Tri-specific antigen binding molecules and uses thereof

Abstract

The present disclosure provides configurations of a trispecific antigen binding molecule, the invention also provides a three-specific antigen binding molecule, a specific binding molecule or a fragment thereof, a specific binding molecule or a fragment thereof, a specific binding molecule or a fragment thereof, and a specific binding molecule or a fragment thereof, wherein the three-specific antigen binding molecule is constructed on the basis of the configuration and aims at two tumor antigens GPRC5D and BCMA and a T cell surface antigen CD3; the invention also relates to a pharmaceutical composition containing the trispecific antigen binding molecule or the specific binding molecule or the fragment thereof, and a related application of the pharmaceutical composition in tumor treatment.

Description

Trispecific antigen binding molecules and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2023101003684 filed on February 7, 2023, and this application cites the full text of the above-mentioned Chinese patent application. Technical Field

[0003] The present disclosure belongs to the field of immunology and primarily relates to a trispecific antigen-binding molecule. More specifically, it relates to a trispecific antigen-binding molecule that specifically binds to two tumor antigens, GPRC5D and BCMA, and the T cell surface antigen CD3, as well as pharmaceutical compositions, preparation methods, and uses comprising the molecule. Background Art

[0004] Multiple myeloma (MM) is a hematologic malignancy and the second most common hematologic malignancy after non-Hodgkin's lymphoma. MM can damage the bones, immune system, kidneys, and red blood cell count, often leading to extensive bone destruction accompanied by osteolytic lesions, osteopenia, and pathological fractures. The disease is common in middle-aged and elderly people, and with the accelerated aging of the population and advances in diagnosis and treatment, the patient population is rapidly increasing.

[0005] Like most cancer treatments, the standard treatment for multiple myeloma is chemotherapy, with melphalan being a common medication. However, these medications often lead to bone marrow suppression and are less than ideal in efficacy, with only approximately 5% achieving complete remission. In recent years, advances in medicine have revolutionized the treatment of multiple myeloma with the development of targeted drugs, such as proteasome inhibitors, immunomodulators, and antibodies. Compared to existing treatment options, these drugs have significantly increased survival rates and significantly improved patient outcomes. However, multiple myeloma remains incurable, and most patients relapse after multiple lines of treatment. The median survival for patients in my country is 24-36 months, with a five-year survival rate of only approximately 24%. Therefore, new treatments are urgently needed.

[0006] B cell maturation antigen (BCMA) is a transmembrane glycoprotein receptor (non-tyrosine kinase receptor) and a member of the tumor necrosis factor (TNF) family. It is primarily expressed on the surface of mature B cells, with smaller amounts expressed on hematopoietic stem cells and other tissue cells. The ligands for BCMA are BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand). Under normal physiological conditions, the BCMA signaling pathway promotes B cell differentiation into plasma cells and maintains homeostasis in long-lived bone marrow plasma cells. BCMA is overexpressed in the bone marrow plasma cells of patients with multiple myeloma (MM). Overexpressed BCMA can stimulate the proliferation of malignant plasma cells and maintain their activity through intracellular AKT, MAPK, and (NF)-κB signaling pathways, thereby promoting disease progression. Blocking the BCMA signaling pathway can inhibit the proliferation of malignant plasma cells and slow / mitigate disease progression. Therefore, BCMA is a promising target for the clinical treatment of MM. In addition, the BCMA protein on the cell membrane can be cleaved by the γ-secretase in the body. The cleaved BCMA protein is free in the serum and is called soluble BCMA protein (sBCMA). sBCMA can form a complex with the ligand, thereby reducing the concentration of the ligand in the serum, blocking the binding of the ligand to membrane-bound BCMA, and inhibiting the BCMA signaling pathway.

[0007] GPRC5D is a G protein coupled receptor C5 family subtype D, an orphan receptor and a seven-transmembrane protein. GPRC5D is specifically and highly expressed in plasma cells of multiple myeloma and is found only in hair follicles in normal tissues. Antibodies and CAR-T cell therapies targeting GPRC5D have shown promising preclinical results, with no hair loss observed in experimental animals. Furthermore, GPRC5D expression is not restricted by BCMA expression. In models of tumor recurrence caused by BCMA antigen loss, GPRC5D-targeted CAR-T therapy can overcome tumor escape. These research results demonstrate that GPRC5D is an ideal clinical target.

[0008] Bispecific antibodies targeting tumor-associated antigens (TAAs) and CD3 antigens can bring T cells and tumor cells closer together, forming synapses and using T cells to specifically kill tumor cells. These antibodies are called T cell engagers (TCE bispecific antibodies). TCE-based treatment strategies rely on the distribution of TAAs on the tumor cells being treated. In addition, studies have also shown that treatments targeting a single TAA site may cause disease recurrence due to tumor escape mechanisms, thereby limiting the effectiveness of treatment.

[0009] Since the expression of GPRC5D and BCMA in tumors is uncorrelated, designing a trispecific antibody that targets GPRC5D and BCMA in combination will cover both patients with GPRC5D-positive tumors and patients with BCMA-positive tumors, while avoiding recurrence caused by loss of a single antigen, thereby achieving the goal of increasing patient coverage and improving treatment efficacy.

[0010] SUMMARY OF THE INVENTION

[0011] To achieve the above objectives, the present disclosure provides a trispecific antigen-binding molecule targeting GPRC5D / BCMA / CD3, and conducts in-depth research on various molecular configurations, successfully achieving multi-target combination and avoiding the inconvenience of multi-drug combination.

[0012] The first aspect of the present disclosure is to provide a trispecific antigen-binding molecule. The trispecific antigen-binding molecule comprises the following polypeptides:

[0013] 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;

[0014] A second polypeptide comprises: a light chain domain of an antigen-binding fragment Fab capable of specifically binding to a first antigen;

[0015] The third polypeptide comprises: (i) a heavy chain single domain antibody (VHH) domain capable of specifically binding to a third antigen and (ii) a second Fc domain.

[0016] 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 the first antigen; the single-chain antibody (scFv) domain forms a second binding site for the second antigen; the heavy chain single-domain antibody (VHH) domain forms a third binding site for the third antigen; and the first Fc domain and the second Fc domain associate with each other.

[0017] Optionally, the trispecific antigen-binding molecule further comprises a fourth polypeptide comprising a light chain domain of an antigen-binding fragment Fab that specifically binds to the first antigen, the light chain domain of the antigen-binding fragment Fab being identical to the light chain domain of the antigen-binding fragment Fab of the second polypeptide, and the third polypeptide further comprises a heavy chain domain of an antigen-binding fragment Fab that specifically binds to the first antigen, the heavy chain domain of the antigen-binding fragment Fab of the third polypeptide being identical to the heavy chain domain of the antigen-binding fragment Fab of the first polypeptide, the C-terminus of which is linked to the N-terminus of the VHH domain, and the heavy chain domain of the Fab of the third polypeptide and the light chain domain of the Fab of the fourth polypeptide forming a fourth binding site for the first antigen.

[0018] In one embodiment, the scFv domain comprises a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region and the light chain variable region of the scFv domain are connected via 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.

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

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

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

[0022] Preferably, to reduce ADCC activity of the antibody, the first and / or second Fc domain comprises amino acid substitutions of L234A, L235A and / or G237A.

[0023] Preferably, the second Fc domain of the third polypeptide comprises an amino acid substitution of H435R.

[0024] Preferably, the Fc domain is derived from IgG1.

[0025] 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.

[0026] In one embodiment, the heavy chain domain of the antigen-binding fragment Fab comprises the heavy chain variable region and the CH1 domain of an immunoglobulin, and the C-terminus of the heavy chain variable region is fused to the N-terminus of the CH1 domain; the light chain domain of the antigen-binding fragment Fab comprises the light chain variable region and the light chain constant region of an immunoglobulin, and the C-terminus of the light chain variable region is fused to the N-terminus of the light chain constant region.

[0027] In one embodiment, the heavy chain domain of the antigen-binding fragment Fab of the first polypeptide is connected 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.

[0028] Preferably, the third linker comprises the amino acid sequence (G4S) n , n is any integer from 1 to 10.

[0029] Optionally, the C-terminus of the heavy chain domain of the antigen-binding fragment Fab of the third polypeptide is connected to the N-terminus of the VHH domain via a fifth linker. Preferably, the fifth linker comprises the amino acid sequence (G4S) n , n is any integer from 1 to 10.

[0030] Preferably, the C-terminus of the heavy chain single-domain antibody (VHH) domain is fused to the N-terminus of the second Fc domain. Preferably, the C-terminus of the 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.

[0031] In one embodiment, the first polypeptide comprises the following structure: Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain.

[0032] Preferably, the first polypeptide comprises the following structure: Fab heavy chain variable region-Fab CH1-third linker-scFv heavy chain variable region-first linker-scFv light chain variable region-second linker-first CH2-first CH3.

[0033] In one embodiment, the second polypeptide comprises the following structure: Fab light chain variable region-light chain constant region.

[0034] In one embodiment, the third polypeptide comprises the following structure: VHH domain-fourth linker-second Fc domain. Preferably, the third polypeptide comprises the following structure: VHH-fourth linker-second CH2-second CH3.

[0035] Optionally, the fourth polypeptide comprises the following structure: Fab light chain variable region-light chain constant region, and the third polypeptide comprises the following structure: Fab heavy chain domain-fifth linker-VHH-fourth linker-second Fc domain, preferably the third polypeptide comprises the following structure: Fab heavy chain variable region-Fab CH1-fifth linker-VHH-fourth linker-second CH2-second CH3.

[0036] In one embodiment, the second antigen is CD3, preferably CD3ε; preferably, the scFv domain comprises a HCDR1 as shown in SEQ ID NO: 27, a HCDR2 as shown in SEQ ID NO: 28, a HCDR3 as shown in SEQ ID NO: 29, a LCDR1 as shown in SEQ ID NO: 30, a LCDR2 as shown in SEQ ID NO: 31 and a LCDR3 as shown in SEQ ID NO: 32.

[0037] More preferably, the scFv domain comprises a heavy chain variable region with a sequence as shown in SEQ ID NO: 25 and a light chain variable region with a sequence as shown in SEQ ID NO: 26.

[0038] More preferably, the scFv domain comprises the amino acid sequence shown in SEQ ID NO: 13.

[0039] In one embodiment, 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.

[0040] In one embodiment, the first antigen is BCMA; preferably, the antigen-binding fragment Fab comprises a HCDR1 with a sequence as shown in SEQ ID NO: 16, a HCDR2 with a sequence as shown in SEQ ID NO: 17, and a HCDR3 with a sequence as shown in SEQ ID NO: 18, and / or a LCDR1 with a sequence as shown in SEQ ID NO: 19, a LCDR2 with a sequence as shown in SEQ ID NO: 20, and a LCDR3 with a sequence as shown in SEQ ID NO: 21.

[0041] More preferably, the heavy chain domain of the antigen-binding fragment Fab comprises a heavy chain variable region with a sequence as shown in SEQ ID NO: 14, and / or the light chain domain of the antigen-binding fragment Fab comprises a light chain variable region with a sequence as shown in SEQ ID NO: 15.

[0042] More preferably, the heavy chain domain of the antigen-binding fragment Fab comprises the amino acid sequence shown in SEQ ID NO: 35, and / or the light chain domain of the antigen-binding fragment Fab comprises the amino acid sequence shown in SEQ ID NO: 7.

[0043] In one embodiment, the third antigen is GPRC5D; preferably, the heavy chain single-domain antibody (VHH) domain capable of specifically binding to the third antigen comprises a HCDR1 as shown in SEQ ID NO: 22, a HCDR2 as shown in SEQ ID NO: 23, and a HCDR3 as shown in SEQ ID NO: 24; more preferably, the VHH domain comprises the sequence shown in SEQ ID NO: 10.

[0044] In one embodiment, the first polypeptide of the trispecific antigen-binding molecule comprises the amino acid sequence shown in SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6; or optionally, the first polypeptide of the trispecific antigen-binding molecule comprises the sequence shown in SEQ ID NO: 5, the second polypeptide and / or the fourth polypeptide comprises the sequence shown in SEQ ID NO: 7, and the third polypeptide comprises the sequence shown in SEQ ID NO: 8.

[0045] The present disclosure also provides nucleic acid molecules encoding the trispecific antigen-binding molecules described herein.

[0046] The present disclosure also provides a vector comprising the nucleic acid molecule.

[0047] The present disclosure also provides a host cell comprising the nucleic acid molecule or vector; preferably, the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is preferably Escherichia coli; the eukaryotic cell is preferably a mammalian cell or yeast; more preferably, the mammalian cell is a CHO cell, Expi293 or HEK293 cell.

[0048] The present disclosure also provides a method for preparing a trispecific antigen-binding molecule, the method comprising: culturing the host cell under suitable conditions.

[0049] The present disclosure also provides an antibody-drug conjugate, which is formed by coupling the aforementioned bispecific antigen-binding molecule with other biologically active molecules; preferably, the other biologically active molecules are small molecule drugs; preferably, the bispecific antigen-binding molecule and the other biologically active molecules are connected via a linker.

[0050] The present disclosure also provides a pharmaceutical composition comprising the aforementioned trispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell and / or antibody-drug conjugate.

[0051] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0052] In one embodiment, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0053] The present disclosure also provides the use of the trispecific antigen binding molecules, nucleic acid molecules, vectors, host cells and / or antibody drug conjugates in the preparation of a medicament for treating, alleviating and / or preventing tumors. Preferably, the tumor is GPRC5D-positive and / or BCMA-positive.

[0054] The present disclosure also provides a method for inducing cell death expressing GPRC5D and / or BCMA, the method comprising contacting the cell with the trispecific antigen binding molecule, nucleic acid molecule, vector, host cell, and / or pharmaceutical composition, wherein the cell expressing GPRC5D3 and / or BCMA is a tumor cell.

[0055] The present disclosure also 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 is recurrent or refractory to treatment with a previous anticancer therapeutic agent. In one embodiment, the method further comprises administering an additional therapeutic agent to the subject.

[0056] Preferably, the tumor or tumor cell of the present disclosure is selected from the group consisting of lymphomas such as multiple myeloma, and metastatic cancers of the above tumors.

[0057] Another aspect of the present disclosure is to provide an antigen-binding molecule or an antigen-binding fragment thereof that specifically binds to GPRC5D, wherein the antigen-binding molecule or the antigen-binding fragment thereof can specifically bind to GPRC5D without non-specific binding to the same family protein GPRC5A.

[0058] Preferably, the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to GPRC5D comprises a heavy chain single-domain antibody (VHH) domain; preferably, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises the HCDR sequence of the heavy chain variable region as shown in SEQ ID NO: 10; preferably, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises a HCDR1 as shown in SEQ ID NO: 22, a HCDR2 as shown in SEQ ID NO: 23, and a HCDR3 as shown in SEQ ID NO: 24; more preferably, the heavy chain single-domain antibody (VHH) comprises the amino acid sequence as shown in SEQ ID NO: 10.

[0059] Preferably, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof further comprises an Fc domain of an immunoglobulin, and the immunoglobulin is selected from IgG1, IgG2, IgG3 or IgG4 forms. Preferably, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof further comprises an Fc domain of IgG1, and further preferably, the Fc domain of IgG1 comprises the amino acid sequence shown in SEQ ID NO: 38.

[0060] In one embodiment, the GPRC5D antigen binding molecule or antigen binding fragment thereof comprises a heavy chain single domain antibody (VHH) domain and an Fc domain of IgG1, preferably, the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of the Fc domain of IgG1, more preferably, the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of the Fc domain of IgG1 via a linker, preferably, the linker comprises the amino acid sequence EPKSS or (G4S) n , n is any integer from 1 to 10. In a preferred embodiment, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof comprises, from N-terminus to C-terminus, a VHH domain having a sequence as shown in SEQ ID NO: 10, a G4S linker, and an IgG1 Fc domain having a sequence as shown in SEQ ID NO: 38.

[0061] The GPRC5D antigen-binding molecule or antigen-binding fragment thereof disclosed herein can be a monoclonal antibody, a bispecific binding molecule, a multispecific binding molecule, a murine antibody, a humanized antibody, a chimeric antibody, a reshaped 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.

[0062] The present disclosure also provides a conjugate, which is formed by conjugating the GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present disclosure with a capture label or a detection label; preferably, the detection label includes a radionuclide, a luminescent substance, a colored substance or an enzyme.

[0063] The present disclosure also provides an antibody-drug conjugate (ADC), which is formed by coupling the GPRC5D antigen-binding molecule or its antigen-binding fragment of the present disclosure to other biologically active molecules; preferably, the other biologically active molecules are small molecule drugs; preferably, the GPRC5D antigen-binding molecule or its antigen-binding fragment and the other biologically active molecules are connected via a linker.

[0064] The present disclosure also provides a fusion protein, wherein one of the fused parts comprises the GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present disclosure.

[0065] The present disclosure also provides a chimeric antigen receptor (CAR) or a cell comprising the chimeric antigen receptor (eg, CAR-T cell), which comprises the GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present disclosure.

[0066] The present disclosure also provides nucleic acids encoding GPRC5D antigen-binding molecules or antigen-binding fragments thereof, as well as recombinant vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors. Preferably, the host cells are prokaryotic cells (preferably Escherichia coli) or eukaryotic cells (preferably mammalian cells or yeast; further preferably, the mammalian cells are CHO cells or HEK293 cells).

[0067] The present disclosure also provides a method for preparing a GPRC5D antigen-binding molecule or an antigen-binding fragment thereof, the method comprising: culturing the above-mentioned host cells under suitable conditions, and purifying the expression product from the cells.

[0068] The present disclosure also provides use of a GPRC5D antigen-binding molecule or an antigen-binding fragment thereof in the preparation of a drug for treating or alleviating tumors.

[0069] In one embodiment, the drug targets tumor cells that aberrantly express GPRC5D.

[0070] In one embodiment, the tumor is selected from the group consisting of lymphomas such as multiple myeloma, and metastases thereof.

[0071] The present disclosure also provides a method for treating a disease associated with expression of GPRC5D in a subject, comprising administering the GPRC5D antigen-binding molecule or antigen-binding fragment thereof to a subject in need thereof.

[0072] Preferably, the disease is a tumor; preferably, the tumor disease is selected from lymphoma such as multiple myeloma, and metastatic cancer of the above tumors;

[0073] More preferably, the method further comprises administering an additional therapeutic agent to the subject.

[0074] The present disclosure also provides use of the GPRC5D antigen-binding molecule or antigen-binding fragment thereof in the preparation of a detection reagent or a diagnostic reagent.

[0075] In one embodiment, the detection reagent is used to detect the expression of GPRC5D; the diagnostic reagent is used to diagnose tumors; preferably, the tumor is selected from: lymphoma such as multiple myeloma, and metastatic cancer of the above tumors.

[0076] The present disclosure also provides a method for detecting GPRC5D expression in a sample, the method comprising:

[0077] (1) contacting a sample with a GPRC5D antigen-binding molecule or antigen-binding fragment thereof disclosed herein;

[0078] (2) detecting the formation of a complex between the GPRC5D antigen-binding molecule or antigen-binding fragment thereof and GPRC5D; optionally, the GPRC5D antigen-binding molecule or antigen-binding fragment thereof is detectably labeled.

[0079] The present disclosure also provides a method of monitoring a GPRC5D-expressing cancer in a subject, the method comprising exposing a sample obtained or derived from the subject to one or more of the antigen-binding molecules or antigen-binding fragments described herein that specifically bind to GPRC5D; determining the amount of GPRC5D present in the sample 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, based on the difference in the amount of GPRC5D in the compared samples, whether the subject's GPRC5D level indicates cancer progression, regression, or stable disease.

[0080] 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, biopsy, fine needle aspirate, or histological preparation.

[0081] The present disclosure also provides a pharmaceutical composition comprising an effective amount of the GPRC5D antigen-binding molecule or antigen-binding fragment thereof of the present disclosure, or an effective amount of the antibody-drug conjugate, fusion protein, CAR-T cell of the present disclosure, or an effective amount of the nucleic acid of the present disclosure, or an effective amount of the recombinant vector of the present disclosure, or an effective amount of the host cell of the present disclosure.

[0082] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0083] Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0084] Another aspect of the present disclosure is to provide an antigen-binding molecule or an antigen-binding fragment thereof that specifically binds to BCMA, wherein the antigen-binding molecule or the antigen-binding fragment thereof comprises the HCDR sequence of the heavy chain variable region as shown in SEQ ID NO: 14, and / or the LCDR sequence of the light chain variable region as shown in SEQ ID NO: 15; preferably, the antigen-binding molecule or the antigen-binding fragment thereof comprises: a HCDR1 as shown in SEQ ID NO: 16, a HCDR2 as shown in SEQ ID NO: 17, and a HCDR3 as shown in SEQ ID NO: 18, and / or a LCHDR1 as shown in SEQ ID NO: 19, a LCDR2 as shown in SEQ ID NO: 20, and a LCDR3 as shown in SEQ ID NO: 21.

[0085] Preferably, the antigen-binding molecule or antigen-binding fragment thereof that specifically binds to BCMA comprises a heavy chain variable region whose sequence is shown 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 shown in SEQ ID NO: 15.

[0086] 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.

[0087] The BCMA antigen-binding molecules or antigen-binding fragments thereof disclosed herein further comprise a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region comprises an Fc; more preferably, the Fc is derived from a mouse or human; more preferably, the sequence of the Fc is native or modified.

[0088] The BCMA antigen-binding molecules or antigen-binding fragments thereof of the present disclosure can be monoclonal antibodies, bispecific binding molecules, multispecific binding molecules, murine antibodies, humanized antibodies, chimeric antibodies, reshaped antibodies, fully human antibodies, full-length antibodies, heavy chain antibodies, nanobodies, Fab, Fv, scFv, F(ab')2, linear antibodies, or heavy chain single domain antibodies.

[0089] The BCMA antigen-binding molecules or antigen-binding fragments thereof of the present disclosure may be full-length antibodies.

[0090] The BCMA antigen-binding molecules or antigen-binding fragments thereof of the present disclosure may be in the form of IgG1, IgG2, IgG3, or IgG4.

[0091] The present disclosure also provides a conjugate, which is formed by conjugating the BCMA antigen-binding molecule or antigen-binding fragment thereof of the present disclosure to a capture label or a detection label; preferably, the detection label comprises a radionuclide, a luminescent substance, a colored substance, or an enzyme.

[0092] The present disclosure also provides an antibody-drug conjugate (ADC), which is formed by coupling the BCMA antigen-binding molecule or its antigen-binding fragment to other bioactive molecules; preferably, the other bioactive molecules are small molecule drugs; preferably, the BCMA antigen-binding molecule or its antigen-binding fragment to the other bioactive molecules are connected via a linker.

[0093] The present disclosure also provides a fusion protein, wherein one of the fused parts comprises the BCMA antigen-binding molecule or antigen-binding fragment thereof of the present disclosure.

[0094] The present disclosure also provides a chimeric antigen receptor (CAR) and a cell comprising the chimeric antigen receptor (such as a CAR-T cell), which comprises the BCMA antigen-binding molecule or antigen-binding fragment thereof of the present disclosure.

[0095] The present disclosure also provides nucleic acids encoding BCMA antigen-binding molecules or antigen-binding fragments thereof, as well as recombinant vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors. Preferably, the host cells are prokaryotic cells (preferably Escherichia coli) or eukaryotic cells (preferably mammalian cells or yeast; further preferably, the mammalian cells are CHO cells or HEK293 cells).

[0096] The present disclosure also provides a method for preparing the BCMA antigen-binding molecule or antigen-binding fragment thereof of the present disclosure, comprising: culturing the above-mentioned host cells under suitable conditions, and purifying the expression product from the cells.

[0097] The present disclosure also provides use of a BCMA antigen-binding molecule or an antigen-binding fragment thereof in the preparation of a medicament for treating or alleviating tumors.

[0098] In one embodiment, the drug targets tumor cells that aberrantly express BCMA.

[0099] In one embodiment, the tumor is selected from the group consisting of lymphomas such as multiple myeloma, and metastases thereof.

[0100] The present disclosure also provides a method for treating a disease associated with BCMA expression in a subject, comprising administering the BCMA antigen-binding molecule or antigen-binding fragment thereof to a subject in need thereof.

[0101] Preferably, the disease is a tumor; preferably, the tumor disease is selected from lymphoma such as multiple myeloma, and metastatic cancer of the above tumors;

[0102] More preferably, the method further comprises administering an additional therapeutic agent to the subject.

[0103] The present disclosure also provides use of a BCMA antigen-binding molecule or an antigen-binding fragment thereof in the preparation of a detection reagent or a diagnostic reagent.

[0104] In one embodiment, the detection reagent is used to detect the expression of BCMA; the diagnostic reagent is used to diagnose a tumor; preferably, the tumor is selected from: lymphoma such as multiple myeloma, and metastatic cancer of the above tumors.

[0105] The present disclosure also provides a method for detecting BCMA expression in a sample, the method comprising:

[0106] (1) contacting a sample with a BCMA antigen-binding molecule or antigen-binding fragment thereof of the present disclosure;

[0107] (2) detecting the formation of a complex between the BCMA antigen-binding molecule or antigen-binding fragment thereof and BCMA; optionally, the BCMA antigen-binding molecule or antigen-binding fragment thereof is detectably labeled.

[0108] The present disclosure also provides methods of monitoring BCMA-expressing cancer in a subject, the methods comprising exposing a sample obtained or derived from the subject to one or more of the BCMA-specific antibodies or antigen-binding fragments described herein; determining the amount of BCMA present in the sample bound by the antibody 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, based on the difference in the amount of BCMA in the compared samples, whether the subject's BCMA level indicates cancer progression, regression, or stable disease.

[0109] 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, biopsy, fine needle aspirate, or histological preparation.

[0110] The present disclosure also provides a pharmaceutical composition comprising an effective amount of the BCMA antigen-binding molecule or antigen-binding fragment thereof, or an effective amount of the antibody-drug conjugate, fusion protein, or CAR-T cell, or an effective amount of the nucleic acid, or an effective amount of the recombinant vector, or an effective amount of the host cell.

[0111] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0112] Preferably, the pharmaceutical composition further comprises one or more additional therapeutic agents.

[0113] The amino acid positions in the substitutions referred to in this disclosure are preferably indicated using the EU numbering system. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The accompanying drawings further illustrate the novel features disclosed in this specification. The features and advantages disclosed in this specification can be better understood with reference to these drawings, but it should be understood that these drawings are only used to illustrate specific implementations of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0115] Figure 1A shows the expression of human GPRC5D on CHOK1 cells verified by anti-GPRC5D positive control antibody detected by flow cytometry; the dotted line in the figure shows the expression of human GPRC5D on CHOK1 mother cells without any plasmid transfection, and the solid line in the figure shows the overexpression of human GPRC5D protein after CHOK1 mother cells were transfected with human GPRC5D plasmid.

[0116] Figure 1B shows the expression of cynomolgus monkey GPRC5D on CHOK1 cells verified by flow cytometry using an anti-GPRC5D positive control antibody; the dotted line in the figure shows the expression of cynomolgus monkey GPRC5D on CHOK1 mother cells without any plasmid transfection, and the solid line in the figure shows the overexpression of cynomolgus monkey GPRC5D protein after CHOK1 mother cells were transfected with cynomolgus monkey GPRC5D plasmid.

[0117] Figure 1C shows the expression of human GPRC5A on CHOK1 cells verified by flow cytometry using an anti-GPRC5A positive control antibody; the dotted line in the figure indicates the expression of human GPRC5A on CHOK1 mother cells without any plasmid transfection, and the solid line in the figure indicates the overexpression of human GPRC5A protein after CHOK1 mother cells were transfected with the human GPRC5A plasmid.

[0118] Figure 2A shows the expression of human BCMA on CHOK1 cells verified by flow cytometry using an anti-BCMA positive control antibody; the dotted line in the figure indicates the expression of human BCMA on CHOK1 mother cells without any plasmid transfection, and the solid line in the figure indicates the overexpression of human BCMA protein after CHOK1 mother cells were transfected with a human BCMA plasmid.

[0119] Figure 2B shows the expression of cynomolgus monkey BCMA on CHOK1 cells verified by flow cytometry using an anti-BCMA positive control antibody; the dotted line in the figure shows the expression of cynomolgus monkey BCMA on CHOK1 mother cells without any plasmid transfection, and the solid line in the figure shows the overexpression of cynomolgus monkey BCMA protein after CHOK1 mother cells were transfected with cynomolgus monkey BCMA plasmid.

[0120] FIG3 shows the structures of two trispecific antibodies constructed in the examples, namely trispecific antibody 1 and trispecific antibody 2.

[0121] Figure 4A shows the binding of Triclosan 1, Triclosan 2, and Teclistamab to the tumor cells NCI-H929 expressing the dual targets BCMA and GPRC5D.

[0122] Figure 4B shows the binding of Triclosan 1, Triclosan 2 and Teclistamab to the tumor cells RPMI8226 expressing the dual targets BCMA and GPRC5D.

[0123] Figure 4C shows the binding of Triple Antibody 1, Triple Antibody 2 and Teclistamab to the stably transfected cell line human GPRC5D CHOK1 cells expressing human GPRC5D.

[0124] Figure 4D shows the binding of Triple Antibody 1, Triple Antibody 2 and Teclistamab to the stably transfected cell line human BCMA CHOK1 cells expressing human BCMA.

[0125] Figure 4E shows the binding of Trial Antibody 1, BGCB491, and TS-F2-5 to the tumor cells NCI-H929 expressing the dual targets BCMA and GPRC5D.

[0126] Figure 4F shows the binding of triple antibody 1, BGCB491, and TS-F2-5 to the tumor cells RPMI8226 expressing the dual targets BCMA and GPRC5D.

[0127] Figure 4G shows the binding of Trial Antibody 1, BGCB491, and TS-F2-5 to the stably transfected cell line human GPRC5D CHOK1 cells expressing human GPRC5D.

[0128] Figure 4H shows the binding of Trial Antibody 1, BGCB491, and TS-F2-5 to the stably transfected human BCMA CHOK1 cell line expressing human BCMA.

[0129] FIG5A shows the binding of Triple Antibody 1, Triple Antibody 2, and Teclistamab to cells expressing human CD3 (Jurkat cells).

[0130] Figure 5B shows the binding of Triple Antibody 1, Triple Antibody 2, and Teclistamab to human PBMC cells.

[0131] Figure 5C shows the binding of Trial Antibody 1, BGCB491, and TS-F2-5 to cells expressing hCD3 (Jurkat cells).

[0132] Figure 6A shows the binding of Triple Antibody 1, Triple Antibody 2 and Teclistamab to the stable cell line cynomolgus monkey GPRC5D CHOK1 cells expressing cynomolgus monkey GPRC5D.

[0133] Figure 6B shows the binding of Triple Antibody 1, Triple Antibody 2 and Teclistamab to the stable cell line cynomolgus macaque BCMA CHOK1 cells expressing cynomolgus macaque BCMA.

[0134] FIG7 shows the binding of Triple Antibody 1, Triple Antibody 2, and Teclistamab to cyno PBMC cells.

[0135] FIG8A shows a T cell-mediated cytotoxicity assay (TDCC) using NCI-H929 as the target cell.

[0136] FIG8B shows a T cell-mediated cytotoxicity assay (TDCC), wherein the target cells were RPMI8226.

[0137] FIG8C shows a T cell-mediated cytotoxicity assay (TDCC) in which the target cells were human BCMA-CHOK1.

[0138] FIG8D shows a T cell-mediated cytotoxicity assay (TDCC) in which the target cells were human GPRC5D CHOK1.

[0139] FIG8E shows a T cell-mediated cytotoxicity assay (TDCC) in which the target cells are a mixed cell system of human BCMA-CHOK1 and human GPRC5D-CHOK1.

[0140] FIG9 shows a T cell-mediated cytotoxicity experiment in the presence of 2500 ng / ml of soluble BCMA (the dotted line indicates the condition of 2500 ng / ml of soluble BCMA).

[0141] FIG10A shows the IL6 release of Triclosan 1, Triclosan 2 and Teclistamab under the co-incubation conditions of PBMC and tumor cells NCI-H929 (PBMC donor number P122080606F).

[0142] FIG10B shows the IL6 release of Triclosan 1, Triclosan 2 and Teclistamab under the co-incubation conditions of PBMC and tumor cells NCI-H929 (PBMC donor number XC11210).

[0143] Figure 11 shows the efficacy experimental results of Triple Antibody 1, Triple Antibody 2, Teclistamab, and the combination of Teclistamab and Talquetamb in a human immune cell reconstructed mouse model - tumor volume changes (tumor cells are NCI-H929).

[0144] Figure 12 shows the efficacy experimental results of Tri-Antibody 1, BGCB491, and TS-F2-5 in a human immune cell-reconstructed mouse model - changes in tumor volume (tumor cells are NCI-H929).

[0145] Figure 13 shows the efficacy experimental results of Tri-Antibody 1, BGCB491, and TS-F2-5 in a human immune cell-reconstructed mouse model - changes in tumor weight (tumor cells are NCI-H929).

[0146] FIG14A shows the binding of the alpaca-derived anti-GPRC5D chimeric antibody of the present disclosure to GPRC5D-expressing cells (human GPRC5D-CHOK1 cell line).

[0147] FIG14B shows the binding of the alpaca-derived anti-GPRC5D chimeric antibody of the present disclosure to GPRC5D-expressing cells (cynomolgus monkey GPRC5D-CHOK1 cell line).

[0148] FIG15A shows the binding of the alpaca-derived anti-GPRC5D humanized antibody of the present disclosure to cells expressing GPRC5D (NCI-H929 tumor cells).

[0149] FIG15B shows the binding of the alpaca-derived anti-GPRC5D humanized antibody of the present disclosure to GPRC5D-expressing cells (cynomolgus monkey GPRC5D-CHOK1 cell line).

[0150] FIG16 shows that the alpaca-derived anti-GPRC5D humanized antibody of the present disclosure does not non-specifically bind to the cognate family member GPRC5A.

[0151] FIG17A shows the binding of the mouse hybridoma-derived anti-BCMA chimeric antibody disclosed herein to cells expressing human BCMA (human BCMA-CHOK1 cell line).

[0152] FIG17B shows the binding of the mouse hybridoma-derived anti-BCMA chimeric antibody of the present disclosure to cells expressing cynomolgus BCMA (cynomolgus BCMA-CHOK1 cell line).

[0153] FIG18A shows the binding of the mouse hybridoma-derived anti-BCMA humanized antibodies disclosed herein to cells expressing human BCMA (human BCMA-CHOK1 cell line).

[0154] FIG18B shows the binding of the mouse hybridoma-derived anti-BCMA humanized antibodies disclosed herein to cells expressing cynomolgus BCMA (cynomolgus BCMA-CHOK1 cell line).

[0155] FIG19A shows the binding of the anti-BCMA humanized antibody variants 19CH-16H2L2-NA and 19CH-16H2L2-QT derived from mouse hybridomas disclosed herein to cells expressing human BCMA (human BCMA-CHOK1 cell line).

[0156] FIG19B shows the binding of the mouse hybridoma-derived anti-BCMA humanized antibody variants 19CH-16H2L2-NA and 19CH-16H2L2-QT of the present disclosure to cynomolgus BCMA-expressing cells (cynomolgus BCMA-CHOK1 cell line).

[0157] Detailed Description of the Invention

[0158] the term

[0159] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0160] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0161] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0162] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0163] The term "antibody" herein may include intact antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragments thereof (i.e., antigen-binding portions) or single chains thereof, and may also include products having antigen-specific binding ability formed by modifications (e.g., linking other peptide segments, rearrangement of functional units, etc.) based on intact antibodies or their antigen-binding fragments or single chains.

[0164] In one embodiment, an antibody typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain comprises a variable domain (VH) and a constant region.

[0165] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM. The corresponding heavy chain constant domains are designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, e.g., IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, designated κ and λ, based on the amino acid sequence of their constant domains.

[0166] In the case of IgG, IgA, and IgD antibodies, the constant region comprises three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a segment of variable length rich in proline and cysteine. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0167] The term "variable region" or "variable domain" shows significant changes in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region of each light chain / heavy chain pair forms an antibody binding site, so that a complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVRs), or more generally, referred to as complementary determining regions (CDRs). VH and VL each have four framework regions, FRs, represented by FR1, FR2, FR3, and FR4, respectively. Therefore, CDR and FR sequences typically appear in the following sequence of a heavy chain variable domain (or light chain variable domain): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0168] The term "antibody fragment" comprises at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or its immunogenicity-determining portion, or a fusion protein product further derived from this fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific antibodies or multispecific antibodies formed by antibody fragments.

[0169] The term "Fab" or "Fab fragment" refers to a monovalent antibody fragment consisting of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain. The term "F(ab')2" or "F(ab')2 fragment" comprises two Fab fragments and a hinge region and is a bivalent antibody fragment.

[0170] The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0171] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, nanobody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain"). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (referred to as "VH domain" in the present disclosure) and the light chain variable domain (referred to as "VL domain" in the present disclosure) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to the epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domain in conventional tetrapeptide chain structure antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by 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" are used interchangeably. "VHH domain" includes but is not limited to natural antibodies produced by camelids, and may also be antibodies produced by camelids that are then humanized, or may be obtained by phage display technology.

[0172] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. As used herein, "amino acid substitution" or "substitution" or "replacement" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution S32A refers to the replacement of serine at position 32 with alanine.

[0173] The multispecific antigen-binding molecules of the present disclosure may also include substitutions or modifications of the constant region (e.g., Fc), including but not limited to amino acid residue substitutions, mutations, and / or modifications, which result in compounds having preferred characteristics including but not limited to altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered Fc ligand binding to Fc receptors (FcRs), enhanced or reduced ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity. In certain aspects, the antibody variants comprise an Fc region having one or more amino acid substitutions that weaken FcγR binding (e.g., substitutions at positions 234 and 235 of the Fc region). In one aspect, the substitutions are L234A and L235A.

[0174] The term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, e.g., an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index.

[0175] The term "knob-into-hole" refers to a modification for promoting the association of the two polypeptide chains of Fc, which comprises a "knob" modification in one of the two polypeptide chains of Fc and a "hole" modification in the other of the two polypeptide chains of Fc. Generally, the method involves introducing a protrusion ("knob") at the interface of the first polypeptide chain and introducing a corresponding cavity ("hole") in the interface of the second polypeptide chain, so that the protrusion can be placed in the cavity to promote heterodimer formation and hinder homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide chain with larger side chains (such as tyrosine or tryptophan). A complementary cavity of the same or similar size as the protrusion is created in the interface of the second polypeptide chain by replacing the large amino acid side chains with smaller amino acid side chains (such as alanine or threonine).

[0176] 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 disclosure, one amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first polypeptide chain, which can be accommodated in a cavity within the CH3 domain of the second polypeptide chain, and in the CH3 domain of the second polypeptide chain of the Fc domain, one amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second polypeptide chain, in which the protuberance within the CH3 domain of the first polypeptide chain can be accommodated. Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0177] The term "linker" refers to any tool used to connect two different functional units (e.g., antigen binding fragments). The types of linkers include, but are not limited to, chemical linkers and polypeptide linkers. The sequence of the polypeptide linker is not limited. The polypeptide linker is preferably non-immunogenic and flexible, such as those comprising serine and glycine sequences. Depending on the specific construct, the linker can be long or short.

[0178] According to the present disclosure, the linker connecting the different functional units preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises the amino acid sequence (G4S) n or (G4S) n A, wherein n is any integer selected from 1 to 10, preferably comprises the amino acid sequence (G4S)3 or (G4S)3A. The linker connecting the VH and VL domains to form the scFv domain of VH-VL or VL-VH preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker. In one embodiment, the linker comprises the amino acid sequence (G4S) n or (G4S) n A, wherein n is any integer selected from 1 to 10, preferably comprises the amino acid sequence (G4S)3 or (G4S).

[0179] As used herein, "antibody" may be used in the broadest sense and may include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

[0180] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced by a single cell clone that is directed against a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or a combination of these techniques.

[0181] It should be noted that the CDR and FR divisions of the variable regions of the antibodies and trispecific antigen-binding molecules disclosed herein are determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies based on the antibody sequences disclosed herein that contain one or more CDRs derived from any naming system are clearly within the scope of this disclosure.

[0182] The term "humanized antibody" refers to an antibody in which all or part of the amino acids outside the CDRs of a non-human antibody (such as a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A "humanized" antibody retains antigenic specificity similar to that of the original antibody.

[0183] The term "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0184] The terms "homologous" and "heterogeneous" used in this article are relative concepts, which can refer to the fact that different elements in a construct have the same or different origins. They can also refer to the fact that after the construction of the construct is completed, some elements that originally had the same origin, i.e., "homologous", have been transformed and changed compared to other original elements that have not been transformed, thus becoming "heterogeneous".

[0185] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, a complete extracellular domain (ECD) or a protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that triggers an immune response. Any form of antigen or a cell or preparation containing the antigen can be used to generate an antibody specific for an antigenic determinant.

[0186] The terms "epitope" and "antigenic determinant" refer to the site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope can be formed by adjacent amino acids or by non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope typically exists in a unique spatial conformation and comprises at least 3-15 amino acids.

[0187] The term "multispecific" refers to an antigen binding molecule that is capable of specifically binding 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 are 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 a molecule comprising an antibody fragment) that is specific for three different antigens (or epitopes), particularly a trispecific antibody.

[0188] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.

[0189] When antibodies, binding molecules, bispecific binding molecules, or multispecific binding molecules are prepared using the variable regions of the present disclosure, the constant regions are not particularly limited, and constant regions known to those skilled in the art or obtained independently can be used. Amino acid mutations (e.g., mutations that increase or decrease binding to Fc receptors or FcRn) can also be introduced into the constant region.

[0190] The method for obtaining the binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules disclosed herein is not particularly limited and may be obtained by any method. The binding molecules, antigen-binding fragments, antibodies, bispecific binding molecules, or multispecific binding molecules of the invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid containing the secreted antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0191] The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0192] The term "stable transfection" or "stable transfection" refers to the introduction and integration of exogenous nucleic acid, DNA or RNA, into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated the foreign DNA into its genomic DNA.

[0193] The term "antibody drug conjugate" (ADC) refers to an antibody to which a therapeutically active substance or active pharmaceutical ingredient (API) has been covalently coupled so that the therapeutically active substance or active pharmaceutical ingredient (API) can be targeted to the binding target of the antibody to exhibit its pharmacological function. The therapeutically active substance or active pharmaceutical ingredient can be a cytotoxin that can kill cells targeted by the ADC, preferably malignant or cancerous cells. Covalent attachment of the therapeutically active substance, active pharmaceutical ingredient or cytotoxin can be performed in a non-site-specific manner using standard chemical linkers that couple payloads to lysine or cysteine ​​residues, or preferably, conjugation is performed in a site-specific manner, which allows complete control over the conjugation site and the drug-to-antibody ratio of the resulting ADC.

[0194] The term "amino acid substitution" or "substitution" or "replacement" means replacing the amino acid at a specific position in the parent polypeptide sequence with another amino acid.

[0195] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). d " refers to the dissociation constant for a specific antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, bio-layer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0196] The term "biological activity" refers to the ability of an antibody to bind to an antigen and result in a measurable biological response, which can be measured in vitro or in vivo.

[0197] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. When a "pharmaceutical composition" is present as a combination of separate formulations containing two or more different active ingredients, they can be administered simultaneously, sequentially, separately, or at intervals, with the goal of exerting the biological activities of the multiple active ingredients for combined treatment of a disease.

[0198] The binding molecules or antigen-binding fragments disclosed herein can be used in combination with other drugs, and the active ingredients can be mixed together to form a single administration unit, or can be independently formed into administration units and used separately.

[0199] The term "effective amount" refers to a dosage of a pharmaceutical formulation of an antibody or fragment of the present disclosure that produces the desired effect in a treated patient after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as ethnic differences; weight, age, and health status; the specific disease involved; the severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0200] As used herein, the term "individual" or "subject" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0201] As used herein, the terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, the term "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance defects (e.g., transplant rejection).

[0202] As used herein, the term "tumor" refers to a disease characterized by pathological proliferation of cells or tissues, and their subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation.

[0203] As used herein, the term "treatment" refers to clinical intervention aimed at altering the course of a disease in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, ameliorating or relieving the condition, and alleviating or improving the prognosis.

[0204] The terms "G protein coupled receptor C5 family subtype D" and "GPRC5D" specifically include human GPRC5D protein, and include variants, subtypes, homologous species and analogs of human GPRC5D that have at least one common epitope with GPRC5D (e.g., human GPRC5D). Exemplary human GPRC5D sequences can be found in GenBank Accession No. BC069341, NCBI Reference Sequence: NP_061124.1 and those described in UniProtKB / Swiss-Prot Accession No. Q9NZD1 (see also Brauner-Osborne, H et al., 2001, Biochim. Biophys. Acta 1518, 237-248).

[0205] The term "BCMA" refers to tumor-associated antigen B-cell maturation antigen, also known as TNFRSF17, and exemplary human BCMA sequences include the human BCMA protein under accession number UniProt Q02223. DETAILED DESCRIPTION

[0206] The present disclosure is further described below with reference to specific examples. It should be understood that these examples are intended only to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Experimental methods in the following examples, where specific conditions are not specified, are generally performed under conventional conditions known in the art or as recommended by the manufacturer.

[0207] Example 1. Human GPRC5D, GPRC5A and cynomolgus monkey GPRC5D antigen information

[0208] The full-length amino acid sequence of human GPRC5D (human-GPRC5D) used in the examples (SEQ ID NO: 1) (Uniprot ID: Q9NZD1) is shown below:

[0209] Note: This protein is a seven-transmembrane protein. The double-lined part is the extracellular region (1-27; 85-93; 145-167; 226-239); the underlined part is the intracellular region (49-63; 115-123; 189-204; 261-345); the italicized part is the transmembrane region (28-48; 64-84; 94-114; 124-144; 168-188; 205-225; 240-260).

[0210] The full-length amino acid sequence of cynomolgus monkey GPRC5D (cyno-GPRC5D) used in the examples (SEQ ID NO: 2) (Uniprot ID: A0A2K5W6I2) is shown below:

[0211] Note: This protein is a seven-transmembrane protein. The double-lined part is the extracellular region (1-27; 85-93; 145-167; 226-239); the underlined part is the intracellular region (49-63; 115-123; 189-204; 261-344); the italicized part is the transmembrane region (28-48; 64-84; 94-114; 124-144; 168-188; 205-225; 240-260).

[0212] The full-length amino acid sequence of human GPRC5A (SEQ ID NO: 36) (Uniprot ID: Q8NFJ5) used in the Examples is shown below:

[0213] Note: This protein is a seven-transmembrane protein. The double-lined part is the extracellular region (1-33; 90-97; 151-176; 234-247); the underlined part is the intracellular region (55-68; 119-129; 198-212; 269-357); the italicized part is the transmembrane region (34-54; 69-89; 98-118; 130-150; 177-197; 213-233; 248-268).

[0214] Example 2. Preparation of cell lines expressing human GPRC5D, human GPRC5A and cynomolgus monkey GPRC5D

[0215] The nucleotide sequence encoding the amino acids of human GPRC5D as shown in SEQ ID NO. 1 was cloned into the pCMV3 vector (SinoBiological, Catalog No. CV011) to obtain a plasmid for constructing a human GPRC5D cell line. This plasmid was transfected into CHOK1 cells (ATCC, Catalog No. CCL-61) to generate a CHOK1 cell line expressing human GPRC5D (abbreviated as: human GPRC5D-CHOK1 cell line).

[0216] The nucleotide sequence encoding the amino acids of cynomolgus monkey GPRC5D as shown in SEQ ID NO. 2 was cloned into the pCMV3 vector to obtain a plasmid for constructing the cynomolgus monkey GPRC5D cell line. This plasmid was transfected into CHOK1 cells to generate the CHOK1 cell line expressing cynomolgus monkey GPRC5D (abbreviated as "cynomolgus monkey GPRC5D-CHOK1 cell line").

[0217] The nucleotide sequence encoding the amino acids of human GPRC5A as shown in SEQ ID NO. 36 was cloned into the pCMV3 vector to obtain a vector for constructing a human GPRC5A cell line. This vector was then transfected into CHOK1 cells to generate a CHOK1 cell line expressing human GPRC5A (abbreviated as: human GPRC5A-CHOK1 cell line).

[0218] The expression of human GPRC5D in the human GPRC5D-CHOK1 cell line obtained above was detected using FACS assay, and a negative control hIgG1 LALA isotype (Bio-Ying Biotechnology, catalog number B109802) was set up in the experiment.

[0219] The expression of cynomolgus monkey GPRC5D in the cynomolgus monkey GPRC5D-CHOK1 cell line obtained above was detected using FACS assay, and a negative control hIgG1 LALA isotype (Bio-Ying Bio, Catalog No. B109802) was set up in the experiment.

[0220] The expression of human GPRC5A in the human GPRC5A-CHOK1 cell line obtained above was detected using FACS assay, and a negative control hIgG1 LALA isotype (Bio-Ying Biotechnology, Catalog No. B109802) was set up in the experiment.

[0221] The results show:

[0222] 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 antibody to human GPRC5D at the cellular level.

[0223] 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, which can be used in subsequent experiments to verify the binding of GPRC5D monoclonal antibody to cynomolgus monkey GPRC5D at the cellular level.

[0224] The expression of human GPRC5A in the human GPRC5A-CHOK1 cell line is shown in Figure 1C. The results indicate that human GPRC5A is well overexpressed in CHOK1 cells, which can be used in subsequent experiments to verify that the GPRC5D monoclonal antibody does not bind nonspecifically to human GPRC5A at the cellular level.

[0225] Example 3. Human BCMA and cynomolgus monkey BCMA antigen information

[0226] The full-length amino acid sequence of human BCMA (human-BCMA) (SEQ ID NO: 3) (Uniprot ID: Q02223) used in the Examples is shown below.

[0227] Note: This protein is a single-pass transmembrane protein. The double-line portion is the extracellular region (1-54); the wavy line portion is the transmembrane region (55-77); and the underlined portion is the intracellular region (78-184).

[0228] The full-length amino acid sequence (SEQ ID NO: 4) (Uniprot ID: G7Q0I4) of cynomolgus monkey BCMA (cyno-BCMA) used in the Examples is shown below.

[0229] Note: This protein is a single-pass transmembrane protein. The double-line portion is the extracellular region (1-53); the wavy line portion is the transmembrane region (54-76); and the underlined portion is the intracellular region (77-183).

[0230] Example 4. Preparation of human BCMA and cynomolgus monkey BCMA cell lines

[0231] The nucleotide sequence encoding the amino acids of human BCMA as shown in SEQ ID NO: 3 was cloned into the pCMV3 vector (SinoBiological, Catalog No. CV011) to obtain a vector for constructing a human BCMA cell line. This vector was transfected into CHOK1 cells (ATCC, Catalog No. CCL-61) to generate a CHOK1 cell line expressing human BCMA (abbreviated as: human BCMA-CHOK1 cell line).

[0232] The nucleotide sequence encoding the amino acids of cynomolgus macaque BCMA as shown in SEQ ID NO: 4 was cloned into the pCDH-CMV-MCS-EF1-Hygro (Honorgene, Catalog No. CD515B-1) vector to obtain a vector for constructing a cynomolgus macaque BCMA cell line. The obtained vector was constructed into a virus and then transfected into CHOK1 cells to obtain a CHOK1 cell line expressing cynomolgus macaque BCMA (abbreviated as "cynomolgus macaque BCMA-CHOK1 cell line").

[0233] The expression of human BCMA in the human BCMA-CHOK1 cell line obtained above was detected using FACS assay, and a negative control hIgG1 LALA isotype (Bio-Ying Bio, Catalog No. B109802) was set up in the experiment.

[0234] The expression of cynomolgus macaque BCMA in the cynomolgus macaque BCMA-CHOK1 cell line obtained above was detected using FACS assay, and a negative control hIgG1 LALA isotype (Bio-Ying Bio, Catalog No. B109802) was set up in the experiment.

[0235] The results show:

[0236] The expression of human BCMA in the human BCMA-CHOK1 cell line is shown in Figure 2A. The results indicate that human BCMA is well overexpressed in CHOK1 cells, which can be used in subsequent experiments to verify the binding of BCMA monoclonal antibodies to human BCMA at the cellular level.

[0237] The expression of cynomolgus monkey BCMA in the cynomolgus monkey BCMA-CHOK1 cell line is shown in Figure 2B. The results show that cynomolgus monkey BCMA is well overexpressed in CHOK1 cells, which can be used in subsequent experiments to verify the binding of BCMA monoclonal antibody and cynomolgus monkey BCMA at the cellular level.

[0238] Example 5. Design and sequence of anti-GPRC5D-BCMA-CD3 trispecific antibody

[0239] An anti-BCMA full-length antibody (BCMA mAb) was obtained through hybridoma screening, the heavy chain sequence of which is shown in SEQ ID NO: 9, and the light chain sequence of which is shown in SEQ ID NO: 7.

[0240] Anti-GPRC5D nanoantibodies (GPRC5D VHH) were screened and obtained by alpaca immunization, and the variable region sequence thereof is shown in SEQ ID NO:10.

[0241] The CD3ε binding domain is derived from the full-length 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 the CD3 mAb were connected via a flexible linker to form a single-chain antibody (scFv) with the structure: VH-(G4S)3-VL, and the sequence is shown in SEQ ID NO:13. The scFv was then fused to the C-terminus of the heavy chain of the Fab segment of the full-length BCMA antibody via a flexible linker.

[0242] CD3scFv was fused to the C-terminus of the BCMA Fab heavy chain CH1 to form one arm of the trispecific antibody. At the same time, another complete GPRC5D nanobody binding domain and an Fc part with a "knob" and "hole" structure were introduced. The resulting trispecific antibody was named Triantibody 1. The sequences of the three heterologous peptide chains after fusion are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. The schematic diagram is shown in Figure 3.

[0243] The CD3scFv was fused to the C-terminus of the heavy chain of the Fab segment of the BCMA full-length antibody to form one arm of the antibody, and the GPRC5D nanobody was fused to the C-terminus of the other Fab segment heavy chain of the BCMA full-length antibody to form the other arm of the antibody. It also contained an Fc part with a "knob" and "hole" structure. The resulting trispecific antibody was named Tri-Anti 2. 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.

[0244] To reduce the ADCC activity of the antibody, the Fc segment of the final trispecific antibody has undergone amino acid substitutions of L234A, L235A, and G237A. The Fc domain in the peptide chain containing the scFv is designed as a "knob" structure, including amino acid substitutions at two sites, S354C and T366W. The Fc domain in the peptide chain not containing the scFv is designed as a "hole" structure, including amino acid substitutions at four sites, Y349C, T366S, L368A, and Y407V. In addition, to facilitate the purification of the trispecific antibody, the heavy chain of the "hole" structure is also replaced with H435R.

[0245] The structures of Trial Antibody 1 and Trial Antibody 2 and their related molecular sequences are summarized in Tables 1 and 2, respectively.

[0246] Table 1 Structural description of trispecific antibodies

[0247] Table 2 Amino acid sequences of trispecific antibodies

[0248] Example 6. Construction of anti-GPRC5D-BCMA-CD3 trispecific antibody and its transient transfection expression in eukaryotic cells

[0249] The coding gene fragments of the aforementioned trispecific antibodies were cloned into the PTT5 expression vector to prepare transfection-grade expression plasmids.

[0250] Cultivation of Expi293F in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, it was further purified using a gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, collect the monomer peak, and exchange the solution into PBS for aliquoting. The final purified antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0251] 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 a GPRC5D binding site and a CD3 binding site) was also expressed and purified.

[0252] At the same time, Janssen's BCMAxCD3 bispecific antibody Teclistamab (derived from patent WO2019220368A1, consisting of sequences SEQ ID NO: 31, 32, 41, 42, containing a BCMA binding site and a CD3 binding site) was also expressed and purified.

[0253] At the same time, Janssen's BGCB491 trispecific antibody (derived from patent WO2022175255A2, consisting of sequences SEQ ID NO: 29, 30, 31, containing a GPRC5D binding site, a BCMA binding site and a CD3 binding site) was also expressed and purified.

[0254] At the same time, Innovent's TS-F2-5 trispecific antibody (derived from patent WO2022174813A1, consisting of SEQ ID NOs: 68, 75, 76, 78, containing a GPRC5D binding site, a BCMA binding site, and a CD3 binding site) was also expressed and purified.

[0255] Example 7. Cell-level affinity testing of anti-GPRC5D-BCMA-CD3 trispecific antibodies

[0256] FACS was used to detect the binding of the anti-GPRC5D-BCMA-CD3 trispecific antibody to NCI-H929 cells and RPMI8226 cells expressing human GPRC5D and human BCMA, human GPRC5D-CHOK1 cells expressing human GPRC5D, human BCMA-CHOK1 cells expressing human BCMA, T lymphocytes (Jurkat) naturally expressing hCD3, and human PBMCs.

[0257] NCI-H929 cells (ATCC, Catalog No. CRL-9068), RPMI8226 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 culture medium for NCI-H929 cells was RPMI1640 + 10% FBS + 0.05 mM mercaptoethanol, the culture medium for RPMI8226 and Jurkat cells was RPMI1640 + 10% FBS, and the culture medium for Human GPRC5D-CHOK1 and Human BCMA-CHOK1 cells was F12K + 10% FBS + 400 μg / ml Hygromycin B. The cells were cultured in a T75 cell culture flask at 37° C. in a 5% CO 2 incubator. When ready to use, place NCI-H929 cells, RPMI8226 cells, and Jurkat cells directly into 50 mL centrifuge tubes without digestion. Human GPRC5D-CHOK1 and Human BCMA-CHOK1 were trypsinized with 0.25% Trpsin-EDTA and digestion was terminated with F12K + 10% FBS + 400 μg / ml Hygromycin B.

[0258] The obtained 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 and adjusted to a cell density of 1E6 / mL. The cells were plated into a 96-well round-bottom culture plate (Corning, Cat. No. 3799) at a volume of 100 μL / well. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells resuspended in 200 μL of 1% BSA (in PBS). The cells were centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate stored at 4°C until ready to use. The test antibody and negative control hIgG1 LALA isotype (Bio-Ying Bio, Cat. No. B109802) were diluted in 1% BSA (in PBS) starting at 100 nM and diluted 5-fold downward to eight concentrations. Resuspend cells with diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash with 160 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Dilute secondary antibody (goat anti-human IgG Fc PE) 1:400 in 1% BSA (in PBS) according to the manufacturer's instructions and resuspend cells with 100 μL / well of the diluted secondary antibody and incubate at 4°C for 0.5 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend cells with 100 μL of 1% BSA (in PBS) and filter through 300-mesh gauze. Measure mean fluorescence intensity in the PE channel by flow cytometry.

[0259] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50 ) and the highest mean fluorescence intensity (Top MFI).

[0260] The results of the binding of the test molecules to tumor antigen cells are shown in Figures 4A-4H and Tables 3 and 4. On the dual-target expressed tumor cell lines NCI-H929 and RPMI8226, the binding of the triple antibody was stronger than that of the single-target dual antibody, and was positively correlated with the expression level of the tumor antigen (the dual-target expression of NCI-H929 cells was higher than that of RPMI8226 cells). On the cell lines expressing a single target, the binding of the triple antibody was weaker than or slightly stronger than the corresponding dual antibody. It is speculated that this result may be caused by the synergistic binding of the dual targets. In addition, on the dual-target expressed tumor cell lines, the binding of triple antibody 1 was stronger than that of Johnson & Johnson BGCB491 and Innovent TS-F2-5 molecules.

[0261] The results of the binding of the test molecules to Jurkat cells and human PBMC cells are shown in Figures 5A-5C and Tables 5 and 6. On Jurkat cells, the binding of the tertiary antibody was weaker than that of Teclistamab and comparable to that of Johnson & Johnson's BGCB491 and Innovent's TS-F2-5.

[0262] Table 3 Results of binding of trispecific antibodies to cells expressing tumor antigens

[0263] Table 4 Results of binding of trispecific antibodies to cells expressing tumor antigens

[0264] Table 5 Results of binding of trispecific antibodies to Jurkat cells and human PBMC cells

[0265] Table 6 Results of binding of trispecific antibodies to Jurkat cells

[0266] Example 8. Species Cross-Reactivity Test of Anti-GPRC5D-BCMA-CD3 Trispecific Antibodies

[0267] The 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 was detected by FACS.

[0268] Cynomolgus macaque GPRC5D-CHOK1 (Example 2) and cynomolgus macaque BCMA-CHOK1 cells (Example 4) were cultured in a medium containing F12K + 10% FBS + 400 μg / ml Hygromycin B in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When the cells were ready for use, cynomolgus macaque GPRC5D-CHOK1 and cynomolgus macaque BCMA-CHOK1 were trypsinized with 0.25% Trpsin-EDTA and digestion was terminated with F12K + 10% FBS + 400 μg / ml Hygromycin B.

[0269] The obtained 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 and adjusted to a cell density of 1E6 / mL. The cells were plated into a 96-well round-bottom culture plate (Corning, Cat. No. 3799) at a volume of 100 μL / well. The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the cells resuspended in 200 μL of 1% BSA in PBS. The cells were centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant discarded, and the plate stored at 4°C until ready to use. The test antibody and negative control hIgG1 LALA isotype (Bio-Ying Bio, Cat. No. B109802) were diluted in 1% BSA in PBS, starting at 100 nM and followed by 8 5-fold dilutions. Resuspend cells with diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash with 160 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Dilute secondary antibody (goat anti-human IgG Fc PE) 1:400 in 1% BSA (in PBS) according to the manufacturer's instructions and resuspend cells with 100 μL / well of the diluted secondary antibody and incubate at 4°C for 0.5 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Resuspend cells with 100 μL of 1% BSA (in PBS) and filter through 300-mesh gauze. Measure mean fluorescence intensity in the PE channel by flow cytometry.

[0270] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50) and the highest mean fluorescence intensity (Top MFI).

[0271] 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 triple antibody molecules have good binding to the corresponding tumor antigen of cynomolgus monkeys.

[0272] The results of the binding of the test molecules to cynomolgus monkey PBMC cells are shown in Figure 7 and Table 8. The triple antibody molecules bind well to cynomolgus monkey PBMC.

[0273] Table 7 Results of trispecific antibodies binding to cells expressing cynomolgus monkey tumor antigens

[0274] Table 8 Results of binding of trispecific antibodies to cyno PBMC cells

[0275] Example 9. In vitro recombinant protein binding affinity and kinetics of anti-GPRC5D-BCMA-CD3 trispecific antibodies

[0276] To determine the affinity and kinetic properties of human CD3 (purchased from Acro, Catalog No. CDD-H52W1) and cynomolgus monkey CD3 (purchased from Acro, Catalog No. CDD-C52W4), a CM5 chip was used to directly immobilize human / cynomolgus monkey CD3 molecules. The CM5 chip was first activated with EDC and NHS. Human / cynomolgus monkey CD3 molecules were diluted to 1 μg / mL in acetate solution, pH 5, and immobilized at a flow rate of 10 μL / min for 60 s. The anti-GPRC5D-BCMA-CD3 trispecific antibody was then blocked with ethanolamine. Two-fold dilutions of the anti-GPRC5D-BCMA-CD3 trispecific antibody were added to a concentration series (100 nM to 0.39 nM) in HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer. Binding was allowed for 90 s and dissociation for 360 s at a flow rate of 50 μL / min.

[0277] After each experimental round, the chip was rinsed with 3M MgCl₂ solution at a flow rate of 30 μL / min for 30 seconds to remove the anti-GPRC5D-BCMA-CD3 trispecific antibody molecules, completing chip regeneration. Raw data were analyzed using Biacore Insight Evaluation Software (3.0.12.15655) and fitted with a (1:1) Langmuir model. The resulting trispecific antibody affinity and kinetics data are shown in Table 9.

[0278] Table 9 Binding affinity and kinetics of anti-GPRC5D-BCMA-CD3 trispecific antibodies to human / cynomolgus monkey CD3 proteins

[0279] The experimental results showed that the two triple antibody molecules bound to both human and crab-eating macaque CD3 with comparable affinity.

[0280] To determine the affinity and kinetics of antibodies binding to human BCMA (purchased from Acro, Catalog No. BCA-H522y) and cynomolgus monkey BCMA (purchased from Acro, Catalog No. BCA-C52H7), an indirect capture assay was used on a CM5 chip. The CM5 chip was activated with EDC and NHS. Anti-human IgG (Fc) antibody (purchased from Cytiva, Catalog No. 10325009) was diluted to 10 μg / mL in 10 mM sodium acetate solution, pH 5, and immobilized for 420 s at a flow rate of 5 μL / min. The chip was then blocked with ethanolamine. The anti-GPRC5D-BCMA-CD3 trispecific antibody was 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 s at a flow rate of 10 μL / min. Human / cynomolgus monkey 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-0.195 nM). Binding was allowed for 90 s at a flow rate of 30 μL / min, and dissociation was allowed for 180 s.

[0281] After each experimental round, the chip was rinsed with 3M MgCl₂ solution at a flow rate of 30 μL / min for 30 seconds to remove the anti-GPRC5D-BCMA-CD3 trispecific antibody molecules, completing chip regeneration. Raw data were analyzed using Biacore Insight Evaluation Software (3.0.12.15655) and fitted with a (1:1) Langmuir model. The resulting trispecific antibody affinity and kinetics data are shown in Table 10.

[0282] Table 10 Binding affinity and kinetics of anti-GPRC5D-BCMA-CD3 trispecific antibodies to human / cynomolgus monkey BCMA proteins

[0283] The experimental results showed that the two triple antibody molecules bound to both human and crab-eating macaque BCMA with comparable affinity.

[0284] Example 10. In vitro functional assay of anti-GPRC5D-BCMA-CD3 trispecific antibody

[0285] AT cell-mediated cytotoxicity assay (TDCC), target cells are NCI-H929

[0286] Culture NCI-H929 cells (ATCC, Cat. No. CRL-9068) in RPMI 1640 medium with 10% FBS and 0.05 mM mercaptoethanol in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When ready to use, place the NCI-H929 cells directly into a 50 mL centrifuge tube without digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI 1640 medium with 2% FBS, count the cells, and adjust the cell density to 1E5 / mL.

[0287] Target cells were plated into a flat-bottom 96-well plate (corning, Cat. No. 3599) at 100 μL / well and incubated overnight at 37°C with 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and adjusted to a cell density of 2E6 / mL in RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C with 5% CO2.

[0288] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 100 nM and dilute 14-fold downwards into 10 concentrations. Add the diluted antibody to the cell culture plate at 50 μL / well and incubate at 37°C with 5% CO2 for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, Cat. No. 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0289] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0290] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0291] The cell killing data were imported into GraphPad Prism, and cell killing / concentration curves were plotted to calculate EC50 values. The results are shown in Table 11 and Figure 8A. Triple Antibody 1 demonstrated a stronger killing ability against NCI-H929 cells than BGCB491 and TS-F2-5.

[0292] BT cell-mediated cytotoxicity assay (TDCC), target cells are RPMI8226

[0293] Culture RPMI8226 cells (ATCC, Cat. No. CCL-155) in RPMI1640 + 10% FBS in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When ready to use, place the RPMI8226 cells directly into a 50 mL centrifuge tube without digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI1640 + 2% FBS, count the cells, and adjust the cell density to 1E5 / mL.

[0294] Target cells were plated into a flat-bottom 96-well plate (corning, Cat. No. 3599) at 100 μL / well and incubated overnight at 37°C with 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and adjusted to a cell density of 2E6 / mL in RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C with 5% CO2.

[0295] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 100 nM and dilute 14-fold downwards into 10 concentrations. Add the diluted antibody to the cell culture plate at 50 μL / well and incubate at 37°C with 5% CO2 for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, Cat. No. 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0296] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0297] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0298] The cell killing data were imported into GraphPad Prism, and cell killing / concentration curves were plotted to calculate EC50 values. The results are shown in Table 11 and Figure 8B. Triple Antibody 1 showed a stronger killing ability against RPMI8226 cells than BGCB491 and TS-F2-5.

[0299] CT cell-mediated cytotoxicity assay (TDCC), target cells are human BCMA-CHOK1

[0300] Human BCMA-CHOK1 (Example 4) was cultured in a T75 cell culture flask at 37°C in a 5% CO2 incubator using F12K + 10% FBS + 400 μg / ml Hygromycin B. When ready for use, trypsinize the human BCMA-CHOK1 cells with 0.25% Trpsin-EDTA and terminate the digestion with F12K + 10% FBS + 400 μg / ml Hygromycin B. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI1640 + 2% FBS, count the cells, and adjust the cell density to 1E5 / mL.

[0301] Target cells were plated into a flat-bottom 96-well plate (corning, Cat. No. 3599) at 100 μL / well and incubated overnight at 37°C with 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and adjusted to a cell density of 2E6 / mL in RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C with 5% CO2.

[0302] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 400 nM and dilute 10-fold downward. Add 50 μL of the diluted antibody to the cell culture plate at a starting concentration of 100 nM. Incubate at 37°C, 5% CO₂ for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, Cat. No. 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0303] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0304] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0305] The cell killing data were imported into GraphPad Prism, and cell killing / concentration curves were plotted and EC50 values ​​were calculated. The results are shown in Table 11 and Figure 8C. Triple Antibody 1 demonstrated comparable cytotoxicity against human BCMA-CHOK1 cells as BGCB491 and greater cytotoxicity than TS-F2-5.

[0306] DT cell-mediated cytotoxicity assay (TDCC), target cells are human GPRC5D-CHOK1

[0307] Human GPRC5D-CHOK1 (Example 2) was cultured in a T75 cell culture flask in a 37°C, 5% CO2 incubator. When ready for use, human BCMA-CHOK1 was trypsinized with 0.25% Trpsin-EDTA and digested with F12K, 10% FBS, and 400 μg / ml Hygromycin B. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant discarded, and the cells resuspended in RPMI1640 medium with 2% FBS. The cells were counted and adjusted to a cell density of 1E5 / mL.

[0308] Target cells were plated into a flat-bottom 96-well plate (corning, Cat. No. 3599) at 100 μL / well and incubated overnight at 37°C with 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and adjusted to a cell density of 2E6 / mL in RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C with 5% CO2.

[0309] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 100 nM and dilute it 5-fold downward to eight concentrations. Add 50 μL of the diluted antibody to the cell culture plate at 50 μL / well and incubate at 37°C, 5% CO2 for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, Cat. No. 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0310] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0311] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0312] The cell killing data were imported into GraphPad Prism, and cell killing / concentration curves were plotted and EC50 values ​​were calculated. The results are shown in Table 11 and Figure 8D. Triple Antibody 1 demonstrated comparable cytotoxicity against human GPRC5D-CHOK1 cells to TS-F2-5 and greater cytotoxicity than BGCB491.

[0313] ET cell-mediated cytotoxicity assay (TDCC), target cells are human BCMA-CHOK1 and human GPRC5D-CHOK1 mixed system

[0314] Human GPRC5D-CHOK1 and Human BCMA-CHOK1 cells were cultured in F12K + 10% FBS + 400 μg / ml Hygromycin B in T75 cell culture flasks at 37°C in a 5% CO2 incubator. When ready for use, trypsinize Human GPRC5D-CHOK1 and Human BCMA-CHOK1 with 0.25% Trpsin-EDTA and terminate digestion with F12K + 10% FBS + 400 μg / ml Hygromycin B. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI1640 + 2% FBS, count the cells, adjust the cell density to 1E5 / mL, and mix the two cells in equal proportions.

[0315] The target cell mixture was plated into a flat-bottom 96-well plate (corning, Cat. No. 3599) at 100 μL / well and incubated overnight at 37°C with 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and the cell density was adjusted to 1.6E6 / mL in RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C with 5% CO2.

[0316] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 100 nM and dilute 14-fold downwards into 10 concentrations. Add the diluted antibody to the cell culture plate at 50 μL / well and incubate at 37°C with 5% CO2 for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, Cat. No. 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0317] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0318] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0319] The cell killing data were imported into GraphPad Prism, and cell killing / concentration curves were plotted and EC50 values ​​were calculated. The results are shown in Table 11 and Figure 8E. Triple Antibody 1 demonstrated a stronger killing ability against the mixed cell system than BGCB491 and TS-F2-5.

[0320] Table 11 T cell-mediated cytotoxicity assay (TDCC), target cells are NCI-H929, RPMI8226, human BCMA-CHOK1, human GPRC5D-CHOK1, human BCMA-CHOK1 and human GPRC5D-CHOK1 mixed cell system

[0321] FT cell-mediated cytotoxicity assay (TDCC), target cells are NCI-H929, in the presence of 2500ng / ml soluble BCMA

[0322] Culture NCI-H929 cells (ATCC, Cat. No. CRL-9068) in RPMI 1640 medium with 10% FBS and 0.05 mM mercaptoethanol in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When ready to use, place the NCI-H929 cells directly into a 50 mL centrifuge tube without digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI 1640 medium with 2% FBS, count the cells, and adjust the cell density to 1E5 / mL. Add soluble BCMA to the cell suspension to a concentration of 2500 ng / mL.

[0323] Target cells containing soluble BCMA were plated into a flat-bottom 96-well plate (corning, Cat. No. 3599) at 100 μL / well and incubated overnight at 37°C with 5% CO2. CD3+ T cells were isolated from fresh PBMC using a T cell negative selection kit (StemCell, Cat. No. 17951). Cells were counted and adjusted to a cell density of 2E6 / mL using RPMI1640 + 2% FBS. Effector cells were plated into a 96-well plate at 50 μL / well and incubated at 37°C with 5% CO2.

[0324] Dilute the antibody in RPMI1640 medium with 2% FBS to a starting concentration of 100 nM and dilute 14-fold downward. Add 50 μL of the diluted antibody to the cell culture plate at a starting concentration of 100 nM. Incubate at 37°C, 5% CO₂ for 24 hours. Centrifuge the plate at 1000 rpm for 5 minutes. Transfer 50 μL of the supernatant to another flat-bottom 96-well plate. Add 50 μL of LDH detection reagent (Romos, Cat. No. 4744934001) to each well, mix thoroughly, centrifuge at 1000 rpm for 5 minutes, and incubate in the dark for 10 minutes. Read the OD492 value on an Envision plate.

[0325] The percentage of cell killing caused by the TDCC effect was calculated using the following formula:

[0326] % cell killing = [sample well - spontaneous release of T cells - spontaneous release of target cells) / (maximum lysis of target cells - spontaneous release of target cells)] * 100%.

[0327] The cell killing data were imported into GraphPad Prism, and cell killing / concentration curves were plotted to calculate EC50 values. The results are shown in Figure 9 (the dotted line indicates the condition of 2500ng / ml soluble BCMA) and Table 12. In the presence of 2500ng / ml soluble BCMA, the killing EC50 value of BGCB491 was reduced by approximately 23 times, the killing EC50 value of TS-F2-5 was basically maintained, and the killing EC50 value of triple antibody 1 was reduced by approximately 3 times. (The killing EC50 value of BCMA dual antibody Teclistamab was reduced by approximately 25 times)

[0328] Table 12 T cell-mediated cytotoxicity assay in the presence of 2500 ng / ml soluble BCMA

[0329] G. IL6 release levels when trispecific antibodies were co-incubated with PBMC and tumor cells NCI-H929 cells

[0330] Culture NCI-H929 cells (ATCC, Cat. No. CRL-9068) in RPMI1640 + 10% FBS + 0.05 mM mercaptoethanol in a T75 cell culture flask at 37°C in a 5% CO2 incubator. When ready to use, place NCI-H929 cells directly into a 50 mL centrifuge tube without digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in RPMI1640 + 2% FBS, count the cells, and adjust the cell density to 1E5 / mL. Plate NCI-H929 cells into a 96-well plate (corning, Cat. No. 3799) at 100 μL / well and incubate at 37°C in 5% CO2.

[0331] Purchase fresh PBMCs, count the cells, and adjust the cell density to 4E6 / mL using RPMI1640 + 2% FBS. Plate the effector cells into a 96-well plate at 50 μL / well and incubate at 37°C with 5% CO2.

[0332] Dilute the antibody in RPMI1640 + 2% FBS medium to a starting concentration of 100 nM and dilute 13-fold downward. Add 50 μL / well of the diluted antibody to the cell culture plate, for a starting and ending concentration of 100 nM. Incubate at 37°C, 5% CO2 for 24 hours.

[0333] The culture plate was centrifuged at 400 g for 10 minutes, and 100 μL of the supernatant was taken and frozen at -80°C for later use.

[0334] Dilute the standard sample according to the instructions of the cytokine assay kit (Biolegend, 430504 (ELISA MAX Deluxe Set Human IL-6)) in two-fold dilutions, with a maximum concentration of 8000 pg / mL and a minimum concentration of 0 pg / mL, for a total of 12 concentration points. Add the prepared standard sample or thawed sample to a 96-well plate at 100 μL / well (samples must be diluted separately) and read the OD450 value on an Envision plate according to the kit instructions.

[0335] The results are shown in Figures 10A-10B. When PBMCs were co-incubated with tumor cells, the level of IL6 cytokine release induced by Triple Antibody 1 was weaker than that induced by Triple Antibody 2.

[0336] Example 11. Stability test of anti-GPRC5D-BCMA-CD3 trispecific antibody

[0337] Differential fluorescence scanning technology was used to detect the thermal stability of the tertiary antibodies in pH 7.4 PBS buffer. The sample concentration was about 1 mg / mL, and the detection was performed using Prometheus NT.Plex (nano DSF). Before detection, each sample was centrifuged at 10,000 g for 10 minutes. 40 μL of sample was added to each well of the sample plate (the instrument loading volume was 10 μL, and each sample had one replicate well). The scanning temperature started from 30°C and ended at 95°C, with a scanning rate of 0.5°C / min. The experimental results are shown in Table 13. Both tertiary antibody molecules showed good thermal stability.

[0338] Table 13 NanoDSF detection results of anti-GPRC5D-BCMA-CD3 trispecific antibody

[0339] Example 12. Pharmacokinetics of anti-GPRC5D-BCMA-CD3 trispecific antibody

[0340] Two naive cynomolgus macaques were used in the experiment, with free access to water. The trispecific antibody was administered at a dose of 1 mg / kg via hindlimb intravenous injection. Blood was collected 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 in polyethylene tubes without anticoagulant, allowed to stand at room temperature for approximately 1 hour, centrifuged at 6000g at 25°C, immediately placed on dry ice, and transferred to a -80°C freezer for long-term storage.

[0341] The concentration of intact GPRC5D-BCMA-CD3 triple antibody in serum was determined by ELISA. A 96-well plate was coated with human GPRC5D protein at a concentration of 2 μg / mL, with 100 μL per well added and incubated at 4°C overnight. The plate was washed three times with 300 μL of PBST per well, and 300 μL of blocking reagent (5% milk powder) was added, followed by incubation at 37°C for 1 hour. The plate was washed three times with 300 μL of PBST per well, and 100 μL of the test sample was added, followed by incubation at 37°C for 1 hour. The plate was washed six times with 300 μL of PBST per well, and 100 μL of Biotin-BCMA reagent was added, followed by incubation at 37°C for 1 hour. The plate was washed six times with 300 μL of PBST per well, and 100 μL of SA-HRP reagent was added, followed by incubation at 37°C for 1 hour. The plate was washed six times with 300 μL of PBST per well, and 100 μL of TMB was added. After incubation in the dark for 10 minutes, 50 μL of stop solution was added to stop the color development reaction. The concentration of intact molecules of the GPRC5D-BCMA-CD3 triple antibody was quantitatively detected based on the color reaction.

[0342] The absorbance at a wavelength of 450 nm was detected using the Envision plate reader from PE, and the data were processed using softmax software.

[0343] Phoenix Winnolin 8.2 software was used to calculate the monkey serum concentration of the trispecific antibody.

[0344] The results showed that the pharmacokinetic properties of Tri-Anti-1 in monkeys were good, consistent with the conventional metabolic characteristics of large molecules.

[0345] Example 13. In vivo efficacy experiment of anti-GPRC5D-BCMA-CD3 trispecific antibody

[0346] A. Mouse model reconstructed with human immune cells, using NCI-H929 tumor cells

[0347] All experimental animals were housed in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 20.0-26.0℃, the humidity was 40-70%, and the light-dark cycle was 12h / 12h.

[0348] NCI-H929 cells (ATCC, catalog number CRL-9068) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 0.05 mM β-ME. Exponentially growing NCI-H929 cells were harvested and resuspended in 0.1 mL (1:1) PBS and Matrigel suspension. 5 × 10 cells were inoculated onto the right anterior dorsal surface of the experimental mice. 6 NCI-H929 cells were used to observe tumor growth regularly. 3 The mice were randomly divided into groups according to tumor size and body weight.

[0349] Donor PBMCs were purchased from AllCells, LLC. 1×10 7 PBMC / 0.1 mL PBS was inoculated intraperitoneally into each mouse on the day of NCI-H929 cell inoculation to establish a mouse model reconstructed with human immune cells.

[0350] Before dosing, all animals were weighed and tumor volume was measured with a vernier caliper. Given that tumor volume can affect treatment effectiveness, mice were randomly assigned to groups based on tumor volume to ensure similar tumor volumes across groups. Grouping was performed using StudyDirector™ (version 3.1.399.19, supplier: Studylog System, Inc., San Francisco, CA, USA) using the Matched Distribution method. The day mice were grouped was designated Day 0, and intraperitoneal injections were administered starting on Day 0, with dosing every three days for five doses. The experimental groups and dosing schedule are shown in Table 14.

[0351] Table 14 Experimental groups and dosing regimen

[0352] After administration, the animals' daily behavior was monitored for 14 days. During the entire experiment, the length and width of the tumors and the tumor volume (mm) were measured twice a week using a vernier caliper. 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2) was calculated. Relative tumor inhibition rate TGI (%): TGI% = (1-T / C) × 100%. T / C% is the relative tumor growth rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the hIgG1 LALA isotype control group at a certain time point. T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the hIgG1 LALA isotype control group at a specific time point, respectively. The experimental results of tumor volume, mouse body weight, tumor weight, etc. of each group of animals are expressed as mean ± standard error (Mean ± SEM). The independent sample T test was used to compare whether there were significant differences between different treatment groups and the control group. The data were analyzed using SPSS. P < 0.05 was considered to be significantly different. The experimental results are shown in Table 15 and Figure 11.

[0353] The 0.004 mg / kg treatment group of the triple antibody molecule demonstrated a highly significant tumor suppression effect (p-value less than 0.001) in the xenograft model of human myeloma NCI-H929 subcutaneously transplanted NPG female mice, significantly outperforming equimolar doses of the dual-antibody molecule teclistamab and the combination of teclistamab and talquetamab. The mice tolerated the triple antibody well, with no significant weight loss or toxicity.

[0354] Table 15 Tumor volume and tumor inhibition rate of each drug group

[0355] B. Human immune cells reconstructed mouse model, tumor cells are NCI-H929

[0356] All experimental animals were housed in independent ventilation boxes with constant temperature and humidity. The temperature of the breeding room was 20.0-26.0℃, the humidity was 40-70%, and the light-dark cycle was 12h / 12h.

[0357] NCI-H929 cells (ATCC, catalog number CRL-9068) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 0.05 mM β-ME. Exponentially growing NCI-H929 cells were harvested and resuspended in 0.1 mL (1:1) PBS and Matrigel suspension. 5 × 10 cells were inoculated onto the right anterior dorsal surface of the experimental mice. 6 NCI-H929 cells were used to observe tumor growth regularly. When the tumor grew to an average volume of 100 mm 3 The mice were randomly divided into groups according to tumor size and body weight.

[0358] Donor PBMCs were purchased from AllCells, LLC. 1×10 7PBMC / 0.2 mL PBS was inoculated intraperitoneally into each mouse on the day of NCI-H929 cell inoculation to establish a mouse model reconstructed with human immune cells.

[0359] Before dosing, all animals were weighed and tumor volume was measured with a vernier caliper. Given that tumor volume can affect treatment effectiveness, mice were randomly assigned to groups based on tumor volume to ensure similar tumor volumes across groups. Day 1 was defined as the day mice were grouped. Dosing was initiated on Day 1 with intraperitoneal injections, twice weekly for a total of four doses. The experimental groups and dosing schedule are shown in Table 16.

[0360] Table 16 Experimental groups and dosing regimen

[0361] After administration, the animals' daily behavior was monitored for 15 days. During the entire experiment, the length and width of the tumors and the tumor volume (mm) were measured twice a week using a vernier caliper. 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ) was calculated. Relative tumor inhibition rate TGI (%): TGI (%) = [1-(Vti-Vt0) / (Vci-Vc0)] 100%. T / C% is the relative tumor growth rate, that is, the percentage value of the relative tumor volume or tumor weight of the treatment group and the hIgG1 LALA isotype control group at a certain time point. T and C are the tumor volume (TV) or tumor weight (TW) of the treatment group and the hIgG1 LALA isotype control group at a specific time point, respectively. The experimental results of tumor volume, mouse body weight, tumor weight, etc. of each group of animals are expressed as mean + standard error (Mean+SEM). The independent sample T test was used to compare whether there were significant differences between different treatment groups and the control group. The data were analyzed using GraphPad prism 8.0.2. P<0.05 was considered to be significantly different. The experimental results are shown in Table 17 and Figures 12 and 13.

[0362] Different doses of Tri-Anti-1 demonstrated significant tumor suppression in the NCI-H929 subcutaneously transplanted NCG female mouse xenograft model (p values ​​less than 0.05). Tri-Anti-1 at 0.006 mg / kg significantly outperformed equimolar doses of BCGB491 and TS-F2-5. The mice tolerated the test drug well, with no significant weight loss or toxicity.

[0363] Table 17 Tumor volume and tumor inhibition rate of each drug group

[0364] Example 14. Construction of an antibody library containing only heavy chains from alpaca immunization

[0365] Alpacas were immunized with CHO-K1 cells that highly expressed human GPRC5D. The immunization dates were day 0, day 21, day 35, day 49, and day 70, for a total of 5 immunizations. Blood samples were drawn on day 28, day 42, and day 63 to separate the serum, and the immune response in the serum was detected using cell-level FACS. Immunization was terminated when the serum titer tended to the plateau phase, and 50 mL of blood samples were drawn from the immunized alpacas. Alpaca PBMCs were separated using Solarbio's lymphocyte separation solution according to the manufacturer's instructions, and total RNA was extracted (OMEGA Cell Total RNA Extraction Kit) and then analyzed using Takara PrimeScript TM II reverse transcription kit was used to synthesize cDNA as a template, and the VHH gene fragment was amplified by nested PCR using the designed specific primers. The VHH fragment was recovered and digested with Sfi I, ligated into the pADL-23c phagemid vector, and electroporated into TG1 electroporation competent cells to construct the GPRC5D alpaca immune library (library capacity 4.07E9).

[0366] Example 15. Screening of Alpaca-derived Anti-GPRC5D Positive Clones

[0367] To obtain antibodies that cross-bind to human GPRC5D and cynomolgus macaque GPRC5D, the above-mentioned library was amplified and assembled into phage using the M13K07 helper phage. Human GPRC5D-CHOK1 cell lines, cynomolgus macaque GPRC5D-CHOK1 cell lines, and CHOK1 cells were cultured to confluence, washed twice with PBS, and fixed with 100 μL of 4% paraformaldehyde 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. After washing three times with PBST, 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-conjugated anti-M13 antibody (diluted 1:10,000 in PBS) was added and incubated at 37°C for 1 hour. Wash the plate six times with 0.1% PBST. Add TMB colorimetric solution (100 μL / well) for development at 37°C for 7 min. Terminate the reaction with stop solution (50 μL / well) and measure the optical density at 450 nm. Sequence the positive clones to obtain the amino acid sequences of five heavy chain antibody variable regions (VHH): clone numbers HHC7, HHC11, HHC22, HHC53, and HHC78. The amino acid sequence of HHC78 is shown below:

[0368]

[0369] Based on the amino acid sequence of HHC78, the CDRs and FRs of the antibody variable region were divided using the Kabat numbering convention. The three CDR sequences of the antibody are shown in the following table:

[0370] Table 18

[0371] Example 16. Construction of Alpaca-derived Anti-GPRC5D Chimeric Antibody and Its Transient Transfection Expression in Eukaryotic Cells

[0372] The sequenced heavy chain antibody variable region of the present invention was spliced ​​with the human IgG1 constant region to generate the target gene fragment, which was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid. The heavy chain antibody variable region can be linked to the human IgG1 constant region via a linker peptide, forming the following sequence: heavy chain antibody variable region-linker peptide-human IgG1 constant region. The linker peptide sequence used in this embodiment is GGGGS.

[0373] The introduced human IgG1 constant region sequence (SEQ ID NO: 38) is as follows:

[0374] Cultivation of Expi293F in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified chimeric antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0375] Example 17. Binding of Alpaca-derived Anti-GPRC5D Chimeric Antibodies to Cells Expressing GPRC5D

[0376] Human GPRC5D-CHOK1 cells and cynomolgus macaque GPRC5D-CHOK1 cells were cultured in a medium containing F12K + 10% FBS + 400 μg / mL hygromycin in T75 cell culture flasks at 37°C in a 5% CO2 incubator. Before use, cells were washed twice with sterile DPBS and digested with 0.25% trypsin-EDTA for approximately 5 minutes before rinsing with complete culture medium.

[0377] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant discarded. Resuspend the cells in 200 μL of 1% BSA in PBS and centrifuge again at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the plate was stored at 4°C until ready to use. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold downward to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Wash the plate with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 in 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in this diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes. Discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0378] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50 ) and the highest mean fluorescence intensity (Top MFI), and the results are shown in Table 19 and Figure 14. The screened anti-GPRC5D heavy chain antibodies had good binding to human GPRC5D and cynomolgus monkey GPRC5D at the cellular level.

[0379] Table 19 Binding of anti-GPRC5D chimeric antibodies to cells expressing GPRC5D

[0380] Example 18. Humanized design of alpaca-derived anti-GPRC5D chimeric antibody

[0381] Through sequence alignment, germline gene sequences with high homology to the candidate heavy chain antibody were selected as VHH grafting framework templates. After the candidate antibody CDR regions were grafted onto the selected human antibody variable region framework, individual amino acid backmutations were performed to obtain humanized antibodies H1, H2, H3, H4, and H5. The sequence of H4 is shown in Table 20:

[0382] Table 20 Amino acid sequences of variable regions of humanized antibodies against GPRC5D from alpaca

[0383] Example 19. Preparation of humanized anti-GPRC5D antibodies from alpaca

[0384] Referring to Example 16, the target gene fragment generated by splicing the variable region of the humanized antibody and the constant region of human IgG1 was cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0385] Cultivation of Expi293F in serum-free medium TM Cells (Thermo Fisher Scientific) were seeded in shake flasks (Corning Inc.) and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified humanized antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0386] Example 20. Binding of Alpaca-derived Humanized Anti-GPRC5D Antibodies to Cells Expressing GPRC5D

[0387] The culture medium for NCI-H929 cells (ATCC, CRL-9068) was RPMI 1640 + 10% FBS + 0.05 mM mercaptoethanol, and the culture medium for cynomolgus macaque GPRC5D-CHOK1 cells was F12K + 10% FBS + 400 μg / mL Hygromycin. T75 cell culture flasks were cultured in a 37°C 5% CO2 incubator.

[0388] When using, NCI-H929 cells were directly transferred to a 50 mL centrifuge tube using a pipette. Cynomolgus monkey GPRC5D-CHOK1 cells were washed twice with sterile DPBS and digested with 0.25% trypsin-EDTA for approximately 5 minutes before terminating with complete culture medium.

[0389] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant discarded. Resuspend the cells in 200 μL of 1% BSA in PBS and centrifuge again at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the plate was stored at 4°C until ready to use. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold downward to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Wash the plate with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 in 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in this diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes. Discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0390] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50 ) and maximum mean fluorescence intensity (Top MFI), as 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, and the binding to NCI-H929 tumor cells was maintained or weakened compared to the parent antibody.

[0391] Table 21 Binding of anti-GPRC5D humanized antibodies to GPRC5D-expressing cells

[0392] Example 21. Binding of Alpaca-derived Humanized Anti-GPRC5D Antibody to the Same Family Member GPRC5A

[0393] The culture medium for human GPRC5A-CHOK1 cells is F12K + 10% FBS + 400 μg / mL Hygromycin. The cells are cultured in T75 cell culture flasks at 37°C in a 5% CO2 incubator. When ready for use, the cells are washed twice with sterile DPBS, digested with 0.25% trypsin EDTA for approximately 5 minutes, and then terminated with complete culture medium.

[0394] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant discarded. Resuspend the cells in 200 μL of 1% BSA in PBS and centrifuge again at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the plate was stored at 4°C until ready to use. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold downward to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Wash the plate with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 in 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in this diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes. Discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0395] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50 ) and the highest mean fluorescence intensity (Top MFI), and the results are shown in FIG16 , indicating that the tested antibodies had no non-specific binding to the same family member GPRC5A.

[0396] Example 22. Obtaining anti-BCMA antibodies from mouse hybridomas

[0397] Anti-BCMA monoclonal antibodies are produced by immunizing mice. The experiments used Balb / c mice, female, 6 weeks old (Charles River Company). Housing environment: SPF grade. After the mice were purchased, they were kept in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. The immunogen was human BCMA ECD protein (Yi Qiao Shenzhou, Cat. No. 10620-H08H), with 25 μg of protein each immunization on days 0, 14, 28, and 42. A booster immunization was performed 2 days before spleen cell fusion. During this period, the mouse serum was tested by ELISA to determine the antibody titer in the mouse serum. After the booster immunization, mice with high antibody titers in the serum and titers approaching a plateau were selected for spleen cell fusion, and the spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells (using an optimized electrofusion step). CRL-8287 TM ) were fused to obtain hybridoma cells.

[0398] After 7-14 days of culture of the fused hybridoma cells, the culture supernatant was taken and the hybridoma supernatant was screened for antibodies using CHO-K1 cells that highly express human BCMA. The positive antibody strains were further screened using CHO-K1 cells that highly express cynomolgus monkey BCMA and blank CHO-K1 cells to exclude non-specific binding antibody hybridoma strains, thereby selecting hybridomas that specifically bind to human / cynomolgus monkey BCMA. The hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM The cDNA obtained by reverse transcription was amplified by PCR using Mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced to obtain the amino acid sequences of the variable regions of the 11 monoclonal antibodies disclosed herein: 19CH-1, 19CH-2, 19CH-3, 19CH-4, 19CH-5, 19CH-7, 19CH-8, 19CH-9, 19CH-11, 19CH-13, and 19CH-16. 19CH-16 is shown in Table 22.

[0399] Table 22 Amino acid sequences of the variable regions of the anti-BCMA 19CH-16 monoclonal antibody derived from mouse hybridoma

[0400] Based on the above amino acid sequence, the CDRs and FRs of the antibody variable region were divided using the Kabat numbering convention. The sequence composition of the six CDRs of 19CH-16 is shown in Table 23 below.

[0401] Table 23 CDR sequences of anti-BCMA monoclonal antibodies derived from mouse hybridomas

[0402] Example 23. Construction of anti-BCMA chimeric antibodies derived from mouse hybridomas and their transient transfection expression in eukaryotic cells

[0403] The sequenced heavy chain variable region and light chain variable region of the monoclonal antibody disclosed herein were spliced ​​with the IgG1 (L234AL235A) heavy chain constant region and the κ light chain constant region to generate target gene fragments, which were cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0404] Expi293F was cultured in Expi293 expression medium (Thermo Fisher, A1435101). TM Cells (Thermo Fisher, A14527) were seeded in shake flasks and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Mabselect Sure column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified chimeric antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0405] Example 24. Binding of Mouse Hybridoma-Derived Anti-BCMA Chimeric Antibodies to BCMA-Expressing Cells

[0406] Both human BCMA-CHOK1 cells and cynomolgus macaque BCMA-CHOK1 cells were cultured in a medium containing F12K + 10% FBS + 400 μg / mL hygromycin in T75 cell culture flasks at 37°C in a 5% CO2 incubator. Before use, cells were washed twice with sterile DPBS and digested with 0.25% trypsin-EDTA for approximately 5 minutes before rinsing with complete culture medium.

[0407] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant discarded. Resuspend the cells in 200 μL of 1% BSA in PBS and centrifuge again at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the plate was stored at 4°C until ready to use. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold downward to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Wash the plate with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 in 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in this diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes. Discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0408] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50 ) and the highest mean fluorescence intensity (Top MFI), and the results are shown in Table 24 and Figure 17. One anti-BCMA chimeric antibody (19CH-16) screened out had good binding to both human BCMA and cynomolgus monkey BCMA at the cellular level.

[0409] Table 24 Binding of anti-BCMA chimeric antibodies derived from mouse hybridomas to BCMA-expressing cells

[0410] Example 25. Humanized Design of Mouse Hybridoma-Derived Anti-BCMA Antibodies

[0411] Based on the results of expression purification test and cell level binding test, the antibody with clone number 19CH-16 was selected for humanized design.

[0412] Humanization of murine anti-human BCMA monoclonal antibodies was performed using methods well-documented in the field. Briefly, human constant domains were substituted for the parental (murine antibody) constant domains, and human antibody sequences were selected based on homology between murine and human antibodies. Based on the representative murine antibody VH / VL CDR structures, the heavy and light chain variable region sequences were compared with a human antibody germline database to obtain human germline templates with high homology.

[0413] The CDR region of the mouse antibody is transplanted onto the selected corresponding humanized template to replace the humanized variable region, and then recombined with the IgG constant region (preferably IgG1 heavy chain and κ light chain). Then, based on the three-dimensional structure of the mouse antibody, back mutations were performed on the buried residues, residues directly interacting with the CDR regions, and residues that have an important influence on the conformation of VL and VH, and antibodies composed of the following combinations of humanized light and heavy chain variable region sequences were designed: 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, among which 19CH-16H2L2 is shown in Table 25.

[0414] Table 25 Amino acid sequence of a humanized antibody variable region derived from mouse hybridoma

[0415] Example 26. Preparation of humanized anti-BCMA antibodies derived from mouse hybridomas

[0416] The target gene fragments generated by splicing the heavy chain variable region and light chain variable region of the humanized antibody with the IgG1 (L234AL235A) heavy chain constant region and the κ light chain constant region were cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.

[0417] Expi293F was cultured in Expi293 expression medium (Thermo Fisher, A1435101). TMCells (Thermo Fisher, A14527) were seeded in shake flasks and cultured on a shaker at 37°C and 8% CO2. The cell density was adjusted, and the recombinant expression vector containing the target gene fragment and the PEI transfection reagent were mixed in an appropriate ratio and added to the cell culture shake flask. After 6 days of cell culture, the expression supernatant was collected, the cell debris was removed by high-speed centrifugation, and affinity purification was performed using a Mabselect Sure column. The column was rinsed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5 and neutralized with 1M Tris-HCl, pH 8.0-9.0. After the eluted sample was appropriately concentrated, the solution was exchanged into PBS for aliquoting. The final purified chimeric antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.

[0418] Example 27. Binding of Mouse Hybridoma-Derived Anti-BCMA Humanized Antibodies to BCMA-Expressing Cells

[0419] The culture medium for human BCMA-CHOK1 cells and cynomolgus macaque BCMA-CHOK1 cells was F12K + 10% FBS + 400 μg / mL Hygromycin. When ready for use, human BCMA-CHOK1 cells and cynomolgus macaque BCMA-CHOK1 cells were washed twice with sterile DPBS, digested with 0.25% trypsin EDTA for approximately 5 minutes, and then terminated with complete culture medium.

[0420] The resulting cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant discarded, and the cells resuspended in 100 μL of 1% BSA in PBS. Count the cells and adjust the cell density to 1E6 / mL. Plate the cells in a 96-well round-bottom culture plate (Corning 3799) and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant discarded. Resuspend the cells in 200 μL of 1% BSA in PBS and centrifuge again at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded and the plate was stored at 4°C until ready to use. Dilute the test antibody sample in 1% BSA in PBS to a starting concentration of 100 nM and dilute 10-fold downward to seven concentrations. Resuspend the cells in the diluted antibody at 100 μL / well and incubate at 4°C for 1 hour. Centrifuge at 1500 rpm for 5 minutes at 4°C and discard the supernatant. Wash the plate with 160 μL of 1% BSA in PBS and centrifuge at 1500 rpm for 5 minutes at 4°C. The supernatant was discarded. Dilute the secondary antibody (goat anti-human IgG Fc PE) 1:400 in 1% BSA (in PBS) according to the manufacturer's instructions. Resuspend the cells in this diluted secondary antibody at 100 μL / well and incubate at 4°C for 0.5 hours. Centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend and wash with 200 μL of 1% BSA (in PBS) and centrifuge at 1500 rpm at 4°C for 5 minutes. Discard the supernatant. Resuspend the cells in 100 μL of 1% BSA (in PBS) and filter through a 300-mesh gauze. Measure the mean fluorescence intensity in the PE channel by flow cytometry.

[0421] The FCS file was exported from the flow cytometer, and the mean fluorescence intensity (MFI) of the PE channel of each sample was analyzed using FlowJo software. The mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cell (EC 50 ) and the highest mean fluorescence intensity (Top MFI), as shown in Table 26 and Figure 18. The anti-BCMA humanized antibody (19CH-16H2L2) prepared and screened in Example 26 had good binding to both human BCMA and cynomolgus monkey BCMA at the cellular level.

[0422] Table 26 Binding of anti-BCMA humanized antibodies derived from mouse hybridomas to BCMA-expressing cells

[0423] Example 28. Preparation of variants of mouse hybridoma-derived anti-BCMA humanized antibodies

[0424] Post-translational modification (PTM) analysis of the CDR region of the anti-BCMA humanized antibody 19CH-16H2L2 revealed a deamidation site in the heavy chain variable region. To mitigate potential risks, site-directed mutagenesis of the 19CH-16H2L2 amino acid sequence was performed at a single site, resulting in two variants: 19CH-16H2L2-NA and 19CH-16H2L2-QT. The variable region amino acid sequences of the 19CH-16H2L2-NA variant are shown in Table 27.

[0425] Table 27 Amino acid sequences of 19CH-16H2L2 humanized antibody variants

[0426] The two variant proteins were prepared by transient expression in Expi293 cells, as described in Example 26. The affinity of the two variants was determined using human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells, as described in Example 27. The results are shown in Table 28 and Figure 19. 19CH-16H2L2-NA maintains cell-binding affinity while eliminating the risk of post-translational modifications.

[0427] Table 28 Binding of 19CH-16H2L2 humanized antibody variants to BCMA-expressing cells

[0428] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

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

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

3. The trispecific antigen-binding molecule of claim 1 or 2, wherein the first Fc domain comprises a first CH2 domain and a first CH3 domain of an immunoglobulin, and the C-terminus of the first CH2 domain is fused to the N-terminus of the first CH3 domain; the second Fc domain comprises a second CH2 domain and a second CH3 domain of an immunoglobulin, and the C-terminus of the second CH2 domain is fused to the N-terminus of the second CH3 domain; Preferably, the first CH3 domain comprises a "knob" structure, and the second CH3 domain comprises a "hole" structure; more preferably, the "knob" structure comprises amino acid substitutions S354C and T366W, and the "hole" structure comprises amino acid substitutions Y349C, T366S, L368A and Y407V; Preferably, the first and / or second Fc domain comprises an amino acid substitution of L234A, L235A and / or G237A; Preferably, the second Fc domain comprises an amino acid substitution of H435R; Preferably, the single-chain antibody (scFv) domain is connected to the first Fc domain via a second linker, wherein the C-terminus of the single-chain antibody (scFv) domain is fused to the N-terminus of the second linker, and the C-terminus of the second linker is fused to the N-terminus of the first Fc domain; More preferably, the second linker comprises the amino acid sequence EPKSS; Preferably, the Fc domain is derived from IgG1.

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

5. The trispecific antigen-binding molecule according to any one of claims 1 to 4, wherein the C-terminus of the heavy chain single-domain antibody (VHH) domain is fused to the N-terminus of the 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.

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

7. The trispecific antigen-binding molecule of any one of claims 1 to 6, wherein the second antigen is CD3, preferably CD3ε; preferably, the single-chain antibody (scFv) domain comprises a HCDR1 as shown in SEQ ID NO: 27, a HCDR2 as shown in SEQ ID NO: 28, a HCDR3 as shown in SEQ ID NO: 29, a LCDR1 as shown in SEQ ID NO: 30, a LCDR2 as shown in SEQ ID NO: 31, and a LCDR3 as shown in SEQ ID NO:

32. More preferably, the single-chain antibody (scFv) domain comprises a heavy chain variable region having a sequence as shown in SEQ ID NO: 25 and a light chain variable region having a sequence as shown in SEQ ID NO: 26; More preferably, the single-chain antibody (scFv) domain comprises the amino acid sequence shown in SEQ ID NO:

13.

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 trispecific antigen-binding molecule according to any one of claims 1 to 8, wherein the first antigen is BCMA; preferably, the antigen-binding fragment Fab comprises a HCDR1 with a sequence as shown in SEQ ID NO: 16, a HCDR2 with a sequence as shown in SEQ ID NO: 17, and a HCDR3 with a sequence as shown in SEQ ID NO: 18; preferably, the antigen-binding fragment Fab comprises a LCDR1 with a sequence as shown in SEQ ID NO: 19, a LCDR2 with a sequence as shown in SEQ ID NO: 20, and a LCDR3 with a sequence as shown in SEQ ID NO: 21; More preferably, the antigen-binding fragment Fab heavy chain domain comprises a heavy chain variable region with a sequence as shown in SEQ ID NO: 14; the antigen-binding fragment Fab light chain domain comprises a light chain variable region with a sequence as shown in SEQ ID NO: 15; More preferably, the Fab heavy chain domain of the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 35; more preferably, the Fab light chain domain of the antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:

7.

10. The trispecific antigen-binding molecule according to any one of claims 1 to 9, wherein the third antigen is GPRC5D; preferably, the heavy chain single domain antibody (VHH) domain capable of specifically binding to the third antigen comprises a HCDR1 as shown in SEQ ID NO: 22, a HCDR2 as shown in SEQ ID NO: 23, and a HCDR3 as shown in SEQ ID NO: 24; more preferably, the heavy chain single domain antibody (VHH) domain comprises the sequence shown in SEQ ID NO:

10.

11. The trispecific antigen-binding molecule of any one of claims 1 to 10, wherein the first polypeptide of the trispecific antigen-binding molecule comprises the amino acid sequence shown in SEQ ID NO: 5, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6; or optionally, the first polypeptide of the trispecific antigen-binding molecule comprises the sequence shown in SEQ ID NO: 5, the second polypeptide and / or the fourth polypeptide comprises the sequence shown in SEQ ID NO: 7, and the third polypeptide comprises the sequence shown in SEQ ID NO:

8.

12. A trispecific antigen-binding molecule, which can bind to an epitope: The epitope is the same as or overlaps with that directed by an antibody comprising HCDR1 as shown in SEQ ID NO:22, HCDR2 as shown in SEQ ID NO:23, and HCDR3 as shown in SEQ ID NO:

24.

13. The trispecific antigen binding molecule of claim 12, wherein the epitope it binds to is: The epitope is the same as or overlaps with the epitope directed by the antibody comprising the amino acid sequence shown in SEQ ID NO:

10.

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

15. A trispecific antigen-binding molecule comprising: (A) a first binding moiety capable of specifically binding to GPRC5D3; (B) a second engaging portion; and (C) a third binding moiety, wherein the first, second and third binding moieties specifically bind to different antigens or epitopes; The first binding portion comprises: The sequence of HCDR1 is shown in SEQ ID NO:22, the sequence of HCDR2 is shown in SEQ ID NO:23, and the sequence of HCDR3 is shown in SEQ ID NO:

24.

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

10.

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

18. A nucleic acid molecule encoding a trispecific antigen binding molecule according to any preceding claim.

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

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

21. A method for preparing a trispecific antigen binding molecule according to any one of claims 1 to 17, the method comprising: The host cell of claim 20 is cultured 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. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable carrier.

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 according to any one of claims 1 to 17, the nucleic acid molecule according to claim 18, the expression vector according to claim 19 and / or the host cell according to claim 20 in the preparation of a drug for treating, alleviating and / or preventing tumors.

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

27. The use according to claim 25 or 26, wherein the tumor is selected from: lymphoma such as multiple myeloma, and metastatic cancer of the above tumors.

28. A method for inducing cell death expressing GPRC5D and / or BCMA, the method comprising contacting the cell with the trispecific antigen binding molecule of any one of claims 1-17, 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 22-24, preferably, the cell expressing GPRC5D and / or BCMA is a tumor cell.

29. The method of claim 28, wherein the tumor cell is a cell selected from the group consisting of a lymphoma such as multiple myeloma, and metastases thereof.

30. A method for treating a disease associated with expression of GPRC5D and / or BCMA in a subject, the method comprising administering to a subject in need thereof the trispecific antigen binding molecule of any one of claims 1-17, 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 22-24.

31. The method of claim 30, wherein the disease is a tumor; Preferably, lymphomas such as multiple myeloma, and metastatic cancers of the above tumors.

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 an antigen-binding fragment thereof, characterized in that: The GPRC5D antigen binding molecule or its antigen binding fragment comprises the HCDR sequence of the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:10; preferably, the GPRC5D antigen binding molecule or its antigen binding fragment comprises a HCDR1 with a sequence as shown in SEQ ID NO:22, a HCDR2 with a sequence as shown in SEQ ID NO:23 and a HCDR3 with a sequence as shown in SEQ ID NO:24; preferably, the GPRC5D antigen binding molecule or its antigen binding fragment comprises an amino acid sequence comprising a sequence as shown in SEQ ID NO:

10.

34. The GPRC5D antigen binding molecule or antigen binding fragment thereof of claim 33, further comprising one or more of the following features: i) it further comprises a heavy chain constant region; preferably, the heavy chain constant region comprises Fc; more preferably, Fc is derived from mouse or human; more preferably, the sequence of Fc is natural or modified; further preferably, the Fc domain of IgG1 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 hybrid molecule, a murine antibody, a humanized antibody, a chimeric antibody, a reshaped antibody, a fully human antibody, a full-length antibody, a heavy chain antibody, a nanobody, Fab, Fv, scFv, F(ab')2, a linear antibody and / or a heavy chain single domain antibody (VHH); and / or iii) it is in the form of IgG1, IgG2, IgG3 or IgG4.

35. The GPRC5D antigen binding molecule or antigen binding fragment thereof of claim 35 or 34, wherein the GPRC5D antigen binding molecule or antigen binding fragment thereof comprises a heavy chain single domain antibody (VHH) domain and an Fc domain of IgG1, preferably, the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of the Fc domain of IgG1, more preferably, the C-terminus of the heavy chain single domain antibody (VHH) domain is fused to the N-terminus of the Fc domain of IgG1 via a linker, preferably, the linker comprises the amino acid sequence EPKSS or (G4S) n , n is any integer between 1 and 10. Preferably, the GPRC5D antigen binding molecule or antigen binding fragment thereof comprises, from N-terminus to C-terminus, a VHH domain with a sequence as shown in SEQ ID NO: 10, a G4S linker and an IgG1 Fc domain with a sequence as shown in SEQ ID NO:

38.

36. A conjugate or fusion protein, characterized in that: The GPRC5D antigen binding molecule or its antigen binding fragment described in any one of claims 33-35 is coupled to a capture marker or a detection marker; preferably, the detection marker comprises a radionuclide, a luminescent substance, a colored substance or an enzyme, or a fused portion of the fusion protein comprises the GPRC5D antigen binding molecule or its antigen binding fragment described in any one of claims 33-35.

37. An antibody-drug conjugate, characterized in that: The GPRC5D antigen binding molecule or its antigen binding fragment according to any one of claims 33 to 35 is coupled with other biologically active molecules; preferably, the other biologically active molecules are small molecule drugs; preferably, the GPRC5D antigen binding molecule or its antigen binding fragment is connected to the other biologically active molecules via a linker.

38. A nucleic acid encoding the GPRC5D antigen binding molecule or its antigen binding fragment described in any one of claims 33-35, or a recombinant vector comprising the nucleic acid, or a host cell comprising the nucleic acid or the recombinant vector; preferably, the host cell is a prokaryotic cell (preferably Escherichia coli), or a eukaryotic cell (preferably a mammalian cell or yeast; further preferably, the mammalian cell is a CHO cell or a HEK293 cell).

39. A method for preparing the GPRC5D antigen binding molecule or antigen binding fragment thereof according to any one of claims 33 to 35, the method comprising: The host cell of claim 38 is cultured under suitable conditions, and the expression product is purified from the cell.

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

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

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

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

44. A chimeric antigen receptor (CAR) or a cell comprising the chimeric antigen receptor, comprising the GPRC5D antigen binding molecule or antigen binding fragment thereof as described in any one of claims 33-35.

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

46. ​​A method of treating a disease associated with expression of GPRC5D in a subject, the method comprising administering to a subject in need thereof a pharmaceutical composition as claimed in claim 43; Preferably, the disease is a tumor; preferably, the tumor disease is selected from lymphoma such as multiple myeloma, and metastatic cancer of the above tumors; More preferably, the method further comprises administering an additional therapeutic agent to the subject.

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