynomolgus BCMA cell line. The resulting vector was converted into a virus and then transfected into CHOK1 cells to obtain a CHOK1 cell line expressing cynomolgus BCMA (abbreviated as cynomolgus BCMA-CHOK1 cell line).

[0191] FACS assay was used to detect the expression of human BCMA in the human BCMA-CHOK1 cell line obtained above, and a negative control, hIgG1 LALA isotype (Hyakuei Bio, product number B109802), was used in the experiment.

[0192] The expression of cynomolgus monkey BCMA in the cynomolgus monkey BBCMA-CHOK1 cell line obtained above was detected using a FACS assay, and a negative control hIgG1 LALA isotype (Hyakuei Bio, product number B109802) was set in the experiment. The results are shown as follows:

[0193] Human BCMA - The expression of human BCMA in the CHOK1 cell line is shown in Figure 2A. This result indicates that human BCMA is well overexpressed in CHOK1 cells, which can be used to verify the binding of BCMA monoclonal antibodies to human BCMA at the cellular level in subsequent experiments.

[0194] The expression of cynomolgus BCMA in the cynomolgus BCMA-CHOK1 cell line is shown in Figure 2B. This result indicates that cynomolgus BCMA is well overexpressed in CHOK1 cells, and can be used in subsequent experiments to verify the binding of BCMA monoclonal antibodies to cynomolgus BCMA at the cellular level.

[0195] Example 5: Design and sequence of anti-GPRC5D-BCMA-CD3 trispecific antibody The anti-BCMA full-length antibody (BCMA mAb) was obtained by hybridoma screening, and its heavy chain sequence is shown in SEQ ID NO:9 and its light chain sequence is shown in SEQ ID NO:7.

[0196] The anti-GPRC5D nanobody (GPRC5D VHH) was obtained by screening using alpaca immunization, and its variable region sequence is shown in SEQ ID NO:10.

[0197] The CD3ε-binding domain was derived from the full-length antibody CD3 mAb, whose heavy chain sequence is shown in SEQ ID NO:11 and whose light chain sequence is shown in SEQ ID NO:12. The heavy and light chain variable regions of CD3 mAb were linked via a flexible linker to form a single-chain antibody scFv with the structure VH-(G4S)3-VL and sequence shown in SEQ ID NO:13. The scFv was further fused via a flexible linker to the C-terminus of the heavy chain of a BCMA full-length antibody Fab segment.

[0198] The CD3scFv was fused to the C-terminus of the BCMA Fab heavy chain CH1 to form one arm of the triabody, while simultaneously introducing another intact GPRC5D nanobody-binding domain and an Fc portion with a "knob" and "hole" structure. The resulting triabody was designated "Triabody 1." The sequences of the three fused heterologous peptide chains are shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, and a schematic diagram is shown in Figure 3.

[0199] The CD3scFv was fused to the C-terminus of the heavy chain of a BCMA full-length antibody Fab segment to form one arm of the antibody, and the GPRC5D nanobody was fused to the C-terminus of the heavy chain of another BCMA full-length antibody Fab segment to form the other arm of the antibody, while also containing an Fc portion with a "knob" and "hole" structure; the resulting trispecific antibody was named "Triabody 2", and the sequences of the fused chains are shown in SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8, and a schematic diagram is shown in Figure 3.

[0200] To reduce the ADCC activity of the antibody, the Fc segment of the final triabody was substituted with the amino acids L234A, L235A, and G237A. The Fc domain of the scFv-containing peptide chain was designed as a "knob" structure containing two amino acid substitutions, S354C and T366W. The Fc domain of the scFv-free peptide chain was designed as a "hole" structure containing four amino acid substitutions, Y349C, T366S, L368A, and Y407V. Furthermore, to facilitate the purification of the triabody, the heavy chain of the "hole" structure needed to be further substituted with H435R.

[0201] The structures of Triabody 1 and Triabody 2 and the associated molecular sequences are summarized in Tables 1 and 2, respectively.

[0202] Structural description of trispecific antibodies [Table 1]

[0203] Amino acid sequence of trispecific antibodies [Table 2] JPEG2026506565000008.jpg226167JPEG2026506565000009.jpg228167JPEG2026506565000010.jpg146166

[0204] Example 6: Construction of anti-GPRC5D-BCMA-CD3 trispecific antibody and its transient transfection and expression in eukaryotic cells Each of the gene fragments encoding the above trispecific antibodies was cloned into a PTT5 expression vector to prepare a transfection-grade expression plasmid.

[0205] Expi293F in serum-free medium TM Cells (Thermo Fisher Scientific) were cultured in shake flasks (Corning Inc.) and grown on a shaker at 37°C in an 8% CO2 environment. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in the appropriate ratio and added to the shake flask for cell culture. After 6 days of cell culture, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purified using a Protein A column. The column was washed with PBS until the A280 reading fell to baseline. The target protein was eluted with an acidic eluent (pH 3.0–3.5) and neutralized with 1 M Tris-HCl (pH 8.0–9.0). After appropriate concentration, the eluted sample was further purified using gel chromatography Superdex 200 (GE) equilibrated with PBS to remove aggregates. The monomer peak was collected, the solution was exchanged into PBS, and aliquots were prepared. The final purified antibody was subjected to SDS-PAGE and HPLC purity analysis, and A280 concentration measurement.

[0206] At the same time, Janssen's GPRC5DxCD3 bispecific antibody Talquetamab (derived from patent WO2018017786A2, consisting of sequences SEQ ID NO: 25, 26, 55, 58, containing one GPRC5D binding site and one CD3 binding site) was also expressed and purified.

[0207] Concurrently, Janssen's BCMAxCD3 bispecific antibody Teclistamab (derived from patent WO29220368A1, consisting of sequences SEQ ID NOs: 31, 32, 41, 42, containing one BCMA binding site and one CD3 binding site) was also expressed and purified.

[0208] Concurrently, Janssen's BGCB491 trispecific antibody (derived from patent WO2022175255A2, consisting of sequences SEQ ID NO: 29, 30, 31, containing one GPRC5D-binding site, one BCMA-binding site, and one CD3-binding site) was also expressed and purified.

[0209] At the same time, the TS-F2-5 trispecific antibody from Innovent Biologics (derived from patent WO2022174813A1, consisting of sequences SEQ ID NO: 68, 75, 76, 78, containing one GPRC5D-binding site, one BCMA-binding site, and one CD3-binding site) was also expressed and purified.

[0210] [Example 7] Cellular affinity test of anti-GPRC5D-BCMA-CD3 trispecific antibody FACS was used to detect binding of the anti-GPRC5D-BCMA-CD3 trispecific antibody to NCI-H929 and RPMI8226 cells expressing the dual targets human GPRC5D and human BCMA, human GPRC5D-CHOK1 cells expressing human GPRC5D, human BCMA-CHOK1 cells expressing human BCMA, and naturally hCD3-expressing T lymphocytes (Jurkat) and human PBMCs.

[0211] NCI-H929 cells (ATCC, Catalog No. CRL-9068), RPMI 8226 cells (ATCC, Catalog No. CCL-155), human GPRC5D-CHOK1 cells (Example 2), human BCMA-CHOK1 cells (Example 4), and Jurkat cells (ATCC, Catalog No. TIB-152) were cultured. The medium for NCI-H929 cells was RPMI1640 + 10% FBS + 0.05 mM mercaptoethanol, the medium for RPMI8226 and Jurkat cells was RPMI1640 + 10% FBS, and the medium for human GPRC5D-CHOK1 and human BCMA-CHOK1 cells was F12K + 10% FBS + 400 μg / mL hygromycin B. Cells were cultured in T75 cell culture flasks in a 5% CO2 incubator at 37°C. When using cells, NCI-H929, RPMI8226, and Jurkat cells were directly placed in 50 mL centrifuge tubes without digestion. Human GPRC5D-CHOK1 and human BCMA-CHOK1 were digested with 0.25% Trpsin-EDTA trypsin, and the digestion was terminated with F12K + 10% FBS + 400 μg / mL hygromycin B.

[0212] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 1% BSA in PBS. Purchased human PBMCs were centrifuged at 1500 rpm for 10 minutes at room temperature, the supernatant discarded, and the cells resuspended in 1% BSA in PBS. The cells were counted, adjusted to a cell density of 1E6 / mL, and seeded at 100 μL per well into a 96-well round-bottom culture plate (Corning, product number 3799). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 200 μL of 1% BSA in PBS. The cells were then centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and stored at 4°C. The test antibodies and negative control hIgG1 LALA isotype (Hyakuei Bio, product number B109802) were diluted in 1% BSA in PBS to a starting concentration of 100 nM, followed by 5-fold serial dilutions to obtain eight concentrations. Cells were resuspended in the diluted antibody and incubated at 100 μL / well for 1 hour at 4°C. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. Cells were washed with 160 μL of 1% BSA in PBS, resuspended, and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The secondary antibody (goat anti-human IgG Fc PE) was diluted 1:400 in 1% BSA in PBS according to the manufacturer's instructions. Cells were resuspended in the diluted antibody and incubated at 100 μL / well for 0.5 hours at 4°C. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The cells were washed by resuspending them in 200 μL of 1% BSA in PBS, centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The cells were then resuspended in 100 μL of 1% BSA in PBS and filtered through a 300-mesh gauze. The mean fluorescence intensity of the PE channel was detected by flow cytometry.

[0213] Export the FCS file from the flow cytometer and analyze the mean fluorescence intensity (MFI) of the PE channel of each sample using flowjo software. Import the MFI data into Graphpad to calculate the half maximal binding concentration (EC) of the antibody to the cells. 50 ) and the highest mean fluorescence intensity (Top MFI) were analyzed.

[0214] The binding results of the test molecules to tumor antigen cells are shown in Figures 4A–4H and Tables 3 and 4. In the dual-target-expressing tumor cell lines NCI-H929 and RPMI8226, binding of the trispecific antibody was stronger than that of the single-target bispecific antibody, and this was positively correlated with the tumor antigen expression level (the dual-target expression level in NCI-H929 cells was higher than that in RPMI8226 cells). In cell lines expressing single targets, binding of the trispecific antibody was weaker or slightly stronger than that of the corresponding bispecific antibody. This result is speculated to be caused by synergistic binding of the dual targets. Furthermore, in tumor cell lines expressing dual targets, binding of Trispecific Antibody 1 was stronger than that of the Janssen BGCB491 and Innovent TS-F2-5 molecules.

[0215] The results of the binding of the test molecules to Jurkat cells and human PBMC cells are shown in Figures 5A to 5C and Tables 5 and 6. The trispecific antibody showed weaker binding to Jurkat cells than teclistamab and was comparable to the Janssen BGCB491 and Innovent TS-F2-5 molecules.

[0216] Consequences of trispecific antibody binding to tumor antigen-expressing cells [Table 3]

[0217] Consequences of trispecific antibody binding to tumor antigen-expressing cells [Table 4]

[0218] Results of trispecific antibody binding to Jurkat cells and human PBMC cells [Table 5]

[0219] Results of trispecific antibody binding to Jurkat cells [Table 6]

[0220] [Example 8] Species cross-reactivity test of anti-GPRC5D-BCMA-CD3 trispecific antibodies FACS was used to detect binding of the anti-GPRC5D-BCMA-CD3 trispecific antibody to cynomolgus monkey GPRC5D-CHOK1 cells expressing cyno GPRC5D, cynomolgus monkey BCMA-CHOK1 cells expressing cyno BCMA, and cyno PBMCs naturally expressing cyno CD3.

[0221] Cynomolgus monkey GPRC5D-CHOK1 cells (Example 2) and cynomolgus monkey BCMA-CHOK1 cells (Example 4) were cultured in a medium of F12K + 10% FBS + 400 μg / mL Hygromycin B, and cultured in a T75 cell culture flask in a 5% CO incubator at 37°C. When using the cells, cynomolgus monkey GPRC5D-CHOK1 and cynomolgus monkey BCMA-CHOK1 were digested with trypsin in 0.25% Trpsin-EDTA, and digestion was terminated with F12K + 10% FBS + 400 μg / mL Hygromycin B.

[0222] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 1% BSA in PBS. Purchased Cyno PBMCs were centrifuged at 1500 rpm for 10 minutes at room temperature, the supernatant discarded, and the cells resuspended in 1% BSA in PBS. The cells were counted, adjusted to a cell density of 1E6 / mL, and seeded at 100 μL per well into a 96-well round-bottom culture plate (Corning, product number 3799). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 200 μL of 1% BSA in PBS. The cells were then centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and stored at 4°C. The test antibodies and negative control hIgG1 LALA isotype (Hyakuei Bio, product number B109802) were diluted in 1% BSA in PBS to a starting concentration of 100 nM, followed by 5-fold serial dilutions to obtain eight concentrations. Cells were resuspended in the diluted antibody and incubated at 100 μL / well for 1 hour at 4°C. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. Cells were washed with 160 μL of 1% BSA in PBS, resuspended, and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The secondary antibody (goat anti-human IgG Fc PE) was diluted 1:400 in 1% BSA in PBS according to the manufacturer's instructions. Cells were resuspended in the diluted antibody and incubated at 100 μL / well for 0.5 hours at 4°C. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The cells were washed by resuspending them in 200 μL of 1% BSA in PBS, centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded. The cells were then resuspended in 100 μL of 1% BSA in PBS and filtered through a 300-mesh gauze. The mean fluorescence intensity of the PE channel was detected by flow cytometry.

[0223] Export the FCS file from the flow cytometer and analyze the mean fluorescence intensity (MFI) of the PE channel of each sample using flowjo software. Import the MFI data into Graphpad to calculate the half maximal binding concentration (EC) of the antibody to the cells. 50 ) and the highest mean fluorescence intensity (Top MFI) were analyzed.

[0224] The results of the binding of the test molecules to the corresponding tumor antigen cells of cynomolgus monkeys are shown in Figures 6A-6B and Table 7. The trispecific antibody molecules showed good binding to the corresponding tumor antigens of cynomolgus monkeys.

[0225] The results of the binding of the test molecules to cynomolgus monkey PBMCs are shown in Figure 7 and Table 8. The trispecific antibody molecule showed good binding to cynomolgus monkey PBMCs.

[0226] Results of trispecific antibody binding to cells expressing cynomolgus monkey tumor antigens [Table 7]

[0227] Results of trispecific antibody binding to cyno PBMC cells [Table 8]

[0228] Example 9: In vitro binding affinity and kinetics of anti-GPRC5D-BCMA-CD3 trispecific antibodies to recombinant proteins To measure the affinity and kinetic properties of human-derived CD3 (purchased from Acro, product number CDD-H52W1) / cynomolgus monkey-derived CD3 (purchased from Acro, product number CDD-C52W4), we directly immobilized human-derived / cynomolgus monkey-derived CD3 molecules using a CM5 chip. 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 on the chip at a flow rate of 10 μL / min for 60 seconds. The chip was then blocked with ethanolamine. Anti-GPRC5D-BCMA-CD3 trispecific antibody molecules were immobilized at a concentration range of 100 nM to 0.39 nM, diluted two-fold in HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer. The binding and dissociation were performed at a flow rate of 50 μL / min for 90 seconds and 360 seconds, respectively.

[0229] After each round of experiment, the chip was washed with 3M MgCl2 solution at a flow rate of 30 μL / min for 30 seconds to remove the anti-GPRC5D-BCMA-CD3 trispecific antibody molecules and complete chip regeneration. The raw data was analyzed using Biacore Insight Evaluation Software (3.0.12.15655) and fitted with a (1:1) Langmuir model. The resulting affinity and kinetic experimental data for the trispecific antibody are shown in Table 9.

[0230] Binding affinity and kinetics of anti-GPRC5D-BCMA-CD3 trispecific antibodies to human / cynomolgus monkey CD3 proteins [Table 9]

[0231] Experimental results showed that both trispecific antibody molecules could bind to human / cynomolgus CD3 with comparable affinity.

[0232] To measure the affinity and kinetics of human BCMA (purchased from Acro, product number BCA-H522y) and cynomolgus BCMA (purchased from Acro, product number BCA-C52H7), antibody molecules were detected by indirect capture using a CM5 chip. The CM5 chip was first activated with EDC and NHS, and then immobilized with anti-human IgG (Fc) antibody (purchased from Cytiva, product number 10325009) diluted to 10 μg / mL in 10 mM sodium acetate solution, pH 5, at a flow rate of 5 μL / min for 420 seconds, followed by blocking with ethanolamine. Anti-GPRC5D-BCMA-CD3 trispecific antibody molecules were diluted to 4 μg / mL in HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer and captured for 60 seconds at a flow rate of 10 μL / min. Human / cynomolgus monkey-derived BCMA 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 (50 nM to 0.195 nM) and allowed to bind for 90 seconds and dissociate for 180 seconds at a flow rate of 30 μL / min.

[0233] After each round of experiment, the chip was washed with 3M MgCl2 solution at a flow rate of 30 μL / min for 30 seconds to remove the anti-GPRC5D-BCMA-CD3 trispecific antibody molecules and complete chip regeneration. The raw data was analyzed using Biacore Insight Evaluation Software (3.0.12.15655) and fitted with a (1:1) Langmuir model. The resulting affinity and kinetic experimental data for the trispecific antibody are shown in Table 10.

[0234] Binding affinity and kinetics of anti-GPRC5D-BCMA-CD3 trispecific antibodies to human / cynomolgus monkey BCMA proteins [Table 10]

[0235] Experimental results showed that both trispecific antibody molecules were able to bind to human / cynomolgus BCMA with comparable affinity.

[0236] [Example 10] In vitro functional experiments of anti-GPRC5D-BCMA-CD3 trispecific antibodies A. T cell-dependent cytotoxicity (TDCC) experiment in which the target cells were NCI-H929 NCI-H929 cells (ATCC, product number CRL-9068) were cultured in RPMI 1640 + 10% FBS + 0.05 mM mercaptoethanol in a T75 cell culture flask in a 37°C, 5% CO2 incubator. For cell use, NCI-H929 cells were directly placed in a 50 mL centrifuge tube without digestion. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI 1640 + 2% FBS medium. The cells were counted and the cell density adjusted to 1E5 / mL.

[0237] Target cells were seeded at 100 μL / well into flat-bottom 96-well plates (Corning, Cat. No. 3599) and cultured overnight at 37°C, 5% CO2. CD3+ T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), counted, and adjusted to a cell density of 2E6 / mL using RPMI 1640 + 2% FBS. Effector cells were seeded at 50 μL / well into 96-well plates and cultured at 37°C, 5% CO2.

[0238] Antibodies were diluted in RPMI 1640 + 2% FBS medium to a starting concentration of 100 nM and serially diluted 14-fold to 10 concentrations. 50 μL of diluted antibody was added to cell culture plates at 50 μL per well and incubated at 37°C, 5% CO2 for 24 hours. The culture plates were centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was aspirated and placed in a separate flat-bottom 96-well plate. 50 μL of LDH detection reagent (Romos, product number 4744934001) was added to each well, mixed evenly, and centrifuged at 1000 rpm for 5 minutes. The plates were then incubated in the dark for 10 minutes. OD492 readings were then taken using an Envision™.

[0239] The cytotoxicity rate due to the TDCC effect was calculated using the following formula. % Cytotoxicity = [Sample well - T cell spontaneous release - target cell spontaneous release) / (maximum target cell lysis - target cell spontaneous release)] x 100%.

[0240] The cytotoxicity data was imported into GraphPad Prism, and the cytotoxicity / concentration curve was plotted to calculate the EC50 value. The results are shown in Table 11 and Figure 8A. The cytotoxicity (cytotoxicity) of one trispecific antibody molecule against NCI-H929 cells was stronger than that of BGCB491 and TS-F2-5.

[0241] B. T cell-dependent cytotoxicity (TDCC) experiment in which target cells were RPMI8226 RPMI8226 cells (ATCC, product number CCL-155) were cultured in RPMI1640 + 10% FBS medium in a T75 cell culture flask in a 37°C, 5% CO2 incubator. When using the cells, RPMI8226 cells were directly placed in a 50 mL centrifuge tube without digestion. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI1640 + 2% FBS medium. The cells were counted and the cell density adjusted to 1E5 / mL.

[0242] Target cells were seeded at 100 μL / well into flat-bottom 96-well plates (Corning, Cat. No. 3599) and cultured overnight at 37°C, 5% CO2. CD3+ T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), counted, and adjusted to a cell density of 2E6 / mL using RPMI 1640 + 2% FBS. Effector cells were seeded at 50 μL / well into 96-well plates and cultured at 37°C, 5% CO2.

[0243] Antibodies were diluted in RPMI 1640 + 2% FBS medium to a starting concentration of 100 nM and serially diluted 14-fold to 10 concentrations. 50 μL of diluted antibody was added to cell culture plates at 50 μL per well and incubated at 37°C, 5% CO2 for 24 hours. The culture plates were centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was aspirated and placed in a separate flat-bottom 96-well plate. 50 μL of LDH detection reagent (Romos, product number 4744934001) was added to each well, mixed evenly, and centrifuged at 1000 rpm for 5 minutes. The plates were then incubated in the dark for 10 minutes. OD492 readings were then taken using an Envision™.

[0244] The cytotoxicity rate due to the TDCC effect was calculated using the following formula. % Cytotoxicity = [Sample well - T cell spontaneous release - target cell spontaneous release) / (maximum target cell lysis - target cell spontaneous release)] x 100%.

[0245] The cytotoxicity data was imported into GraphPad Prism, and the cytotoxicity / concentration curve was plotted to calculate the EC50 value. The results are shown in Table 11 and Figure 8B. The cytotoxicity (cytotoxicity) of one trispecific antibody molecule against RPMI8226 cells was stronger than that of BGCB491 and TS-F2-5.

[0246] C. T cell-dependent cytotoxicity (TDCC) experiment using human BCMA-CHOK1 target cells Human BCMA-CHOK1 cells (Example 4) were cultured in F12K + 10% FBS + 400 μg / mL Hygromycin B medium and placed in a T75 cell culture flask in a 37°C, 5% CO2 incubator. When using the cells, human BCMA-CHOK1 cells were digested with 0.25% Trpsin-EDTA trypsin and terminated with F12K + 10% FBS + 400 μg / mL Hygromycin B medium. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI 1640 + 2% FBS medium. The cells were counted and the cell density adjusted to 1E5 / mL.

[0247] Target cells were seeded at 100 μL / well into flat-bottom 96-well plates (Corning, Cat. No. 3599) and cultured overnight at 37°C, 5% CO2. CD3+ T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), counted, and adjusted to a cell density of 2E6 / mL using RPMI 1640 + 2% FBS. Effector cells were seeded at 50 μL / well into 96-well plates and cultured at 37°C, 5% CO2.

[0248] Antibodies were diluted in RPMI 1640 + 2% FBS medium to a starting concentration of 400 nM and serially diluted 10-fold. 50 μL of diluted antibody was added to cell culture plates at a final concentration of 100 nM. Cells were cultured at 37°C, 5% CO2 for 24 hours. The culture plates were centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was aspirated and placed in a separate flat-bottom 96-well plate. 50 μL of LDH detection reagent (Romos, product number 4744934001) was added to each well, mixed evenly, and centrifuged at 1000 rpm for 5 minutes. The plates were then incubated in the dark for 10 minutes. OD492 readings were then taken using an Envision™.

[0249] The cytotoxicity rate due to the TDCC effect was calculated using the following formula. % Cytotoxicity = [Sample well - T cell spontaneous release - target cell spontaneous release) / (maximum target cell lysis - target cell spontaneous release)] x 100%.

[0250] The cytotoxicity data was imported into GraphPad Prism, and cytotoxicity / concentration curves were plotted to calculate EC50 values. The results are shown in Table 11 and Figure 8C. The cytotoxicity (cytotoxic potential) of the trispecific antibody 1 molecule against human BCMA-CHOK1 cells was comparable to that of BGCB491 and stronger than that of TS-F2-5.

[0251] D. T cell-dependent cytotoxicity (TDCC) experiment using human GPRC5D-CHOK1 as target cells Human GPRC5D-CHOK1 cells (Example 2) were cultured in F12K + 10% FBS + 400 μg / mL Hygromycin B medium and placed in a T75 cell culture flask in a 5% CO2 incubator at 37°C. When using the cells, human BCMA-CHOK1 cells were digested with 0.25% Trpsin-EDTA trypsin and terminated with F12K + 10% FBS + 400 μg / mL Hygromycin B medium. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI 1640 + 2% FBS medium. The cells were counted and the cell density adjusted to 1E5 / mL.

[0252] Target cells were seeded at 100 μL / well into flat-bottom 96-well plates (Corning, Cat. No. 3599) and cultured overnight at 37°C, 5% CO2. CD3+ T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), counted, and adjusted to a cell density of 2E6 / mL using RPMI 1640 + 2% FBS. Effector cells were seeded at 50 μL / well into 96-well plates and cultured at 37°C, 5% CO2.

[0253] Antibodies were diluted in RPMI 1640 + 2% FBS medium to a starting concentration of 100 nM and serially diluted 5-fold to eight concentrations. 50 μL of diluted antibody was added to cell culture plates at 50 μL per well and incubated at 37°C, 5% CO2 for 24 hours. The culture plates were centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was aspirated and placed in a separate flat-bottom 96-well plate. 50 μL of LDH detection reagent (Romos, product number 4744934001) was added to each well, mixed evenly, and centrifuged at 1000 rpm for 5 minutes. The plates were then incubated in the dark for 10 minutes. OD492 readings were then taken using an Envision™ system.

[0254] The cytotoxicity rate due to the TDCC effect was calculated using the following formula. % Cytotoxicity = [Sample well - T cell spontaneous release - target cell spontaneous release) / (maximum target cell lysis - target cell spontaneous release)] x 100%.

[0255] The cytotoxicity data was imported into GraphPad Prism, and cytotoxicity / concentration curves were plotted to calculate EC50 values. The results are shown in Table 11 and Figure 8D. The cytotoxicity (cytotoxic potential) of one trispecific antibody molecule against human GPRC5D-CHOK1 cells was comparable to that of TS-F2-5 and stronger than that of BGCB491.

[0256] E. T cell-dependent cytotoxicity (TDCC) experiment using a mixture of human BCMA-CHOK1 and human GPRC5D-CHOK1 target cells Human GPRC5D-CHOK1 cells and human BCMA-CHOK1 cells were cultured in F12K + 10% FBS + 400 μg / mL hygromycin B medium in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When using the cells, human GPRC5D-CHOK1 and human BCMA-CHOK1 cells were digested with trypsin containing 0.25% Trpsin-EDTA and terminated with F12K + 10% FBS + 400 μg / mL hygromycin B medium. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the cells were resuspended in RPMI 1640 + 2% FBS medium. The cell density of the two types was adjusted to 1E5 / mL, and the two types of cells were mixed in equal proportions.

[0257] The target cell mixture was seeded into a flat-bottom 96-well plate (Corning, Cat. No. 3599) at 100 μL / well and cultured overnight at 37°C, 5% CO2. CD3+ T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), counted, and adjusted to a cell density of 1.6E6 / mL using RPMI 1640 + 2% FBS. Effector cells were seeded into a 96-well plate at 50 μL / well and cultured at 37°C, 5% CO2.

[0258] Antibodies were diluted in RPMI 1640 + 2% FBS medium to a starting concentration of 100 nM and serially diluted 14-fold to 10 concentrations. 50 μL of diluted antibody was added to cell culture plates at 50 μL per well and incubated at 37°C, 5% CO2 for 24 hours. The culture plates were centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was aspirated and placed in a separate flat-bottom 96-well plate. 50 μL of LDH detection reagent (Romos, product number 4744934001) was added to each well, mixed evenly, and centrifuged at 1000 rpm for 5 minutes. The plates were then incubated in the dark for 10 minutes. OD492 readings were then taken using an Envision™.

[0259] The cytotoxicity rate due to the TDCC effect was calculated using the following formula. % Cytotoxicity = [Sample well - T cell spontaneous release - target cell spontaneous release) / (maximum target cell lysis - target cell spontaneous release)] x 100%.

[0260] The cytotoxicity data was imported into GraphPad Prism, and the cytotoxicity / concentration curve was plotted to calculate the EC50 value. The results are shown in Table 11 and Figure 8E. The cytotoxicity (cytotoxicity) of one trispecific antibody molecule against RPMI8226 cells was stronger than that of BGCB491 and TS-F2-5.

[0261] T cell-dependent cytotoxicity (TDCC) experiments using target cells NCI-H929, RPMI8226, human BCMA-CHOK1, human GPRC5D-CHOK1, and a mixed cell line of human BCMA-CHOK1 and human GPRC5D-CHOK1. [Table 11]

[0262] F. T cell-dependent cytotoxicity (TDCC) experiment using NCI-H929 target cells in the presence of 2500 ng / mL soluble BCMA NCI-H929 cells (ATCC, Cat. No. CRL-9068) were cultured in RPMI 1640 + 10% FBS + 0.05 mM mercaptoethanol in a T75 cell culture flask at 37°C in a 5% CO2 incubator. For cell use, NCI-H929 cells were directly placed in a 50 mL centrifuge tube without digestion. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded and the cells were resuspended in RPMI 1640 + 2% FBS medium. The cells were counted and adjusted to a cell density of 1E5 / mL. Soluble BCMA was added to the cell suspension at a concentration of 2500 ng / mL.

[0263] Target cells containing soluble BCMA were seeded at 100 μL / well in a flat-bottom 96-well plate (Corning, Cat. No. 3599) and cultured overnight at 37°C, 5% CO2. CD3+ T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), counted, and adjusted to a cell density of 2E6 / mL in RPMI 1640 + 2% FBS. Effector cells were seeded at 50 μL / well in a 96-well plate and cultured at 37°C, 5% CO2.

[0264] Antibodies were diluted in RPMI 1640 + 2% FBS medium to a starting concentration of 100 nM and serially diluted 14-fold. 50 μL of diluted antibody was added to cell culture plates at a final concentration of 100 nM. Cells were cultured at 37°C, 5% CO2 for 24 hours. The culture plates were centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was aspirated and placed in a separate flat-bottom 96-well plate. 50 μL of LDH detection reagent (Romos, product number 4744934001) was added to each well, mixed evenly, and centrifuged at 1000 rpm for 5 minutes. The plates were then incubated in the dark for 10 minutes. OD492 readings were then taken using an Envision™.

[0265] The cytotoxicity rate due to the TDCC effect was calculated using the following formula. % cell damage = [sample well - T cell spontaneous release - target cell spontaneous release) / (maximum target cell lysis - target cell spontaneous release)] x 100%.

[0266] The cytotoxicity data was imported into GraphPad Prism, and the cytotoxicity / concentration curve was plotted to calculate the EC50 value. The results are shown in Figure 9 (the dotted line represents the 2500 ng / mL soluble BCMA condition) and Table 12. In the presence of 2500 ng / mL soluble BCMA, the cytotoxicity EC50 value of BGCB491 was reduced approximately 23-fold, the cytotoxicity EC50 value of TS-F2-5 was essentially maintained, and the cytotoxicity EC50 value of trispecific antibody 1 was reduced approximately 3-fold. (The cytotoxicity EC50 value of the BCMA bispecific antibody teclistamab was reduced approximately 25-fold.)

[0267] T cell-dependent cytotoxicity experiments in the presence of 2500ng / mL soluble BCMA [Table 12]

[0268] G. IL6 release levels under co-incubation conditions of trispecific antibodies with PBMCs and tumor cells NCI-H929 cells NCI-H929 cells (ATCC, Cat. No. CRL-9068) were cultured in RPMI 1640 + 10% FBS + 0.05 mM mercaptoethanol in a T75 cell culture flask in an incubator at 37°C with 5% CO2. For cell use, NCI-H929 cells were directly placed in a 50 mL centrifuge tube without digestion and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in RPMI 1640 + 2% FBS medium. The cells were counted and adjusted to a cell density of 1E5 / mL. NCI-H929 cells were seeded into a 96-well plate (Corning, Cat. No. 3799) at 100 μL / well and cultured at 37°C with 5% CO2.

[0269] Fresh PBMCs were purchased, counted, and adjusted to a cell density of 4E6 / mL in RPMI 1640 + 2% FBS. Effector cells were seeded into 96-well plates at 50 μL / well and cultured at 37°C, 5% CO2.

[0270] Antibodies were diluted in RPMI 1640 + 2% FBS medium to a starting concentration of 100 nM and serially diluted 13-fold. 50 μL of diluted antibody was added to cell culture plates at a resulting starting concentration of 100 nM. Cells were cultured at 37°C, 5% CO for 24 hours.

[0271] The culture plate was centrifuged at 400 g for 10 minutes, and 100 μL of the supernatant was collected and frozen at −80°C for storage.

[0272] According to the instructions of the cytokine detection kit (Biolegend, 430504 (ELISA MAX Deluxe Set Human IL-6)), the standard was diluted two-fold to a total of 12 concentrations, with the highest concentration being 8000 pg / mL and the lowest being 0 pg / mL. The prepared standards or thawed samples (each sample required a separate dilution) were added to a 96-well plate at 100 μL / well, and the procedure was carried out according to the kit protocol. OD450 values ​​were read using Envision.

[0273] The results are shown in Figures 10A and 10B. Under the conditions of co-incubation of PBMCs and tumor cells, the level of IL6 cytokine release induced by one trispecific antibody molecule was weaker than that induced by two trispecific antibody molecules.

[0274] [Example 11] Stability test of anti-GPRC5D-BCMA-CD3 trispecific antibody The thermal stability of the trispecific antibodies in PBS buffer at pH 7.4 was detected using differential fluorescence scanning technology. The sample concentration was approximately 1 mg / mL, and Prometheus NT.Plex (nano DSF) was used for detection. Prior to 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 duplicate wells). The scan temperature ranged from 30°C to 95°C, and the scan rate was 0.5°C / min. The experimental results are shown in Table 13. Both trispecific antibody molecules showed good thermal stability.

[0275] NanoDSF detection results of anti-GPRC5D-BCMA-CD3 trispecific antibody [Table 13]

[0276] Example 12: Pharmacokinetics of anti-GPRC5D-BCMA-CD3 trispecific antibody Two naive cynomolgus monkeys were used for this study, with free access to water. The trispecific antibody was injected intravenously into the hind leg at a dose of 1 mg / kg. Blood samples were collected at pre-dose, 5 minutes, 2 hours, 6 hours, 24 hours, 72 hours, 168 hours, 264 hours, 336 hours, 504 hours, 672 hours, and 840 hours. Whole blood samples were collected into anticoagulant-free polyethylene tubes, left at room temperature for approximately 1 hour, centrifuged at 6000 g and 25°C, immediately placed on dry ice, and then transferred to a -80°C refrigerator for long-term storage.

[0277] The concentration of intact GPRC5D-BCMA-CD3 trispecific antibody molecules in serum was detected using an ELISA method. Human GPRC5D protein was coated onto a 96-well plate at a concentration of 2 μg / mL, 100 μL per well, and incubated overnight at 4°C. The plate was washed three times with 300 μL per well of PBST, followed by the addition of 300 μL of blocking reagent containing 5% milk powder and incubation at 37°C for 1 hour. The plate was washed three times with 300 μL per well of PBST, followed by the addition of 100 μL of test sample and incubation at 37°C for 1 hour. The plate was washed six times with 300 μL per well of PBST, followed by the addition of 100 μL of Biotin-BCMA reagent and incubation at 37°C for 1 hour. The plate was washed six times with 300 μL / well of PBST, followed by the addition of 100 μL of SA-HRP reagent and incubation at 37°C for 1 hour. The plate was washed six times with 300 μL / well of PBST, followed by the addition of 100 μL of TMB and incubation in the dark for 10 minutes. The color reaction was then stopped by the addition of 50 μL of stop solution. The concentration of intact GPRC5D-BCMA-CD3 trispecific antibody molecules was quantitatively determined by the color reaction.

[0278] The absorbance values ​​were detected at a wavelength of 450 nm using a PE Envision plate reader, and the data were processed using softmax software.

[0279] Monkey serum concentrations of the trispecific antibody were calculated using Phoenix Winnolin 8.2 software. The results showed that the pharmacokinetic properties of trispecific antibody 1 in monkeys were favorable and consistent with the conventional metabolic properties of macromolecules.

[0280] [Example 13] In vivo efficacy experiment of anti-GPRC5D-BCMA-CD3 trispecific antibody A. Human immune cell-reconstituted mouse model in which tumor cells are NCI-H929 All experimental animals were housed in independently ventilated cages with constant temperature and humidity, and the temperature of the breeding room was 20.0 to 26.0°C, humidity was 40 to 70%, and the light / dark cycle (light / dark) was 12 hours / 12 hours.

[0281] NCI-H929 cells (ATCC, Cat. No. CRL-9068) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.05 mM β-ME. Exponentially growing NCI-H929 cells were harvested and resuspended in 0.1 mL of a 1:1 suspension of PBS and Matrigel. 5 × 10 6 NCI-H929 cells were inoculated into the right dorsal anterior of experimental mice, and tumor growth was monitored periodically until tumors reached an average volume of 83 mm. 3 When the tumors grew to 100%, the mice were randomly assigned to groups according to tumor size and mouse weight and administered the treatment.

[0282] Donor PBMCs were purchased from AllCells, LLC. On the day of NCI-H929 cell inoculation, 1 × 10 7 PBMCs / 0.1 mL PBS were injected intraperitoneally to establish a human immune cell-reconstituted mouse model.

[0283] Before the start of treatment, all animals were weighed and tumor volumes were measured with calipers. Considering that tumor volume may affect the efficacy of treatment, mice were grouped according to tumor volume using a randomized grouping design to ensure similar tumor volumes across different groups. Mice were grouped using StudyDirector™ (version 3.1.399.19, provider: Studylog System, Inc., San Francisco, CA, USA) with the matched distribution method selected. The day of grouping was defined as day 0. Treatment began on day 0, with intraperitoneal injections, once every three days for a total of five doses. The experimental grouping and treatment regimen are shown in Table 14.

[0284] Experimental grouping and dosing regimen [Table 14]

[0285] The animals' daily behavior was monitored daily for 14 days after administration. Throughout the entire experimental period, the tumor length and width were measured twice a week using a vernier caliper, and tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 Relative tumor inhibition rate (TGI) (%): TGI% = (1-T / C) x 100%. T / C% is the relative tumor growth rate, i.e., the rate of tumor growth after treatment at a certain time point. group The mean values ​​are the percentages of the relative tumor volume or tumor weight of the treatment group and the hIgG1 LALA isotype control group. T and C refer to the tumor volume (TV) or tumor weight (TW) of the treatment group and the hIgG1 LALA isotype control group, respectively, at a specific time point. Experimental results, such as tumor volume, mouse body weight, and tumor weight of animals in each group, were expressed as mean ± standard error (mean ± SEM). An independent sample t-test was used to compare whether there were significant differences between different treatment groups and the control group. Data were analyzed using SPSS. P<0.05 was considered significant. The experimental results are shown in Table 15 and Figure 11.

[0286] The trispecific antibody single molecule at 0.004 mg / kg demonstrated highly significant tumor suppression (p<0.001) compared with equimolar doses of the bispecific antibody molecule teclistamab and the combination of teclistamab and talquetamab in a subcutaneous xenograft model of NPG female mice bearing the human myeloma NCI-H929. Mice tolerated the test trispecific antibody molecule well, with no significant weight loss or toxicity observed.

[0287] Tumor volume and tumor suppression rate in each treatment group [Table 15]

[0288] B. Human immune cell-reconstituted mouse model in which tumor cells are NCI-H929 All experimental animals were housed in independently ventilated cages with constant temperature and humidity, and the temperature of the breeding room was 20.0 to 26.0°C, humidity was 40 to 70%, and the light / dark cycle (light / dark) was 12 hours / 12 hours.

[0289] NCI-H929 cells (ATCC, Cat. No. CRL-9068) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.05 mM β-ME. Exponentially growing NCI-H929 cells were harvested and resuspended in 0.1 mL of a 1:1 suspension of PBS and Matrigel. 5 × 10 6 NCI-H929 cells were inoculated into the right dorsal anterior of experimental mice, and tumor growth was monitored periodically until tumors reached an average volume of 100 mm. 3 When the tumors grew to 100%, the mice were randomly assigned to groups according to tumor size and mouse weight and administered the treatment.

[0290] Donor PBMCs were purchased from AllCells, LLC. Each mouse received 1 × 10 7 A human immune cell-reconstituted mouse model was established by intraperitoneal injection of PBMCs in 0.2 mL of PBS.

[0291] Before the start of treatment, all animals were weighed and tumor volumes were measured with calipers. Considering that tumor volume may affect the efficacy of treatment, mice were grouped according to tumor volume using a randomized grouping design to ensure similar tumor volumes across different groups. The day of grouping was defined as day 1, and treatment began on day D1 via intraperitoneal injection, twice weekly for a total of four doses. The experimental grouping and treatment regimen are shown in Table 16.

[0292] Experimental grouping and dosing regimen [Table 16]

[0293] The animals' daily behavior was monitored daily for 15 days after administration. Throughout the entire experimental period, the tumor length and width were measured twice a week using a vernier caliper, and tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 ) was calculated. Relative tumor inhibition rate TGI (%): TGI (%) = [1-(Vti-Vt0) / (Vci-Vc0)] 100%. T / C% is the relative tumor growth rate, i.e., the rate of tumor growth after treatment at a certain time point. group The values ​​are the percentages of the relative tumor volume or tumor weight of the treatment group and the hIgG1 LALA isotype control group. T and C refer to the tumor volume (TV) or tumor weight (TW) of the treatment group and the hIgG1 LALA isotype control group, respectively, at a specific time point. Experimental results, such as tumor volume, mouse body weight, and tumor weight of animals in each group, were expressed as mean + standard error (mean + SEM). An independent sample t-test was used to compare whether there were significant differences between different treatment groups and the control group. Data were analyzed using GraphPad Prism 8.0.2. P<0.05 was considered significant. The experimental results are shown in Table 17 and Figures 12 and 13.

[0294] Different doses of trispecific antibody 1 demonstrated highly significant tumor suppression effects (all p values ​​less than 0.05) in a subcutaneously implanted NCI-H929 human myeloma xenograft model in NCG female mice. The trispecific antibody 1 dose of 0.006 mg / kg was significantly superior to equimolar doses of BCGB491 and TS-F2-5. Mice tolerated the test trispecific antibody molecule well, with no significant weight loss or toxicity observed.

[0295] Tumor volume and tumor suppression rate in each treatment group [Table 17]

[0296] [Example 14] Construction of an alpaca immunized, heavy chain-only antibody immune library Alpacas were immunized with CHO-K1 cells highly expressing human GPRC5D. Immunizations were administered on days 0, 21, 35, 49, and 70, for a total of five immunizations. Blood samples were collected on days 28, 42, and 63, respectively, and serum was isolated. Cell-level FACS was used to detect immune responses in the serum. When serum titers approached the plateau phase, immunization was terminated. 50 mL of blood samples were collected again from the immunized alpacas. Alpaca PBMCs were isolated using Solarbio's lymphocyte separation solution according to the manufacturer's instructions. Total RNA (OMEGA Cell Total RNA Extraction Kit) was then extracted using Takara PrimeScript. TM cDNA was synthesized using the II reverse transcription kit as a template, and nested PCR was performed using designed specific primers to amplify VHH gene fragments. The VHH fragments were recovered and then ligated into the pADL-23c phagemid vector by Sfi I digestion, followed by electroporation into TG1 electrocompetent cells to construct a GPRC5D alpaca immune library (library capacity: 4.07E9).

[0297] [Example 15] Screening of positive anti-GPRC5D clones derived from alpaca To obtain positive antibodies capable of cross-linking to human GPRC5D and cynomolgus monkey GPRC5D, the above library was amplified and added to M13K07 helper phage to assemble the phage. Human GPRC5D-CHOK1 cell line, cynomolgus monkey GPRC5D-CHOK1 cell line, and CHOK1 cells were cultured until confluent and washed twice with PBS. Cells were fixed with 100 μL of 4% paraformaldehyde and incubated at 25°C for 20–30 minutes. After washing twice with PBS, 300 μL of 5% skim milk was added to each well and blocked at 37°C for 1 hour. The plate was then washed three times with PBST, and 50 μL of 5% skim milk and 50 μL of phage supernatant were added to each well and incubated at 37°C for 1 hour. After washing five times with 0.1% PBST, 100 μL / well of horseradish peroxidase-labeled anti-M13 antibody (diluted 1:10,000 in PBS) was added and incubated at 37°C for 1 hour. The plate was washed six times with 0.1% PBST. 100 μL of TMB color development solution was added to each well to develop the color, and the plate was incubated at 37°C for 7 minutes. The reaction was stopped by adding stop solution, and the optical density was measured at 450 nm with 50 μL / well. Positive clones were sequenced to obtain the amino acid sequences of the five heavy chain antibody variable regions (VHH). The clone numbers were HHC7, HHC11, HHC22, HHC53, and HHC78, respectively. The amino acid sequence of HHC78 is as follows:

[0298] QLQLVESGGGLVQPGGSLRLSCAGSGLFFASSDMSWFRQPPGKERELVAEITSVGNNIKYADSVEGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCSARRRVRYWGQGTQVTVSS(SEQ ID NO:37) Based on the amino acid sequence of HHC78 described above, the CDRs and FRs of the variable region of the antibody were divided according to the Kabat numbering system, and the composition of the three CDR sequences of the antibody is shown in the table below. [Table 18]

[0299] [Example 16] Construction of alpaca-derived anti-GPRC5D chimeric antibody and its transient transfection expression in eukaryotic cells The target gene fragment generated by linking the sequenced heavy chain antibody variable region of the present invention to a human IgG1 constant region was cloned into a pTT5 expression vector to prepare a transfection-grade expression plasmid. The heavy chain antibody variable region was linked to the human IgG1 constant region via a short peptide linker, i.e., forming the heavy chain antibody variable region-short peptide-human IgG1 constant region. The short peptide linker sequence used in this example was GGGGS.

[0300] The introduced human IgG1 constant region sequence (SEQ ID NO: 38) is as follows:

[0301] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Expi293F in serum-free medium TMCells (Thermo Fisher Scientific) were cultured in shake flasks (Corning Inc.) and grown on a shaker at 37°C in an 8% CO2 environment. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in the appropriate ratio and added to the shake flask for cell culture. After 6 days of cell culture, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purified using a Protein A column. The column was washed with PBS until the A280 reading fell to baseline. The target protein was eluted with an acidic eluent (pH 3.0-3.5) and neutralized with 1 M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was buffer exchanged with PBS and prepared in aliquots. The final purified chimeric antibody was analyzed for purity by SDS-PAGE and HPLC, and its A280 concentration was measured.

[0302] [Example 17] Binding of alpaca-derived anti-GPRC5D chimeric antibody to GPRC5D-expressing cells Human GPRC5D-CHOK1 cells and cynomolgus monkey GPRC5D-CHOK1 cells were cultured in a T75 cell culture flask in F12K + 10% FBS + 400 μg / mL hygromycin medium at 37°C in a 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS and digested with 0.25% trypsin-EDTA for approximately 5 minutes, followed by quenching with complete medium.

[0303] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and resuspended in 100 μL of 1% BSA in PBS. The cells were counted, adjusted to a cell density of 1E6 / mL, and seeded into a 96-well round-bottom culture plate (Corning, product number 3799). The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 200 μL of 1% BSA in PBS. The plate was centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate was stored at 4°C. Test antibody samples were diluted in 1% BSA in PBS to a starting concentration of 100 nM, followed by 10-fold serial dilutions to seven concentrations. The diluted antibody was used to resuspend the cells and incubated at 4°C for 1 hour at 100 μL per well. The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant discarded. The cells were washed by resuspending in 160 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The secondary antibody (goat anti-human IgG Fc PE) was diluted 1:400 in 1% BSA in PBS according to the manufacturer's instructions. The cells were resuspended in the diluted secondary antibody at 100 μL / well and incubated at 4°C for 0.5 hours. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were washed by resuspending in 200 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA in PBS and filtered through a 300-mesh gauze. The mean fluorescence intensity of the PE channel was detected by flow cytometry.

[0304] Export the FCS file from the flow cytometer and analyze the mean fluorescence intensity (MFI) of the PE channel of each sample using flowjo software. Import the MFI data into Graphpad to calculate the half maximal binding concentration (EC) of the antibody to the cells. 50 The results are shown in Table 19 and Figure 14. The screened anti-GPRC5D heavy chain antibodies bound well to both human GPRC5D and cynomolgus monkey GPRC5D at the cellular level.

[0305] Binding of anti-GPRC5D chimeric antibodies to GPRC5D-expressing cells [Table 19]

[0306] [Example 18] Humanized design of alpaca-derived anti-GPRC5D chimeric antibody Germline gene sequences highly homologous to the candidate heavy chain antibodies were selected by sequence alignment and used as templates for VHH-grafted frameworks. The CDR regions of the candidate antibodies were grafted onto the selected human antibody variable region frameworks, followed by backmutation of individual amino acids to obtain humanized antibodies H1, H2, H3, H4, and H5 (the H4 sequence is shown in Table 20).

[0307] Amino acid sequences of the variable regions of a humanized anti-GPRC5D antibody derived from alpaca [Table 20]

[0308] [Example 19] Preparation of alpaca-derived humanized anti-GPRC5D antibody As described in Example 16, the target gene fragment generated by linking the variable region of the humanized antibody with the human IgG1 constant region was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0309] Expi293F in serum-free medium TMCells (Thermo Fisher Scientific) were cultured in shake flasks (Corning Inc.) and grown on a shaker at 37°C in an 8% CO2 environment. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in the appropriate ratio and added to the shake flask for cell culture. After 6 days of cell culture, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purified using a Protein A column. The column was washed with PBS until the A280 reading fell to baseline. The target protein was eluted with an acidic eluent (pH 3.0-3.5) and neutralized with 1 M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was buffer exchanged with PBS and prepared in aliquots. The final purified human antibody was analyzed for purity by SDS-PAGE and HPLC, and its A280 concentration was measured.

[0310] [Example 20] Binding of alpaca-derived anti-GPRC5D humanized antibody to GPRC5D-expressing cells The culture medium for NCI-H929 cells (ATCC, CRL-9068) was RPMI1640 + 10% FBS + 0.05 mM mercaptoethanol, and the culture medium for cynomolgus monkey GPRC5D-CHOK1 cells was F12K + 10% FBS + 400 μg / mL hygromycin. They were cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator.

[0311] When using NCI-H929 cells, transfer the cells directly to a 50 mL centrifuge tube using a pipette. Before using cynomolgus monkey GPRC5D-CHOK1 cells, wash the cells twice with sterile DPBS, digest them with 0.25% trypsin-EDTA for approximately 5 minutes, and then terminate them with complete medium.

[0312] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and resuspended in 100 μL of 1% BSA in PBS. The cells were counted, adjusted to a cell density of 1E6 / mL, and seeded into a 96-well round-bottom culture plate (Corning, product number 3799). The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 200 μL of 1% BSA in PBS. The plate was centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate was stored at 4°C. Test antibody samples were diluted in 1% BSA in PBS to a starting concentration of 100 nM, followed by 10-fold serial dilutions to seven concentrations. The diluted antibody was used to resuspend the cells and incubated at 4°C for 1 hour at 100 μL per well. The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant discarded. The cells were washed by resuspending in 160 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The secondary antibody (goat anti-human IgG Fc PE) was diluted 1:400 in 1% BSA in PBS according to the manufacturer's instructions. The cells were resuspended in the diluted secondary antibody at 100 μL / well and incubated at 4°C for 0.5 hours. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were washed by resuspending in 200 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA in PBS and filtered through a 300-mesh gauze. The mean fluorescence intensity of the PE channel was detected by flow cytometry.

[0313] Export the FCS file from the flow cytometer and analyze the mean fluorescence intensity (MFI) of the PE channel of each sample using flowjo software. Import the MFI data into Graphpad to calculate the half maximal binding concentration (EC) of the antibody to the cells. 50 The mean fluorescence intensity (MFI) and the highest mean fluorescence intensity (Top MFI) were analyzed, and the results are shown in Table 21 and Figure 15. The binding of the clone HHC78 humanized antibody to cynomolgus monkey GPRC5D-CHOK1 cells was comparable to that of the parent antibody, but the binding to NCI-H929 tumor cells was maintained or attenuated compared to the parent antibody.

[0314] Binding of anti-GPRC5D humanized antibodies to GPRC5D-expressing cells [Table 21]

[0315] [Example 21] Binding of alpaca-derived anti-GPRC5D humanized antibody to its family member GPRC5A Human GPRC5A-CHOK1 cells were cultured in F12K + 10% FBS + 400 μg / mL hygromycin medium in a T75 cell culture flask in a 37°C, 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin-EDTA for approximately 5 minutes, and then digested with complete medium.

[0316] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and resuspended in 100 μL of 1% BSA in PBS. The cells were counted, adjusted to a cell density of 1E6 / mL, and seeded into a 96-well round-bottom culture plate (Corning, product number 3799). The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 200 μL of 1% BSA in PBS. The plate was centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate was stored at 4°C. Test antibody samples were diluted in 1% BSA in PBS to a starting concentration of 100 nM, followed by 10-fold serial dilutions to seven concentrations. The diluted antibody was used to resuspend the cells and incubated at 4°C for 1 hour at 100 μL per well. The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant discarded. The cells were washed by resuspending in 160 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The secondary antibody (goat anti-human IgG Fc PE) was diluted 1:400 in 1% BSA in PBS according to the manufacturer's instructions. The cells were resuspended in the diluted secondary antibody at 100 μL / well and incubated at 4°C for 0.5 hours. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were washed by resuspending in 200 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA in PBS and filtered through a 300-mesh gauze. The mean fluorescence intensity of the PE channel was detected by flow cytometry.

[0317] Export the FCS file from the flow cytometer and analyze the mean fluorescence intensity (MFI) of the PE channel of each sample using flowjo software. Import the MFI data into Graphpad to calculate the half maximal binding concentration (EC) of the antibody to the cells. 50 The results are shown in FIG. 16, demonstrating that the measured antibodies did not bind nonspecifically to the family member GPRC5A.

[0318] [Example 22] Obtaining anti-BCMA antibodies from mouse hybridomas Anti-BCMA monoclonal antibodies were produced by immunization of mice. Laboratory Balb / c mice, female, 6 weeks old (Charles River). The breeding environment was SPF grade. After purchase, the mice were housed in a laboratory environment with a 12 / 12-hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60% for one week. The immunogen was human BCMA ECD protein (Yiqiao Shenzhou, product number 10620-H08H). Each immunization was with 25 μg of protein. The immunization times were days 0, 14, 28, and 42. Booster immunizations were administered two days before splenocyte fusion. During this period, mouse serum was detected using ELISA to determine the antibody titer in the serum. After the booster immunization, mice with high serum antibody titers approaching a plateau were selected and subjected to splenocyte fusion. Spleen lymphocytes and myeloma cells Sp2 / 0 cells (ATCC® CRL-8287) were fused using an optimized electrofusion procedure. TM ) were fused to obtain hybridoma cells.

[0319] After culturing the fused hybridoma cells for 7 to 14 days, the culture supernatant was collected and subjected to antibody screening in a FACS experiment using CHO-K1 cells that highly express human BCMA. To exclude nonspecific binding hybridoma lines, the resulting positive antibody lines were further screened using CHO-K1 cells that highly express cynomolgus BCMA and blank CHO-K1 cells to select hybridomas that specifically bind to human / cynomolgus BCMA. Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript). TMThe cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB 326 Rev.B 0503) and then sequenced to obtain the amino acid sequences of the variable regions of 11 monoclonal antibodies according to the present invention: 19CH-1, 19CH-2, 19CH-3, 19CH-4, 19CH-5, 19CH-7, 19CH-8, 19CH-9, 19CH-11, 19CH-13, and 19CH-16 (of which, 19CH-16 is shown in Table 22).

[0320] Amino acid sequence of the variable region of the anti-BCMA 19CH-16 monoclonal antibody derived from a murine hybridoma [Table 22]

[0321] Based on the above amino acid sequence, the CDRs and FRs of the antibody variable region are divided according to the Kabat numbering system, and the composition of the six CDR sequences of 19CH-16 is shown in Table 23 below.

[0322] CDR sequences of anti-BCMA monoclonal antibodies derived from mouse hybridomas [Table 23]

[0323] Example 23: Construction of anti-BCMA chimeric antibodies derived from mouse hybridomas and their transient transfection expression in eukaryotic cells The heavy and light chain variable regions of the sequenced monoclonal antibody according to the present invention were ligated to the heavy chain constant region and the κ light chain constant region of IgG1 (L234AL235A), respectively, to generate target gene fragments. These fragments were then cloned into the pTT5 expression vector to prepare transfection-grade expression plasmids.

[0324] Expi293F in Expi293 expression medium (ThermoFisher, A1435101) TM Cells (ThermoFisher, A14527) were cultured and inoculated into shake flasks and grown on a shaker at 37°C in an 8% CO2 environment. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in the appropriate ratio and added to the shake flask for cell culture. After 6 days of cell culture, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purified using a Mabselect Sure column. The column was washed with PBS until the A280 reading fell to baseline. The target protein was eluted with an acidic eluent (pH 3.0-3.5) and neutralized with 1 M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was buffer exchanged with PBS and prepared in aliquots. The final purified chimeric antibody was analyzed for purity by SDS-PAGE and HPLC, and its A280 concentration was measured.

[0325] Example 24: Binding of anti-BCMA chimeric antibodies derived from mouse hybridomas to BCMA-expressing cells Human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells were cultured in a T75 cell culture flask in F12K + 10% FBS + 400 μg / mL hygromycin medium at 37°C in a 5% CO2 incubator. Before use, the cells were washed twice with sterile DPBS and digested with 0.25% trypsin-EDTA for approximately 5 minutes, followed by quenching with complete medium.

[0326] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and resuspended in 100 μL of 1% BSA in PBS. The cells were counted, adjusted to a cell density of 1E6 / mL, and seeded into a 96-well round-bottom culture plate (Corning, product number 3799). The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 200 μL of 1% BSA in PBS. The plate was centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate was stored at 4°C. Test antibody samples were diluted in 1% BSA in PBS to a starting concentration of 100 nM, followed by 10-fold serial dilutions to seven concentrations. The diluted antibody was used to resuspend the cells and incubated at 4°C for 1 hour at 100 μL per well. The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant discarded. The cells were washed by resuspending in 160 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The secondary antibody (goat anti-human IgG Fc PE) was diluted 1:400 in 1% BSA in PBS according to the manufacturer's instructions. The cells were resuspended in the diluted secondary antibody at 100 μL / well and incubated at 4°C for 0.5 hours. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were washed by resuspending in 200 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA in PBS and filtered through a 300-mesh gauze. The mean fluorescence intensity of the PE channel was detected by flow cytometry.

[0327] Export the FCS file from the flow cytometer and analyze the mean fluorescence intensity (MFI) of the PE channel of each sample using flowjo software. Import the MFI data into Graphpad to calculate the half maximal binding concentration (EC) of the antibody to the cells. 50 ) and highest mean fluorescence intensity (Top MFI) were analyzed, and the results are shown in Table 24 and Figure 17. One anti-BCMA chimeric antibody screened (19CH-16) bound well to both human BCMA and cynomolgus BCMA at the cellular level.

[0328] Binding of anti-BCMA chimeric antibodies derived from mouse hybridomas to BCMA-expressing cells [Table 24]

[0329] [Example 25] Humanized design of anti-BCMA antibodies derived from mouse hybridomas Based on the results of expression purification tests and cell-level binding tests, antibody clone number 19CH-16 was selected for humanization design.

[0330] Humanization of the mouse anti-human BCMA monoclonal antibody was performed as described in many publications in the art. Briefly, human constant domains were used instead of the parent (mouse antibody) constant domains, and human germline antibody sequences were selected based on the homology between mouse and human antibodies. Based on the typical structures of the obtained mouse antibody VH / VL CDRs, the heavy and light chain variable region sequences were compared with a human antibody germline database to obtain highly homologous human germline templates.

[0331] The CDR regions of the mouse antibody were grafted onto the selected corresponding humanized template, replacing the humanized variable regions, and then recombined with an IgG constant region (preferably an IgG1 heavy chain and a κ light chain). Next, based on the three-dimensional structure of the mouse antibody, back mutations of buried residues, residues directly interacting with the CDR regions, and residues that have a significant effect on the conformation of the VL and VH were used to design antibodies composed of the following combinations of humanized light and heavy chain variable region sequences: 19CH-16H1L1, 19CH-16H1L2, 19CH-16H1L3, 19CH-16H2L1, 19CH-16H2L2, 19CH-16H2L3, 19CH-16H3L1, 19CH-16H3L2, 19CH-16H3L3, 19CH-16H4L1, 19CH-16H4L2, and 19CH-16H4L3. Of these, 19CH-16H2L2 is shown in Table 25.

[0332] Amino acid sequence of the variable region of one humanized antibody derived from a murine hybridoma [Table 25]

[0333] [Example 26] Preparation of anti-BCMA humanized antibodies derived from mouse hybridomas The target gene fragments generated by linking the heavy and light chain variable regions of the humanized antibody to the heavy and kappa light chain constant regions of IgG1 (L234AL235A), respectively, were cloned into the pTT5 expression vector to prepare transfection-grade expression plasmids.

[0334] Expi293F in Expi293 expression medium (ThermoFisher, A1435101) TM Cells (ThermoFisher, A14527) were cultured and inoculated into shake flasks and grown on a shaker at 37°C in an 8% CO2 environment. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and PEI transfection reagent were mixed in the appropriate ratio and added to the shake flask for cell culture. After 6 days of cell culture, the expression supernatant was collected, cell debris was removed by high-speed centrifugation, and affinity purified using a Mabselect Sure column. The column was washed with PBS until the A280 reading fell to baseline. The target protein was eluted with an acidic eluent (pH 3.0-3.5) and neutralized with 1 M Tris-HCl (pH 8.0-9.0). After appropriate concentration, the eluted sample was buffer exchanged with PBS and prepared in aliquots. The final purified chimeric antibody was analyzed for purity by SDS-PAGE and HPLC, and its A280 concentration was measured.

[0335] Example 27: Binding of anti-BCMA humanized antibodies derived from mouse hybridomas to BCMA-expressing cells The culture medium for human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells was F12K + 10% FBS + 400 μg / mL hygromycin. Before use, human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells were washed twice with sterile DPBS, digested with 0.25% trypsin-EDTA for approximately 5 minutes, and then digested with complete culture medium.

[0336] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and resuspended in 100 μL of 1% BSA in PBS. The cells were counted, adjusted to a cell density of 1E6 / mL, and seeded into a 96-well round-bottom culture plate (Corning, product number 3799). The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells were resuspended in 200 μL of 1% BSA in PBS. The plate was centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate was stored at 4°C. Test antibody samples were diluted in 1% BSA in PBS to a starting concentration of 100 nM, followed by 10-fold serial dilutions to seven concentrations. The diluted antibody was used to resuspend the cells and incubated at 4°C for 1 hour at 100 μL per well. The plate was centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant discarded. The cells were washed by resuspending in 160 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The secondary antibody (goat anti-human IgG Fc PE) was diluted 1:400 in 1% BSA in PBS according to the manufacturer's instructions. The cells were resuspended in the diluted secondary antibody at 100 μL / well and incubated at 4°C for 0.5 hours. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were washed by resuspending in 200 μL of 1% BSA in PBS and centrifuged at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. The cells were resuspended in 100 μL of 1% BSA in PBS and filtered through a 300-mesh gauze. The mean fluorescence intensity of the PE channel was detected by flow cytometry.

[0337] Export the FCS file from the flow cytometer and analyze the mean fluorescence intensity (MFI) of the PE channel of each sample using flowjo software. Import the MFI data into Graphpad to calculate the half maximal binding concentration (EC) of the antibody to the cells. 50 ) and highest mean fluorescence intensity (Top MFI) were analyzed, and the results are shown in Table 26 and Figure 18. One anti-BCMA humanized antibody (19CH-16H2L2) prepared and screened in Example 26 bound well to both human BCMA and cynomolgus BCMA at the cellular level.

[0338] Binding of anti-BCMA humanized antibodies derived from murine hybridomas to BCMA-expressing cells [Table 26]

[0339] Example 28: Preparation of anti-BCMA humanized antibody mutants derived from mouse hybridomas Post-translational modification (PTM) analysis of the CDR regions of the anti-BCMA humanized antibody 19CH-16H2L2 described above revealed the presence of one deamidation site in the heavy chain variable region. To eliminate this potential risk, single-site-directed mutagenesis was performed on the amino acid sequence of 19CH-16H2, resulting in two mutants, 19CH-16H2L2-NA and 19CH-16H2L2-QT. The amino acid sequence of the variable region of the 19CH-16H2L2-NA mutant is shown in Table 27.

[0340] Amino acid sequence of the 19CH-16H2L2 humanized antibody variant [Table 27]

[0341] Referring to the description in Example 26, the above two mutant proteins were prepared by transient transfection expression in Expi293 cells. Referring to the description in Example 27, the affinity of the two mutants was measured using human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells. The results are shown in Table 28 and Figure 19. 19CH-16H2L2-NA can maintain affinity at the cell binding level while eliminating the risk of post-translational modification.

[0342] Binding of the 19CH-16H2L2 humanized antibody variant to BCMA-expressing cells [Table 28]

[0343] The above-described embodiments of the invention are merely illustrative, and those skilled in the art will recognize or be able to determine, using no more than routine experimentation, numerous equivalents to specific compounds, materials, and procedures, all of which are within the scope of the present invention and encompassed by the claims.

Claims

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

2. the single chain antibody (scFv) domain comprises a heavy chain variable region and a light chain variable region, and preferably the heavy chain variable region of the single chain antibody (scFv) domain is linked to the light chain variable region of the single chain antibody (scFv) domain by 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 has the amino acid sequence (G ４ S) ｎ wherein n is any integer from 1 to 10; The trispecific binding molecule of claim 1 .

3. the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, the C-terminus of the first CH2 domain being 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, the C-terminus of the second CH2 domain being 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 first and / or second Fc domain comprises the amino acid substitutions L234A, L235A and / or G237A; Preferably, the second Fc domain comprises the amino acid substitution H435R; Preferably, the single chain antibody (scFv) domain is linked 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, said second linker comprises the amino acid sequence EPKSS, Preferably, the Fc domain is derived from IgG1. The trispecific antigen-binding molecule of claim 1 or 2.

4. the heavy chain domain of the antigen-binding fragment Fab comprises an immunoglobulin heavy chain variable region and a CH1 domain, and the C-terminus of the heavy chain variable region is fused to the N-terminus of the CH1 domain; the light chain domain of the antigen-binding fragment Fab comprises an immunoglobulin light chain variable region and a light chain constant region, and the C-terminus of the light chain variable region is fused to the N-terminus of the light chain constant region; Preferably, the heavy chain domain of the antigen-binding fragment Fab of the first polypeptide is linked to the single chain antibody (scFv) domain via a third linker, wherein the C-terminus of the heavy chain domain of the antigen-binding fragment Fab 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; Optionally, the fragment antigen-binding Fab heavy chain domain of said third polypeptide is linked to said VHH domain via a fifth linker, wherein the C-terminus of the fragment antigen-binding Fab heavy chain domain is fused to the N-terminus of said fifth linker and the C-terminus of said fifth linker is fused to the N-terminus of the VHH domain. Preferably, the third linker and / or the fifth linker has the amino acid sequence (G ４ S) ｎ wherein n is any integer from 1 to 10; The trispecific antigen-binding molecule of any one of claims 1 to 3.

5. the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of a second Fc domain, preferably the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of the second Fc domain via a fourth linker, more preferably the fourth linker comprises the amino acid sequence EPKSS; The trispecific antigen-binding molecule of any one of claims 1 to 4.

6. the first polypeptide comprises a structure of a Fab heavy chain domain-an scFv domain-a first Fc domain; Preferably, the first polypeptide comprises a structure of 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; wherein the second polypeptide comprises a structure of a Fab light chain variable region-light chain constant region, and / or the third polypeptide comprises a structure of a VHH domain-a second Fc domain, preferably the third polypeptide comprises a structure of a VHH domain-a fourth linker-a second CH2-a second CH3; Optionally, the fourth polypeptide comprises a structure of Fab light chain variable region-light chain constant region, and the third polypeptide comprises a structure of Fab heavy chain domain-fifth linker-VHH-fourth linker-second Fc domain, more preferably, the third polypeptide comprises a structure of Fab heavy chain variable region-Fab CH1-fifth linker-VHH-fourth linker-second CH2-second CH3; The trispecific antigen-binding molecule of any one of claims 1 to 5.

7. the second antigen is CD3, preferably CD3ε, and preferably the single chain antibody (scFv) domain comprises an HCDR1 having the sequence set forth in SEQ ID NO: 27, an HCDR2 having the sequence set forth in SEQ ID NO: 28, an HCDR3 having the sequence set forth in SEQ ID NO: 29, an LCDR1 having the sequence set forth in SEQ ID NO: 30, an LCDR2 having the sequence set forth in SEQ ID NO: 31, and an LCDR3 having the sequence set forth in SEQ ID NO: 32; More preferably, the single chain antibody (scFv) domain comprises a heavy chain variable region whose sequence is set forth in SEQ ID NO: 25 and a light chain variable region whose sequence is set forth in SEQ ID NO: 26; More preferably, the single chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:

13. The trispecific antigen-binding molecule of any one of claims 1 to 6.

8. The trispecific 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: 33 and the second Fc domain comprises the amino acid sequence shown in SEQ ID NO:

34.

9. the first antigen is BCMA, and preferably the antigen-binding fragment Fab comprises an HCDR1 whose sequence is set forth in SEQ ID NO: 16, an HCDR2 whose sequence is set forth in SEQ ID NO: 17, and an HCDR3 whose sequence is set forth in SEQ ID NO: 18; and preferably the antigen-binding fragment Fab comprises an LCDR1 whose sequence is set forth in SEQ ID NO: 19, an LCDR2 whose sequence is set forth in SEQ ID NO: 20, and an LCDR3 whose sequence is set forth in SEQ ID NO: 21; More preferably, the heavy chain domain of the antigen-binding fragment Fab comprises a heavy chain variable region set forth in SEQ ID NO: 14, and the light chain domain of the antigen-binding fragment Fab comprises a light chain variable region whose sequence is set forth in SEQ ID NO: 15; More preferably, the heavy chain domain of said antigen-binding fragment Fab comprises the amino acid sequence set forth in SEQ ID NO: 35, and more preferably, the light chain domain of said antigen-binding fragment Fab comprises the amino acid sequence set forth in SEQ ID NO:

7. The trispecific antigen-binding molecule of any one of claims 1 to 8.

10. The third antigen is GPRC5D, and preferably, the heavy chain single domain antibody (VHH) domain capable of specifically binding to the third antigen comprises an HCDR1 shown in SEQ ID NO: 22, an HCDR2 shown in SEQ ID NO: 23, and an HCDR3 shown in SEQ ID NO: 24, and more preferably, the heavy chain single domain antibody (VHH) domain comprises the sequence shown in SEQ ID NO:

10. The trispecific antigen-binding molecule of any one of claims 1 to 9.

11. the first polypeptide of the trispecific antigen-binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7, and the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6; or, optionally, the first polypeptide of the trispecific antigen-binding molecule comprises the sequence set forth in SEQ ID NO: 5, the second polypeptide and / or the fourth polypeptide comprises the sequence set forth in SEQ ID NO: 7, and the third polypeptide comprises the sequence set forth in SEQ ID NO:

8. The trispecific antigen-binding molecule of any one of claims 1 to 10.

12. 1. A trispecific antigen-binding molecule comprising: A trispecific antigen-binding molecule capable of binding to an epitope identical to or overlapping with an epitope for an antibody comprising an HCDR1 whose sequence is set forth in SEQ ID NO: 22, an HCDR2 whose sequence is set forth in SEQ ID NO: 23, and an HCDR3 whose sequence is set forth in SEQ ID NO:

24.

13. capable of binding to an epitope identical to or overlapping with an epitope for an antibody whose sequence comprises the amino acid sequence set forth in SEQ ID NO: 10; The trispecific antigen-binding molecule of claim 12.

14. The trispecific antigen-binding molecule of claim 12 or 13, which is capable of binding to CD3 and BCMA, preferably CD3ε and BCMA.

15. 1. A trispecific antigen-binding molecule comprising: (A) a first binding moiety capable of specifically binding to GPRC5D3; (B) a second binding moiety, and (C) a third binding moiety; the antigens or epitopes that specifically bind to the first, second, and third binding moieties are different from one another; The first binding moiety is comprising an HCDR1 whose sequence is set forth in SEQ ID NO: 22, an HCDR2 whose sequence is set forth in SEQ ID NO: 23, and an HCDR3 whose sequence is set forth in SEQ ID NO: 24; Trispecific antigen binding molecule.

16. 16. The trispecific antigen-binding molecule of claim 15, wherein the first binding moiety comprises the amino acid sequence set forth in SEQ ID NO:

10.

17. 17. The bispecific antigen-binding molecule of claim 15 or 16, wherein the second binding moiety is capable of binding to CD3, preferably CD3ε, and / or the third binding moiety is capable of binding to BCMA.

18. A nucleic acid molecule encoding the trispecific antigen-binding molecule of any of the preceding claims.

19. 19. An expression vector comprising the nucleic acid molecule of claim 18.

20. 20. A host cell comprising the nucleic acid molecule of claim 18 or the expression vector of claim 19, preferably wherein the host cell is a prokaryotic or eukaryotic cell, wherein the prokaryotic cell is preferably E. coli, and wherein the eukaryotic cell is preferably a mammalian cell or yeast, more preferably wherein the mammalian cell is a CHO cell, Expi293 or HEK293 cell.

21. A method for producing the trispecific antigen-binding molecule of any one of claims 1 to 17, comprising culturing the host cell of claim 20 under suitable conditions.

22. A pharmaceutical composition comprising the trispecific antigen-binding molecule of any one of claims 1 to 17, the nucleic acid molecule of claim 18, the expression vector of claim 19, and / or the host cell of claim 20.

23. 23. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable carrier.

24. 24. The pharmaceutical composition of claim 22 or 23, further comprising one or more additional therapeutic agents.

25. Use of the trispecific antigen-binding molecule of any one of claims 1 to 17, the nucleic acid molecule of claim 18, the expression vector of claim 19, and / or the host cell of claim 20 in the manufacture of a medicament for the treatment, alleviation, and / or prevention of tumors.

26. 26. The use according to claim 25, wherein the tumor is a GPRC5D-positive and / or BCMA-positive tumor.

27. 27. The use according to claim 25 or 26, wherein the tumor is selected from the group consisting of lymphomas, such as multiple myeloma, and metastases of said tumors.

28. 1. A method of inducing death of a cell expressing GPRC5D and / or BCMA, comprising:

26. A method comprising contacting said cell with the trispecific antigen-binding molecule of any one of claims 1 to 17, the nucleic acid molecule of claim 18, the expression vector of claim 19, the host cell of claim 20, and / or the pharmaceutical composition of any one of claims 22 to 24, wherein preferably the GPRC5D and / or BCMA-expressing cell is a tumor cell.

29. 29. The method of claim 28, wherein the tumor cells are cells of a tumor selected from the group consisting of lymphomas, such as multiple myeloma, and metastatic cancers of said tumors.

30. 26. A method for treating a disease associated with expression of GPRC5D and / or BCMA in a subject, comprising administering to a subject in need thereof the trispecific antigen-binding molecule of any one of claims 1 to 17, the nucleic acid molecule of claim 18, the expression vector of claim 19, the host cell of claim 20, and / or the pharmaceutical composition of any one of claims 22 to 24.

31. 31. The method of claim 30, wherein the disease is a tumor, preferably selected from the group consisting of lymphomas such as multiple myeloma, and metastases of said tumors.

32. 32. The method of claim 30 or 31, further comprising administering to the subject an additional therapeutic agent.

33. A GPRC5D antigen-binding molecule or antigen-binding fragment thereof, wherein the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises the HCDR sequence of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 10, and preferably the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises the HCDR1 having the sequence set forth in SEQ ID NO: 22, the HCDR2 having the sequence set forth in SEQ ID NO: 23, and the HCDR3 having the sequence set forth in SEQ ID NO: 24, and preferably the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:

10.

34. i) further comprising a heavy chain constant region, preferably said heavy chain constant region comprising Fc, more preferably Fc is of murine or human origin, more preferably the sequence of Fc is natural or modified, even more preferably said IgG1 Fc domain comprises the amino acid sequence shown in SEQ ID NO: 38; ii) The GPRC5D antigen-binding molecule or antigen-binding fragment thereof is a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a murine antibody, a humanized antibody, a chimeric antibody, a modified antibody, a fully human antibody, a full-length antibody, a heavy chain antibody, a nanobody, Fab, Fv, scFv, F(ab') ２ , linear antibodies, and / or heavy chain single domain antibodies (VHH), and / or iii) the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA further has one or more of the following characteristics: it is an IgG1, IgG2, IgG3, or IgG4 isotype; 34. The GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to claim 33.

35. The GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises a heavy chain single domain antibody (VHH) domain and an IgG1 Fc domain, and preferably the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of the IgG1 Fc domain, more preferably the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of the IgG1 Fc domain via a linker, and preferably the linker has the amino acid sequence EPKSS or (G ４ S) ｎ where n is any integer from 1 to 10; Preferably, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises, from the N-terminus to the C-terminus, a VHH domain whose sequence is set forth in SEQ ID NO: 10, a G ４ S linker, and an IgG1 Fc domain whose sequence is set forth in SEQ ID NO: 38; A GPRC5D antigen-binding molecule or an antigen-binding fragment thereof according to claim 35 or 34.

36. A conjugate or fusion protein formed by binding the GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35 to a capture or detection marker, preferably wherein the detection marker comprises a radionuclide, a luminescent substance, a colored substance, or an enzyme; or One fused moiety of the fusion protein comprises a GPRC5D antigen-binding molecule or an antigen-binding fragment thereof according to any one of claims 33 to 35. Conjugates or fusion proteins.

37. An antibody-drug conjugate, the antibody-drug conjugate being formed by binding the GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35 to another biologically active molecule, wherein the other biologically active molecule is preferably a small molecule drug, and the GPRC5D antigen-binding molecule or antigen-binding fragment thereof is preferably linked to the other biologically active molecule via a linker.

38. A nucleic acid encoding the GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35, or a recombinant vector comprising said nucleic acid, or a host cell comprising said nucleic acid or said recombinant vector. Preferably, the host cell is a prokaryotic cell (preferably E. coli) or a eukaryotic cell (preferably a mammalian cell or yeast, more preferably the mammalian cell is a CHO cell or a HEK293 cell).

39. A method for producing the GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35, comprising culturing the host cell according to claim 38 under appropriate conditions and purifying the expression product from the cell.

40. Use of a GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35, a conjugate or fusion protein according to claim 36, an antibody-drug conjugate according to claim 37, or a nucleic acid, recombinant vector or host cell according to claim 38 in the manufacture of a medicament for treating or alleviating tumors. Preferably, the drug targets tumor cells in which GPRC5D is aberrantly expressed, and preferably, the tumor cells are cells of a tumor selected from the group consisting of lymphomas such as multiple myeloma, and metastatic cancers of said tumors.

41. Use of a GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35, a conjugate or fusion protein according to claim 36, an antibody-drug conjugate according to claim 37, or a nucleic acid, recombinant vector or host cell according to claim 38 in the manufacture of a detection reagent or a diagnostic reagent. Preferably, the detection reagent is used to detect the expression of GPRC5D, and the diagnostic reagent is used to diagnose a tumor, and preferably, the tumor cells are cells of a tumor selected from the group consisting of lymphomas such as multiple myeloma, and metastatic cancers of said tumors.

42. 1. A method for detecting expression of GPRC5D in a sample, comprising: (1) contacting a sample with a GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35; (2) detecting the formation of a conjugate between the GPRC5D antigen-binding molecule or its antigen-binding fragment and GPRC5D, wherein optionally the GPRC5D antigen-binding molecule or its antigen-binding fragment is detectably labeled; method.

43. 1. A pharmaceutical composition comprising: An effective amount of a GPRC5D antigen-binding molecule or antigen-binding fragment thereof according to any one of claims 33 to 35, a conjugate or fusion protein according to claim 36, an antibody-drug conjugate according to claim 37, or a nucleic acid, recombinant vector or host cell according to claim 38, Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises one or more additional other therapeutic agents. Pharmaceutical compositions.

44. A chimeric antigen receptor (CAR) or a cell containing a chimeric antigen receptor, comprising the GPRC5D antigen-binding molecule or an antigen-binding fragment thereof according to any one of claims 33 to 35.

45. 1. A method for inducing death of a cell expressing GPRC5D, comprising: contacting the cell with the pharmaceutical composition of claim 43, wherein the GPRC5D-expressing cell is a tumor cell; Preferably, the tumor cells are cells of a tumor selected from the group consisting of lymphomas, such as multiple myeloma, and metastases of the tumors. method.

46. 1. A method of treating a disease associated with expression of GPRC5D in a subject, comprising:

44. A method for treating a rheumatoid arthritis comprising administering the pharmaceutical composition of claim 43 to a subject in need thereof, Preferably, the disease is a tumor, and preferably, the tumor disease is selected from the group consisting of lymphomas, such as multiple myeloma, and metastases of the tumors; More preferably, the method further comprises administering an additional therapeutic agent to the subject. method.

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