WO2024165031A1 - 三特异性抗原结合分子及其应用 - Google Patents

三特异性抗原结合分子及其应用 Download PDF

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WO2024165031A1
WO2024165031A1 PCT/CN2024/076521 CN2024076521W WO2024165031A1 WO 2024165031 A1 WO2024165031 A1 WO 2024165031A1 CN 2024076521 W CN2024076521 W CN 2024076521W WO 2024165031 A1 WO2024165031 A1 WO 2024165031A1
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antigen
domain
antibody
binding
seq
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French (fr)
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杨柳青
钱宏亮
罗娟
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Shanghai Qilu Pharmaceutical Research and Development Centre Ltd
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Shanghai Qilu Pharmaceutical Research and Development Centre Ltd
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Priority to EP24752886.2A priority Critical patent/EP4663662A1/en
Priority to AU2024218657A priority patent/AU2024218657A1/en
Priority to CN202480008241.2A priority patent/CN120603856A/zh
Priority to KR1020257026203A priority patent/KR20250141153A/ko
Priority to JP2025545874A priority patent/JP2026504747A/ja
Publication of WO2024165031A1 publication Critical patent/WO2024165031A1/zh
Priority to MX2025009228A priority patent/MX2025009228A/es
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure belongs to the field of immunology, and mainly 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 a T cell surface antigen CD3, as well as a pharmaceutical composition, preparation method and use thereof.
  • MM Multiple myeloma
  • MM is a malignant tumor of the blood system and the second most common hematological malignancy after non-Hodgkin's lymphoma.
  • Multiple myeloma can damage bones, the immune system, kidneys and red blood cells, usually leading to extensive bone destruction, accompanied by osteolytic lesions, osteopenia and pathological fractures.
  • the disease is common in middle-aged and elderly people. With the acceleration of aging and the advancement of diagnosis and treatment methods, the patient population is showing a rapid growth trend.
  • BCMA B cell maturation antigen
  • BAFF B cell activating factor
  • APRIL a proliferation-inducing ligand
  • BCMA can stimulate the proliferation of malignant plasma cells and maintain their activity through the intracellular AKT, MAPK and (NF)- ⁇ B signaling pathways, thereby promoting the progression of the disease. Blocking the BCMA signaling pathway can inhibit the proliferation of malignant plasma cells and inhibit/alleviate the progression of the disease. Therefore, BCMA is a good target for the clinical treatment of MM.
  • the BCMA protein on the cell membrane can also be cleaved by the ⁇ -secretase in the body. The cleaved BCMA protein is in a free state 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 and membrane-bound BCMA, and preventing the BCMA signaling pathway.
  • GPRC5D is a G protein coupled receptor C5 family subtype D, an orphan Receptor, a 7-transmembrane protein. GPRC5D is specifically highly expressed in plasma cells of multiple myeloma, and only in hair follicles in normal tissues. Antibodies and CAR-T cell therapies targeting GPRC5D have shown optimistic effects in preclinical trials, and animals did not experience hair loss in the experiment. Moreover, the expression of GPRC5D is not limited to the expression of BCMA. In the tumor recurrence model caused by BCMA antigen loss, GPRC5D-targeted CAR-T therapy can overcome tumor escape. These research results all indicate that GPRC5D is an ideal target clinically.
  • TAA tumor-associated antigens
  • CD3 antigens can bring T cells and tumor cells closer in space to form synapses, and use T cells to achieve specific killing of tumor cells.
  • TCE bispecific antibody T cell engager
  • TCE-based treatment strategies rely on the distribution of TAA on the tumor cells to be treated.
  • studies have also shown that treatment forms targeting a single TAA site may cause disease recurrence due to the tumor's escape mechanism, thereby limiting the effectiveness of treatment.
  • the present disclosure provides a trispecific antigen binding molecule targeting GPRC5D/BCMA/CD3, and conducts in-depth research on various molecular configurations, successfully achieving a multi-target combination and avoiding the inconvenience of multi-drug combination.
  • the first aspect of the present disclosure is to provide a trispecific antigen binding molecule.
  • the trispecific antigen binding molecule comprises the following polypeptides:
  • a first polypeptide comprising: (i) a heavy chain domain of an antigen-binding fragment Fab capable of specifically binding to a first antigen, (ii) a single-chain antibody (scFv) domain capable of specifically binding to a second antigen, and (iii) a first Fc domain;
  • a second polypeptide comprises: a light chain domain of an antigen-binding fragment Fab capable of specifically binding to a first antigen;
  • 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.
  • VHH heavy chain single domain antibody
  • 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;
  • the first Fc domain and the second Fc domain are associated with each other.
  • 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 Fab of the third polypeptide and the light chain domain of the Fab of the fourth polypeptide form a fourth binding site for the first antigen.
  • a fourth polypeptide which comprises a light chain domain of an antigen-binding fragment Fab that specifically
  • the scFv domain comprises a heavy chain variable region and a light chain variable region; preferably, the heavy chain variable region of the scFv domain
  • the heavy chain variable region is connected to the light chain variable region through 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.
  • the first linker comprises an amino acid sequence (G 4 S) n , where n is any integer from 1-10.
  • 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.
  • the first CH3 domain comprises a "knob” structure
  • the second CH3 domain comprises a "hole” structure
  • the "knob” structure comprises amino acid substitutions S354C and T366W
  • the "hole” structure comprises amino acid substitutions Y349C, T366S, L368A and Y407V.
  • the first and/or second Fc domain comprises the amino acid substitutions of L234A, L235A and/or G237A.
  • the second Fc domain of the third polypeptide comprises an amino acid substitution of H435R.
  • the Fc domain is derived from IgG1.
  • 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.
  • the heavy chain domain of the antigen-binding fragment Fab 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 light chain domain of the antigen-binding fragment Fab 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.
  • 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.
  • the third linker comprises an amino acid sequence (G 4 S) n , where n is any integer from 1-10.
  • 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.
  • the fifth linker comprises an amino acid sequence (G 4 S) n , where n is any integer from 1-10.
  • 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.
  • the first polypeptide comprises the following structure: Fab heavy chain domain-third linker-scFv domain-second linker-first Fc domain.
  • the first polypeptide comprises the following structure: Fab heavy chain variable region-Fab CH1-third linker-scFv heavy chain variable region-first linker-scFv Light chain variable region-second linker-first CH2-first CH3.
  • the second polypeptide comprises the following structure: Fab light chain variable region-light chain constant region.
  • the third polypeptide comprises the following structure: VHH domain-fourth linker-second Fc domain.
  • the third polypeptide comprises the following structure: VHH-fourth linker-second CH2-second CH3.
  • the fourth polypeptide comprises the following structure: Fab light chain variable region-light chain constant region
  • the third polypeptide comprises the following structure: Fab heavy chain domain-fifth linker-VHH-fourth linker-second Fc domain
  • the third polypeptide comprises the following structure: Fab heavy chain variable region-Fab CH1-fifth linker-VHH-fourth linker-second CH2-second CH3.
  • the second antigen is CD3, preferably CD3 ⁇ ; preferably, the scFv domain comprises HCDR1 as shown in SEQ ID NO: 27, HCDR2 as shown in SEQ ID NO: 28, HCDR3 as shown in SEQ ID NO: 29, LCDR1 as shown in SEQ ID NO: 30, LCDR2 as shown in SEQ ID NO: 31 and LCDR3 as shown in SEQ ID NO: 32.
  • the scFv domain comprises a heavy chain variable region as shown in SEQ ID NO: 25 and a light chain variable region as shown in SEQ ID NO: 26.
  • the scFv domain comprises the amino acid sequence shown in SEQ ID NO: 13.
  • the first Fc domain comprises the amino acid sequence shown in SEQ ID NO: 33
  • the second Fc domain comprises the amino acid sequence shown in SEQ ID NO: 34.
  • the first antigen is BCMA; preferably, the antigen binding fragment Fab comprises HCDR1 with a sequence as shown in SEQ ID NO: 16, HCDR2 with a sequence as shown in SEQ ID NO: 17 and HCDR3 with a sequence as shown in SEQ ID NO: 18, and/or LCDR1 with a sequence as shown in SEQ ID NO: 19, LCDR2 with a sequence as shown in SEQ ID NO: 20 and LCDR3 with a sequence as shown in SEQ ID NO: 21.
  • the heavy chain domain of the antigen-binding fragment Fab comprises a heavy chain variable region whose sequence is shown in SEQ ID NO: 14, and/or the light chain domain of the antigen-binding fragment Fab comprises a light chain variable region whose sequence is shown in SEQ ID NO: 15.
  • 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.
  • the third antigen is GPRC5D; preferably, the heavy chain single domain antibody (VHH) domain capable of specifically binding to the third antigen comprises HCDR1 with a sequence as shown in SEQ ID NO:22, HCDR2 with a sequence as shown in SEQ ID NO:23 and HCDR3 with a sequence as shown in SEQ ID NO:24; more preferably, the VHH domain comprises the sequence shown in SEQ ID NO:10.
  • VHH heavy chain single domain antibody
  • 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
  • the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 6
  • 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
  • the third polypeptide comprises the sequence shown in SEQ ID NO: 8.
  • the present disclosure also provides nucleic acid molecules encoding the trispecific antigen-binding molecules described in the present disclosure.
  • the present disclosure also provides a vector comprising the nucleic acid molecule.
  • the present disclosure also provides a host cell, which comprises 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.
  • the present disclosure also provides a method for preparing a trispecific antigen binding molecule, the method comprising: culturing the host cell under suitable conditions.
  • 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.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned trispecific antigen-binding molecule, nucleic acid molecule, expression vector, host cell and/or antibody-drug conjugate.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more additional therapeutic agents.
  • 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 drugs for treating, alleviating and/or preventing tumors.
  • the tumor is a GPRC5D-positive and/or BCMA-positive tumor.
  • 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.
  • the present disclosure also provides a method for treating a disease associated with expressing 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.
  • the disease is a tumor.
  • the subject is recurrent or refractory to treatment with a previous anticancer therapeutic agent.
  • the method further comprises administering an additional therapeutic agent to the subject.
  • the tumor or tumor cell of the present disclosure is selected from: lymphoma such as multiple myeloma, and metastatic cancer of the above tumors.
  • 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.
  • the antigen binding molecule or its antigen binding fragment that specifically binds to GPRC5D comprises a heavy chain single domain antibody (VHH) domain; preferably, the GPRC5D antigen binding molecule or its antigen binding fragment comprises the HCDR sequence of the heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:10; preferably, the GPRC5D antigen binding molecule or its antigen binding fragment comprises a HCDR1 whose sequence is shown in SEQ ID NO:22, a HCDR2 whose sequence is shown in SEQ ID NO:23 and a HCDR3 whose sequence is shown in SEQ ID NO:24; more preferably, the heavy chain single domain antibody (VHH) comprises the amino acid sequence shown in SEQ ID NO:10.
  • VHH heavy chain single domain antibody
  • the GPRC5D antigen binding molecule or antigen binding fragment thereof further comprises an Fc domain of an immunoglobulin, wherein the immunoglobulin is selected from the form of IgG1, IgG2, IgG3 or IgG4.
  • the GPRC5D antigen binding molecule or antigen binding fragment thereof further comprises an Fc domain of IgG1. Domain, further preferably, the Fc domain of IgG1 comprises the amino acid sequence shown in SEQ ID NO:38.
  • the GPRC5D antigen binding molecule or its antigen binding fragment 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 an amino acid sequence EPKSS or (G 4 S) n , n is any integer from 1 to 10.
  • the GPRC5D antigen binding molecule or its antigen binding fragment comprises, from N-terminus to C-terminus, a VHH domain of sequence such as SEQ ID NO: 10, a G 4 S linker, and an Fc domain of IgG1 of sequence such as SEQ ID NO: 38.
  • 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.
  • 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 marker or a detection marker; preferably, the detection marker includes a radionuclide, a luminescent substance, a colored substance or an enzyme.
  • 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 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 and the other biologically active molecules are connected via a linker.
  • ADC antibody-drug conjugate
  • 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.
  • 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.
  • CAR chimeric antigen receptor
  • the present disclosure also provides nucleic acids encoding GPRC5D antigen binding molecules or antigen binding fragments thereof, and recombinant vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors.
  • 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).
  • 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 host cell under suitable conditions, and purifying the expression product from the cell.
  • 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 a tumor.
  • the drug targets tumor cells in which GPRC5D is abnormally expressed.
  • the tumor is selected from the group consisting of lymphomas such as multiple myeloma, and metastases of the above tumors.
  • the present disclosure also provides a method for treating a disease associated with the expression of GPRC5D in a subject, comprising administering the GPRC5D antigen binding molecule or antigen binding fragment thereof to a subject in need thereof.
  • the disease is a tumor; preferably, the tumor disease is selected from lymphoma such as multiple myeloma, and metastatic cancer of the above tumors;
  • the method further comprises administering an additional therapeutic agent to the subject.
  • the present disclosure also provides use of the GPRC5D antigen binding molecule or the antigen binding fragment thereof in preparing a detection reagent or a diagnostic reagent.
  • the detection reagent is used to detect the expression of GPRC5D; 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.
  • the present disclosure also provides a method for detecting GPRC5D expression in a sample, the method comprising:
  • the present disclosure also provides a method for monitoring GPRC5D-expressing cancer in a subject, the method comprising exposing a sample obtained from 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.
  • 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.
  • 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.
  • the present disclosure also provides a pharmaceutical composition, which comprises an effective amount of the GPRC5D antigen binding molecule or its antigen binding fragment of the present disclosure, or comprises an effective amount of the antibody drug conjugate, fusion protein, CAR-T cell of the present disclosure, or comprises an effective amount of the nucleic acid of the present disclosure, or comprises an effective amount of the recombinant vector of the present disclosure, or comprises an effective amount of the host cell of the present disclosure.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more additional other therapeutic agents.
  • 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 a HCDR sequence of a heavy chain variable region as shown in SEQ ID NO: 14, and/or a LCDR sequence of a 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.
  • the antigen binding molecule or its antigen binding fragment 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 its antigen binding fragment that specifically binds to BCMA comprises a light chain variable region whose sequence is shown in SEQ ID NO: 15.
  • the antigen-binding molecule or its antigen-binding fragment 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 its antigen-binding fragment that specifically binds to BCMA comprises the light chain amino acid sequence shown in SEQ ID NO: 7.
  • 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 Fc; more preferably, Fc is derived from mouse or human; more preferably, the sequence of Fc is natural or modified.
  • the BCMA antigen-binding molecules or antigen-binding fragments thereof disclosed herein may be monoclonal antibodies, bispecific binding molecules, multispecific binding molecules, Murine antibody, humanized antibody, chimeric antibody, modified antibody, fully human antibody, full-length antibody, heavy chain antibody, nanobody, Fab, Fv, scFv, F(ab')2, linear antibody or heavy chain single domain antibody.
  • the BCMA antigen-binding molecules or antigen-binding fragments thereof of the present disclosure may be full-length antibodies.
  • 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.
  • 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 with a capture marker or a detection marker; preferably, the detection marker comprises a radionuclide, a luminescent substance, a colored substance or an enzyme.
  • 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 of the present disclosure with other biologically active molecules; preferably, the other biologically active molecules are small molecule drugs; preferably, the BCMA antigen-binding molecule or its antigen-binding fragment and the other biologically active molecules are connected via a linker.
  • ADC antibody-drug conjugate
  • 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.
  • 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.
  • CAR chimeric antigen receptor
  • the present disclosure also provides nucleic acids encoding BCMA antigen-binding molecules or antigen-binding fragments thereof, and recombinant vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors.
  • 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).
  • the present disclosure also provides a method for preparing the BCMA antigen-binding molecule or antigen-binding fragment thereof of the present disclosure, the method comprising: culturing the above-mentioned host cells under suitable conditions, and purifying the expression product from the cells.
  • the present disclosure also provides use of a BCMA antigen-binding molecule or an antigen-binding fragment thereof in the preparation of a drug for treating or alleviating a tumor.
  • the drug targets tumor cells that aberrantly express BCMA.
  • the tumor is selected from the group consisting of lymphomas such as multiple myeloma, and metastases of the above tumors.
  • the present disclosure also provides a method for treating a disease associated with the expression of BCMA in a subject, comprising administering the BCMA antigen-binding molecule or the antigen-binding fragment thereof to a subject in need thereof.
  • the disease is a tumor; preferably, the tumor disease is selected from lymphoma such as multiple myeloma, and metastatic cancer of the above tumors;
  • the method further comprises administering an additional therapeutic agent to the subject.
  • 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.
  • 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.
  • the present disclosure also provides a method for detecting BCMA expression in a sample, the method comprising:
  • the present disclosure also provides a method of monitoring a BCMA-expressing cancer in a subject, the method 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 to 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.
  • 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.
  • 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.
  • the present disclosure also provides a pharmaceutical composition, which comprises an effective amount of the BCMA antigen-binding molecule or antigen-binding fragment thereof of the present disclosure, or comprises an effective amount of the antibody-drug conjugate, fusion protein, CAR-T cell of the present disclosure, or comprises an effective amount of the nucleic acid of the present disclosure, or comprises an effective amount of the recombinant vector of the present disclosure, or comprises an effective amount of the host cell of the present disclosure.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises one or more additional other therapeutic agents.
  • amino acid positions in the substitutions referred to in the present disclosure are preferably indicated using the EU numbering system.
  • 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 transfer, and the solid line in the figure shows the overexpression of human GPRC5D protein after CHOK1 mother cells were transfected with human GPRC5D plasmid.
  • 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 transfer, 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.
  • Figure 1C shows the expression of human GPRC5A on CHOK1 cells verified by anti-GPRC5A positive control antibody detected by flow cytometry; the dotted line in the figure shows the expression of human GPRC5A on CHOK1 mother cells without any plasmid transfection, and the solid line in the figure shows the overexpression of human GPRC5A protein after CHOK1 mother cells were transfected with human GPRC5A plasmid.
  • Figure 2A shows the expression of human BCMA on CHOK1 cells verified by anti-BCMA positive control antibody detected by flow cytometry; the dotted line in the figure shows the expression of human BCMA on CHOK1 mother cells without any plasmid transfection, and the solid line in the figure shows the overexpression of human BCMA protein after CHOK1 mother cells were transfected with human BCMA plasmid.
  • 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.
  • FIG3 shows the structures of two trispecific antibodies constructed in the examples, namely, trispecific antibody 1 and trispecific antibody 2.
  • Figure 4A shows the binding of Triclosan 1, Triclosan 2 and Teclistamab to tumor cells NCI-H929 expressing dual targets BCMA and GPRC5D.
  • Figure 4B shows the binding of Triclosan 1, Triclosan 2 and Teclistamab to the tumor cells RPMI8226 expressing the dual targets BCMA and GPRC5D.
  • Figure 4C shows the binding of Triclosan 1, Triclosan 2 and Teclistamab to the stably transfected cell line human GPRC5D CHOK1 cells expressing human GPRC5D.
  • Figure 4D shows the binding of Triclosan 1, Triclosan 2 and Teclistamab to the stably transfected cell line human BCMA CHOK1 cells expressing human BCMA.
  • Figure 4E shows the binding of triple antibody 1, BGCB491, and TS-F2-5 to tumor cells NCI-H929 expressing dual targets BCMA and GPRC5D.
  • Figure 4F shows the binding of triple antibody 1, BGCB491, and TS-F2-5 to tumor cells RPMI8226 expressing dual targets BCMA and GPRC5D.
  • Figure 4G shows the binding of triple antibody 1, BGCB491, TS-F2-5 and human GPRC5D CHOK1 cells, a stably transfected cell line expressing human GPRC5D.
  • Figure 4H shows the binding of Tri-Antibody 1, BGCB491, and TS-F2-5 to the stably transfected cell line human BCMA CHOK1 cells expressing human BCMA.
  • Figure 5A shows the binding of Triple Antibody 1, Triple Antibody 2 and Teclistamab to cells expressing human CD3 (Jurkat cells).
  • Figure 5B shows the binding of Triple Antibody 1, Triple Antibody 2 and Teclistamab to human PBMC cells.
  • Figure 5C shows the binding of Tri-Antibody 1, BGCB491, and TS-F2-5 to cells expressing hCD3 (Jurkat cells).
  • Figure 6A shows the binding of Triclosan 1, Triclosan 2 and Teclistamab to the stable cell line Cynomolgus monkey GPRC5D CHOK1 cells expressing Cynomolgus monkey GPRC5D.
  • Figure 6B shows the binding of Triclosan 1, Triclosan 2 and Teclistamab to the stable cell line cynomolgus macaque BCMA CHOK1 cells expressing cynomolgus macaque BCMA.
  • Figure 7 shows the binding of Triple Antibody 1, Triple Antibody 2 and Teclistamab to cyno PBMC cells.
  • FIG8A shows a T cell-mediated cytotoxicity assay (TDCC), with the target cells being NCI-H929.
  • FIG8B shows a T cell-mediated cytotoxicity assay (TDCC), wherein the target cells are RPMI8226.
  • Figure 8C shows a T cell-mediated cytotoxicity assay (TDCC) with human BCMA CHOK1 as the target cell.
  • FIG8D shows a T cell-mediated cytotoxicity assay (TDCC) in which the target cell is human GPRC5D CHOK1.
  • Figure 8E shows a T cell-mediated cytotoxicity experiment (TDCC), in which the target cells are a mixed cell system of human BCMA CHOK1 and human GPRC5D CHOK1.
  • Figure 9 shows the T cell-mediated cytotoxicity experiment in the presence of 2500 ng/ml of soluble BCMA (the dotted line indicates 2500 ng/ml of soluble BCMA conditions).
  • FIG. 10A shows the IL6 release of Triclosan 1, Triclosan 2 and Teclistamab under the co-incubation condition of PBMC and tumor cells NCI-H929 (PBMC donor number P122080606F).
  • FIG. 10B shows the IL6 release of Triclosan 1, Triclosan 2 and Teclistamab under the co-incubation condition of PBMC and tumor cells NCI-H929 (PBMC donor number XC11210).
  • Figure 11 shows the results of the efficacy experiment of Triple Antibody 1, Triple Antibody 2, Teclistamab, and the combination of Teclistamab and Talquetamb in the human immune cell reconstructed mouse model - tumor volume changes (tumor cells are NCI-H929).
  • Figure 12 shows the efficacy test results of Tri-Antibody 1, BGCB491, and TS-F2-5 in the human immune cell-reconstructed mouse model - changes in tumor volume (tumor cells are NCI-H929).
  • Figure 13 shows the results of the efficacy experiments of Tri-Antibody 1, BGCB491, and TS-F2-5 in the human immune cell-reconstructed mouse model - changes in tumor weight (tumor cells are NCI-H929).
  • FIG. 14A shows the binding of the alpaca-derived anti-GPRC5D chimeric antibody of the present disclosure to GPRC5D-expressing cells (human GPRC5D-CHOK1 cell line).
  • FIG. 14B 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).
  • FIG. 15A shows the binding of the alpaca-derived anti-GPRC5D humanized antibody of the present disclosure to cells expressing GPRC5D (NCI-H929 tumor cells).
  • FIG. 15B shows the binding of the alpaca-derived anti-GPRC5D humanized antibodies of the present disclosure to GPRC5D-expressing cells (cynomolgus monkey GPRC5D-CHOK1 cell line).
  • FIG. 16 shows that the alpaca-derived anti-GPRC5D humanized antibodies of the present disclosure do not non-specifically bind to the cognate family member GPRC5A.
  • FIG. 17A shows the binding of the mouse hybridoma-derived anti-BCMA chimeric antibody of the present disclosure to cells expressing human BCMA (human BCMA-CHOK1 cell line).
  • FIG. 17B shows the binding of the mouse hybridoma-derived anti-BCMA chimeric antibody of the present disclosure to cells expressing cynomolgus monkey BCMA (cynomolgus monkey BCMA-CHOK1 cell line).
  • FIG. 18A shows the binding of the mouse hybridoma-derived anti-BCMA humanized antibodies disclosed herein to cells expressing human BCMA (human BCMA-CHOK1 cell line).
  • FIG. 18B shows the binding of the mouse hybridoma-derived anti-BCMA humanized antibodies of the present disclosure to cells expressing cynomolgus monkey BCMA (cynomolgus monkey BCMA-CHOK1 cell line).
  • FIG. 19A 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).
  • FIG. 19B 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).
  • 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.
  • 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., connection to other peptide segments, rearrangement of functional units, etc.) based on intact antibodies or their antigen-binding fragments or their single chains.
  • 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.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • IgG and IgA can be further divided into different subclasses, for example, IgG can be divided into IgG1, IgG2, IgG3, 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 based on the amino acid sequence of their constant domains. are called ⁇ and ⁇ .
  • the constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain, CH4).
  • CH1 and CH2 domains are separated by a flexible hinge region, which is a proline- and cysteine-rich segment of variable length.
  • Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.
  • variable region or “variable domain” shows a significant change 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 4 framework regions FR, represented by FR1, FR2, FR3, and FR4, respectively.
  • CDR and FR sequences are usually present in the following sequence of the heavy chain variable domain (or light chain variable domain): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.
  • antibody fragment comprises at least a portion of a complete antibody.
  • 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 in 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, etc.
  • 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.
  • F(ab') 2 or "F(ab') 2 fragment” comprises two Fab fragments and a hinge region, which is a bivalent antibody fragment.
  • single-chain antibody refers to a fusion protein comprising at least one variable region antibody fragment including a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light chain 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 a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • 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.
  • VHH domain also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, nanobody, is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody” (i.e., an "antibody lacking a light chain”).
  • VHH domain is used to distinguish the variable domain from the heavy chain variable domain (which is referred to as “VH domain” in the present disclosure) and the light chain variable domain (which is referred to as "VL domain” in the present disclosure) present in conventional tetrapeptide chain structure antibodies.
  • the VHH domain specifically binds to an epitope without the need for other antigen-binding domains (this is in contrast to the VH or VL domains 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.
  • the "VHH domain” includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids that are then humanized, or antibodies obtained by screening using phage display technology.
  • amino acid modification includes amino acid substitutions, insertions and/or deletions in a polypeptide sequence.
  • amino acid substitution or “substitution” or “replacement” herein means replacing an amino acid at a particular position in a parent polypeptide sequence with another amino acid.
  • substitution S32A means that the serine at position 32 is replaced by alanine.
  • 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 produce compounds having the following preferred characteristics, including but not limited to: altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered Fc ligand binding to Fc receptors (FcRs), enhanced or reduced ADCC or CDC, altered glycosylation and/or disulfide bonds, and modified binding specificity.
  • the antibody variant comprises an Fc region having one or more amino acid substitutions that weaken Fc ⁇ R binding (e.g., substitutions at positions 234 and 235 of the Fc region).
  • the substitutions are L234A and L235A.
  • Fc is used to define the C-terminal region of an immunoglobulin heavy chain, which region includes at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an IgG heavy chain can 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 includes the IgG CH2 and IgG CH3 constant domains.
  • 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.
  • knock-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.
  • the method involves introducing a protuberance ("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 protuberance can be placed in the cavity to promote heterodimer formation and hinder homodimer formation.
  • the protuberance is constructed by replacing the small amino acid side chain from the interface of the first polypeptide chain with a larger side chain (e.g., tyrosine or tryptophan).
  • a complementary cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide chain, which is performed by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).
  • one amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance in the CH3 domain of the first polypeptide chain, which can be placed in a cavity in the CH3 domain of the second polypeptide chain, and in the CH3 domain of the second polypeptide chain of the Fc domain, one amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second polypeptide chain, wherein the protuberance in the CH3 domain of the first polypeptide chain can be placed.
  • 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).
  • 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).
  • linker refers to any tool for connecting 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.
  • the linker connecting different functional units preferably comprises a flexible peptide linker, such as a glycine-serine peptide linker.
  • the linker comprises an amino acid sequence (G 4 S) n or (G 4 S) n A, wherein n is any integer selected from 1 to 10, preferably comprising an amino acid sequence (G 4 S) 3 or (G 4 S) 3 A.
  • 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.
  • the linker comprises the amino acid sequence (G 4 S) n or (G 4 S) n A, wherein n is any integer selected from 1 to 10, preferably comprises the amino acid sequence (G 4 S) 3 or (G 4 S).
  • antibody may include 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 mutant proteins and their variants), etc.
  • monoclonal antibody refers to an antibody that is essentially homogeneous and directed only against a specific antigenic epitope and is produced by a single cell clone.
  • 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 the above techniques.
  • humanized antibody refers to an antibody in which all or part of the amino acids outside the CDR of a non-human antibody (such as a mouse antibody) are replaced by corresponding amino acids derived from human immunoglobulins. A small amount of addition, deletion, insertion, substitution or modification of amino acids is permitted as long as they do not eliminate the ability of the antibody to bind to a specific antigen. A “humanized” antibody retains an antigen specificity similar to that of the original antibody.
  • 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.
  • homologous and heterologous used in this article are a set of relative concepts, which can refer to the same or different sources of different elements in the construct, or they can refer to the fact that after the construction of the construct is completed, some elements of the same source, i.e., "homologous”, have been transformed and changed compared with other original elements that have not been transformed, thus becoming “heterologous”.
  • an antigen refers to a substance that is recognized and specifically bound by an antibody or antibody binding fragment.
  • 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.
  • ECD extracellular domain
  • 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 antibodies that are specific to an antigenic determinant.
  • epitope and "antigenic determinant” refer to sites on an antigen that specifically bind to an immunoglobulin or antibody.
  • An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are usually retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost after treatment with denaturing solvents.
  • An epitope usually exists in a unique spatial conformation and includes at least 3-15 amino acids.
  • multispecific refers to an antigen binding molecule that is able to specifically bind to multiple different antigenic determinants.
  • 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.
  • 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.
  • telomere 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.
  • the constant region is not particularly limited, and constant regions known to those skilled in the art or constant regions obtained by themselves can be used. Amino acid mutations (e.g., mutations that increase or decrease binding to Fc receptors or FcRn) can also be introduced into the constant region portion.
  • the method for obtaining the binding molecules, antigen binding fragments, antibodies, bispecific binding molecules or multispecific binding molecules disclosed in the present invention is not particularly limited and can 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 by conventional methods.
  • cDNA sequences encoding heavy chains and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially 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 and cultured in serum-free medium in a bioreactor to produce antibodies. The culture fluid secreting the antibodies can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • transfection 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.
  • stable transfection or “stable transfection” refers to the introduction and integration of foreign nucleic acid, DNA or RNA, into the genome of the transfected cell.
  • stable transfectant refers to a cell that has stably integrated the foreign DNA into its genomic DNA.
  • ADC antibody drug conjugate
  • ADC 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 capable of killing cells targeted by the ADC, preferably malignant or cancerous cells.
  • the 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, the conjugation is performed in a site-specific manner, which allows complete control of the conjugation site and the drug-to-antibody ratio of the resulting ADC.
  • amino acid substitution or “substitution” or “replacement” means replacing the amino acid at a particular position in a parent polypeptide sequence with another amino acid.
  • 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).
  • Kd 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, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration, and the like. Quantitative calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry, etc.
  • biological activity refers to the ability of an antibody to bind an antigen and result in a measurable biological response, which can be measured in vitro or in vivo.
  • composition refers to a preparation or a combination of preparations 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 preparation is administered.
  • pharmaceutical composition is in the form of a combination of separate preparations containing different, two or more active ingredients, it can be administered simultaneously, sequentially, separately or at intervals, with the purpose of exerting the biological activities of multiple active ingredients and being used together to treat diseases.
  • 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 they can be independently formed into administration units and used separately.
  • the term "effective amount" refers to the dosage of the pharmaceutical preparation of the antibody or fragment of the present disclosure, which produces the desired effect in the treated patient after being administered to the patient in a single or multiple doses.
  • the effective amount can be easily determined by the attending physician who is a person skilled in the art by considering a variety of factors such as racial 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 preparation; the selected dosing regimen; and the use of any concomitant therapy.
  • the term "individual” or “subject” as used herein 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 monkeys or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • non-human primates e.g., cynomolgus monkeys or rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • disease refers to any change or disorder that damages or interferes with the normal function of cells, tissues or organs.
  • disease includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T cell dysfunction diseases, or immune tolerance defects (such as transplant rejection).
  • 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.
  • treatment refers to clinical intervention in an attempt to change the disease process caused by an individual or a cell, which can be either preventive or intervention in the clinical pathological process.
  • the therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc.
  • 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 UniProtKB/Swiss-Prot Accession No. Q9NZD1 (see also Brauner-Osborne, H et al., 2001, Biochim. Biophys. Acta 1518, 237-248).
  • 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.
  • 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 italic part is the transmembrane region (28-48; 64-84; 94-114; 124-144; 168-188; 205-225; 240-260).
  • cyno-GPRC5D used in the examples (SEQ ID NO: 2) (Uniprot ID: A0A2K5W6I2) is as follows:
  • 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 italic part is the transmembrane region (28-48; 64-84; 94-114; 124-144; 168-188; 205-225; 240-260).
  • 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. region (55-68; 119-129; 198-212; 269-357); the italic parts are the transmembrane regions (34-54; 69-89; 98-118; 130-150; 177-197; 213-233; 248-268).
  • Example 2 Preparation of cell lines expressing human GPRC5D, human GPRC5A and cynomolgus monkey GPRC5D
  • the nucleotide sequence encoding the amino acid of human GPRC5D shown in SEQ ID NO.1 was cloned into the pCMV3 (SinoBiological, Catalog No. CV011) vector to obtain a plasmid for constructing a human GPRC5D cell line.
  • the obtained plasmid was transfected into CHOK1 cells (ATCC, Catalog No. CCL-61) to obtain a CHOK1 cell line expressing human GPRC5D (referred to as: human GPRC5D-CHOK1 cell line).
  • the nucleotide sequence encoding the amino acid of cynomolgus monkey GPRC5D shown in SEQ ID NO.2 was cloned into the pCMV3 vector to obtain a plasmid for constructing the cynomolgus monkey GPRC5D cell line.
  • the obtained plasmid was transfected into CHOK1 cells to obtain a CHOK1 cell line expressing cynomolgus monkey GPRC5D (abbreviated as: cynomolgus monkey GPRC5D-CHOK1 cell line).
  • the nucleotide sequence encoding the amino acid of human GPRC5A shown in SEQ ID NO.36 was cloned into the pCMV3 vector to obtain a vector for constructing a human GPRC5A cell line.
  • the obtained vector was transfected into CHOK1 cells to obtain a CHOK1 cell line expressing human GPRC5A (referred to as: human GPRC5A-CHOK1 cell line).
  • the FACS assay method was used to detect the expression of human GPRC5D in the human GPRC5D-CHOK1 cell line obtained above, and a negative control hIgG1 LALA isotype (Bai Ying Bio, catalog number B109802) was set up in the experiment.
  • the FACS assay method was used to detect the expression of crab-eating macaque GPRC5D in the crab-eating macaque GPRC5D-CHOK1 cell line obtained above, and a negative control hIgG1 LALA isotype (Bai Ying Bio, catalog number B109802) was set up in the experiment.
  • the FACS assay method was used to detect the expression of human GPRC5A in the human GPRC5A-CHOK1 cell line obtained above, and a negative control hIgG1 LALA isotype (Bai Ying Bio, catalog number B109802) was set up in the experiment.
  • This protein is a single-pass transmembrane protein.
  • the double-line part is the extracellular region (1-54); the wavy line part is the transmembrane region (55-77); and the dashed line part is the intracellular region (78-184).
  • SEQ ID NO: 4 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.
  • This protein is a single-pass transmembrane protein.
  • the double-line part is the extracellular region (1-53); the wavy line part is the transmembrane region (54-76); and the underlined part is the intracellular region (77-183).
  • the nucleotide sequence encoding the amino acids of human BCMA shown in SEQ ID NO: 3 was cloned into the pCMV3 (SinoBiological, Catalog No. CV011) vector to obtain a vector for constructing a human BCMA cell line.
  • the obtained vector was transfected into CHOK1 cells (ATCC, Catalog No. CCL-61) to obtain a CHOK1 cell line expressing human BCMA (referred to as: human BCMA-CHOK1 cell line).
  • the nucleotide sequence encoding the amino acid of cynomolgus monkey BCMA shown in SEQ ID NO: 4 was cloned into the pCDH-CMV-MCS-EF1-Hygro (Honorgene, catalog number CD515B-1) vector to obtain a vector for constructing a cynomolgus monkey 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 monkey BCMA (abbreviated as: cynomolgus monkey BCMA-CHOK1 cell line).
  • the FACS assay method was used to detect the expression of human BCMA in the human BCMA-CHOK1 cell line obtained above, and a negative control hIgG1 LALA isotype (Bio-Ying Bio, catalog number B109802) was set up in the experiment.
  • the FACS assay method was used to detect the expression of BCMA in the BCMA-CHOK1 cell line obtained above, and a negative control hIgG1 LALA isotype (Bio-Yin Biotechnology, catalog number B109802) was set up in the experiment.
  • the anti-BCMA full-length antibody (BCMA mAb) was obtained through hybridoma screening, whose heavy chain sequence is shown in SEQ ID NO:9, and the light chain sequence is shown in SEQ ID NO:7.
  • anti-GPRC5D nanoantibody GPRC5D VHH
  • its variable region sequence is shown in SEQ ID NO:10.
  • the CD3 ⁇ binding domain is from the full-length antibody CD3mAb, whose heavy chain sequence is shown in SEQ ID NO: 11, and the light chain sequence is shown in SEQ ID NO: 12.
  • the heavy chain variable region and light chain variable region of CD3mAb are connected via a flexible linker to form a single-chain antibody scFv, whose structure is: VH-(G 4 S) 3 -VL, and the sequence is shown in SEQ ID NO: 13.
  • the scFv is then fused to the C-terminus of the heavy chain of the Fab segment of the full-length BCMA antibody via a flexible linker.
  • CD3scFv was fused to the C-terminus of BCMA’s Fab heavy chain CH1 to form one arm of the trispecific antibody.
  • BCMA BCMA’s Fab heavy chain CH1
  • Another complete GPRC5D nanoantibody binding domain and an Fc part with a “knob” and “hole” structure were introduced.
  • the trispecific antibody formed was named Tri-Antibody 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.
  • CD3scFv was fused to the C-terminus of the heavy chain of the Fab segment of the full-length BCMA antibody to form one arm of the antibody
  • the GPRC5D nanobody was fused to the C-terminus of the other Fab segment heavy chain of the full-length BCMA antibody to form the other arm of the antibody, and it also contained an Fc part with a "knob" and "hole” structure.
  • the trispecific antibody formed was named Tri-Anti 2, and the sequences of the fused chains are shown in SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8.
  • the schematic diagram is shown in Figure 3.
  • 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 scFv is designed as a "knob" structure, including amino acid substitutions at two sites, S354C and T366W.
  • the Fc domain in the peptide chain without scFv is designed as a "hole” structure, including amino acid substitutions at four sites, Y349C, T366S, L368A and Y407V.
  • the heavy chain of the "hole" structure must also be replaced with H435R.
  • Example 6 Construction of anti-GPRC5D-BCMA-CD3 trispecific antibody and its transient transfection expression in eukaryotic cells
  • the coding gene fragments of the aforementioned three specific antibodies were cloned into the PTT5 expression vector respectively to prepare transfection-grade expression plasmids.
  • Expi293F TM cells (Thermo Fisher Scientific) were cultured in serum-free medium, inoculated in shake flasks (Corning Inc.), and cultured on a shaker at 37°C and 8% CO 2. 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 change the solution to PBS for aliquoting. The final purified antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.
  • GE gel chromatography Superdex200
  • 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.
  • the TS-F2-5 trispecific antibody of Innovent (derived from patent WO2022174813A1, composed of SEQ ID NO: 68, 75, 76, 78, containing a GPRC5D binding site, a BCMA binding site and a CD3 binding site).
  • 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 dual targets, human GPRC5D-CHOK1 cells expressing human GPRC5D, human BCMA-CHOK1 cells expressing human BCMA, T lymphocytes (Jurkat) naturally expressing hCD3, and Human PBMCs.
  • 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
  • Jurkat cells ATCC, Catalog No. TIB-152
  • the culture medium for NCI-H929 cells was RPMI1640+10% FBS+0.05mM mercaptoethanol
  • the culture medium for RPMI8226 and Jurkat cells was RPMI1640+10% FBS
  • the culture medium for Human GPRC5D-CHOK1 and Human BCMA-CHOK1 cells was F12K+10% FBS+400 ⁇ g/ml Hygromycin B
  • T75 cell culture flasks were placed in a 37°C 5% CO 2 incubator for culture.
  • NCI-H929 cells, RPMI8226 cells and Jurkat cells are placed directly into 50 mL centrifuge tubes without digestion.
  • Human GPRC5D-CHOK1 and Human BCMA-CHOK1 are digested with 0.25% Trpsin-EDTA trypsin, and digestion is terminated with F12K+10% FBS+400 ⁇ g/ml Hygromycin B.
  • the obtained cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 1% BSA (in PBS).
  • the purchased Human PBMC was centrifuged at 1500 rpm for 10 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 1% BSA (in PBS).
  • the cells were counted and the cell density was adjusted to 1E6/mL, and spread into a 96-well round-bottom culture plate (corning, catalog number 3799), 100 ⁇ L/well.
  • the FCS file was exported from the flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of the PE channel of each sample was analyzed using flowjo software.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cells (hereinafter referred to as EC 50 ) and the highest mean fluorescence intensity (Top MFI).
  • Example 8 Species cross-activity test of anti-GPRC5D-BCMA-CD3 trispecific antibodies
  • FACS was used to detect the binding of anti-GPRC5D-BCMA-CD3 trispecific antibodies to cynomolgus monkey GPRC5D-CHOK1 cells expressing Cyno GPRC5D, cynomolgus monkey BCMA-CHOK1 cells expressing Cyno BCMA, and Cyno PBMCs naturally expressing Cyno CD3.
  • Cynomolgus monkey GPRC5D-CHOK1 (Example 2) and cynomolgus monkey BCMA-CHOK1 cells (Example 4) were cultured in a medium of F12K + 10% FBS + 400 ⁇ g / ml Hygromycin B, and cultured in a T75 cell culture flask at 37 ° C 5% CO 2 incubator.
  • cynomolgus monkey GPRC5D-CHOK1 and cynomolgus monkey BCMA-CHOK1 were digested with 0.25% Trpsin-EDTA trypsin, and digestion was terminated with F12K + 10% FBS + 400 ⁇ g / ml Hygromycin B.
  • the obtained cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 1% BSA (in PBS).
  • the purchased Cyno PBMC was centrifuged at 1500 rpm for 10 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 1% BSA (in PBS).
  • the cells were counted and the cell density was adjusted to 1E6/mL, and spread into a 96-well round-bottom culture plate (corning, catalog number 3799), 100 ⁇ L/well.
  • the FCS file was exported from the flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of the PE channel of each sample was analyzed using flowjo software.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half-binding concentration of the antibody to the cells (hereinafter referred to as EC 50 ) and the highest mean fluorescence intensity (Top MFI).
  • Example 9 In vitro recombinant protein binding affinity and kinetics of anti-GPRC5D-BCMA-CD3 trispecific antibodies
  • the anti-GPRC5D-BCMA-CD3 trispecific antibody molecules were diluted twice to a series of concentrations (100nM-0.39nM) with HBS-EP+ (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% P20) buffer, and bound for 90s at a flow rate of 50 ⁇ L/min, and dissociated for 360s.
  • HBS-EP+ 10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% P20
  • the chip was rinsed with 3M MgCl 2 solution at a flow rate of 30 ⁇ L/min for 30 s to remove the anti-GPRC5D-BCMA-CD3 trispecific antibody molecules and complete the regeneration of the chip.
  • the raw data were analyzed using Biacore Insight Evaluation Software (3.0.12.15655) and fitted with the (1:1) Langmuir model. The obtained trispecific antibody affinity and kinetic experimental data are shown in Table 9.
  • the CM5 chip was used to detect the affinity and kinetic properties of antibody molecules and human/cynomolgus monkey BCMA using the indirect capture method.
  • the anti-GPRC5D-BCMA-CD3 trispecific antibody molecule was diluted to 4 ⁇ g/mL with 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 ⁇ L/min.
  • Human/cynomolgus monkey BCMA was diluted two-fold to a series of concentrations (50 nM-0.195 nM) with HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20) buffer, bound for 90 s at a flow rate of 30 ⁇ L/min, and dissociated for 180 s.
  • HBS-EP+ 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20
  • the chip was rinsed with 3M MgCl 2 solution at a flow rate of 30 ⁇ L/min for 30 s to remove the anti-GPRC5D-BCMA-CD3 trispecific antibody molecules and complete the regeneration of the chip.
  • the raw data were analyzed using Biacore Insight Evaluation Software (3.0.12.15655) and fitted with the (1:1) Langmuir model. The obtained trispecific antibody affinity and kinetic experimental data are shown in Table 10.
  • T cell-mediated cytotoxicity assay (TDCC) target cells are NCI-H929
  • NCI-H929 cells (ATCC, catalog number CRL-9068) were cultured in RPMI1640 + 10% FBS + 0.05mM mercaptoethanol in a T75 cell culture flask placed in a 37°C 5% CO 2 incubator. When the cells were to be used, the NCI-H929 cells were placed directly into a 50mL centrifuge tube without Digestion is required. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend and count the cells in RPMI1640 + 2% FBS medium, and adjust the cell density to 1E5/mL.
  • Target cells were plated in a flat-bottom 96-well plate (corning, Cat. No. 3599), 100 ⁇ L/well, and cultured overnight at 37°C 5% CO 2.
  • CD3+T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), cells were counted, and the cell density was adjusted to 2E6/mL with RPMI1640+2% FBS. Effector cells were plated in a 96-well plate, 50 ⁇ L/well, and cultured at 37°C 5% CO 2 .
  • Cell killing % [sample well - T cell spontaneous release - target cell spontaneous release) / (target cell maximum lysis - target cell spontaneous release)] * 100%.
  • the cell killing data were imported into GraphPad Prism, the cell killing/concentration curve was drawn, and the EC50 value was calculated. The results are shown in Table 11 and Figure 8A.
  • the killing ability of the triple antibody 1 molecule on NCI-H929 cells was stronger than that of BGCB491 and TS-F2-5.
  • T cell-mediated cytotoxicity assay target cells are RPMI8226
  • RPMI8226 cells (ATCC, catalog number CCL-155) in RPMI1640+10% FBS medium, and culture in a T75 cell culture flask at 37°C in a 5% CO 2 incubator. When the cells are ready for 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 and count the cells in RPMI1640+2% FBS medium, and adjust the cell density to 1E5/mL.
  • Target cells were plated in a flat-bottom 96-well plate (corning, Cat. No. 3599), 100 ⁇ L/well, and cultured overnight at 37°C 5% CO 2.
  • CD3+T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), cells were counted, and the cell density was adjusted to 2E6/mL with RPMI1640+2% FBS. Effector cells were plated in a 96-well plate, 50 ⁇ L/well, and cultured at 37°C 5% CO 2 .
  • Cell killing % [sample well - T cell spontaneous release - target cell spontaneous release) / (target cell maximum lysis - target cell spontaneous release)] * 100%.
  • the cell killing data were imported into GraphPad Prism, the cell killing/concentration curve was drawn, and the EC50 value was calculated. The results are shown in Table 11 and Figure 8B.
  • the killing ability of the triple antibody 1 molecule on RPMI8226 cells was stronger than that of BGCB491 and TS-F2-5.
  • T cell-mediated cytotoxicity assay (TDCC) target cells are human BCMA-CHOK1
  • Human BCMA-CHOK1 (Example 4) was cultured in a medium of F12K+10% FBS+400 ⁇ g/ml Hygromycin B, and a T75 cell culture flask was used to culture in a 37°C 5% CO 2 incubator. When the cells were to be used, Human BCMA-CHOK1 was digested with trypsin containing 0.25% Trpsin-EDTA, and digestion was terminated with F12K+10% FBS+400 ⁇ g/ml Hygromycin B medium. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend and count the cells in RPMI1640+2% FBS medium, and adjust the cell density to 1E5/mL.
  • Target cells were plated in a flat-bottom 96-well plate (corning, Cat. No. 3599), 100 ⁇ L/well, and cultured overnight at 37°C 5% CO 2.
  • CD3+T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), cells were counted, and the cell density was adjusted to 2E6/mL with RPMI1640+2% FBS. Effector cells were plated in a 96-well plate, 50 ⁇ L/well, and cultured at 37°C 5% CO 2 .
  • Cell killing % [sample well - T cell spontaneous release - target cell spontaneous release) / (target cell maximum lysis - target cell spontaneous release)] * 100%.
  • the cell killing data were imported into GraphPad Prism, the cell killing/concentration curve was drawn, and the EC50 value was calculated. The results are shown in Table 11 and Figure 8C.
  • the killing ability of the triple anti-1 molecule on Human BCMA-CHOK1 cells is comparable to that of BGCB491 and stronger than that of TS-F2-5.
  • T cell-mediated cytotoxicity assay target cells are human GPRC5D-CHOK1
  • Human GPRC5D-CHOK1 (Example 2) was cultured in a medium of F12K+10% FBS+400 ⁇ g/ml Hygromycin B, and a T75 cell culture flask was used to culture in a 37°C 5% CO 2 incubator.
  • Human BCMA-CHOK1 was digested with trypsin containing 0.25% Trpsin-EDTA, and digestion was terminated with F12K+10% FBS+400 ⁇ g/ml Hygromycin B medium. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend and count the cells using RPMI1640+2% FBS medium, and adjust the cell density to 1E5/mL.
  • Target cells were plated in a flat-bottom 96-well plate (corning, Cat. No. 3599), 100 ⁇ L/well, and cultured overnight at 37°C 5% CO 2.
  • CD3+T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), cells were counted, and the cell density was adjusted to 2E6/mL with RPMI1640+2% FBS. Effector cells were plated in a 96-well plate, 50 ⁇ L/well, and cultured at 37°C 5% CO 2 .
  • Cell killing % [sample well - T cell spontaneous release - target cell spontaneous release) / (target cell maximum lysis - target cell spontaneous release)] * 100%.
  • the cell killing data were imported into GraphPad Prism, the cell killing/concentration curve was drawn, and the EC50 value was calculated. The results are shown in Table 11 and Figure 8D.
  • the killing ability of the triple anti-1 molecule on Human GPRC5D-CHOK1 cells is comparable to that of TS-F2-5 and stronger than that of BGCB491.
  • T cell-mediated cytotoxicity assay target cells are human BCMA-CHOK1 and human GPRC5D-CHOK1 mixed system
  • Human GPRC5D-CHOK1 and Human BCMA-CHOK1 cells were cultured in F12K+10% FBS+400 ⁇ g/ml Hygromycin B medium in a T75 cell culture flask at 37°C in a 5% CO 2 incubator.
  • Human GPRC5D-CHOK1 and Human BCMA-CHOK1 were digested with 0.25% Trpsin-EDTA trypsin, and digestion was terminated with F12K+10% FBS+400 ⁇ g/ml Hygromycin B medium.
  • the target cell mixture was plated into a flat-bottom 96-well plate (corning, Cat. No. 3599), 100 ⁇ L/well, and cultured overnight at 37°C 5% CO 2.
  • CD3+T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), the cells were counted, and the cell density was adjusted to 1.6E6/mL with RPMI1640+2% FBS. Effector cells were plated into a 96-well plate, 50 ⁇ L/well, and cultured at 37°C 5% CO 2 .
  • Cell killing % [sample well - T cell spontaneous release - target cell spontaneous release) / (target cell maximum lysis - target cell spontaneous release)] * 100%.
  • the cell killing data were imported into GraphPad Prism, the cell killing/concentration curve was drawn, and the EC50 value was calculated. The results are shown in Table 11 and Figure 8E.
  • the killing ability of the triple antibody 1 molecule on the mixed cell system was stronger than that of BGCB491 and TS-F2-5.
  • T cell-mediated cytotoxicity assay target cells are NCI-H929, RPMI8226, human BCMA-CHOK1, human GPRC5D-CHOK1, human BCMA-CHOK1 and human GPRC5D-CHOK1 mixed cell system
  • T cell-mediated cytotoxicity assay (TDCC) target cells are NCI-H929, in the presence of 2500ng/ml soluble BCMA
  • NCI-H929 cells (ATCC, CRL-9068) were cultured in RPMI1640+10% FBS+0.05mM mercaptoethanol in a T75 cell culture flask placed in a 37°C 5% CO 2 incubator. When the cells are ready for use, place the NCI-H929 cells directly into a 50mL centrifuge tube without digestion. Centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend and count the cells in RPMI1640+2% FBS medium, and adjust the cell density to 1E5/mL. Add soluble BCMA to the cell suspension to a concentration of 2500ng/ml.
  • Target cells containing soluble BCMA were plated into a flat-bottom 96-well plate (corning, Cat. No. 3599), 100 ⁇ L/well, and cultured overnight at 37°C 5% CO 2.
  • CD3+T cells were sorted from fresh PBMCs using a T cell negative selection kit (StemCell, Cat. No. 17951), cells were counted, and the cell density was adjusted to 2E6/mL with RPMI1640+2% FBS. Effector cells were plated into a 96-well plate, 50 ⁇ L/well, and cultured at 37°C 5% CO 2 .
  • Cell killing % [sample well - T cell spontaneous release - target cell spontaneous release) / (target cell maximum lysis - target cell spontaneous release)] * 100%.
  • the cell killing data was imported into GraphPad Prism, the cell killing/concentration curve was drawn, and the EC50 value was calculated.
  • the results are shown in Figure 9 (the dotted line indicates the condition of 2500ng/ml soluble BCMA) and Table 12.
  • the killing EC50 value of BGCB491 was weakened by about 23 times
  • the killing EC50 value of TS-F2-5 was basically maintained
  • the killing EC50 value of triple antibody 1 was weakened by about 3 times.
  • the killing EC50 value of BCMA dual antibody Teclistamab was weakened by about 25 times
  • NCI-H929 cells (ATCC, catalog number CRL-9068) were cultured in RPMI1640 + 10% FBS + 0.05mM mercaptoethanol in a T75 cell culture flask placed in a 37°C 5% CO 2 incubator. When the cells were to be used, the NCI-H929 cells were placed directly into a 50mL centrifuge tube without Digestion is required, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend and count the cells in RPMI1640 + 2% FBS medium, and adjust the cell density to 1E5/mL. NCI-H929 cells were plated in a 96-well plate (corning, catalog number 3799), 100 ⁇ L/well, and cultured at 37°C with 5% CO 2 .
  • the culture plate was centrifuged at 400 g for 10 minutes, and 100 ⁇ L of the supernatant was frozen at -80°C for later use.
  • the thermal stability of the three antibodies in pH 7.4 PBS buffer was detected by differential fluorescence scanning technology.
  • the sample concentration was about 1 mg/mL, and Prometheus NT.Plex (nano DSF) was used for detection. Before detection, each sample was centrifuged at 10000g for 10 minutes. 40 ⁇ L of sample was added to each well of the sample plate (the instrument loading volume was 10 ⁇ L, and each sample had a duplicate well).
  • the scanning temperature started from 30°C and ended at 95°C, and the scanning rate was 0.5°C/min.
  • Table 13 Both three antibody molecules showed good thermal stability.
  • the trispecific antibody was administered at a dose of 1 mg/kg, injected intravenously into the hind limb.
  • the blood was collected at pre-dose, 5 min, 2 hr, 6 hr, 24 hr, 72 hr, 168 hr, 264 hr, 336 hr, 504 hr, 672 hr, and 840 hr.
  • Whole blood samples were collected in polyethylene tubes without anticoagulants, placed at room temperature for about 1 hour, centrifuged at 6000 g, 25 °C, immediately placed on dry ice, and transferred to a -80 °C refrigerator for long-term storage.
  • the concentration of the complete GPRC5D-BCMA-CD3 tri-antibody in serum was detected by ELISA.
  • Human GPRC5D protein was used to coat a 96-well plate at a concentration of 2 ⁇ g/mL, 100 ⁇ L per well, and placed at 4°C overnight; the plate was washed 3 times with 300 ⁇ L PBST per well, 300 ⁇ L of blocking reagent 5% milk powder was added, and incubated at 37°C for 1 hour; the plate was washed 3 times with 300 ⁇ L PBST per well, 100 ⁇ L of the sample to be tested was added, and incubated at 37°C for 1 hour; the plate was washed 6 times with 300 ⁇ L PBST per well, 100 ⁇ L of the sample to be tested was added, and incubated at 37°C for 1 hour.
  • Biotin-BCMA reagent incubate at 37°C for 1h; wash the plate 6 times with 300 ⁇ L PBST per well, add 100 ⁇ L SA-HRP reagent, incubate at 37°C for 1h; wash the plate 6 times with 300 ⁇ L PBST per well, add 100 ⁇ L TMB, place in dark for 10min, and add 50 ⁇ L stop solution to stop the color reaction.
  • concentration of the complete GPRC5D-BCMA-CD3 triple antibody molecule was quantitatively detected according to the color reaction.
  • 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.
  • the experimental animals were kept in independent ventilation boxes with constant temperature and humidity.
  • the temperature in the breeding room was 20.0-26.0°C, the humidity was 40-70%, and the light-dark alternation time was 12h/12h.
  • NCI-H929 cells (ATCC, catalog number CRL-9068) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.05 mM ⁇ -ME. NCI-H929 cells in the exponential growth phase were collected and resuspended in 0.1 mL (1:1) PBS and matrix gel suspension. 5 ⁇ 10 6 NCI-H929 cells were inoculated in the front of the right back of the experimental mice. The tumor growth was observed regularly. When the tumor grew to an average volume of 83 mm 3 , the mice were randomly divided into groups and given drugs according to the tumor size and mouse weight.
  • Donor PBMCs were purchased from AllCells, LLC. 1 ⁇ 10 7 PBMCs/0.1 mL PBS were intraperitoneally injected into each mouse on the day of NCI-H929 cell inoculation to establish a mouse model of human immune cell reconstruction.
  • Use StudyDirectorTM version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA
  • the day when mice were grouped was defined as day 0, and intraperitoneal injections were started on D0, once every three days, for 5 times.
  • the experimental groups and dosing schedule are shown in Table 14.
  • 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.
  • the 0.004 mg/kg treatment group of the triple antibody 1 molecule showed a very significant tumor inhibition effect in the human myeloma NCI-H929 subcutaneously transplanted NPG female mouse Xenograft model (p value less than 0.001), and was significantly better than the equimolar dose of the dual antibody molecule Teclistamab and the combination of Teclistamab and Talquetamab.
  • the mice tolerated the test drug triple antibody molecule well, and there was no obvious weight loss or toxicity in the mice.
  • the experimental animals were kept in independent ventilation boxes with constant temperature and humidity.
  • the temperature of the breeding room was 20.0-26.0°C, the humidity was 40-70%, and the light-dark alternation time was 12h/12h.
  • NCI-H929 cells (ATCC, catalog number CRL-9068) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 0.05 mM ⁇ -ME. NCI-H929 cells in the exponential growth phase were collected and resuspended in 0.1 mL (1:1) PBS and matrix gel suspension. 5 ⁇ 10 6 NCI-H929 cells were inoculated in the front of the right back of the experimental mice. The tumor growth was observed regularly. When the tumor grew to an average volume of 100 mm 3 , the mice were randomly divided into groups and given drugs according to the tumor size and mouse weight.
  • Donor PBMCs were purchased from AllCells, LLC. 1 ⁇ 10 7 PBMCs/0.2 mL PBS were intraperitoneally injected into each mouse on the day of NCI-H929 cell inoculation to establish a mouse model of human immune cell reconstruction.
  • mice were grouped according to their tumor volume using a random grouping design method to ensure that the tumor volume between different groups was similar.
  • the day when the mice were grouped was defined as day 1, and intraperitoneal injection was started on day D1, twice a week, for 4 times.
  • the experimental grouping and dosing schedule are shown in Table 16.
  • 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). Independent sample T test was used to compare whether there were significant differences between different treatment groups and the control group. Data were analyzed using GraphPad prism 8.0.2. P ⁇ 0.05 was considered to be significantly different. The experimental results are shown in Table 17 and Figures 12 and 13.
  • Tri-Anti-1 showed a very significant tumor inhibition effect in the human myeloma NCI-H929 subcutaneously transplanted NCG female mouse Xenograft model (p values were all less than 0.05), among which Tri-Anti-1 molecule 0.006mg/kg was significantly better than equimolar doses of BCGB491 and TS-F2-5.
  • the mice tolerated the test drug Tri-Anti-1 molecule well, and no obvious weight loss or toxicity was observed in the mice.
  • 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 days 28, 42, and 63 to separate serum, and the immune response in the serum was detected by cell-level FACS. Immunization was terminated when the serum titer tended to the plateau phase, and 50 mL of blood was drawn again from the immunized alpacas.
  • Solarbio's lymphocyte separation solution was used to separate alpaca PBMCs according to the manufacturer's instructions, and total RNA was extracted (OMEGA Cell Total RNA Extraction Kit).
  • VHH fragments were recovered and digested with Sfi I, ligated to the pADL-23c phagemid vector, and electroporated into TG1 electroporation competent cells to construct the GPRC5D alpaca immune library (with a library capacity of 4.07E9).
  • Washed twice with PBS 300 ⁇ L of 5% skim milk was added to each well, blocked at 37°C for 1h, washed three times with PBST, 50 ⁇ L of 5% skim milk and 50 ⁇ L of phage supernatant were added to each well, and incubated at 37°C for 1h.
  • Washed five times with 0.1% PBST and horseradish peroxidase-labeled anti-M13 antibody (diluted 1:10000 with PBS) was added, 100 ⁇ L/well, and incubated at 37°C for 1h. Wash the plate 6 times with 0.1% PBST.
  • Example 16 Construction of alpaca-derived anti-GPRC5D chimeric antibody and its transient transfection expression in eukaryotic cells
  • the target gene fragment generated by splicing the sequenced heavy chain antibody variable region of the present disclosure with the human IgG1 constant region is cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.
  • the heavy chain antibody variable region can be connected to the human IgG1 constant region through a connecting short peptide, that is, forming: heavy chain antibody variable region-connecting short peptide-human IgG1 constant region.
  • the connecting short peptide sequence used in this implementation is GGGGS.
  • the introduced human IgG1 constant region sequence (SEQ ID NO: 38) is as follows:
  • Expi293F TM cells (Thermo Fisher Scientific) were cultured in serum-free medium, inoculated 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 changed to PBS for aliquoting. The final purified chimeric antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.
  • the culture medium for human GPRC5D-CHOK1 cells and cynomolgus monkey GPRC5D-CHOK1 cells was F12K + 10% FBS + 400 ⁇ g/mL Hygromycin, and T75 cell culture flasks were placed in a 37°C 5% CO 2 incubator for culture. When ready for use, the cells were washed twice with sterile DPBS, digested with 0.25% trypsin EDTA for about 5 minutes, and then terminated with complete culture medium.
  • the obtained cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 100 ⁇ L 1% BSA (in PBS). The cells were counted and the cell density was adjusted to 1E6/mL, and the cells were plated in a 96-well round-bottom culture plate (corning 3799). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended with 200 ⁇ L 1% BSA (in PBS). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were placed at 4°C for later use.
  • the antibody sample to be tested was diluted with 1% BSA (in PBS) with a starting concentration of 100 nM and diluted 10 times to 7 concentrations.
  • the cells were resuspended with the diluted antibody, 100 ⁇ L/well, and incubated at 4°C for 1 hour.
  • the cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • the cells were resuspended and washed with 160 ⁇ L 1% BSA (in PBS), centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • Dilute the secondary antibody (goat anti human IgG Fc PE) with 1% BSA (in PBS) at 1:400 according to the instructions, resuspend the cells with the diluted secondary antibody, 100 ⁇ L/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 ⁇ L 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend the cells with 100 ⁇ L 1% BSA (in PBS), filter the cells with 300 mesh gauze, and detect the average fluorescence intensity of the PE channel by flow cytometry.
  • the FCS file was exported from the flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of the PE channel of each sample was analyzed using flowjo software.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half binding concentration of the antibody to the cell (hereinafter referred to as EC 50 ) and the highest mean fluorescence intensity (Top MFI).
  • EC 50 half binding concentration of the antibody to the cell
  • Top MFI top MFI
  • Example 18 Humanized design of alpaca-derived anti-GPRC5D chimeric antibody
  • 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.
  • Expi293F TM cells (Thermo Fisher Scientific) were cultured in serum-free medium, inoculated in shake flasks (Corning Inc.), and cultured on a shaker at 37°C and 8% CO 2. 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 changed to PBS for aliquoting. The final purified humanized antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.
  • Example 20 Binding of alpaca-derived anti-GPRC5D humanized antibodies to cells expressing GPRC5D
  • the culture medium of NCI-H929 cells was RPMI 1640 + 10% FBS + 0.05 mM mercaptoethanol, and the culture medium of cynomolgus monkey GPRC5D-CHOK1 cells was F12K + 10% FBS + 400 ⁇ g/mL Hygromycin. T75 cell culture flasks were placed in a 37°C 5% CO 2 incubator for culture.
  • NCI-H929 cells When using NCI-H929 cells, transfer them directly to a 50 mL centrifuge tube with a pipette. When using cynomolgus monkey GPRC5D-CHOK1 cells, wash the cells twice with sterile DPBS, digest with 0.25% trypsin EDTA for about 5 minutes, and then terminate with complete culture medium.
  • the obtained cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 100 ⁇ L 1% BSA (in PBS). The cells were counted and the cell density was adjusted to 1E6/mL, and the cells were plated in a 96-well round-bottom culture plate (corning 3799). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended with 200 ⁇ L 1% BSA (in PBS). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were placed at 4°C for later use.
  • the antibody sample to be tested was diluted with 1% BSA (in PBS) with a starting concentration of 100 nM and diluted 10 times to 7 concentrations.
  • the cells were resuspended with the diluted antibody, 100 ⁇ L/well, and incubated at 4°C for 1 hour.
  • the cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • the cells were resuspended and washed with 160 ⁇ L 1% BSA (in PBS), centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • Dilute the secondary antibody (goat anti human IgG Fc PE) with 1% BSA (in PBS) at 1:400 according to the instructions, resuspend the cells with the diluted secondary antibody, 100 ⁇ L/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 ⁇ L 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend the cells with 100 ⁇ L 1% BSA (in PBS), filter the cells with 300 mesh gauze, and detect the average fluorescence intensity of the PE channel by flow cytometry.
  • the FCS file was exported from the flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of the PE channel of each sample was analyzed using flowjo software.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half binding concentration of the antibody to the cells (hereinafter referred to as EC 50 ) and the highest mean fluorescence intensity (Top MFI).
  • EC 50 half binding concentration of the antibody to the cells
  • Top MFI top MFI
  • Example 21 Binding of alpaca-derived anti-GPRC5D humanized antibody to the same family member GPRC5A
  • the culture medium of human GPRC5A-CHOK1 cells is F12K+10% FBS+400 ⁇ g/mL Hygromycin.
  • the cells are cultured in a T75 cell culture flask 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 about 5 minutes, and then terminated with complete culture medium.
  • the obtained cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 100 ⁇ L 1% BSA (in PBS). The cells were counted and the cell density was adjusted to 1E6/mL, and the cells were plated in a 96-well round-bottom culture plate (corning 3799). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended with 200 ⁇ L 1% BSA (in PBS). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were placed at 4°C for later use.
  • the antibody sample to be tested was diluted with 1% BSA (in PBS) with a starting concentration of 100 nM and diluted 10 times to 7 concentrations.
  • the cells were resuspended with the diluted antibody, 100 ⁇ L/well, and incubated at 4°C for 1 hour.
  • the cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • the cells were resuspended and washed with 160 ⁇ L 1% BSA (in PBS), centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • Dilute the secondary antibody (goat anti human IgG Fc PE) with 1% BSA (in PBS) at 1:400 according to the instructions, resuspend the cells with the diluted secondary antibody, 100 ⁇ L/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 ⁇ L 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend the cells with 100 ⁇ L 1% BSA (in PBS), filter the cells with 300 mesh gauze, and detect the average fluorescence intensity of the PE channel by flow cytometry.
  • the FCS file was exported from the flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of the PE channel of each sample was analyzed using flowjo software.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half binding concentration of the antibody to the cells (hereinafter referred to as EC 50 ) and the highest mean fluorescence intensity (Top MFI). The results are shown in Figure 16, indicating that the tested antibody had no non-specific binding to the same family member GPRC5A.
  • Example 22 Obtaining anti-BCMA antibodies from mouse hybridomas
  • 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 level. 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 is human BCMA ECD protein (Sino Biological, Cat. No. 10620-H08H), with 25 ⁇ g of protein each immunization on days 0, 14, 28, and 42.
  • Booster immunization was performed 2 days before spleen cell fusion. During this period, the mouse serum was detected by ELISA to determine the antibody titer in the mouse serum.
  • mice with high antibody titers in the serum and titers tending to a plateau were selected for spleen cell fusion, and the spleen lymphocytes were fused with myeloma cells using an optimized electrofusion step. Sp2/0 cells The fusion was performed to obtain hybridoma cells.
  • the culture supernatant was taken and the hybridoma supernatant was screened for antibodies using CHO-K1 cells that highly expressed human BCMA by FACS.
  • the positive antibody strains obtained were further screened using CHO-K1 cells that highly expressed 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.
  • Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScriptTM Reverse Transcriptase, Takara #2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced to obtain the amino acid sequences of the 11 monoclonal antibody variable regions disclosed in the present invention: 19CH-1, 19CH-2, 19CH-3, 19CH-4, 19CH-5, 19CH-7, 19CH-8, 19CH-9, 19CH-11, 19CH-13 and 19CH-16, among which 19CH-16 is shown in Table 22.
  • Example 23 Construction of anti-BCMA chimeric antibodies derived from mouse hybridomas and their transient transfection expression in eukaryotic cells
  • sequenced heavy chain variable region and light chain variable region of the monoclonal antibody disclosed herein were respectively spliced with the IgG1 (L234AL235A) heavy chain constant region and the ⁇ light chain constant region to generate the target gene fragments cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.
  • Expi293F TM cells (Thermo Fisher, A14527) were cultured in Expi293 expression medium (Thermo Fisher, A1435101), the cells were inoculated in a shake flask, and cultured on a shaker at 37°C, 8% CO2. The cell density was adjusted, 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.
  • Example 24 Binding of anti-BCMA chimeric antibodies derived from mouse hybridomas to cells expressing BCMA
  • the culture medium for human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells was F12K + 10% FBS + 400 ⁇ g/mL Hygromycin, and cultured in a T75 cell culture flask at 37°C in a 5% CO 2 incubator. When ready for use, wash the cells twice with sterile DPBS, digest with 0.25% trypsin EDTA for about 5 minutes, and then terminate with complete culture medium.
  • the obtained cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 100 ⁇ L 1% BSA (in PBS). The cells were counted and the cell density was adjusted to 1E6/mL, and the cells were plated in a 96-well round-bottom culture plate (corning 3799). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended with 200 ⁇ L 1% BSA (in PBS). The cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were placed at 4°C for later use.
  • the antibody sample to be tested was diluted with 1% BSA (in PBS) with a starting concentration of 100 nM and diluted 10 times to 7 concentrations.
  • the cells were resuspended with the diluted antibody, 100 ⁇ L/well, and incubated at 4°C for 1 hour.
  • the cells were centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • the cells were resuspended and washed with 160 ⁇ L 1% BSA (in PBS), centrifuged at 1500 rpm for 5 minutes at 4°C, and the supernatant was discarded.
  • Dilute the secondary antibody (goat anti human IgG Fc PE) with 1% BSA (in PBS) at 1:400 according to the instructions, resuspend the cells with the diluted secondary antibody, 100 ⁇ L/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 ⁇ L 1% BSA (in PBS), centrifuge at 1500 rpm at 4°C for 5 minutes and discard the supernatant. Resuspend the cells with 100 ⁇ L 1% BSA (in PBS), filter the cells with 300 mesh gauze, and detect the average fluorescence intensity of the PE channel by flow cytometry.
  • the FCS file was exported from the flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of the PE channel of each sample was analyzed using flowjo software.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half binding concentration of the antibody to the cell (hereinafter referred to as EC 50 ) and the highest mean fluorescence intensity (Top MFI).
  • the results are shown in Table 24 and Figure 17.
  • the screened anti-BCMA chimeric antibody (19CH-16) has good binding to both human BCMA and cynomolgus monkey BCMA at the cell level.
  • Example 25 Humanized design of anti-BCMA antibodies derived from mouse hybridomas
  • the antibody with clone number 19CH-16 was selected for humanization design.
  • mouse anti-human BCMA monoclonal antibodies The humanization of mouse anti-human BCMA monoclonal antibodies was performed as disclosed in many literatures in the art. In brief, the parent (mouse antibody) constant domain was replaced with a human constant domain, and the human antibody sequence was selected based on the homology between the mouse antibody and the human antibody. On the basis of typical structures, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain human germline templates with high homology.
  • 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 for the heavy chain and ⁇ for the light chain). Then, based on the three-dimensional structure of the mouse antibody, the buried residues, the residues directly interacting with the CDR region, and the residues that have an important influence on the conformation of VL and VH were back mutated, and an antibody composed of the following combination of humanized light and heavy chain variable region sequences was 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.
  • the IgG constant region preferably IgG
  • Example 26 Preparation of anti-BCMA humanized antibodies derived from mouse hybridoma
  • the target gene fragments generated by splicing the heavy chain variable region and light chain variable region of the humanized antibody with the heavy chain constant region of IgG1 (L234AL235A) and the kappa light chain constant region were cloned into the pTT5 expression vector to prepare a transfection-grade expression plasmid.
  • Expi293F TM cells (Thermo Fisher, A14527) were cultured in Expi293 expression medium (Thermo Fisher, A1435101), inoculated in a shake flask, 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 pH3.0-pH3.5 and neutralized with 1M Tris-HCl, pH8.0-9.0. After the eluted sample was appropriately concentrated, the solution was changed to PBS for aliquoting. The final purified chimeric antibody was subjected to SDS-PAGE and HPLC purity analysis and A280 concentration determination.
  • Example 27 Binding of anti-BCMA humanized antibodies derived from mouse hybridomas to cells expressing BCMA
  • the culture medium for human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells is F12K + 10% FBS + 400 ⁇ g/mL Hygromycin.
  • human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells are washed twice with sterile DPBS, digested with 0.25% trypsin EDTA for about 5 minutes, and then terminated with complete culture medium.
  • the obtained cells were centrifuged at 1000 rpm for 5 minutes at room temperature, the supernatant was discarded, and the cells were resuspended with 100 ⁇ L 1% BSA (in PBS).
  • the cells were counted and the cell density was adjusted to 1E6/mL, and the cells were plated in a 96-well round-bottom culture plate (corning 3799).
  • the cells were centrifuged at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended with 200 ⁇ L 1% BSA (in PBS).
  • the cells were centrifuged again at 1500 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were placed at 4°C for later use.
  • the antibody sample to be tested was diluted with 1% BSA (in PBS) with a starting concentration of 100 nM and 7 concentrations diluted 10 times.
  • the cells were resuspended with the diluted antibody, 100 ⁇ L/well, and incubated at 4°C Incubate for 1 hour. Centrifuge at 1500rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash with 160 ⁇ L 1% BSA (in PBS), centrifuge at 1500rpm for 5 minutes at 4°C and discard the supernatant.
  • Dilute the secondary antibody (goat anti human IgG Fc PE) with 1% BSA (in PBS) at 1:400 according to the instructions, resuspend the cells with the diluted secondary antibody, 100 ⁇ L/well, and incubate at 4°C for 0.5 hour. Centrifuge at 1500rpm for 5 minutes at 4°C and discard the supernatant. Resuspend and wash with 200 ⁇ L 1% BSA (in PBS), centrifuge at 1500rpm for 5 minutes at 4°C and discard the supernatant. Resuspend the cells with 100 ⁇ L 1% BSA (in PBS), filter the cells with 300-mesh gauze, and detect the average fluorescence intensity of the PE channel by flow cytometry.
  • the FCS file was exported from the flow cytometer, and the mean fluorescence intensity (hereinafter referred to as MFI) of the PE channel of each sample was analyzed using flowjo software.
  • MFI mean fluorescence intensity
  • the mean fluorescence intensity obtained by the analysis was imported into Graphpad to analyze the half binding concentration of the antibody to the cell (hereinafter referred to as EC 50 ) and the highest mean fluorescence intensity (Top MFI).
  • EC 50 half binding concentration of the antibody to the cell
  • Top MFI top MFI
  • Example 28 Preparation of variants of anti-BCMA humanized antibodies derived from mouse hybridomas
  • Post-translational modification (PTM) analysis of the CDR region of the anti-BCMA humanized antibody 19CH-16H2L2 revealed that there was a deamidation site in the heavy chain variable region.
  • PTM Post-translational modification
  • a single site-directed mutagenesis of the 19CH-16H2 amino acid sequence was performed to obtain two variants, 19CH-16H2L2-NA and 19CH-16H2L2-QT.
  • the variable region amino acid sequence of the 19CH-16H2L2-NA variant is shown in Table 27.
  • Example 26 the above two variant proteins were prepared by transient expression in Expi293 cells.
  • Example 27 the affinity of the two variants was measured using human BCMA-CHOK1 cells and cynomolgus monkey BCMA-CHOK1 cells. The results are shown in Table 28 and Figure 19. 19CH-16H2L2-NA can maintain affinity at the cell binding level while removing the risk of post-translational modification.

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Abstract

提供了三特异性抗原结合分子的构型，还提供了基于该构型所构建的针对两种肿瘤抗原GPRC5D和BCMA以及T细胞表面抗原CD3的三特异性抗原结合分子、针对GPRC5D的特异性结合分子或其片段、针对BCMA的特异性结合分子或其片段、包含所述三特异性抗原结合分子或所述特异性结合分子或其片段的药物组合物及其在治疗肿瘤方面的相关应用。

Description

三特异性抗原结合分子及其应用
相关申请的交叉引用
本申请要求于2023年2月7日提交的中国专利申请2023101003684号的优先权，本申请引用上述中国专利申请的全文。
技术领域
本公开属于免疫学领域，主要涉及一种三特异性抗原结合分子。更具体地，涉及特异性结合GPRC5D和BCMA两种肿瘤抗原和T细胞表面抗原CD3的三特异性抗原结合分子，以及包含所述分子的药物组合物、制备方法和用途。
背景技术
多发性骨髓瘤(multiple myeloma，MM)是一种血液系统的恶性肿瘤，是仅次于非霍奇金淋巴瘤的第二大常见血液学恶性肿瘤。多发性骨髓瘤可破坏骨骼、免疫系统、肾脏及红细胞数量，通常导致广泛的骨骼破坏，并伴有溶骨性病变、骨质减少及病理性骨折。该病常见于中老年人，随着老龄化的加速以及随着诊疗手段的进步，患者人群呈现快速增长的趋势。
与绝大多数的癌症治疗方式一样，多发性骨髓瘤的标准治疗为化疗，普遍用药是马法兰(melphalan)等，通常服用这类药后会导致骨髓抑制，而且疗效不甚理想，治疗后完全缓解率只有约5％。近几年随着医学的进步，靶向药物，例如蛋白酶体抑制剂，免疫调节剂及抗体类的药物开发为多发性骨髓瘤的治疗带来了变革，与原有治疗方案相比，大大提高了存活率，显著改善了患者的预后，但多发性骨髓瘤仍然无法治愈，而且大多数患者会在多线治疗后复发，我国患者的中位生存期为24-36个月，五年生存率仅约24％，因此，亟需新的治疗方式。
B细胞成熟抗原(B cell maturation antigen，BCMA)是一种跨膜的糖蛋白受体(非酪氨酸激酶受体)，是肿瘤坏死因子家族的成员。其主要在成熟的B细胞表面表达，少量的在造血干细胞或其他组织细胞中表达。BCMA的配体分子为BAFF(B cell activating factor)和APRIL(a proliferation-inducing ligand)。BCMA的信号通路在正常的生理状态下可以促进B细胞分化成浆细胞，维持长生命周期骨髓浆细胞的稳态。BCMA在MM患者的骨髓浆细胞中过量表达，过量表达的BCMA可以通过细胞内的AKT、MAPK以及(NF)-κB信号通路，刺激恶性浆细胞的增殖，维持其活性，从而促进疾病的进展。阻断BCMA信号通路后，可以抑制恶性浆细胞的增殖，抑制/缓解病情的进展，因此BCMA是MM临床治疗的一个很好的靶点。另外，细胞膜上的BCMA蛋白还可以被体内的γ-分泌酶切割，切割后的BCMA蛋白在血清中处于游离状态，被称为可溶的BCMA蛋白(sBCMA)。sBCMA可以和配体形成复合物，从而降低血清中配体的浓度、阻断配体和膜结合型BCMA的结合，阻止BCMA信号通路。
GPRC5D是G蛋白偶联受体C5家族亚型D(G protein coupled receptor C5 family subtype D)，属于一种孤儿 受体，为7次跨膜蛋白。GPRC5D特异性地在多发性骨髓瘤的浆细胞高表达，在正常组织中只有毛囊有。针对GPRC5D的抗体及CAR-T细胞疗法，临床前均显示了乐观的效果，而在实验中动物并没有出现脱毛的现象。而且GPRC5D的表达不受限于BCMA的表达，在BCMA抗原丢失导致的肿瘤复发模型中，GPRC5D靶向的CAR-T疗法可以克服肿瘤逃逸。这些研究结果均表明GPRC5D在临床上是一个理想靶标。
靶向肿瘤相关抗原(tumor-associated antigen,TAA)和CD3抗原的双特异性抗体能够将T细胞与肿瘤细胞在空间上拉近，形成突触，利用T细胞对肿瘤细胞实现特异性的杀伤作用，这类抗体被称为T细胞衔接器(T cell-engager,TCE双抗)。基于TCE的治疗策略依赖于TAA在待治疗的肿瘤细胞上的分布。此外，研究也显示，靶向单个TAA位点的治疗形式可能会因肿瘤的逃逸机制而引起疾病的复发，从而限制治疗的有效性。
由于GPRC5D和BCMA在肿瘤中的表达呈非相关性，设计组合靶向GPRC5D和BCMA的三特异性抗体将既可以覆盖GPRC5D阳性肿瘤患者，也可以覆盖BCMA阳性肿瘤患者，同时可以避免由于单一抗原丢失带来的复发，从而达到提高患者覆盖率和改善治疗功效的目的。
发明概述
为了实现上述目的，本公开提供了靶向GPRC5D/BCMA/CD3的三特异性抗原结合分子，并对多种分子构型进行了深入研究，成功实现了多靶点组合，避免了多药联用的不便。
本公开的第一个方面是提供一种三特异性抗原结合分子。该三特异性抗原结合分子种构型包含以下多肽：
第一多肽，包含：(i)能够特异性结合第一抗原的抗原结合片段Fab的重链结构域，(ii)能够特异性结合第二抗原的单链抗体(scFv)结构域，和(iii)第一Fc结构域；
第二多肽，包含：能够特异性结合第一抗原的抗原结合片段Fab的轻链结构域；
第三多肽，包含：(i)能够特异性结合第三抗原的重链单域抗体(VHH)结构域和(ii)第二Fc结构域。
所述第一多肽的抗原结合片段Fab的重链结构域与所述第二多肽的抗原结合片段Fab的轻链结构域形成针对第一抗原的第一结合位点；所述单链抗体(scFv)结构域形成针对第二抗原的第二结合位点；所述重链单域抗体(VHH)结构域形成针对第三抗原的第三结合位点；所述第一Fc结构域和所述第二Fc结构域相互缔合。
任选地，所述三特异性抗原结合分子还包含第四多肽，其包含特异性结合第一抗原的抗原结合片段Fab的轻链结构域，所述抗原结合片段Fab的轻链结构域与第二多肽的抗原结合片段Fab的轻链结构域相同，且所述第三多肽还包含特异性结合第一抗原的抗原结合片段Fab的重链结构域，所述第三多肽的抗原结合片段Fab的重链结构域与第一多肽的抗原结合片段Fab的重链结构域相同，其C端与VHH结构域的N端相连，所述第三多肽的Fab的重链结构域与第四多肽的Fab的轻链结构域形成针对第一抗原的第四结合位点。
在一个实施方案中，所述scFv结构域包含重链可变区和轻链可变区；优选地，所述scFv结构域的重链可变区 与轻链可变区通过第一接头连接，其中重链可变区的C端与第一接头的N端融合，第一接头的C端与轻链可变区的N端融合。
更优选地，所述第一接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
在一个实施方案中，所述第一Fc结构域包含免疫球蛋白的第一CH2结构域和第一CH3结构域，所述第一CH2结构域的C端与第一CH3结构域的N端融合；所述第二Fc结构域包含免疫球蛋白的第二CH2结构域和第二CH3结构域，所述第二CH2结构域的C端与第二CH3结构域的N端融合。
优选地，所述第一CH3结构域包含“节”(knob)结构，所述第二CH3结构域包含“穴”(hole)结构；更优选地，所述“节”(knob)结构包含氨基酸取代S354C和T366W，所述“穴”(hole)结构包含氨基酸取代Y349C、T366S、L368A和Y407V。
优选地，为降低抗体的ADCC活性，第一和/或第二Fc结构域包含L234A、L235A和/或G237A的氨基酸取代。
优选地，所述第三多肽的第二Fc结构域包含H435R的氨基酸取代。
优选地，所述Fc结构域来源于IgG1。
在一个实施方案中，所述第一Fc结构域的N端与所述scFv结构域的C端融合，优选地，所述第一Fc结构域的N端通过第二接头与所述scFv结构域的C端融合，更优选地，所述第二接头包含氨基酸序列EPKSS。
在一个实施方案中，所述抗原结合片段Fab的重链结构域包含免疫球蛋白的重链可变区和CH1结构域，所述重链可变区的C端与CH1结构域的N端融合；抗原结合片段Fab的轻链结构域包含免疫球蛋白的轻链可变区和轻链恒定区，所述轻链可变区的C端与轻链恒定区的N端融合。
在一个实施方案中，所述第一多肽的抗原结合片段Fab的重链结构域与所述scFv结构域通过第三接头连接，其中抗原结合片段Fab的重链结构域的C端与第三接头的N端融合，第三接头的C端与scFv结构域的N端融合。
优选地，所述第三接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
任选地，所述第三多肽的抗原结合片段Fab的重链结构域的C端通过第五接头与VHH结构域的N端相连。优选地，所述第五接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
优选地，所述重链单域抗体(VHH)结构域的C端与第二Fc结构域的N端融合，优选地，所述VHH结构域的C端通过第四接头与第二Fc结构域的N端融合，更优选地，所述第四接头包含氨基酸序列EPKSS。
在一个实施方案中，第一多肽包含如下结构：Fab重链结构域-第三接头-scFv结构域-第二接头-第一Fc结构域。
优选地，所述第一多肽包含如下结构：Fab重链可变区-Fab CH1-第三接头-scFv的重链可变区-第一接头-scFv的 轻链可变区-第二接头-第一CH2-第一CH3。
在一个实施方案中，第二多肽包含如下结构：Fab轻链可变区-轻链恒定区。
在一个实施方案中，第三多肽包含如下结构：VHH结构域-第四接头-第二Fc结构域。优选地，所述第三多肽包含如下结构：VHH-第四接头-第二CH2-第二CH3。
任选地，第四多肽包含如下结构：Fab轻链可变区-轻链恒定区，且第三多肽包含如下结构：Fab重链结构域-第五接头-VHH-第四接头-第二Fc结构域，优选地所述第三多肽包含如下结构：Fab重链可变区-Fab CH1-第五接头-VHH-第四接头-第二CH2-第二CH3。
在一个实施方案中，所述第二抗原为CD3，优选为CD3ε；优选地，所述scFv结构域包含序列如SEQ ID NO：27所示的HCDR1、序列如SEQ ID NO：28所示的HCDR2、序列如SEQ ID NO：29所示的HCDR3、序列如SEQ ID NO：30所示的LCDR1、序列如SEQ ID NO：31所示的LCDR2和序列如SEQ ID NO：32所示的LCDR3。
更优选地，所述scFv结构域包含序列如SEQ ID NO：25所示的重链可变区和序列如SEQ ID NO：26所示的轻链可变区。
更优选地，所述scFv结构域包含SEQ ID NO：13所示的氨基酸序列。
在一个实施方案中，所述第一Fc结构域包含SEQ ID NO：33所示的氨基酸序列，所述第二Fc结构域包含SEQ ID NO：34所示的氨基酸序列。
在一个实施方式中，所述第一抗原为BCMA；优选地，所述抗原结合片段Fab包含序列如SEQ ID NO：16所示的HCDR1、序列如SEQ ID NO：17所示的HCDR2和序列如SEQ ID NO：18所示的HCDR3，和/或，序列如SEQ ID NO:19所示的LCDR1、序列如SEQ ID NO:20所示的LCDR2和序列如SEQ ID NO:21所示的LCDR3。
更优选地，所述抗原结合片段Fab的重链结构域包含序列如SEQ ID NO：14所示的重链可变区，和/或，所述抗原结合片段Fab的轻链结构域包含序列如SEQ ID NO：15所示的轻链可变区。
更优选地，所述抗原结合片段Fab的重链结构域包含如SEQ ID NO：35所示的氨基酸序列，和/或，所述抗原结合片段Fab的轻链结构域包含SEQ ID NO:7所示的氨基酸序列。
在一个实施方式中，所述第三抗原为GPRC5D；优选地，所述能够特异性结合第三抗原的重链单域抗体(VHH)结构域包含序列如SEQ ID NO:22所示的HCDR1、序列如SEQ ID NO:23所示的HCDR2和序列如SEQ ID NO:24所示的HCDR3；更优选地，所述VHH结构域包含SEQ ID NO:10所示的序列。
在一个实施方案中，所述三特异性抗原结合分子的第一多肽包含SEQ ID NO：5所示的氨基酸序列，所述第二多肽包含SEQ ID NO:7所示的氨基酸序列，所述第三多肽包含SEQ ID NO:6所示的氨基酸序列；或者任选地，所述三特异性抗原结合分子的第一多肽包含SEQ ID NO:5所示的序列，所述第二多肽和/或第四多肽包含SEQ ID NO:7所示的序列，所述第三多肽包含SEQ ID NO:8所示的序列。
本公开还提供了核酸分子，其编码本公开所述的三特异性抗原结合分子。
本公开还提供了载体，其包含所述的核酸分子。
本公开还提供了宿主细胞，其包含所述的核酸分子或者载体；优选地，所述宿主细胞为原核细胞或真核细胞；所述原核细胞优选大肠杆菌；所述真核细胞优选哺乳动物细胞或酵母；更优选地，所述哺乳动物细胞为CHO细胞、Expi293或HEK293细胞。
本公开还提供了制备三特异性抗原结合分子的方法，所述方法包括：在适合的条件下培养所述宿主细胞。
本公开还提供了抗体药物偶联物，其是将前述双特异性抗原结合分子与其他生物活性分子偶联形成；优选地，所述其他生物活性分子为小分子药物；优选地，所述双特异性抗原结合分子与所述其他生物活性分子通过接头连接。
本公开还提供了药物组合物，其包含前述三特异性抗原结合分子、核酸分子、表达载体、宿主细胞和/或抗体药物偶联物。
在一个实施方案中，所述药物组合物还包含药学上可接受的载体。
在一个实施方案中，所述药物组合物还包含一种或多种额外的治疗剂。
本公开还提供了所述三特异性抗原结合分子、核酸分子、载体、宿主细胞和/或抗体药物偶联物在制备治疗、缓解和/或预防肿瘤的药物中的用途。优选地，所述肿瘤是GPRC5D阳性和/或BCMA阳性的肿瘤。
本公开还提供一种诱导表达GPRC5D和/或BCMA的细胞死亡的方法，所述方法包括使所述细胞与所述三特异性抗原结合分子、核酸分子、载体、宿主细胞、和/或药物组合物接触，所述表达GPRC5D3和/或BCMA的细胞是肿瘤细胞。
本公开还提供一种治疗受试者中与表达GPRC5D和/或BCMA相关的疾病的方法，所述方法包括向有需要的受试者施用所述三特异性抗原结合分子、核酸分子、载体、宿主细胞、和/或药物组合物。优选地，所述疾病是肿瘤。优选地，其中受试者对于用先前的抗癌治疗剂进行的治疗具有复发性或难治性。在一个实施方案中，所述方法还包括向所述受试者给予额外的治疗剂。
优选地，本公开所述肿瘤或肿瘤细胞选自：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
本公开的另一个方面是提供一种特异性结合GPRC5D的抗原结合分子或其抗原结合片段，所述抗原结合分子或其抗原结合片段能够特异性地结合GPRC5D，而对同家族蛋白GPRC5A没有非特异结合。
优选地，所述特异性结合GPRC5D的抗原结合分子或其抗原结合片段包含重链单域抗体(VHH)结构域；优选地，所述GPRC5D抗原结合分子或其抗原结合片段包含氨基酸序列如SEQ ID NO:10所示的重链可变区的HCDR序列；优选地，所述GPRC5D抗原结合分子或其抗原结合片段包含序列如SEQ ID NO:22所示的HCDR1、序列如SEQ ID NO:23所示的HCDR2和序列如SEQ ID NO:24所示的HCDR3；更优选地，所述重链单域抗体(VHH)包含SEQ ID NO:10所示的氨基酸序列。
优选地，所述所述GPRC5D抗原结合分子或其抗原结合片段还包含免疫球蛋白的Fc结构域，所述免疫球蛋白选自IgG1、IgG2、IgG3或IgG4形式，优选地，所述GPRC5D抗原结合分子或其抗原结合片段还包含IgG1的Fc 结构域，进一步优选地，所述IgG1的Fc结构域包含如SEQ ID NO:38所示的氨基酸序列。
在一个实施方案中，所述GPRC5D抗原结合分子或其抗原结合片段包含重链单域抗体(VHH)结构域和IgG1的Fc结构域，优选地，所述重链单域抗体(VHH)结构域的C端融合到IgG1的Fc结构域的N端，更优选地，所述重链单域抗体(VHH)结构域的C端通过接头融合到IgG1的Fc结构域的N端，优选地，所述接头包含氨基酸序列EPKSS或(G4S)n，n为1-10中的任意整数。在一个优选的实施方案中，所述GPRC5D抗原结合分子或其抗原结合片段由N端至C端包含序列如SEQ ID NO:10的VHH结构域、G4S接头和序列如SEQ ID NO:38所示的IgG1的Fc结构域。
本公开的GPRC5D抗原结合分子或其抗原结合片段可以是单克隆抗体、双特异性结合分子、多特异性结合分子、鼠源抗体、人源化抗体、嵌合抗体、改型抗体、全人源抗体、全长抗体、重链抗体、纳米抗体、Fab、Fv、scFv、F(ab’)2、线性抗体或重链单域抗体。
本公开还提供一种偶联物，是将本公开的GPRC5D抗原结合分子或其抗原结合片段与捕获标记物或检测标记物偶联形成；优选地，所述检测标记物包括放射性核素、发光物质、有色物质或酶。
本公开还提供一种抗体药物偶联物(ADC)，是将本公开的GPRC5D抗原结合分子或其抗原结合片段与其他生物活性分子偶联形成；优选地，所述其他生物活性分子为小分子药物；优选地，所述GPRC5D抗原结合分子或其抗原结合片段与所述其他生物活性分子通过接头连接。
本公开还提供一种融合蛋白，其中的一个被融合的部分包含本公开的GPRC5D抗原结合分子或其抗原结合片段。
本公开还提供一种嵌合抗原受体(CAR)或包含所述嵌合抗原受体的细胞(例如CAR-T细胞)，其包含本公开的GPRC5D抗原结合分子或其抗原结合片段。
本公开还提供编码GPRC5D抗原结合分子或其抗原结合片段的核酸，以及包含该核酸的重组载体，以及包含上述核酸或载体的宿主细胞。优选地，所述宿主细胞为原核细胞(优选大肠杆菌)，或者真核细胞(优选哺乳动物细胞或酵母；进一步优选地，所述哺乳动物细胞为CHO细胞或HEK293细胞)。
本公开还提供制备GPRC5D抗原结合分子或其抗原结合片段的方法，所述方法包括：在适合的条件下培养上述宿主细胞，并从所述细胞中纯化获得表达产物。
本公开还提供GPRC5D抗原结合分子或其抗原结合片段在制备治疗或缓解肿瘤的药物中的用途。
在一个实施方案中，所述药物靶向GPRC5D异常表达的肿瘤细胞。
在一个实施方案中，所述肿瘤选自：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
本公开还提供一种治疗受试者中与表达GPRC5D相关的疾病的方法，包括向有此需要的受试者施用所述GPRC5D抗原结合分子或其抗原结合片段。
优选地，所述疾病是肿瘤；优选地，所述肿瘤疾病选自淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌；
更优选地，还包括向所述受试者给予额外的治疗剂。
本公开还提供所述的GPRC5D抗原结合分子或其抗原结合片段在制备检测试剂或诊断试剂中的用途。
在一个实施方案中，所述检测试剂用于检测GPRC5D的表达；所述诊断试剂用于诊断肿瘤；优选地，所述肿瘤选自：淋巴瘤诸如多发性骨髓瘤、以及上述肿瘤的转移癌。
本公开还提供检测样品中GPRC5D表达的方法，所述方法包括：
(1)将样品与本公开的GPRC5D抗原结合分子或其抗原结合片段接触；
(2)检测所述GPRC5D抗原结合分子或其抗原结合片段与GPRC5D的复合物的形成；任选地，所述GPRC5D抗原结合分子或其抗原结合片段是被可检测地标记的。
本公开还提供了监测受试者中GPRC5D表达型癌症的方法，所述方法包括将从受试者中获得或来源于受试者的样品暴露于本文所述的特异性结合GPRC5D的抗原结合分子或抗原结合片段中的一种或多种；测定存在于由抗体或其抗原结合片段结合的样品中的GPRC5D的量；将存在于样品中的GPRC5D的量与已知标准品或参照样品或先前从受试者中获得的类似样品中的GPRC5D的量进行比较；以及基于所比较的样品中GPRC5D的量的差异，确定受试者的GPRC5D水平是否指示癌症进展、消退或稳定疾病。
从受试者中获得或来源于受试者的样品是生物样品，诸如尿液、血液、血清、血浆、唾液、腹水、循环细胞、循环肿瘤细胞、非组织缔合的细胞、组织、手术切除的肿瘤组织、活体组织切片、细针抽吸样品或组织学制备物。
本公开还提供一种药物组合物，其包含有效量的本公开的GPRC5D抗原结合分子或其抗原结合片段、或者包含有效量的本公开的抗体药物偶联物、融合蛋白、CAR-T细胞、或者包含有效量的本公开的核酸、或者包含有效量的本公开的重组载体、或者包含有效量的本公开的宿主细胞。
在一个实施方案中，所述药物组合物还包含药学上可接受的载体。
优选地，所述药物组合物还包含一种或多种额外的其他治疗剂。
本公开的另一个方面是提供一种特异性结合BCMA的抗原结合分子或其抗原结合片段，所述抗原结合分子或其抗原结合片段包含序列如SEQ ID NO：14所示的重链可变区的HCDR序列，和/或，序列如SEQ ID NO:15所示的轻链可变区的LCDR序列；优选地，所述抗原结合分子或其抗原结合片段包含：序列如SEQ ID NO：16所示的HCDR1、序列如SEQ ID NO：17所示的HCDR2和序列如SEQ ID NO：18所示的HCDR3，和/或序列如SEQ ID NO:19所示的LCHDR1、序列如SEQ ID NO:20所示的LCDR2和序列如SEQ ID NO:21所示的LCDR3。
优选地，所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含序列如SEQ ID NO：14所示的重链可变区，和/或所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含序列如SEQ ID NO：15所示的轻链可变区。
更优选地，所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含SEQ ID NO：9所示的重链氨基酸序列，和/或，所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含SEQ ID NO:7所示的轻链氨基酸序列。
本公开的BCMA抗原结合分子或其抗原结合片段还包含重链恒定区和/或轻链恒定区；优选地，所述重链恒定区包含Fc；更优选地，Fc来源于鼠或人；更优选地，Fc的序列是天然的或经过修饰的。
本公开的BCMA抗原结合分子或其抗原结合片段可以是单克隆抗体、双特异性结合分子、多特异性结合分子、 鼠源抗体、人源化抗体、嵌合抗体、改型抗体、全人源抗体、全长抗体、重链抗体、纳米抗体、Fab、Fv、scFv、F(ab’)2、线性抗体或重链单域抗体。
本公开的BCMA抗原结合分子或其抗原结合片段可以是全长抗体。
本公开的BCMA抗原结合分子或其抗原结合片段可以是IgG1、IgG2、IgG3或IgG4形式。
本公开还提供一种偶联物，是将本公开的BCMA抗原结合分子或其抗原结合片段与捕获标记物或检测标记物偶联形成；优选地，所述检测标记物包括放射性核素、发光物质、有色物质或酶。
本公开还提供一种抗体药物偶联物(ADC)，是将本公开的BCMA抗原结合分子或其抗原结合片段与其他生物活性分子偶联形成；优选地，所述其他生物活性分子为小分子药物；优选地，所述BCMA抗原结合分子或其抗原结合片段与所述其他生物活性分子通过接头连接。
本公开还提供一种融合蛋白，其中的一个被融合的部分包含本公开的BCMA抗原结合分子或其抗原结合片段。
本公开还提供一种嵌合抗原受体(CAR)以及包含所述嵌合抗原受体的细胞(如CAR-T细胞)，其包含本公开的BCMA抗原结合分子或其抗原结合片段。
本公开还提供编码BCMA抗原结合分子或其抗原结合片段的核酸，以及包含该核酸的重组载体，以及包含上述核酸或载体的宿主细胞。优选地，所述宿主细胞为原核细胞(优选大肠杆菌)，或者真核细胞(优选哺乳动物细胞或酵母；进一步优选地，所述哺乳动物细胞为CHO细胞或HEK293细胞)。
本公开还提供制备本公开BCMA抗原结合分子或其抗原结合片段的方法，所述方法包括：在适合的条件下培养上述宿主细胞，并从所述细胞中纯化获得表达产物。
本公开还提供了BCMA抗原结合分子或其抗原结合片段在制备治疗或缓解肿瘤的药物中的用途。
在一个实施方案中，所述药物靶向BCMA异常表达的肿瘤细胞。
在一个实施方案中，所述肿瘤选自：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
本公开还提供一种治疗受试者中与表达BCMA相关的疾病的方法，包括向有此需要的受试者施用所述BCMA抗原结合分子或其抗原结合片段。
优选地，所述疾病是肿瘤；优选地，所述肿瘤疾病选自淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌；
更优选地，还包括向所述受试者给予额外的治疗剂。
本公开还提供了BCMA抗原结合分子或其抗原结合片段在制备检测试剂或诊断试剂中的用途。
在一个实施方案中，所述检测试剂用于检测BCMA的表达；所述诊断试剂用于诊断肿瘤；优选地，所述肿瘤选自：淋巴瘤诸如多发性骨髓瘤、以及上述肿瘤的转移癌。
本公开还提供检测样品中BCMA表达的方法，所述方法包括：
(1)将样品与本公开的BCMA抗原结合分子或其抗原结合片段接触；
(2)检测所述BCMA抗原结合分子或其抗原结合片段与BCMA的复合物的形成；任选地，所述BCMA抗原 结合分子或其抗原结合片段是被可检测地标记的。
本公开还提供了监测受试者中BCMA表达型癌症的方法，所述方法包括将从受试者中获得或来源于受试者的样品暴露于本文所述的BCMA特异性抗体或抗原结合片段中的一种或多种；测定存在于由抗体或其抗原结合片段结合的样品中的BCMA的量；将存在于样品中的BCMA的量与已知标准品或参照样品或先前从受试者中获得的类似样品中的BCMA的量进行比较；以及基于所比较的样品中BCMA的量的差异，确定受试者的BCMA水平是否指示癌症进展、消退或稳定疾病。
从受试者中获得或来源于受试者的样品是生物样品，诸如尿液、血液、血清、血浆、唾液、腹水、循环细胞、循环肿瘤细胞、非组织缔合的细胞、组织、手术切除的肿瘤组织、活体组织切片、细针抽吸样品或组织学制备物。
本公开还提供一种药物组合物，其包含有效量的本公开BCMA抗原结合分子或其抗原结合片段、或者包含有效量的本公开的抗体药物偶联物、融合蛋白、CAR-T细胞、或者包含有效量的本公开的核酸、或者包含有效量的本公开的重组载体、或者包含有效量的本公开的宿主细胞。
在一个实施方案中，所述药物组合物还包含药学上可接受的载体。
优选地，所述药物组合物还包含一种或多种额外的其他治疗剂。
本公开所称的取代中的氨基酸位置优选采用EU编号系统表示。
附图说明
附图更进一步说明了本说明书所公开的新特性。参照这些附图将能更好地理解本说明书中所公开的特性和优点，但应当理解，这些附图仅用于说明本文所公开原理的具体的实施方案，而非意欲对所附权利要求的范围加以限制。
图1A显示了流式细胞术检测的抗GPRC5D阳性对照抗体验证CHOK1细胞上人GPRC5D的表达情况；图中虚线所示为未转任何质粒的CHOK1母本细胞上人GPRC5D的表达情况，图中实线所示为CHOK1母本细胞转染人GPRC5D质粒后，人GPRC5D蛋白过表达情况。
图1B显示了流式细胞术检测的抗GPRC5D阳性对照抗体验证CHOK1细胞上食蟹猴GPRC5D的表达情况；图中虚线所示为未转任何质粒的CHOK1母本细胞上食蟹猴GPRC5D的表达情况，图中实线所示为CHOK1母本细胞转染食蟹猴GPRC5D质粒后，食蟹猴GPRC5D蛋白过表达情况。
图1C显示了流式细胞术检测的抗GPRC5A阳性对照抗体验证CHOK1细胞上人GPRC5A的表达情况；图中虚线所示为未转任何质粒的CHOK1母本细胞上人GPRC5A的表达情况，图中实线所示为CHOK1母本细胞转染人GPRC5A质粒后，人GPRC5A蛋白过表达情况。
图2A显示了流式细胞术检测的抗BCMA阳性对照抗体验证CHOK1细胞上人BCMA的表达情况；图中虚线所示为未转任何质粒的CHOK1母本细胞上人BCMA的表达情况，图中实线所示为CHOK1母本细胞转染人BCMA质粒后，人BCMA蛋白过表达情况。
图2B显示了流式细胞术检测的抗BCMA阳性对照抗体验证CHOK1细胞上食蟹猴BCMA的表达情况；图中虚线所示为未转任何质粒的CHOK1母本细胞上食蟹猴BCMA的表达情况，图中实线所示为CHOK1母本细胞转染食蟹猴BCMA质粒后，食蟹猴BCMA蛋白过表达情况。
图3显示了实施例中构建的2个三特异性抗体的结构，分别为三抗1和三抗2。
图4A显示了三抗1、三抗2及Teclistamab与表达双靶点BCMA和GPRC5D的肿瘤细胞NCI-H929的结合情况。
图4B显示了三抗1、三抗2及Teclistamab与表达双靶点BCMA和GPRC5D的肿瘤细胞RPMI8226的结合情况。
图4C显示了三抗1、三抗2及Teclistamab与表达人GPRC5D的稳转细胞系人GPRC5D CHOK1细胞的结合情况。
图4D显示了三抗1、三抗2及Teclistamab与表达人BCMA的稳转细胞系人BCMA CHOK1细胞的结合情况。
图4E显示了三抗1及BGCB491,TS-F2-5与表达双靶点BCMA和GPRC5D的肿瘤细胞NCI-H929的结合情况。
图4F显示了三抗1及BGCB491,TS-F2-5与表达双靶点BCMA和GPRC5D的肿瘤细胞RPMI8226的结合情况。
图4G显示了三抗1及BGCB491,TS-F2-5与表达人GPRC5D的稳转细胞系人GPRC5D CHOK1细胞的结合情况。
图4H显示了三抗1及BGCB491,TS-F2-5与表达人BCMA的稳转细胞系人BCMA CHOK1细胞的结合情况。
图5A显示了三抗1、三抗2及Teclistamab与表达human CD3细胞(Jurkat细胞)的结合情况。
图5B显示了三抗1、三抗2及Teclistamab与human PBMC细胞的结合情况。
图5C显示了三抗1及BGCB491,TS-F2-5与表达hCD3细胞(Jurkat细胞)的结合情况。
图6A显示了三抗1、三抗2及Teclistamab与表达食蟹猴GPRC5D的稳转细胞系食蟹猴GPRC5D CHOK1细胞的结合情况。
图6B显示了三抗1、三抗2及Teclistamab与表达食蟹猴BCMA的稳转细胞系食蟹猴BCMA CHOK1细胞的结合情况。
图7显示了三抗1、三抗2及Teclistamab与cyno PBMC细胞的结合情况。
图8A显示了T细胞介导的细胞毒性实验(TDCC)，靶细胞为NCI-H929。
图8B显示了T细胞介导的细胞毒性实验(TDCC)，靶细胞为RPMI8226。
图8C显示了T细胞介导的细胞毒性实验(TDCC)，靶细胞为人BCMA CHOK1。
图8D显示了T细胞介导的细胞毒性实验(TDCC)，靶细胞为人GPRC5D CHOK1。
图8E显示了T细胞介导的细胞毒性实验(TDCC)，靶细胞为人BCMA CHOK1与人GPRC5D CHOK1混合细胞体系。
图9显示了在2500ng/ml的soluble BCMA存在下，T细胞介导的细胞毒性实验(虚线表示2500ng/ml的soluble BCMA条件)。
图10A显示了三抗1、三抗2及Teclistamab在PBMC及肿瘤细胞NCI-H929共孵育条件下IL6释放(PBMC捐献者号P122080606F)。
图10B显示了三抗1、三抗2及Teclistamab在PBMC及肿瘤细胞NCI-H929共孵育条件下IL6释放(PBMC捐献者号XC11210)。
图11显示了三抗1、三抗2及Teclistamab、Teclistamab与Talquetamb联合在人源免疫细胞重建小鼠模型上的药效实验结果-肿瘤体积变化(肿瘤细胞为NCI-H929)。
图12显示了三抗1及BGCB491,TS-F2-5在人源免疫细胞重建小鼠模型上的药效实验结果-肿瘤体积变化(肿瘤细胞为NCI-H929)。
图13显示了三抗1及BGCB491,TS-F2-5在人源免疫细胞重建小鼠模型上的药效实验结果-肿瘤重量变化(肿瘤细胞为NCI-H929)。
图14A显示了本公开的羊驼来源的抗GPRC5D嵌合抗体与表达GPRC5D细胞(人GPRC5D-CHOK1细胞系)的结合情况。
图14B显示了本公开的羊驼来源的抗GPRC5D嵌合抗体与表达GPRC5D细胞(食蟹猴GPRC5D-CHOK1细胞系)的结合情况。
图15A显示了本公开的羊驼来源的抗GPRC5D人源化抗体与表达GPRC5D细胞(NCI-H929肿瘤细胞)的结合情况。
图15B显示了本公开的羊驼来源的抗GPRC5D人源化抗体与表达GPRC5D细胞(食蟹猴GPRC5D-CHOK1细胞系)的结合情况。
图16显示了本公开的羊驼来源的抗GPRC5D人源化抗体与同家族成员GPRC5A无非特异结合。
图17A显示了本公开的小鼠杂交瘤来源的抗BCMA嵌合抗体与表达人BCMA细胞(人BCMA-CHOK1细胞系)的结合情况。
图17B显示了本公开的小鼠杂交瘤来源的抗BCMA嵌合抗体与表达食蟹猴BCMA细胞(食蟹猴BCMA-CHOK1细胞系)的结合情况。
图18A显示了本公开的小鼠杂交瘤来源的抗BCMA人源化抗体与表达人BCMA细胞(人BCMA-CHOK1细胞系)的结合情况。
图18B显示了本公开的小鼠杂交瘤来源的抗BCMA人源化抗体与表达食蟹猴BCMA细胞(食蟹猴BCMA-CHOK1细胞系)的结合情况。
图19A显示来本公开的小鼠杂交瘤来源的抗BCMA人源化抗体变体19CH-16H2L2-NA和19CH-16H2L2-QT与表达人BCMA细胞(人BCMA-CHOK1细胞系)的结合情况。
图19B显示本公开的小鼠杂交瘤来源的抗BCMA人源化抗体变体19CH-16H2L2-NA和19CH-16H2L2-QT表达食蟹猴BCMA细胞(食蟹猴BCMA-CHOK1细胞系)的结合情况。
发明详述
术语
本说明书中提及的所有公布、专利和专利申请都以引用的方式并入本文，所述引用的程度就如同已特定地和个别地指示将各个别公布、专利或专利申请以引用的方式并入本文。
在下文详细描述本公开前，应理解本公开不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本公开的范围。除非另外定义，本文中使用的所有技术和科学术语与本公开所属领域中普通技术人员通常的理解具有相同的含义。
本文所公开的某些实施方案包含了数值范围，并且本公开的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本公开的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。不论所述数值的宽窄，上述原则均同等适用。当采用范围描述时，该范围包括范围的端点。
当涉及可测量值比如量、暂时持续时间等时，术语“约”是指包括指定值的±20％、或在某些情况下±10％、或在某些情况下±5％、或在某些情况下±1％、或在某些情况下±0.1％的变化。
本文中的术语“抗体”可以包含完整抗体(例如全长单克隆抗体)及其任何抗原结合片段(即抗原结合部分)或其单链，还可以包含在完整抗体或其抗原结合片段或其单链的基础上进行改造(例如连接其他肽段、功能单位重排等)而形成的具有抗原特异性结合能力的产物。
在一个实施方案中，抗体典型是指包含通过共价二硫键和非共价相互作用保持在一起的两条重(H)多肽链和两条轻(L)多肽链的Y型四聚蛋白。天然IgG抗体即具有这样的结构。每条轻链由一个可变结构域(VL)和一个恒定结构域(CL)组成。每条重链包含一个可变结构域(VH)和恒定区。
本领域已知五个主要类别的抗体：IgA，IgD，IgE，IgG和IgM，对应的重链恒定结构域分别被称为α，δ，ε，γ和μ，IgG和IgA可以进一步分为不同的亚类，例如IgG可分为IgG1，IgG2，IgG3，IgG4，IgA可分为IgA1和IgA2。来自任何脊椎动物物种的抗体的轻链基于其恒定结构域的氨基酸序列可以被分配到两种明显相异的类型之一， 称为κ和λ。
在IgG、IgA和IgD抗体的情形中，该恒定区包含称为CH1、CH2和CH3的三个结构域(IgM和IgE具有第四结构域CH4)。在IgG、IgA和IgD类别中，CH1和CH2结构域被柔性铰链区分离，该铰链区是可变长度的富含脯氨酸和半胱氨酸的区段。每类抗体进一步包含由配对半胱氨酸残基形成的链间和链内二硫键。
术语“可变区”或“可变结构域”显示出从一种抗体到另一种抗体的氨基酸组成的显著变化，并且主要负责抗原识别和结合。每个轻链/重链对的可变区形成抗体结合位点，使得完整的IgG抗体具有两个结合位点(即它是二价的)。重链的可变区(VH)和轻链的可变区(VL)结构域各包含具有极端变异性的三个区域，被称为高变区(HVR)，或更通常地，被称为互补决定区(CDR)，VH和VL各有4个骨架区FR，分别用FR1，FR2，FR3，FR4表示。因此，CDR和FR序列通常出现在重链可变结构域(或轻链可变结构域)的以下序列中：FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
术语“抗体片段”包含完整抗体的至少一部分。如在此所使用，抗体分子的“片段”包括抗体的“抗原结合片段”，并且术语“抗原结合片段”是指免疫球蛋白或抗体中与所选抗原或其免疫原性决定部分特异性结合或反应的多肽片段，或由此片段进一步衍生的融合蛋白产物，例如单链抗体，嵌合抗原受体中的胞外结合区等。示例性的抗体片段或其抗原结合片段包括但不限于：可变轻链片段、可变重链片段、Fab片段、F(ab’)2片段、Fd片段、Fv片段、单结构域抗体、线性抗体、单链抗体(scFv)及由抗体片段形成的双特异性抗体或多特异性抗体等。
术语“Fab”或“Fab片段”是指由重链的VH和CH1结构域以及轻链的VL和CL结构域组成的单价的抗体片段。术语“F(ab’)2”或“F(ab’)2片段”包含2个Fab片段以及铰链区，其为二价的抗体片段。
术语“单链抗体”或“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，scFv可以以任何顺序(例如相对于多肽的N-末端和C-末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
“VHH结构域”，亦称为重链单域抗体、VHH、VHH抗体片段、VHH抗体、纳米抗体，是称为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白的可变结构域。使用术语“VHH结构域”以将所述可变结构域与存在于常规四肽链结构抗体中的重链可变结构域(其在本公开中称为“VH结构域”)以及轻链可变结构域(其在本公开中称为“VL结构域”)进行区分。VHH结构域特异性结合表位而无需其他抗原结合结构域(此与常规四肽链结构抗体中的VH或VL结构域相反，在该情况下表位由VL结构域与VH结构域一起识别)。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。术语“重链单域抗体”、“VHH结构域”、“VHH”、“VHH结构域”、“VHH抗体片段”、“VHH抗体”以及“重链抗体可变区”可互换使用。“VHH结构域”包括但不限于经骆驼科动物产生的天然抗体，也可以是骆驼科动物产生的抗体后再经人源化的，也可以是经噬菌体体展示技术筛选获得的。
术语“氨基酸修饰”(或“修饰的氨基酸”)包括在多肽序列中的氨基酸取代、插入和/或缺失。本文中的“氨基酸取代”或“取代”或“替代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代S32A指第32位的丝氨酸被丙氨酸替换。
本公开的多特异性抗原结合分子还可以包括恒定区(例如Fc)的取代或修饰，包括但不限于氨基酸残基取代、突变和/或修饰，它们产生具有以下优选的特征的化合物，这些优选的特征包括但不限于：改变的药物代谢动力学、增加的血清半衰期、增加的结合亲和力、降低的免疫原性、增加的产量、与Fc受体(FcR)的改变的Fc配体结合、增强或减弱的ADCC或CDC、改变的糖基化和/或二硫键以及修饰的结合特异性。在某些方面，抗体变体包含具有一处或多处削弱FcγR结合的氨基酸替代(例如Fc区的位置234和235处的替代)的Fc区。在一个方面，该替代是L234A和L235A。
术语“Fc”用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。虽然IgG重链的Fc区的边界可以略微变化，但是人IgG重链Fc区通常定义为自Cys226或Pro230延伸至重链的羧基端，例如，IgG Fc域包含IgG CH2和IgG CH3恒定域。除非本文中另外指定，Fc区或恒定区中氨基酸残基的编号方式依照EU编号系统，也称作EU索引。
术语“节-入-穴”(Knob-into-Hole)是指一种用于促进Fc的两条多肽链缔合的修饰，其包含在Fc的两个多肽链之一中的“节”(knob)修饰和在Fc的两个多肽链之另一中的“穴”(hole)修饰。一般地，该方法牵涉在第一多肽链的界面处引入隆起(“节”)并在第二多肽链的界面中引入相应的空腔(“穴”)，使得隆起可以置于空腔中从而促进异二聚体形成并阻碍同二聚体形成。通过将来自第一多肽链界面的小氨基酸侧链用更大的侧链(例如酪氨酸或色氨酸)替换来构建隆起。在第二多肽链的界面中创建具有与隆起相同或相似大小的互补性空腔，其通过将大氨基酸侧链用更小的氨基酸侧链(例如丙氨酸或苏氨酸)替换进行。
因而，在一个具体的实施方案中，在本公开的三特异性抗原结合分子的Fc域的第一多肽链的CH3域中，一个氨基酸残基用具有更大侧链体积的氨基酸残基替换，由此在第一多肽链的CH3域内生成隆起，其可安置于第二多肽链的CH3域内的空腔中，而且在Fc域的第二多肽链的CH3域中，一个氨基酸残基用具有更小侧链体积的氨基酸残基替换，由此在第二多肽链的CH3域内生成空腔，其中可安置第一多肽链的CH3域内的隆起。优选地，所述具有更大侧链体积的氨基酸残基选自下组：精氨酸(R)，苯丙氨酸(F)，酪氨酸(Y)，和色氨酸(W)。优选地，所述具有更小侧链体积的氨基酸残基选自下组：丙氨酸(A)，丝氨酸(S)，苏氨酸(T)，和缬氨酸(V)。
术语“接头”是指用于连接两个不同功能单元(例如抗原结合片段)的任何工具。接头的类型包括但不限于化学接头和多肽接头。多肽接头的序列不受限制。多肽接头优选是非免疫原性和柔性的，例如包含丝氨酸和甘氨酸序列的那些。取决于具体的构建体，接头可以长或短。
根据本公开，连接不同功能单元的接头优选包含柔性肽接头，例如甘氨酸-丝氨酸肽接头。在一个实施方案中，接头包含氨基酸序列(G4S)n或(G4S)nA，其中n是1-10中的任意整数选择，优选包含氨基酸序列(G4S)3或(G4S)3A。 连接VH和VL结构域以形成VH-VL或VL-VH的scFv结构域的接头优选包含柔性肽接头，例如甘氨酸-丝氨酸肽接头。在一个实施方案中，接头包含氨基酸序列(G4S)n或(G4S)nA，其中n是1-10中的任意整数选择，优选包含氨基酸序列(G4S)3或(G4S)。
本文中“抗体”可在最广的意义上使用，可包括如多克隆抗体(polyclonal antibodies)、单克隆抗体、嵌合抗体、人源化抗体及灵长类化抗体、CDR移植抗体(CDR-grafted antibody)、人类抗体(包括重组产生的人类抗体)、重组产生的抗体、胞内抗体、多特异性抗体、双特异性抗体、单价抗体、多价抗体、抗个体基因型抗体、合成抗体(包括突变蛋白及其变体)等等。
术语“单克隆抗体”(或称“单抗”)指由单一细胞克隆产生的基本均质、仅针对某一特定抗原表位的抗体。单克隆抗体可以使用本领域中已知的多种技术制备，包括杂交瘤技术、重组技术、噬菌体展示技术、转基因动物、合成技术或上述技术的组合等。
需说明的是，本公开的抗体和三特异性抗原结合分子的可变区的CDR和FR的划分是根据Kabat定义确定的。而其他命名和编号系统，例如Chothia、IMGT或AHo等，也是本领域技术人员已知的。因此，以本公开的抗体序列为基础，包含任何命名系统衍生的一种或多种CDR的人源化抗体均明确地保持在本公开的范围内。
术语“人源化抗体”是指其中非人抗体(如小鼠抗体)CDR以外的所有或部分氨基酸被源自人免疫球蛋白的相应氨基酸置换的抗体。氨基酸的少量添加、缺失、插入、取代或修饰是容许的，只要它们不消除抗体结合特定抗原的能力。“人源化”抗体保持与原抗体类似的抗原特异性。
术语“嵌合抗体”是指其中可变区源自一个物种而恒定区源自另一物种的抗体，例如其中可变区源自小鼠抗体而恒定区源自人抗体的抗体。
本文所使用的“同源”和“异源”是一组相对的概念，既可以指构建体中不同要素的来源相同或不同，也可以指构建体构建完成以后，原本来源相同即“同源”的多个要素之间，有的要素进行了改造，与未进行改造的其他原要素相比发生了改变，从而变成“异源”。
术语“抗原”是指被抗体或抗体结合片段识别并特异性结合的物质，广义上，抗原可以包括所选靶标的任何免疫原性片段或决定簇，包括单表位、多表位、单结构域、多结构域、完整的胞外结构域(ECD)或蛋白质。肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合均可构成抗原。非限制性示例性抗原包括肿瘤抗原或病原体抗原等。“抗原”也可以指引发免疫反应的分子。任何形式的抗原或含有该抗原的细胞或制剂都可以用于生成对抗原决定簇具有特异性的抗体。
术语“表位”、“抗原决定簇”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象存在并且包括至少3-15个氨基酸。
术语“多特异性的”是指抗原结合分子能够特异性结合多个不同的抗原决定簇。术语“抗原结合分子”在其最广泛的含义上指特异性结合抗原决定簇的分子。抗原结合分子的实例是免疫球蛋白及其衍生物，例如片段。术语“三特异性抗原结合分子”或“三特异性结合分子”指对三种不同抗原(或表位)具有特异性的结合分子(例如抗体或包含抗体片段的分子)，特别是三特异性抗体。
术语“特异性结合”是指该结合对抗原具有选择性，且可以与不想要或非特异的相互作用区分开。可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术来测定抗体结合特异性抗原决定簇的能力，
以本公开中的可变区制作抗体、结合分子、双特异性结合分子或多特异性结合分子时，恒定区没有特别限定，可以使用本领域技术人员公知的恒定区或者自行获得的恒定区，还可以在恒定区部分导入氨基酸突变(例如提高或降低与Fc与受体或FcRn的结合的突变)。
获得本公开的结合分子、抗原结合片段、抗体、双特异性结合分子或多特异性结合分子的方法没有特别限制，可通过任意方法获得。发明的结合分子、抗原结合片段、抗体、双特异性结合分子或多特异性结合分子可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。
本文所用的术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“稳定转染”或“稳转”是指将外源核酸、DNA或RNA引入和整合到转染细胞的基因组中。术语“稳定转染体”(stable transfectant)是指将外来DNA稳定地整合到基因组DNA中的细胞。
术语“抗体药物偶联物”(ADC)是指已共价偶联治疗活性物质或活性药物成分(API)的抗体，从而治疗活性物质或活性药物成分(API)可以靶向至抗体的结合靶标以表现出其药理学功能。治疗活性物质或活性药物成分可以是能够杀死ADC靶向的细胞的细胞毒素，优选恶性或癌细胞。治疗活性物质、活性药物成分或细胞毒素的共价连接可以以非位点特异性方式利用偶联有效载荷至赖氨酸或半胱氨酸残基的标准化学接头进行，或者优选地，缀合以位点特异性方式进行，其允许完全控制缀合位点以及产生的ADC的药物比抗体比例。
术语“氨基酸取代”或“取代”或“替代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。
术语“亲和力”或“结合亲和力”指分子(例如抗体)的单一结合位点与其结合配偶体(例如抗原)之间全部非共价相互作用总和的强度。术语“Kd”是指特定的抗体-抗原相互作用的解离常数。可以使用本领域已知的各种技术来确定结合亲和力，例如表面等离子体共振、生物层干涉法、双极化干涉法、静态光散射、动态光散射、等温滴 定量热法、ELISA、分析超速离心和流式细胞术等。
术语“生物学活性”指抗体结合抗原并导致可测量的生物学反应的能力，所述生物学反应可以在体外或体内进行测量。
术语“药物组合物”是指包含一种、两种或多种活性成分的制剂或制剂的组合形式，其允许包含在其中的活性成分以生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。当“药物组合物”以含有不同的、两种以上活性成分的单独制剂的组合形式存在时，可以同时、依次、分别或间隔给药，其目的是发挥多种活性成分的生物活性，共同用于治疗疾病。
本公开的结合分子或抗原结合片段可与其他药物联合使用，活性成分可以混合在一起形成单一的给药单元，也可分别独立成为给药单元，分别使用。
术语“有效量”指本公开的抗体或片段的药物制剂的剂量，其以单一或多次剂量施用患者后，在治疗的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如人种差异；体重、年龄和健康状况；涉及的具体疾病；疾病的严重程度；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
本文所用的术语“个体”或“受试者”是指任何动物，例如哺乳动物或有袋动物。本公开的个体包括但不限于人类、非人类灵长类动物(例如食蟹猴或恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“疾病”或“病症”或“紊乱”等是指任何损害或干扰细胞、组织或器官的正常功能的改变或失调。例如，所述的“疾病”包括但不限于：肿瘤、病原体感染、自身免疫性疾病、T细胞功能障碍性疾病、或免疫耐受能力缺陷(如移植排斥)。
本文所用的术语“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病，及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的，不诱导或抑制正常细胞增殖。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的的疾病过程中的临床干预，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
术语“G蛋白偶联受体C5家族亚型D”和“GPRC5D”特别包括人GPRC5D蛋白，并且包括人GPRC5D的变体、亚型、同源种类和与GPRC5D(例如人GPRC5D)具有至少一个共同表位的类似物，示例性的人GPRC5D序列可见，GenBank登录号BC069341、NCBI参考序列：NP_061124.1和UniProtKB/Swiss-Prot登录号Q9NZD1中所描述的那些(还可参见Brauner-Osborne,H等人，2001,Biochim.Biophys.Acta 1518,237-248)。
术语“BCMA”指肿瘤相关抗原B细胞成熟抗原，也称为TNFRSF17，示例性的人BCMA序列包括登录号UniProt Q02223下的人BCMA蛋白。
具体实施方式
下面结合具体实施例，进一步阐述本公开。应理解，这些实施例仅用于说明本公开而不用于限制本公开的范围。下列实施例中未注明具体条件的实验方法，通常按照本领域公知的常规条件或按照制造厂商所建议的条件。
实施例1.人GPRC5D、GPRC5A及食蟹猴GPRC5D抗原信息
实施例中使用的人GPRC5D(human-GPRC5D)的全长氨基酸序列(SEQ ID NO：1)(Uniprot ID：Q9NZD1)如下所示：
注释：该蛋白为七次跨膜蛋白，双横线部分为胞外区(1-27；85-93；145-167；226-239)；划横线部分为胞内区(49-63；115-123；189-204；261-345)；斜体部分为跨膜区(28-48；64-84；94-114；124-144；168-188；205-225；240-260)。
实施例中使用的食蟹猴GPRC5D(cyno-GPRC5D)的全长氨基酸序列(SEQ ID NO：2)(Uniprot ID：A0A2K5W6I2)如下所示：
注释：该蛋白为七次跨膜蛋白，双横线部分为胞外区(1-27；85-93；145-167；226-239)；划横线部分为胞内区(49-63；115-123；189-204；261-344)；斜体部分为跨膜区(28-48；64-84；94-114；124-144；168-188；205-225；240-260)。
实施例中使用的人GPRC5A的全长氨基酸序列(SEQ ID NO：36)(Uniprot ID：Q8NFJ5)如下所示：
注释：该蛋白为七次跨膜蛋白，双横线部分为胞外区(1-33；90-97；151-176；234-247)；划横线部分为胞内 区(55-68；119-129；198-212；269-357)；斜体部分为跨膜区(34-54；69-89；98-118；130-150；177-197；213-233；248-268)。
实施例2.表达人GPRC5D、人GPRC5A及食蟹猴GPRC5D细胞系的制备
将编码SEQ ID NO.1所示的人GPRC5D氨基酸的核苷酸序列，克隆至pCMV3(SinoBiological，货号CV011)载体中，从而获得用于人GPRC5D细胞系构建的质粒。将所获得的质粒转染至CHOK1细胞(ATCC，货号CCL-61)中，获得表达人GPRC5D的CHOK1细胞系(简称：人GPRC5D-CHOK1细胞系)。
将编码SEQ ID NO.2所示的食蟹猴GPRC5D氨基酸的核苷酸序列，克隆至pCMV3载体中，获得用于食蟹猴GPRC5D细胞系构建的质粒。将所获得的质粒转染至CHOK1细胞中，获得表达食蟹猴GPRC5D的CHOK1细胞系(简称：食蟹猴GPRC5D-CHOK1细胞系)。
将编码SEQ ID NO.36所示的人GPRC5A氨基酸的核苷酸序列，克隆至pCMV3载体中，获得用于人GPRC5A细胞系构建的载体。将所获得的载体转染至CHOK1细胞中，获得表达人GPRC5A的CHOK1细胞系(简称：人GPRC5A-CHOK1细胞系)。
使用FACS测定方法，检测上述获得的人GPRC5D-CHOK1细胞系中人GPRC5D的表达情况，并在实验中设立阴性对照hIgG1 LALA isotype(百英生物，货号B109802)。
使用FACS测定方法，检测上述获得的食蟹猴GPRC5D-CHOK1细胞系中食蟹猴GPRC5D的表达情况，并在实验中设立阴性对照hIgG1 LALA isotype(百英生物，货号B109802)。
使用FACS测定方法，检测上述获得的人GPRC5A-CHOK1细胞系中人GPRC5A的表达情况，并在实验中设立阴性对照hIgG1 LALA isotype(百英生物，货号B109802)。
结果显示：
人GPRC5D-CHOK1细胞系中人GPRC5D的表达情况如图1A所示，结果表明人GPRC5D在CHOK1细胞中有很好的过表达现象，可以用于后续实验验证GPRC5D单抗与人GPRC5D在细胞水平的结合情况。
食蟹猴GPRC5D-CHOK1细胞系中食蟹猴GPRC5D的表达情况如图1B所示，结果表明食蟹猴GPRC5D在CHOK1细胞中有很好的过表达现象，可以用于后续实验验证GPRC5D单抗与食蟹猴GPRC5D在细胞水平的结合情况。
人GPRC5A-CHOK1细胞系中人GPRC5A的表达情况如图1C所示，结果表明人GPRC5A在CHOK1细胞中有很好的过表达现象，可以用于后续实验验证GPRC5D单抗与人GPRC5A在细胞水平没有非特异结合现象。
实施例3.人BCMA及食蟹猴BCMA抗原信息
实施例中使用的人BCMA(human-BCMA)的全长氨基酸序列(SEQ ID NO：3)(Uniprot ID：Q02223)如下所示。
注释：该蛋白为单次跨膜蛋白，双横线部分为胞外区(1-54)；波浪线部分为跨膜区(55-77)；划横线部分为胞内区(78-184)。
实施例中使用的食蟹猴BCMA(cyno-BCMA)的全长氨基酸序列(SEQ ID NO：4)(Uniprot ID：G7Q0I4)如下所示。
注释：该蛋白为单次跨膜蛋白，双横线部分为胞外区(1-53)；波浪线部分为跨膜区(54-76)；划横线部分为胞内区(77-183)。
实施例4.人BCMA及食蟹猴BCMA细胞系的制备
将编码SEQ ID NO：3所示的人BCMA氨基酸的核苷酸序列，克隆至pCMV3(SinoBiological，货号CV011)载体中，从而获得用于人BCMA细胞系构建的载体。将所获得的载体转染至CHOK1细胞(ATCC，货号CCL-61)中，获得表达人BCMA的CHOK1细胞系(简称：人BCMA-CHOK1细胞系)。
将编码SEQ ID NO：4所示的食蟹猴BCMA氨基酸的核苷酸序列，克隆至pCDH-CMV-MCS-EF1-Hygro(Honorgene，货号CD515B-1)载体中，获得用于食蟹猴BCMA细胞系构建的载体。将所获得的载体构建成病毒后转染至CHOK1细胞中，获得表达食蟹猴BCMA的CHOK1细胞系(简称：食蟹猴BCMA-CHOK1细胞系)。
使用FACS测定方法，检测上述获得的人BCMA-CHOK1细胞系中人BCMA的表达情况，并在实验中设立阴性对照hIgG1 LALA isotype(百英生物，货号B109802)。
使用FACS测定方法，检测上述获得的食蟹猴BCMA-CHOK1细胞系中食蟹猴BCMA的表达情况，并在实验中设立阴性对照hIgG1 LALA isotype(百英生物，货号B109802)。
结果显示：
人BCMA-CHOK1细胞系中人BCMA的表达情况如图2A所示，结果表明人BCMA在CHOK1细胞中有很好的过表达现象，可以用于后续实验验证BCMA单抗与人BCMA在细胞水平的结合情况。
食蟹猴BCMA-CHOK1细胞系中食蟹猴BCMA的表达情况如图2B所示，结果表明食蟹猴BCMA在CHOK1细胞中有很好的过表达现象，可以用于后续实验验证BCMA单抗与食蟹猴BCMA在细胞水平的结合情况。
实施例5.抗GPRC5D-BCMA-CD3三特异性抗体的设计及序列
通过杂交瘤筛选获得抗BCMA全长抗体(BCMA mAb)，其重链序列如SEQ ID NO:9所示，轻链序列如SEQ ID NO:7所示。
通过羊驼免疫，筛选获得抗GPRC5D纳米抗体，(GPRC5D VHH)，其可变区序列如SEQ ID NO:10所示。
CD3ε结合结构域来自于全长抗体CD3mAb，其重链序列如SEQ ID NO:11所示，轻链序列如SEQ ID NO:12所示。将CD3mAb的重链可变区和轻链可变区经由柔性接头连接形成单链抗体scFv，其结构为：VH-(G4S)3-VL，序列如SEQ ID NO:13所示。所述scFv再通过柔性连接头融合到BCMA全长抗体Fab段重链的C末端。
将CD3scFv融合至BCMA的Fab重链CH1的C末端形成三特异性抗体的一条臂，同时引入包含另一完整的GPRC5D纳米抗体结合结构域以及带有“节”“穴”结构的Fc部分，所形成的三特异性抗体被命名为三抗1，融合后的三条异源的肽链的序列如SEQ ID NO:5，SEQ ID NO:6，SEQ ID NO:7所示，示意图见图3。
将CD3scFv融合至BCMA全长抗体Fab段重链的C末端形成抗体的一条臂，且GPRC5D纳米抗体融合至BCMA全长抗体另一Fab段重链的C末端形成抗体的另一条臂，同时包含带有“节”“穴”结构的Fc部分，所形成的三特异性抗体被命名为三抗2，融合后的链的序列如SEQ ID NO:5，SEQ ID NO:7，SEQ ID NO:8所示，示意图见图3。
为降低抗体的ADCC活性，最终构建的三特异性抗体的Fc段均已进行了L234A、L235A和G237A的氨基酸取代。含有scFv的肽链中的Fc结构域被设计为“节”(knob)结构，包括S354C和T366W两个位点的氨基酸取代。不含有scFv的肽链中的Fc结构域被设计为“穴”(hole)结构，包括Y349C、T366S、L368A和Y407V四个位点的氨基酸取代。以及，为便于三特异性抗体的纯化，“穴”结构的重链还要进行H435R的取代。
三抗1和三抗2的结构以及相关的分子序列分别总结于表1和表2。
表1三特异性抗体的结构描述

表2三特异性抗体的氨基酸序列



实施例6.抗GPRC5D-BCMA-CD3三特异性抗体的构建及其在真核细胞中的瞬时转染表达
将前述三特异性抗体的编码基因片段分别克隆到PTT5表达载体中，制备转染级别的表达质粒。
在无血清培养基中培养Expi293FTM细胞(Thermo Fisher Scientific)，将细胞接种在摇瓶(Corning Inc.)中，并在37℃，8％CO2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的重组表达载体和PEI转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，细胞培养6天后收集表达上清，高速离心去除细胞碎片，用Protein A柱进行亲和纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，换液到PBS分装备用。对最终纯化的抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
同时，还表达纯化了Janssen的GPRC5DxCD3双特异性抗体Talquetamab(来源于专利WO2018017786A2，由序列SEQ ID NO:25,26,55,58组成，包含一个GPRC5D结合位点和一个CD3结合位点)。
同时，还表达纯化了Janssen的BCMAxCD3双特异性抗体Teclistamab(来源于专利WO2019220368A1，由序列SEQ ID NO:31,32,41,42组成，包含一个BCMA结合位点和一个CD3结合位点)。
同时，还表达纯化了Janssen的BGCB491三特异性抗体(来源于专利WO2022175255A2，由序列SEQ ID NO:29,30,31组成，包含一个GPRC5D结合位点，一个BCMA结合位点和一个CD3结合位点)。
同时，还表达纯化了信达的TS-F2-5三特异性抗体(来源于专利WO2022174813A1，由序列SEQ ID NO:68，75， 76，78组成，包含一个GPRC5D结合位点，一个BCMA结合位点和一个CD3结合位点)。
实施例7.抗GPRC5D-BCMA-CD3三特异性抗体的细胞水平亲和力测试
使用FACS检测抗GPRC5D-BCMA-CD3三特异性抗体与表达human GPRC5D和human BCMA双靶点的NCI-H929细胞和RPMI8226细胞、表达human GPRC5D的人GPRC5D-CHOK1细胞，表达human BCMA的人BCMA-CHOK1细胞，天然表达hCD3的T淋巴细胞(Jurkat)和Human PBMC的结合情况。
培养NCI-H929细胞(ATCC，货号CRL-9068)，RPMI8226细胞(ATCC，货号CCL-155)，Human GPRC5D-CHOK1细胞(实施例2)，Human BCMA-CHOK1细胞(实施例4)和Jurkat细胞(ATCC，货号TIB-152)。NCI-H929细胞的培养基为RPMI1640+10％FBS+0.05mM mercaptoethanol，RPMI8226和Jurkat细胞的培养基为RPMI1640+10％FBS，Human GPRC5D-CHOK1和Human BCMA-CHOK1细胞的培养基为F12K+10％FBS+400μg/ml Hygromycin B，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，将NCI-H929细胞、RPMI8226细胞和Jurkat细胞分别直接放入50mL离心管中，无需消化。将Human GPRC5D-CHOK1和Human BCMA-CHOK1用0.25％Trpsin-EDTA的胰酶消化，F12K+10％FBS+400μg/ml Hygromycin B终止消化。
将得到的细胞以1000rpm转速常温离心5分钟，弃上清，用1％BSA(in PBS)重悬细胞。将购买的Human PBMC以1500rpm转速常温离心10分钟，弃上清，用1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL，铺到96孔圆底培养板(corning，货号3799)中，100μL/孔。1500rpm转速4℃离心5分钟，弃上清，用200μL 1％BSA(in PBS)重悬细胞，再次1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。用1％BSA(in PBS)稀释待测抗体及阴性对照hIgG1 LALA isotype(百英生物，货号B109802)，起始浓度为100nM，5倍往下稀释8个浓度。使用稀释好的抗体重悬细胞，100μL/孔，4℃孵育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:400稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。200μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)和最高平均荧光强度(Top MFI)。
待测分子与肿瘤抗原细胞结合的结果如图4A-4H以及表3和表4所示。在双靶点表达的肿瘤细胞系NCI-H929和RPMI8226上，三抗的结合强于单一靶点双抗，且与肿瘤抗原的表达量正相关(NCI-H929细胞双靶点表达均高于RPMI8226细胞)。在单靶点表达的细胞系上，三抗的结合弱于或略强于相应双抗。推测该结果可 能由双靶点的协同结合引起。另在双靶点表达的肿瘤细胞系上，三抗1的结合强于强生BGCB491和信达TS-F2-5分子。
待测分子与Jurkat细胞及human PBMC细胞结合的结果如图5A-5C，表5和表6所示。在Jurkat细胞上的结合，三抗弱于Teclistamab，与强生BGCB491和信达TS-F2-5分子相当。
表3三特异性抗体与表达肿瘤抗原细胞结合的结果
表4三特异性抗体与表达肿瘤抗原细胞结合的结果
表5三特异性抗体与Jurkat细胞及human PBMC细胞结合的结果
表6三特异性抗体与Jurkat细胞结合的结果

实施例8.抗GPRC5D-BCMA-CD3三特异性抗体的种属交叉活性测试
使用FACS检测抗GPRC5D-BCMA-CD3三特异性抗体与表达Cyno GPRC5D的食蟹猴GPRC5D-CHOK1细胞，表达Cyno BCMA的食蟹猴BCMA-CHOK1细胞，天然表达Cyno CD3的Cyno PBMC的结合情况。
培养食蟹猴GPRC5D-CHOK1(实施例2)和食蟹猴BCMA-CHOK1细胞(实施例4)，培养基为F12K+10％FBS+400μg/ml Hygromycin B，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，将食蟹猴GPRC5D-CHOK1和食蟹猴BCMA-CHOK1用0.25％Trpsin-EDTA的胰酶消化，F12K+10％FBS+400μg/ml Hygromycin B终止消化。
将得到的细胞以1000rpm转速常温离心5分钟，弃上清，用1％BSA(in PBS)重悬细胞。将购买的Cyno PBMC以1500rpm转速常温离心10分钟，弃上清，用1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL，铺到96孔圆底培养板(corning，货号3799)中，100μL/孔。1500rpm转速4℃离心5分钟，弃上清，用200μL 1％BSA(in PBS)重悬细胞，再次1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。用1％BSA(in PBS)稀释待测抗体及阴性对照hIgG1 LALA isotype(百英生物，货号B109802)，起始浓度为100nM，5倍往下稀释8个浓度。使用稀释好的抗体重悬细胞，100μL/孔，4℃孵育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:400稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。200μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)和最高平均荧光强度(Top MFI)。
待测分子与食蟹猴相应肿瘤抗原细胞结合的结果如图6A-6B和表7所示。三抗分子与食蟹猴相应肿瘤抗原有很好的的结合。
待测分子与食蟹猴PBMC细胞结合的结果如图7和表8所示。三抗分子与食蟹猴PBMC有很好的的结合。
表7三特异性抗体与表达食蟹猴肿瘤抗原细胞结合的结果
表8三特异性抗体与cyno PBMC细胞结合的结果
实施例9.抗GPRC5D-BCMA-CD3三特异性抗体的体外重组蛋白结合亲和力和动力学
为测定与人源CD3(购自Acro，货号CDD-H52W1)/食蟹猴源CD3(购自Acro，货号CDD-C52W4)的亲和力与动力学性质，采用CM5芯片直接固化人源/食蟹猴源CD3分子的方法。CM5芯片先用EDC和NHS活化，人源/食蟹猴源CD3分子用pH＝5的醋酸盐溶液稀释至1μg/mL，以10μL/min的流速固化60s，再用乙醇胺封闭抗GPRC5D-BCMA-CD3三特异性抗体分子用HBS-EP+(10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05％P20)缓冲液两倍比稀释至系列浓度(100nM-0.39nM)，以50μL/min的流速结合90s，解离360s。
每一轮实验结束后，使用3M MgCl2溶液冲洗，以30μL/min的流速冲洗30s，将抗GPRC5D-BCMA-CD3三特异性抗体分子去除，完成芯片的再生。原始数据使用Biacore Insight Evaluation Software(3.0.12.15655)软件进行分析，以(1∶1)Langmuir模型进行拟合，得到的三特异性抗体亲和力和动力学实验数据如表9所示。
表9抗GPRC5D-BCMA-CD3三特异性抗体与人/食蟹猴CD3蛋白的结合亲和力和动力学

实验结果显示，两个三抗分子对人源/食蟹猴源CD3均有结合，且亲和力相当。
为测定与人源BCMA(购自Acro，货号BCA-H522y)/食蟹猴源BCMA(购自Acro，货号BCA-C52H7)的亲和力与动力学性质，采用CM5芯片，使用间接捕获法检测抗体分子和人源/食蟹猴源BCMA的亲和力和动力学性质。CM5芯片先用EDC和NHS活化，Anti-human IgG(Fc)抗体(购自cytiva，货号10325009)用pH＝5的10mM醋酸钠溶液稀释至10μg/mL，以5μL/min的流速固化420s，再用乙醇胺封闭。抗GPRC5D-BCMA-CD3三特异性抗体分子用HBS-EP+(10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05％P20)缓冲液稀释至4μg/mL，以10μL/min的流速捕获60s。人源/食蟹猴源BCMA用HBS-EP+(10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05％P20)缓冲液两倍比稀释至系列浓度(50nM-0.195nM)，以30μL/min的流速结合90s，解离180s。
每一轮实验结束后，使用3M MgCl2溶液冲洗，以30μL/min的流速冲洗30s，将抗GPRC5D-BCMA-CD3三特异性抗体分子去除，完成芯片的再生。原始数据使用Biacore Insight Evaluation Software(3.0.12.15655)软件进行分析，以(1∶1)Langmuir模型进行拟合，得到的三特异性抗体亲和力和动力学实验数据如表10所示。
表10抗GPRC5D-BCMA-CD3三特异性抗体与人/食蟹猴BCMA蛋白的结合亲和力和动力学
实验结果显示，两个三抗分子对人源/食蟹猴源BCMA均有结合，且亲和力相当。
实施例10.抗GPRC5D-BCMA-CD3三特异性抗体的体外功能实验
A.T细胞介导的细胞毒性实验(TDCC)，靶细胞为NCI-H929
培养NCI-H929细胞(ATCC，货号CRL-9068)，培养基为RPMI1640+10％FBS+0.05mM mercaptoethanol，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，将NCI-H929细胞直接放入50mL离心管中，无 需消化。1000rpm转速离心5分钟，弃上清，使用RPMI1640+2％FBS培养基重悬并计数细胞，将细胞密度调整到1E5/mL。
将靶细胞铺到平底96孔板(corning，货号3599)中，100μL/孔，37℃5％CO2培养过夜。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+2％FBS将细胞密度调整到2E6/mL。将效应细胞铺到96孔板中，50μL/孔，37℃5％CO2培养。
用RPMI1640+2％FBS培养基稀释抗体，起始浓度为100nM，14倍往下稀释10个浓度。将稀释好的抗体加入到细胞培养板中，50μL/孔，37℃5％CO2培养24小时。将培养板1000rpm转速离心5分钟，吸取上清50μL置另一平底96孔板中，每孔加入LDH检测试剂(Romos，货号4744934001)50μL混匀，1000rpm转速离心5分钟后避光孵育10分钟，Envision上读取OD492值。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
细胞杀伤％＝[样品孔-T细胞自发释放-靶细胞自发释放)/(靶细胞最大裂解-靶细胞自发释放)]*100％。
将细胞杀伤数据导入GraphPad Prism，绘制细胞杀伤/浓度曲线，计算EC50值。结果如表11及图8A所示。三抗1分子对NCI-H929细胞杀伤能力强于BGCB491及TS-F2-5。
B.T细胞介导的细胞毒性实验(TDCC)，靶细胞为RPMI8226
培养RPMI8226细胞(ATCC，货号CCL-155)，培养基为RPMI1640+10％FBS，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，将RPMI8226细胞直接放入50mL离心管中，无需消化。1000rpm转速离心5分钟，弃上清，使用RPMI1640+2％FBS培养基重悬并计数细胞，将细胞密度调整到1E5/mL。
将靶细胞铺到平底96孔板(corning，货号3599)中，100μL/孔，37℃5％CO2培养过夜。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+2％FBS将细胞密度调整到2E6/mL。将效应细胞铺到96孔板中，50μL/孔，37℃5％CO2培养。
用RPMI1640+2％FBS培养基稀释抗体，起始浓度为100nM，14倍往下稀释10个浓度。将稀释好的抗体加入到细胞培养板中，50μL/孔，37℃5％CO2培养24小时。将培养板1000rpm转速离心5分钟，吸取上清50μL置另一平底96孔板中，每孔加入LDH检测试剂(Romos，货号4744934001)50μL混匀，1000rpm转速离心5分钟后避光孵育10分钟，Envision上读取OD492值。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
细胞杀伤％＝[样品孔-T细胞自发释放-靶细胞自发释放)/(靶细胞最大裂解-靶细胞自发释放)]*100％。
将细胞杀伤数据导入GraphPad Prism，绘制细胞杀伤/浓度曲线，计算EC50值。结果如表11及图8B所示。三抗1分子对RPMI8226细胞杀伤能力强于BGCB491及TS-F2-5。
C.T细胞介导的细胞毒性实验(TDCC)，靶细胞为human BCMA-CHOK1
培养Human BCMA-CHOK1(实施例4)，培养基为F12K+10％FBS+400μg/ml Hygromycin B，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，用0.25％Trpsin-EDTA的胰酶消化Human BCMA-CHOK1，F12K+10％FBS+400μg/ml Hygromycin B培养基终止消化。1000rpm转速离心5分钟，弃上清，使用RPMI1640+2％FBS培养基重悬并计数细胞，将细胞密度调整到1E5/mL。
将靶细胞铺到平底96孔板(corning，货号3599)中，100μL/孔，37℃5％CO2培养过夜。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+2％FBS将细胞密度调整到2E6/mL。将效应细胞铺到96孔板中，50μL/孔，37℃5％CO2培养。
用RPMI1640+2％FBS培养基稀释抗体，起始浓度为400nM，10倍往下稀释。将稀释好的抗体加入到细胞培养板中，50μL/孔，故起始终浓度为100nM。37℃5％CO2培养24小时。将培养板1000rpm转速离心5分钟，吸取上清50μL置另一平底96孔板中，每孔加入LDH检测试剂(Romos，货号4744934001)50μL混匀，1000rpm转速离心5分钟后避光孵育10分钟，Envision上读取OD492值。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
细胞杀伤％＝[样品孔-T细胞自发释放-靶细胞自发释放)/(靶细胞最大裂解-靶细胞自发释放)]*100％。
将细胞杀伤数据导入GraphPad Prism，绘制细胞杀伤/浓度曲线，计算EC50值。结果如表11及图8C所示。三抗1分子对Human BCMA-CHOK1细胞杀伤能力与BGCB491相当，强于TS-F2-5。
D.T细胞介导的细胞毒性实验(TDCC)，靶细胞为human GPRC5D-CHOK1
培养Human GPRC5D-CHOK1(实施例2)，培养基为F12K+10％FBS+400μg/ml Hygromycin B，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，用0.25％Trpsin-EDTA的胰酶消化Human BCMA-CHOK1，F12K+10％FBS+400μg/ml Hygromycin B培养基终止消化。1000rpm转速离心5分钟，弃上清，使用RPMI1640+2％FBS培养基重悬并计数细胞，将细胞密度调整到1E5/mL。
将靶细胞铺到平底96孔板(corning，货号3599)中，100μL/孔，37℃5％CO2培养过夜。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+2％FBS将细胞密度调整到2E6/mL。将效应细胞铺到96孔板中，50μL/孔，37℃5％CO2培养。
用RPMI1640+2％FBS培养基稀释抗体，起始浓度为100nM，5倍往下稀释8个浓度。将稀释好的抗体加入到细胞培养板中，50μL/孔，37℃5％CO2培养24小时。将培养板1000rpm转速离心5分钟，吸取上清50μL置另一平底96孔板中，每孔加入LDH检测试剂(Romos，货号4744934001)50μL混匀，1000rpm转速离心5分钟后避光孵育10分钟，Envision上读取OD492值。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
细胞杀伤％＝[样品孔-T细胞自发释放-靶细胞自发释放)/(靶细胞最大裂解-靶细胞自发释放)]*100％。
将细胞杀伤数据导入GraphPad Prism，绘制细胞杀伤/浓度曲线，计算EC50值。结果如表11及图8D所示。三抗1分子对Human GPRC5D-CHOK1细胞杀伤能力与TS-F2-5相当，强于BGCB491。
E.T细胞介导的细胞毒性实验(TDCC)，靶细胞为human BCMA-CHOK1和human GPRC5D-CHOK1混合体系
培养Human GPRC5D-CHOK1和Human BCMA-CHOK1细胞，培养基为F12K+10％FBS+400μg/ml Hygromycin B，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，用0.25％Trpsin-EDTA的胰酶消化Human GPRC5D-CHOK1和Human BCMA-CHOK1，F12K+10％FBS+400μg/ml Hygromycin B培养基终止消化。1000rpm转速离心5分钟，弃上清，使用RPMI1640+2％FBS培养基重悬并计数细胞，将两种细胞密度调整到1E5/mL，将两种细胞等比混合。
将靶细胞混合物铺到平底96孔板(corning，货号3599)中，100μL/孔，37℃5％CO2培养过夜。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+2％FBS将细胞密度调整到1.6E6/mL。将效应细胞铺到96孔板中，50μL/孔，37℃5％CO2培养。
用RPMI1640+2％FBS培养基稀释抗体，起始浓度为100nM，14倍往下稀释10个浓度。将稀释好的抗体加入到细胞培养板中，50μL/孔，37℃5％CO2培养24小时。将培养板1000rpm转速离心5分钟，吸取上清50μL置另一平底96孔板中，每孔加入LDH检测试剂(Romos，货号4744934001)50μL混匀，1000rpm转速离心5分钟后避光孵育10分钟，Envision上读取OD492值。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
细胞杀伤％＝[样品孔-T细胞自发释放-靶细胞自发释放)/(靶细胞最大裂解-靶细胞自发释放)]*100％。
将细胞杀伤数据导入GraphPad Prism，绘制细胞杀伤/浓度曲线，计算EC50值。结果如表11及图8E所示。三抗1分子对混合细胞体系的杀伤能力强于BGCB491和TS-F2-5。
表11 T细胞介导的细胞毒性实验(TDCC)，靶细胞为NCI-H929，RPMI8226，人BCMA-CHOK1，人GPRC5D-CHOK1，人BCMA-CHOK1与人GPRC5D-CHOK1混合细胞体系
F.T细胞介导的细胞毒性实验(TDCC)，靶细胞为NCI-H929，2500ng/ml soluble BCMA存在情况下
培养NCI-H929细胞(ATCC，货号CRL-9068)，培养基为RPMI1640+10％FBS+0.05mM mercaptoethanol，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，将NCI-H929细胞直接放入50mL离心管中，无需消化。1000rpm转速离心5分钟，弃上清，使用RPMI1640+2％FBS培养基重悬并计数细胞，将细胞密度调整到1E5/mL。往细胞悬液中加入soluble BCMA，使其浓度为2500ng/ml。
将含soluble BCMA的靶细胞铺到平底96孔板(corning，货号3599)中，100μL/孔，37℃5％CO2培养过夜。用T细胞阴选试剂盒(StemCell，货号17951)从新鲜PBMC中分选CD3+T细胞，计数细胞并用RPMI1640+2％FBS将细胞密度调整到2E6/mL。将效应细胞铺到96孔板中，50μL/孔，37℃5％CO2培养。
用RPMI1640+2％FBS培养基稀释抗体，起始浓度为100nM，14倍往下稀释。将稀释好的抗体加入到细胞培养板中，50μL/孔，故起始终浓度为100nM。37℃5％CO2培养24小时。将培养板1000rpm转速离心5分钟，吸取上清50μL置另一平底96孔板中，每孔加入LDH检测试剂(Romos，货号4744934001)50μL混匀，1000rpm转速离心5分钟后避光孵育10分钟，Envision上读取OD492值。
TDCC效应引起的细胞杀伤百分比采用以下公式进行计算：
细胞杀伤％＝[样品孔-T细胞自发释放-靶细胞自发释放)/(靶细胞最大裂解-靶细胞自发释放)]*100％。
将细胞杀伤数据导入GraphPad Prism，绘制细胞杀伤/浓度曲线，计算EC50值。结果如图9(虚线表示2500ng/ml的soluble BCMA条件)和表12所示。在2500ng/ml的soluble BCMA存在下，BGCB491杀伤EC50值减弱约23倍，TS-F2-5杀伤EC50值基本维持，三抗1杀伤EC50值减弱约3倍。(BCMA双抗Teclistamab杀伤EC50值减弱约25倍)
表12在2500ng/ml的soluble BCMA存在下，T细胞介导的细胞毒性实验
G.三特异性抗体与PBMC及肿瘤细胞NCI-H929细胞共孵育条件下，IL6释放水平
培养NCI-H929细胞(ATCC，货号CRL-9068)，培养基为RPMI1640+10％FBS+0.05mM mercaptoethanol，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待细胞使用时，将NCI-H929细胞直接放入50mL离心管中，无 需消化，1000rpm转速离心5分钟，弃上清，使用RPMI1640+2％FBS培养基重悬并计数细胞，将细胞密度调整到1E5/mL。将NCI-H929细胞铺到96孔板(corning，货号3799)中，100μL/孔，37℃5％CO2培养。
购买新鲜PBMC，细胞计数，并用RPMI1640+2％FBS将细胞密度调整到4E6/mL。将效应细胞铺到96孔板中，50μL/孔，37℃5％CO2培养。
用RPMI1640+2％FBS培养基稀释抗体，起始浓度为100nM，13倍往下稀释。将稀释好的抗体加入到细胞培养板中，50μL/孔，故起始终浓度为100nM。37℃5％CO2培养24小时。
将培养板400g转速离心10分钟，取100μL上清冻存在-80℃备用。
按照细胞因子检测试剂盒(Biolegend，430504(ELISA MAX Deluxe Set Human IL-6))说明书稀释标准品，两倍稀释，最高浓度为8000pg/mL，最低点浓度为0pg/mL，一共12个浓度点。将配置好的标准品或者解冻后的样品加到96孔板中，100μL/孔(其中样品均需要另外稀释)，按照试剂盒方法操作，于Envision上读取0D450值。
结果如图10A-10B所示。在PBMC与肿瘤细胞共孵育的情况下，三抗1分子诱导IL6细胞因子释放的水平均弱于三抗2分子。
实施例11.抗GPRC5D-BCMA-CD3三特异性抗体的稳定性测试
利用差示荧光扫描技术检测三抗在pH7.4PBS缓冲液中的热稳定性。样品浓度在1mg/mL左右，利用Prometheus NT.Plex(nano DSF)进行检测。检测前，将各个样品10000g离心10分钟。样品板每个孔加入40μL样品(仪器上样量为10μL，每个样品均有一个复孔)。扫描温度从30℃开始到95℃结束，扫描速率0.5℃/min。实验结果如表13所示。两个三抗分子均表现出良好的热稳定性。
表13抗GPRC5D-BCMA-CD3三特异性抗体的NanoDSF检测结果
实施例12.抗GPRC5D-BCMA-CD3三特异性抗体的药代动力学
实验用naive食蟹猴两只，自由饮水。三特异性抗体给药剂量为1mg/kg，后肢静脉注射。采血时间点为Pre-dose，5min，2hr，6hr，24hr，72hr，168hr，264hr，336hr，504hr，672hr，840hr。将全血样品收集在无抗凝剂的聚乙烯管中，室温放置约1小时，6000g，25℃离心，立即置于干冰上，转移至-80℃冰箱长期保存。
采用ELISA法检测血清中的GPRC5D-BCMA-CD3三抗完整分子的浓度。使用人源GPRC5D蛋白按2μg/mL浓度包被96孔板，每孔100μL，4℃放置过夜；每孔300μL PBST洗板3次，加入300μL封闭试剂5％奶粉，37℃孵育1h；每孔300μL PBST洗板3次，加入100μL待测样品，37℃孵育1h；每孔300μL PBST洗板6次，加入100μL  Biotin-BCMA试剂，37℃孵育1h；每孔300μL PBST洗板6次，加入100μL SA-HRP试剂，37℃孵育1h；每孔300μL PBST洗板6次，再加入100μL TMB，避光放置10min后，加入50μL终止液停止显色反应。根据颜色反应定量检测GPRC5D-BCMA-CD3三抗完整分子的浓度。
使用PE公司的Envision读板仪检测450nm波长的吸光度值，并使用softmax软件处理数据。
采用Phoenix Winnolin 8.2软件对三特异性抗体的猴血清浓度进行计算。
结果显示三抗1在猴体内的药代性质良好，符合大分子常规代谢特征。
实施例13.抗GPRC5D-BCMA-CD3三特异性抗体的体内药效实验
A.人源免疫细胞重建小鼠模型，肿瘤细胞为NCI-H929
实验动物均饲养于恒温恒湿的独立通风盒内，饲养室温度20.0-26.0℃，湿度40-70％，昼夜明暗交替时间12h/12h。
NCI-H929细胞(ATCC，货号CRL-9068)培养在含10％胎牛血清和0.05mMβ-ME的RPMI 1640培养液中。收集指数生长期的NCI-H929细胞，细胞重悬在0.1mL(1:1)PBS与基质胶的混悬液中，于实验小鼠右侧背部前方接种5×106NCI-H929细胞，定期观察肿瘤生长情况，待肿瘤生长至平均体积83mm3时根据肿瘤大小和小鼠体重随机分组给药。
捐赠者PBMC购于AllCells,LLC。1×107PBMC/0.1mL PBS于NCI-H929细胞接种当天腹腔注射接种每只小鼠，建立人源免疫细胞重建小鼠模型。
在给药开始前，称量所有动物的体重，并用游标卡尺测量肿瘤体积。鉴于肿瘤体积会影响治疗的有效性，所以用随机分组设计的方法，根据鼠的肿瘤体积对其分组，以保证不同组别间的肿瘤体积相似。使用StudyDirectorTM(版本号3.1.399.19，供应商Studylog System,Inc.,S.San Francisco,CA,USA)选择Matched distribution方法进行分组。小鼠分组当天定义为第0天，并于D0天开始腹腔注射给药，每三天一次，给药5次。实验分组及给药方案见表14。
表14实验分组及给药方案
给药后每天监测动物日常行为表现，共进行14天。整个实验过程中，用游标卡尺每周测量2次肿瘤长径和宽径，肿瘤体积(mm3)＝0.5×(肿瘤长径×肿瘤短径2)计算。相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。 T/C％为相对肿瘤增值率，即在某一时间点，治疗组和hIgG1 LALA isotype对照组相对肿瘤体积或瘤重的百分比值。T和C分别为治疗组和hIgG1 LALA isotype对照组在某一特定时间点的肿瘤体积(TV)或瘤重(TW)。各组动物的肿瘤体积、小鼠体重、肿瘤重量等实验结果以平均值±标准误差(Mean±SEM)表示。采用独立样本T检验比较不同治疗组与对照组相比有无显著性差异。数据使用SPSS进行分析。P<0.05为具有显著性差异。实验结果见表15及图11。
三抗1分子0.004mg/kg处理组在人源骨髓瘤NCI-H929皮下移植NPG雌性小鼠Xenograft模型中显现出了非常显著的抑制肿瘤效果(p值小于0.001)，且显著优于等摩尔剂量的双抗分子Teclistamab和Teclistamab与Talquetamab联用。小鼠对测试药三抗分子的耐受良好，未出现明显的小鼠体重下降或毒性。
表15各给药组肿瘤体积大小及抑瘤率
B.人源免疫细胞重建小鼠模型，肿瘤细胞为NCI-H929
实验动物均饲养于恒温恒湿的独立通风盒内，饲养室温度20.0-26.0℃，湿度40-70％，昼夜明暗交替时间12h/12h。
NCI-H929细胞(ATCC，货号CRL-9068)培养在含10％胎牛血清和0.05mMβ-ME的RPMI 1640培养液中。收集指数生长期的NCI-H929细胞，细胞重悬在0.1mL(1:1)PBS与基质胶的混悬液中，于实验小鼠右侧背部前方接种5×106NCI-H929细胞，定期观察肿瘤生长情况，待肿瘤生长至平均体积100mm3时根据肿瘤大小和小鼠体重随机分组给药。
捐赠者PBMC购于AllCells,LLC。1×107PBMC/0.2mL PBS于NCI-H929细胞接种当天腹腔注射接种每只小鼠，建立人源免疫细胞重建小鼠模型。
在给药开始前，称量所有动物的体重，并用游标卡尺测量肿瘤体积。鉴于肿瘤体积会影响治疗的有效性，所以用随机分组设计的方法，根据鼠的肿瘤体积对其分组，以保证不同组别间的肿瘤体积相似。小鼠分组当天定义为第1天，并于D1天开始腹腔注射给药，每周两次，给药4次。实验分组及给药方案见表16。
表16实验分组及给药方案
给药后每天监测动物日常行为表现，共进行15天。整个实验过程中，用游标卡尺每周测量2次肿瘤长径和宽径，肿瘤体积(mm3)＝0.5×(肿瘤长径×肿瘤短径2)计算。相对肿瘤抑制率TGI(％)：TGI(％)＝[1-(Vti-Vt0)/(Vci-Vc0)]100％。T/C％为相对肿瘤增值率，即在某一时间点，治疗组和hIgG1 LALA isotype对照组相对肿瘤体积或瘤重的百分比值。T和C分别为治疗组和hIgG1 LALA isotype对照组在某一特定时间点的肿瘤体积(TV)或瘤重(TW)。各组动物的肿瘤体积、小鼠体重、肿瘤重量等实验结果以平均值+标准误差(Mean+SEM)表示。采用独立样本T检验比较不同治疗组与对照组相比有无显著性差异。数据使用GraphPad prism 8.0.2进行分析。P<0.05为具有显著性差异。实验结果见表17及图12和13。
三抗1的不同剂量处理组在人源骨髓瘤NCI-H929皮下移植NCG雌性小鼠Xenograft模型中均显现出了非常显著的抑制肿瘤效果(p值均小于0.05)，其中三抗1分子0.006mg/kg显著优于等摩尔剂量的BCGB491和TS-F2-5。小鼠对测试药三抗分子的耐受良好，未出现明显的小鼠体重下降或毒性。
表17各给药组肿瘤体积大小及抑瘤率
实施例14.羊驼免疫，仅重链的抗体免疫文库的构建
用高表达人GPRC5D的CHO-K1细胞对羊驼进行免疫。免疫的日期分别为第0天、第21天、第35天、第49天，第70天，共免疫5次。分别在第28天、第42天、第63天抽取血样分离出血清，利用细胞水平FACS检测血清中免疫应答反应情况。检测血清效价趋于平台期时结束免疫，对免疫的羊驼重新抽取血样50mL，按制造商说明书使用Solarbio公司淋巴细胞分离液分别分离羊驼PBMC，提取总RNA(OMEGA细胞总RNA提取试剂盒)后用Takara PrimeScriptTMII反转录试剂盒合成cDNA做模板，用设计的特异性引物进行巢式PCR扩增出VHH基因片 段，回收VHH片段后通过Sfi I酶切连接到pADL-23c噬菌粒载体上，电转化TG1电转化感受态细胞建成GPRC5D羊驼免疫文库(库容为4.07E9)。
实施例15.羊驼来源的抗GPRC5D的阳性克隆的筛选
为了获得可与人GPRC5D和食蟹猴GPRC5D交叉结合的阳性抗体，将上述文库扩增加入M13K07 helper phage组装成噬菌体。培养人GPRC5D-CHOK1细胞系、食蟹猴GPRC5D-CHOK1细胞系、CHOK1细胞至铺满整版，PBS洗涤2次后加入100μL 4％多聚甲醛固定细胞，25℃作用20-30min。PBS洗涤2次，每孔加入300μL 5％脱脂牛奶，37℃封闭1h，PBST洗涤3次，每孔加入50μL 5％脱脂牛奶和50μL噬菌体上清，37℃孵育1h。0.1％PBST洗涤5次，加入辣根过氧化物酶标记的抗M13抗体(用PBS按1:10000稀释)，100μL/孔，37℃作用1h。0.1％PBST洗板6次。加入TMB显色液显色，100μL/孔，37℃，7min，加入终止液终止反应，50μL/孔，于450nm下测光密度。将阳性克隆进行测序，获得5个重链抗体可变区(VHH)的氨基酸序列，克隆号分别为HHC7、HHC11、HHC22、HHC53和HHC78，其中HHC78的氨基酸序列如下所示：
在上述HHC78的氨基酸序列的基础上，利用Kabat编号规则划分抗体可变区的CDR和FR，该抗体的3个CDR序列组成如下表所示：
表18
实施例16.羊驼来源的抗GPRC5D嵌合抗体的构建及其在真核细胞中的瞬时转染表达
将测序完成的本公开的重链抗体可变区与人IgG1恒定区拼接后生成的目的基因片段克隆到pTT5表达载体中，制备转染级别的表达质粒。重链抗体可变区可以通过连接短肽与人IgG1恒定区连接，即形成：重链抗体可变区-连接短肽-人IgG1恒定区。本实施中采用的连接短肽序列为GGGGS。
引入的人IgG1恒定区序列(SEQ ID NO：38)如下：
在无血清培养基中培养Expi293FTM细胞(Thermo Fisher Scientific)，将细胞接种在摇瓶(Corning Inc.)中，并在37℃，8％CO2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的重组表达载体和PEI转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，细胞培养6天后收集表达上清，高速离心去除细胞碎片，用Protein A柱进行亲和纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，换液到PBS分装备用。对最终纯化的嵌合抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
实施例17.羊驼来源的抗GPRC5D嵌合抗体与表达GPRC5D细胞的结合
人GPRC5D-CHOK1细胞及食蟹猴GPRC5D-CHOK1细胞的培养基均为F12K+10％FBS+400μg/mL Hygromycin，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待使用时用无菌DPBS洗细胞两遍，0.25％胰酶EDTA消化约5分钟后用完全培养基终止。
将得到的细胞以1000rpm转速常温离心5分钟，弃上清，用100μL 1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL，铺到96孔圆底培养板(corning 3799)中，1500rpm转速4℃离心5分钟，弃上清，用200μL 1％BSA(in PBS)重悬细胞，再次1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。用1％BSA(in PBS)稀释待测抗体样品，起始浓度为100nM，10倍往下稀释7个浓度。使用稀释好的抗体重悬细胞，100μL/孔，4℃孵育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:400稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。200μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)和最高平均荧光强度(Top MFI)，结果如表19及附图14所示。筛选到的抗GPRC5D重链抗体与人GPRC5D及食蟹猴GPRC5D在细胞水平均有较好的结合。
表19抗GPRC5D嵌合抗体与表达GPRC5D细胞的结合
实施例18.羊驼来源的抗GPRC5D嵌合抗体的人源化设计
通过序列比对选择与候选重链抗体同源性较高的胚系基因序列作为VHH移植框架模板。将候选抗体CDR区嫁接至选定的人抗体可变区框架后，进行个别氨基酸的回复突变，得到人源化抗体H1、H2、H3、H4和H5，其中H4序列如表20所示：
表20羊驼来源的抗GPRC5D的人源化抗体可变区的氨基酸序列

实施例19.羊驼来源的抗GPRC5D人源化抗体的制备
参照实施例16所述，将人源化抗体的可变区与人IgG1恒定区拼接后生成的目的基因片段克隆到pTT5表达载体中，制备转染级别的表达质粒。
在无血清培养基中培养Expi293FTM细胞(Thermo Fisher Scientific)，将细胞接种在摇瓶(Corning Inc.)中，并在37℃，8％CO2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的重组表达载体和PEI转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，细胞培养6天后收集表达上清，高速离心去除细胞碎片，用Protein A柱进行亲和纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，换液到PBS分装备用。对最终纯化的人源化抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
实施例20.羊驼来源的抗GPRC5D人源化抗体与表达GPRC5D细胞的结合
NCI-H929细胞(ATCC,CRL-9068)的培养基为RPMI 1640+10％FBS+0.05mM mercaptoethanol，食蟹猴GPRC5D-CHOK1细胞的培养基为F12K+10％FBS+400μg/mL Hygromycin，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。
NCI-H929细胞使用时用移液管直接转移到50mL离心管中。食蟹猴GPRC5D-CHOK1细胞待使用时使用无菌DPBS洗细胞两遍，0.25％胰酶EDTA消化约5分钟后用完全培养基终止。
将得到的细胞以1000rpm转速常温离心5分钟，弃上清，用100μL 1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL，铺到96孔圆底培养板(corning 3799)中，1500rpm转速4℃离心5分钟，弃上清，用200μL 1％BSA(in PBS)重悬细胞，再次1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。用1％BSA(in PBS)稀释待测抗体样品，起始浓度为100nM，10倍往下稀释7个浓度。使用稀释好的抗体重悬细胞，100μL/孔，4℃孵育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:400稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。200μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)和最高平均荧光强度(Top MFI)，结果如表21及附图15所示。克隆HHC78人源化抗体和食蟹猴GPRC5D-CHOK1细胞的结合与母本抗体相当，和NCI-H929肿瘤细胞的结合相比母本维持或减弱。
表21抗GPRC5D人源化抗体与表达GPRC5D细胞的结合
实施例21.羊驼来源的抗GPRC5D人源化抗体与同家族成员GPRC5A的结合
人GPRC5A-CHOK1细胞的培养基为F12K+10％FBS+400μg/mL Hygromycin，使用T75细胞培养瓶置于37℃5％CO2培养箱培养，待使用时用无菌DPBS洗细胞两遍，0.25％胰酶EDTA消化约5分钟后用完全培养基终止。
将得到的细胞以1000rpm转速常温离心5分钟，弃上清，用100μL 1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL，铺到96孔圆底培养板(corning 3799)中，1500rpm转速4℃离心5分钟，弃上清，用200μL 1％BSA(in PBS)重悬细胞，再次1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。用1％BSA(in PBS)稀释待测抗体样品，起始浓度为100nM，10倍往下稀释7个浓度。使用稀释好的抗体重悬细胞，100μL/孔，4℃孵育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:400稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。200μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)和最高平均荧光强度(Top MFI)，结果如附图16所示，显示所测抗体与同家族成员GPRC5A无非特异结合。
实施例22.小鼠杂交瘤来源的抗BCMA抗体的获得
抗BCMA单克隆抗体通过免疫小鼠产生。实验用Balb/c小鼠，雌性，6周龄(Charles River公司)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。免疫原为人BCMA ECD蛋白(义翘神州，货号10620-H08H)，每次免疫25μg蛋白，时间为第0、14、28、42天，在进行脾细胞融合前2天进行加强免疫。期间用ELISA方法检测小鼠血清，确定小鼠血清中的抗体滴度。加强免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合，采用优化的电融合步骤将脾淋巴细胞与骨髓瘤细胞 Sp2/0细胞进行融合得到杂交瘤细胞。
融合后的杂交瘤细胞培养7-14天后，取培养基上清，使用高表达人BCMA的CHO-K1细胞，FACS实验对杂交瘤上清进行抗体筛选，得到的阳性抗体株进一步使用高表达食蟹猴BCMA的CHO-K1细胞，空白CHO-K1细胞以排除非特异性结合抗体杂交瘤株，从而选定特异性结合人/食蟹猴BCMA的杂交瘤。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA并反转录(PrimeScriptTM Reverse Transcriptase，Takara#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后测序，获得本公开的11个单克隆抗体可变区的氨基酸序列：19CH-1、19CH-2、19CH-3、19CH-4、19CH-5、19CH-7、19CH-8、19CH-9、19CH-11、19CH-13和19CH-16，其中19CH-16如表22所示。
表22小鼠杂交瘤来源的抗BCMA的19CH-16单克隆抗体可变区的氨基酸序列
在上述氨基酸序列的基础上，利用Kabat编号规则划分抗体可变区的CDR和FR，19CH-16的6个CDR序列组成如下表23所示。
表23小鼠杂交瘤来源的抗BCMA的单克隆抗体的CDR序列
实施例23.小鼠杂交瘤来源的抗BCMA嵌合抗体的构建及其在真核细胞中的瞬时转染表达
将测序完成的本公开的单抗重链可变区及轻链可变区分别与IgG1(L234AL235A)重链恒定区以及κ轻链恒定区拼接后生成的目的基因片段克隆到pTT5表达载体中，制备转染级别的表达质粒。
在Expi293表达培养基(赛默飞，A1435101)中培养Expi293FTM细胞(赛默飞，A14527)，将细胞接种在摇瓶中，并在37℃，8％CO2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的重组表达载体和PEI转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，细胞培养6天后收集表达上清，高速离心去除细胞碎片，用Mabselect Sure柱进行亲和纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱 目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，换液到PBS分装备用。对最终纯化的嵌合抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
实施例24.小鼠杂交瘤来源的抗BCMA嵌合抗体与表达BCMA细胞的结合
人BCMA-CHOK1细胞及食蟹猴BCMA-CHOK1细胞的培养基均为F12K+10％FBS+400μg/mL Hygromycin，使用T75细胞培养瓶置于37℃5％CO2培养箱培养。待使用时用无菌DPBS洗细胞两遍，0.25％胰酶EDTA消化约5分钟后用完全培养基终止。
将得到的细胞以1000rpm转速常温离心5分钟，弃上清，用100μL 1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL，铺到96孔圆底培养板(corning 3799)中，1500rpm转速4℃离心5分钟，弃上清，用200μL 1％BSA(in PBS)重悬细胞，再次1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。用1％BSA(in PBS)稀释待测抗体样品，起始浓度为100nM，10倍往下稀释7个浓度。使用稀释好的抗体重悬细胞，100μL/孔，4℃孵育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:400稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。200μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)和最高平均荧光强度(Top MFI)，结果如表24及附图17所示。筛选到的1个抗BCMA嵌合抗体(19CH-16)与人BCMA及食蟹猴BCMA在细胞水平均有较好的结合。
表24小鼠杂交瘤来源的抗BCMA嵌合抗体与表达BCMA细胞的结合
实施例25.小鼠杂交瘤来源的抗BCMA抗体的人源化设计
根据表达纯化测试及细胞水平结合测试的结果，选择克隆号为19CH-16的抗体进行人源化设计。
鼠源抗人BCMA单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种抗体序列。在所获得的鼠源抗体VH/VL CDR 典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同源性高的人种系模板。
将鼠源抗体的CDR区移植到选择好的相应人源化模板上，替换人源化可变区，再与IgG恒定区(优选重链为IgG1，轻链为κ)重组。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，设计了由如下人源化轻重链可变区序列组合而成的抗体：19CH-16H1L1、19CH-16H1L2、19CH-16H1L3、19CH-16H2L1、19CH-16H2L2、19CH-16H2L3、19CH-16H3L1、19CH-16H3L2、19CH-16H3L3、19CH-16H4L1、19CH-16H4L2和19CH-16H4L3，其中19CH-16H2L2如表25所示。
表25小鼠杂交瘤来源的1个人源化抗体可变区的氨基酸序列
实施例26.小鼠杂交瘤来源的抗BCMA人源化抗体的制备
将人源化抗体的重链可变区及轻链可变区分别与IgG1(L234AL235A)重链恒定区以及κ轻链恒定区拼接后生成的目的基因片段克隆到pTT5表达载体中，制备转染级别的表达质粒。
在Expi293表达培养基(赛默飞，A1435101)中培养Expi293FTM细胞(赛默飞，A14527)，将细胞接种在摇瓶中，并在37℃，8％CO2的环境中置于摇床上培养。调整细胞密度，将含有目的基因片段的重组表达载体和PEI转染试剂按照合适的比例混合，并添加进细胞培养摇瓶中，细胞培养6天后收集表达上清，高速离心去除细胞碎片，用Mabselect Sure柱进行亲和纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，换液到PBS分装备用。对最终纯化的嵌合抗体进行SDS-PAGE及HPLC纯度分析和A280浓度测定。
实施例27.小鼠杂交瘤来源的抗BCMA人源化抗体与表达BCMA细胞的结合
人BCMA-CHOK1细胞及食蟹猴BCMA-CHOK1细胞的培养基为F12K+10％FBS+400μg/mL Hygromycin，人BCMA-CHOK1细胞及食蟹猴BCMA-CHOK1细胞待使用时用无菌DPBS洗细胞两遍，0.25％胰酶EDTA消化约5分钟后用完全培养基终止。
将得到的细胞以1000rpm转速常温离心5分钟，弃上清，用100μL 1％BSA(in PBS)重悬细胞。细胞计数，并将细胞密度调整到1E6/mL，铺到96孔圆底培养板(corning 3799)中，1500rpm转速4℃离心5分钟，弃上清，用200μL 1％BSA(in PBS)重悬细胞，再次1500rpm转速4℃离心5分钟，弃上清，4℃放置备用。用1％BSA(in PBS)稀释待测抗体样品，起始浓度为100nM，10倍往下稀释7个浓度。使用稀释好的抗体重悬细胞，100μL/孔，4℃孵 育1小时。1500rpm转速4℃离心5分钟，弃上清。160μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。用1％BSA(in PBS)按照说明书1:400稀释二抗(goat anti human IgG Fc PE)，用稀释好的二抗重悬细胞，100μL/孔，4℃孵育0.5小时。1500rpm转速4℃离心5分钟，弃上清。200μL 1％BSA(in PBS)重悬洗涤，1500rpm转速4℃离心5分钟，弃上清。100μL 1％BSA(in PBS)重悬细胞，300目纱布过滤细胞，流式细胞仪检测PE通道平均荧光强度。
从流式细胞仪导出FCS文件，用flowjo软件分析每个样本的PE通道平均荧光强度(以下简称MFI)，将分析得出的平均荧光强度导入Graphpad分析抗体与细胞的半数结合浓度(以下简称EC50)和最高平均荧光强度(Top MFI)，结果如表26及附图18所示。实施例26中制备的、筛选到的1个抗BCMA人源化抗体(19CH-16H2L2)与人BCMA及食蟹猴BCMA在细胞水平均有较好的结合。
表26小鼠杂交瘤来源的抗BCMA人源化抗体与表达BCMA细胞的结合
实施例28.制备小鼠杂交瘤来源的抗BCMA人源化抗体的变体
对上述的抗BCMA人源化抗体19CH-16H2L2的CDR区进行翻译后修饰(PTM)分析发现，重链可变区中存在1个脱酰胺位点，为了消除潜在的风险，对19CH-16H2氨基酸序列进行单个位点的定点突变，得到19CH-16H2L2-NA和19CH-16H2L2-QT两个变体。19CH-16H2L2-NA变体的可变区氨基酸序列如表27所示。
表27 19CH-16H2L2人源化抗体变体的氨基酸序列
参照实施例26所述，通过Expi293细胞瞬转表达制备得到上述2个变体蛋白。参照实施例27所述，使用人BCMA-CHOK1细胞及食蟹猴BCMA-CHOK1细胞对两个变体的亲和力进行测定。结果如表28及附图19所示。19CH-16H2L2-NA能够保持细胞结合水平上的亲和力同时去除翻译后修饰的风险。
表28 19CH-16H2L2人源化抗体变体与表达BCMA细胞的结合
上文所述的本公开的实施方案仅为示例性的，任何本领域技术人员都可以认识到或者可以确定无数的特定化合物、材料和操作的等价物，而不需要进行超出常规的试验。所有这些等价物都是在本公开范围之内的，并且被权利要求所包含。

Claims (47)

  1. 一种三特异性抗原结合分子，包含以下多肽：
    第一多肽，包含：(i)能够特异性结合第一抗原的抗原结合片段Fab的重链结构域，(ii)能够特异性结合第二抗原的单链抗体(scFv)结构域，和(iii)第一Fc结构域，
    第二多肽，包含：能够特异性结合第一抗原的抗原结合片段Fab的轻链结构域，和，
    第三多肽，包含：(i)能够特异性结合第三抗原的重链单域抗体(VHH)结构域和(ii)第二Fc结构域，
    其中所述第一多肽的抗原结合片段Fab的重链结构域与所述第二多肽的抗原结合片段Fab的轻链结构域形成针对第一抗原的第一结合位点；所述单链抗体(scFv)结构域形成针对第二抗原的第二结合位点；所述重链单域抗体(VHH)结构域形成针对第三抗原的第三结合位点；所述第一Fc结构域和所述第二Fc结构域相互缔合；
    任选地，所述三特异性抗原结合分子还包含第四多肽，其包含特异性结合第一抗原的抗原结合片段Fab的轻链结构域，所述抗原结合片段Fab的轻链结构域与第二多肽的抗原结合片段Fab的轻链结构域相同，且所述第三多肽还包含特异性结合第一抗原的抗原结合片段Fab的重链结构域，所述第三多肽的抗原结合片段Fab的重链结构域与第一多肽的抗原结合片段Fab的重链结构域相同，其C端与VHH结构域的N端相连，所述第三多肽的抗原结合片段Fab的重链结构域与第四多肽的抗原结合片段Fab的轻链结构域形成针对第一抗原的第四结合位点。
  2. 如权利要求1所述的三特异性结合分子，其中所述单链抗体(scFv)结构域包含重链可变区和轻链可变区；优选地，所述单链抗体(scFv)结构域的重链可变区与所述单链抗体(scFv)结构域的轻链可变区通过第一接头连接，其中单链抗体(scFv)结构域的重链可变区的C端与第一接头的N端融合，第一接头的C端与单链抗体(scFv)结构域的轻链可变区的N端融合；更优选地，所述第一接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
  3. 如权利要求1或2所述的三特异性抗原结合分子，其中所述第一Fc结构域包含免疫球蛋白的第一CH2结构域和第一CH3结构域，所述第一CH2结构域的C端与第一CH3结构域的N端融合；所述第二Fc结构域包含免疫球蛋白的第二CH2结构域和第二CH3结构域，所述第二CH2结构域的C端与第二CH3结构域的N端融合；
    优选地，所述第一CH3结构域包含“节”(knob)结构，所述第二CH3结构域包含“穴”(hole)结构；更优选地，所述“节”(knob)结构包含氨基酸取代S354C和T366W，所述“穴”(hole)结构包含氨基酸取代Y349C、T366S、L368A和Y407V；
    优选地，第一和/或第二Fc结构域包含L234A、L235A和/或G237A的氨基酸取代；
    优选地，所述第二Fc结构域包含H435R的氨基酸取代；
    优选地，所述单链抗体(scFv)结构域与所述第一Fc结构域通过第二接头连接，其中单链抗体(scFv)结构域的C端与第二接头的N端融合，第二接头的C端与第一Fc结构域的N端融合；
    更优选地，所述第二接头包含氨基酸序列EPKSS；
    优选地，其中所述Fc结构域来源于IgG1。
  4. 如权利要求1-3任一项所述的三特异性抗原结合分子，其中所述抗原结合片段Fab重链结构域包含免疫球蛋白的重链可变区和CH1结构域，所述重链可变区的C端与CH1结构域的N端融合；所述抗原结合片段Fab轻链结构域包含免疫球蛋白的轻链可变区和轻链恒定区，所述轻链可变区的C端与轻链恒定区的N端融合；
    优选地，所述第一多肽的抗原结合片段Fab重链结构域与所述单链抗体(scFv)结构域通过第三接头连接，其中抗原结合片段Fab重链结构域的C端与第三接头的N端融合，第三接头的C端与单链抗体(scFv)结构域的N端融合；
    任选地，所述第三多肽的抗原结合片段Fab重链结构域与所述VHH结构域通过第五接头连接，其中抗原结合片段Fab重链结构域的C端与第五接头的N端融合，第五接头的C端与VHH结构域的N端融合。
    优选地，所述第三接头和/或第五接头包含氨基酸序列(G4S)n，n为1-10中的任意整数。
  5. 如权利要求1-4任一项所述的三特异性抗原结合分子，所述重链单域抗体(VHH)结构域的C端与第二Fc结构域的N端融合，优选地，所述重链单域抗体(VHH)结构域的C端通过第四接头与与第二Fc结构域的N端融合；更优选地，所述第四接头包含氨基酸序列EPKSS。
  6. 如权利要求1-5任一项所述的三特异性抗原结合分子，其中所述第一多肽包含如下结构：Fab重链结构域-scFv结构域-第一Fc结构域，优选所述第一多肽包含如下结构：Fab重链可变区-Fab CH1-第三接头-scFv的重链可变区-第一接头-scFv的轻链可变区-第二接头-第一CH2-第一CH3；
    其中第二多肽包含如下结构：Fab轻链可变区-轻链恒定区；
    和/或，其中第三多肽包含如下结构：VHH结构域-第二Fc结构域，优选所述第三多肽包含如下结构：VHH结构域-第四接头-第二CH2-第二CH3；
    任选地，所述第四多肽包含如下结构：Fab轻链可变区-轻链恒定区，且所述第三多肽包含如下结构：Fab重链结构域-第五接头-VHH-第四接头-第二Fc结构域，进一步优选地，所述第三多肽包含如下结构：Fab重链可变区-Fab CH1-第五接头-VHH-第四接头-第二CH2-第二CH3。
  7. 如权利要求1-6任一项所述的三特异性抗原结合分子，其中所述第二抗原为CD3，优选为CD3ε；优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：27所示的HCDR1、序列如SEQ ID NO：28所示的HCDR2、序列如SEQ ID NO：29所示的HCDR3、序列如SEQ ID NO：30所示的LCDR1、序列如SEQ ID NO：31所示的LCDR2和序列如SEQ ID NO：32所示的LCDR3。
    更优选地，所述单链抗体(scFv)结构域包含序列如SEQ ID NO：25所示的重链可变区和序列如SEQ ID NO：26所示的轻链可变区；
    更优选地，所述单链抗体(scFv)结构域包含SEQ ID NO：13所示的氨基酸序列。
  8. 如权利要求1-7任一项所述的三特异性抗原结合分子，其中所述第一Fc结构域包含SEQ ID NO：33所示的氨基酸序列，所述第二Fc结构域包含SEQ ID NO：34所示的氨基酸序列。
  9. 如权利要求1-8任一项所述的三特异性抗原结合分子，所述第一抗原为BCMA；优选地，所述抗原结合片段Fab包含序列如SEQ ID NO：16所示的HCDR1、序列如SEQ ID NO：17所示的HCDR2和序列如SEQ ID NO：18所示的HCDR3；优选地，所述抗原结合片段Fab包含序列如SEQ ID NO:19所示的LCDR1、序列如SEQ ID NO:20所示的LCDR2和序列如SEQ ID NO:21所示的LCDR3；
    更优选地，所述抗原结合片段Fab重链结构域包含序列如SEQ ID NO：14所示的重链可变区；所述抗原结合片段Fab轻链结构域包含序列如SEQ ID NO：15所示的轻链可变区；
    更优选地，所述抗原结合片段Fab重链结构域包含SEQ ID NO：35所示的氨基酸序列；更优选地，所述抗原结合片段Fab轻链结构域包含SEQ ID NO:7所示的氨基酸序列。
  10. 如权利要求1-9任一项所述的三特异性抗原结合分子，所述第三抗原为GPRC5D；优选地，所述能够特异性结合第三抗原的重链单域抗体(VHH)结构域包含序列如SEQ ID NO:22所示的HCDR1、序列如SEQ ID NO:23所示的HCDR2和序列如SEQ ID NO:24所示的HCDR3；更优选地，所述重链单域抗体(VHH)结构域包含SEQ ID  NO:10所示的序列。
  11. 如权利要求1-10任一项所述的三特异性抗原结合分子，所述三特异性抗原结合分子的第一多肽包含SEQ ID NO：5所示的氨基酸序列，所述第二多肽包含SEQ ID NO:7所示的氨基酸序列，所述第三多肽包含SEQ ID NO:6所示的氨基酸序列；或者任选地，所述三特异性抗原结合分子的第一多肽包含SEQ ID NO:5所示的序列，所述第二多肽和/或第四多肽包含SEQ ID NO:7所示的序列，所述第三多肽包含SEQ ID NO:8所示的序列。
  12. 一种三特异性抗原结合分子，其能够结合的表位：
    与包含序列如SEQ ID NO:22所示的HCDR1、序列如SEQ ID NO:23所示的HCDR2和序列如SEQ ID NO:24所示的HCDR3的抗体针对的表位相同或重叠。
  13. 如权利要求12所述的三特异性抗原结合分子，其能够结合的表位：
    与包含序列如SEQ ID NO：10所示的氨基酸序列的抗体针对的表位相同或重叠。
  14. 如权利要求12或13所述的三特异性抗原结合分子，其能与CD3和BCMA结合，其中优选能与CD3ε和BCMA结合。
  15. 一种三特异性抗原结合分子，其包含：
    (A)能够特异性结合GPRC5D3的第一结合部分；
    (B)第二接合部分；以及
    (C)第三结合部分，所述第一、第二和第三结合部分特异性结合的抗原或表位互不相同；
    所述第一结合部分包含：
    序列如SEQ ID NO:22所示的HCDR1、序列如SEQ ID NO:23所示的HCDR2和序列如SEQ ID NO:24所示的HCDR3。
  16. 如权利要求15所述的三特异性抗原结合分子，其中所述第一结合部分包含：
    序列如SEQ ID NO：10所示的氨基酸序列。
  17. 如权利要求15或16所述的双特异性抗原结合分子，所述第二结合部分能与CD3结合，优选能与CD3ε结合，和/或第三结合部分能与BCMA结合。
  18. 核酸分子，其编码前述任一项权利要求所述的三特异性抗原结合分子。
  19. 表达载体，其包含权利要求18所述的核酸分子。
  20. 宿主细胞，其包含权利要求18所述的核酸分子或者权利要求19所述的表达载体；优选地，所述宿主细胞为原核细胞或真核细胞；所述原核细胞优选大肠杆菌；所述真核细胞优选哺乳动物细胞或酵母；更优选地，所述哺乳动物细胞为CHO细胞、Expi293或HEK293细胞。
  21. 制备权利要求1-17任一项所述的三特异性抗原结合分子的方法，所述方法包括：在适合的条件下培养权利要求20所述的宿主细胞。
  22. 药物组合物，其包含权利要求1-17任一项所述的三特异性抗原结合分子、权利要求18所述的核酸分子、权利要求19所述的表达载体和/或权利要求20所述的宿主细胞。
  23. 如权利要求22所述的药物组合物，其还包含药学上可接受的载体。
  24. 如权利要求22或23所述的药物组合物，其还包含一种或多种额外的治疗剂。
  25. 权利要求1-17任一项所述的三特异性抗原结合分子、权利要求18所述的核酸分子、权利要求19所述的表达载体和/或权利要求20所述的宿主细胞在制备治疗、缓解和/或预防肿瘤的药物中的用途。
  26. 如权利要求25所述的用途，其中所述肿瘤是GPRC5D和/或BCMA阳性的肿瘤。
  27. 如权利要求25或26所述的用途，其中所述肿瘤选自：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
  28. 一种诱导表达GPRC5D和/或BCMA的细胞死亡的方法，所述方法包括使所述细胞与权利要求1-17任一项所述的三特异性抗原结合分子、权利要求18所述的核酸分子、权利要求19所述的表达载体、权利要求20所述的宿主细胞物和/或权利要求22-24任一项所述的药物组合物接触，优选地，所述表达GPRC5D和/或BCMA的细胞是肿瘤细胞。
  29. 如权利要求28所述的方法，其中所述肿瘤细胞是选自以下肿瘤的细胞：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
  30. 一种治疗受试者中与表达GPRC5D和/或BCMA相关的疾病的方法，所述方法包括向有需要的受试者施用权利要求1-17任一项所述的三特异性抗原结合分子、权利要求18所述的核酸分子、权利要求19所述的表达载体、权利要求20所述的宿主细胞和/或权利要求22-24任一项所述的药物组合物。
  31. 如权利要求30所述的方法，其中所述疾病是肿瘤；
    优选地，淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
  32. 如权利要求30或31所述的方法，其还包括向所述受试者给予额外的治疗剂。
  33. 一种特异性结合BCMA的抗原结合分子或其抗原结合片段，其特征在于：所述抗原结合分子或其抗原结合片段包含序列如SEQ ID NO：14所示的重链可变区的HCDR序列和/或序列如SEQ ID NO:15所示的轻链可变区的LCDR序列；优选地，所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含序列如SEQ ID NO：16所示的HCDR1、序列如SEQ ID NO：17所示的HCDR2和序列如SEQ ID NO：18所示的HCDR3，和/或，所述轻链可变区包含序列如SEQ ID NO:19所示的LCHDR1、序列如SEQ ID NO:20所示的LCDR2和序列如SEQ ID NO:21所示的LCDR3。
  34. 权利要求33所述的特异性结合BCMA的抗原结合分子或其抗原结合片段，所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含序列如SEQ ID NO：14所示的重链可变区，和/或序列如SEQ ID NO：15所示的轻链可变区。
  35. 权利要求33或34所述的特异性结合BMCA的抗原结合分子或其抗原结合片段，其还具有以下特征的一种或多种：
    i)其还包含重链恒定区和/或轻链恒定区；优选地，所述重链恒定区包含Fc；更优选地，Fc来源于鼠或人；更优选地，Fc的序列是天然的或经过修饰的；进一步优选地，所述IgG1的Fc结构域包含如SEQ ID NO:38所示的氨基酸序列；
    ii)所述BCMA抗原结合分子或其抗原结合片段为单克隆抗体、双特异性结合分子、多特异性结合分子、鼠源抗体、人源化抗体、嵌合抗体、改型抗体、全人源抗体、全长抗体、重链抗体、纳米抗体、Fab、Fv、scFv、F(ab’)2、线性抗体和/或重链单域抗体；和/或
    iii)其为IgG1、IgG2、IgG3或IgG4形式。
  36. 权利要求33-35任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段，所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含SEQ ID NO：9所示的重链氨基酸序列，和/或，所述特异性结合BCMA的抗原结合分子或其抗原结合片段包含SEQ ID NO:7所示的轻链氨基酸序列。
  37. 一种偶联物或融合蛋白，其特征在于：将权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段与捕获标记物或检测标记物偶联形成；优选地，所述检测标记物包括放射性核素、发光物质、有色物质或酶，或者，所述融合蛋白的一个被融合的部分包含权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段。
  38. 一种抗体药物偶联物，其特征在于：将权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段与其他生物活性分子偶联形成；优选地，所述其他生物活性分子为小分子药物；优选地，所述特异性结合BMCA的抗原结合分子或其抗原结合片段与所述其他生物活性分子通过接头连接。
  39. 编码权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段的核酸，或者包含所述核酸的重组载体，或者包含所述核酸或所述重组载体的宿主细胞；优选地，所述宿主细胞为原核细胞(优选大肠杆菌)，或者真核细胞(优选哺乳动物细胞或酵母；进一步优选地，所述哺乳动物细胞为CHO细胞或HEK293细胞)。
  40. 制备权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段的方法，所述方法包括：在适合的条件下培养权利要求53中所述的宿主细胞，并从所述细胞中纯化获得表达产物。
  41. 权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段、权利要求37所述的偶联物或融合蛋白、权利要求38所述的抗体偶联药物或权利要求39所述的核酸、重组载体或宿主细胞在制备治疗或缓解肿瘤的药物中的用途；
    优选地，所述药物靶向BCMA异常表达的肿瘤细胞；优选地所述肿瘤细胞是选自以下肿瘤的细胞：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
  42. 权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段、权利要求37所述的偶联物或融合蛋白、权利要求38所述的抗体偶联药物或权利要求39所述的核酸、重组载体或宿主细胞在制备检测试剂或诊断试剂中的用途；
    优选地，所述检测试剂用于检测BCMA的表达；所述诊断试剂用于诊断肿瘤；优选地，所述肿瘤细胞是选自以下肿瘤的细胞：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
  43. 一种检测样品中BCMA表达的方法，所述方法包括：
    (1)将样品与权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段接触；
    (2)检测所述特异性结合BMCA的抗原结合分子或其抗原结合片段与BCMA的复合物的形成；任选地，所述特异性结合BMCA的抗原结合分子或其抗原结合片段是被可检测地标记的。
  44. 一种药物组合物，其包含有效量的权利要求33-36任一项所述的特异性结合BMCA的抗原结合分子或其抗原结合片段、权利要求37所述的偶联物或融合蛋白、权利要求38所述的抗体偶联药物或权利要求39所述的核酸、重组载体或宿主细胞；
    优选地，其还包含药学上可接受的载体；优选地，其还包含一种或多种额外的其他治疗剂。
  45. 一种嵌合抗原受体(CAR)或包含所述嵌合抗原受体的细胞，其包含权利要求33-36任一项所述的特异性 结合BMCA的抗原结合分子或其抗原结合片段。
  46. 一种诱导表达BCMA的细胞死亡的方法，所述方法包括使所述细胞与权利要求44所述的药物组合物接触，所述表达BCMA的细胞是肿瘤细胞；
    优选地，所述肿瘤细胞是选自以下肿瘤的细胞：淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌。
  47. 一种治疗受试者中与表达BCMA相关的疾病的方法，所述方法包括向有此需要的受试者施用如权利要求44所述的药物组合物；
    优选地，所述疾病是肿瘤；优选地，所述肿瘤疾病选自淋巴瘤诸如多发性骨髓瘤，以及上述肿瘤的转移癌；
    更优选地，还包括向所述受试者给予额外的治疗剂。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120424217A (zh) * 2025-04-30 2025-08-05 立凌生物制药(苏州)有限公司 靶向b细胞抗原和t细胞抗原的多特异性抗体及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018017786A2 (en) 2016-07-20 2018-01-25 Janssen Pharmaceutica Nv Anti- gprc5d antibodies, bispecific antigen binding molecules that bind gprc5d and cd3, and uses thereof
WO2019220368A1 (en) 2018-05-16 2019-11-21 Janssen Biotech, Inc. Bcma/cd3 and gprdc5d/cd3 bispecific antibodies for use in cancer therapy
WO2022174813A1 (zh) 2021-02-19 2022-08-25 信达生物制药(苏州)有限公司 抗GPRC5DxBCMAxCD3三特异性抗体及其用途
WO2022175255A2 (en) 2021-02-16 2022-08-25 Janssen Pharmaceutica Nv Trispecific antibody targeting bcma, gprc5d, and cd3
US20220411533A1 (en) * 2021-06-17 2022-12-29 Boehringer Ingelheim International Gmbh Novel Tri-specific Binding Molecules

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN121591910A (zh) * 2017-11-21 2026-03-03 诺华股份有限公司 针对肿瘤相关抗原的三特异性结合分子及其用途
AU2019277029C1 (en) * 2018-06-01 2024-01-04 Novartis Ag Binding molecules against BCMA and uses thereof
MX2021000305A (es) * 2018-07-11 2021-09-08 Momenta Pharmaceuticals Inc Composiciones y métodos relacionados con constructos de dominio de unión a antígeno-fc modificados genéticamente.
WO2022002033A1 (zh) * 2020-06-30 2022-01-06 和铂医药(上海)有限责任公司 具有H2L2与HCAb结构的结合蛋白
CN116368154A (zh) * 2020-10-08 2023-06-30 阿菲姆德股份有限公司 三特异性结合剂
US20240076411A1 (en) * 2020-12-30 2024-03-07 WuXi Biologics Ireland Limited Multispecific antigen binding proteins
CN112794916B (zh) * 2021-04-08 2021-08-10 正大天晴药业集团南京顺欣制药有限公司 三特异性抗原结合构建体及构建方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018017786A2 (en) 2016-07-20 2018-01-25 Janssen Pharmaceutica Nv Anti- gprc5d antibodies, bispecific antigen binding molecules that bind gprc5d and cd3, and uses thereof
WO2019220368A1 (en) 2018-05-16 2019-11-21 Janssen Biotech, Inc. Bcma/cd3 and gprdc5d/cd3 bispecific antibodies for use in cancer therapy
WO2022175255A2 (en) 2021-02-16 2022-08-25 Janssen Pharmaceutica Nv Trispecific antibody targeting bcma, gprc5d, and cd3
WO2022174813A1 (zh) 2021-02-19 2022-08-25 信达生物制药(苏州)有限公司 抗GPRC5DxBCMAxCD3三特异性抗体及其用途
US20220411533A1 (en) * 2021-06-17 2022-12-29 Boehringer Ingelheim International Gmbh Novel Tri-specific Binding Molecules

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. BC069341
"Master’s Thesis", vol. 2022, 16 January 2022, CHINA MEDICAL UNIVERSITY, China, article WU, MING: "Basic Study on the Therapeutic Function of Trispecific Antibodies Targeting CD3-TROP2-PDL1 in Triple-negative Breast Cancer", pages: 1 - 43, XP009556737, DOI: 10.27652/d.cnki.gzyku.2021.001313 *
"NCBI", Database accession no. NP 061124.1
"UniProt", Database accession no. Q02223
"UniProtKB", Database accession no. Q9NZD 1
BRAUNER-OSBORNE, H ET AL., BIOCHIM.BIOPHYS.ACTA, vol. 1518, 2001, pages 237 - 248
See also references of EP4663662A1
ZHANG RUI; LI DA-LI: "Structure Design and Function Research of the Trispecific Antibody", PHARMACEUTICAL BIOTECHNOLOGY, vol. 29, no. 3, 31 December 2022 (2022-12-31), pages 227 - 233, XP009556731, ISSN: 1005-8915, DOI: 10.19526/j.cnki.1005-8915.20220302 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120424217A (zh) * 2025-04-30 2025-08-05 立凌生物制药(苏州)有限公司 靶向b细胞抗原和t细胞抗原的多特异性抗体及其应用

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