WO2023131328A1 - 一种抗体及其用途 - Google Patents

一种抗体及其用途 Download PDF

Info

Publication number
WO2023131328A1
WO2023131328A1 PCT/CN2023/071314 CN2023071314W WO2023131328A1 WO 2023131328 A1 WO2023131328 A1 WO 2023131328A1 CN 2023071314 W CN2023071314 W CN 2023071314W WO 2023131328 A1 WO2023131328 A1 WO 2023131328A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
antibody
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2023/071314
Other languages
English (en)
French (fr)
Chinese (zh)
Other versions
WO2023131328A9 (zh
Inventor
孙建明
黄潇
秦玉蓉
张鹏
房行行
尚红岩
凌虹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Leads Biolabs Co Ltd
Original Assignee
Nanjing Leads Biolabs Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Leads Biolabs Co Ltd filed Critical Nanjing Leads Biolabs Co Ltd
Priority to JP2024541191A priority Critical patent/JP2025504795A/ja
Priority to CN202380016649.XA priority patent/CN118510810A/zh
Priority to US18/728,022 priority patent/US20250092135A1/en
Priority to EP23737190.1A priority patent/EP4464722A4/en
Publication of WO2023131328A1 publication Critical patent/WO2023131328A1/zh
Publication of WO2023131328A9 publication Critical patent/WO2023131328A9/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/60—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
    • C07K2317/70—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

Definitions

  • the invention provides an antibody or its antigen-binding protein, and an asymmetric bispecific antibody constructed based on it.
  • the present invention further provides nucleic acid molecules encoding the antibodies, expression vectors for expressing the antibodies, host cells and preparation methods thereof.
  • the invention also provides diagnostic and therapeutic methods using the antibodies of the invention.
  • CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation.
  • Functional CD3 is formed by the dimeric association of two of four different chains: ⁇ , ⁇ , ⁇ , and ⁇ .
  • the CD3 dimer arrangement consists of ⁇ / ⁇ , ⁇ / ⁇ , and ⁇ / ⁇ .
  • Antibodies against CD3 have been shown to aggregate CD3 on T cells, causing T cell activation in a manner similar to the engagement of TCRs by peptide-loaded MHC molecules. Accordingly, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation.
  • bispecific antibodies that can bind to CD3 and target tumor surface antigens can engage tumor cells and T cells, thereby directly activating T cells, releasing granzymes, perforin, and cytokines to kill tumors, thereby achieving tumor suppression. therapeutic purposes.
  • G protein-coupled receptor family C group 5 member D (GPRC5D), as an orphan receptor, was found to be highly expressed in plasma cells of multiple myeloma patients and was associated with patient survival (Atamaniuk, J., et al .(2012). "Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma.” Eur J Clin Invest 42(9):953-960.). Further studies have shown that GPRC5D is expressed on the surface of plasma cells in blood cells of normal people, and the expression of GPRC5D on the surface of other blood cells is negative.
  • CD38+CD138+ plasma cells isolated from multiple myeloma patients have high expression of GPRC5D on the surface of plasma cells (Kodama , T., et al.(2019). "Anti-GPRC5D/CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma.”Mol Cancer Ther 18(9):1555-1564.), for patients with multiple myeloma Analysis of myeloma cells found that the expression of GPRC5D is not correlated with BCMA (Smith, E.L., et al. (2019).
  • GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Sci Transl Med 11 (485).)(Pillarisetti, K., et al.(2020).′′A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma.′′Blood 135(15) : 1232-1243.), so GPRC5D can be used as a tumor-specific target for multiple myeloma. In addition, GPRC5D can also serve as a tumor-specific target for other tumors.
  • MUC16 protein has been used as a specific marker of ovarian cancer since its discovery, and is also considered to be associated with poor prognosis of ovarian cancer.
  • MUC16 is a type of transmembrane glycoprotein with huge molecular weight, which is composed of extracellular domain, transmembrane segment and intracellular part; the extracellular domain is divided into two parts, the first half of which is highly glycosylated, and the second half of the extracellular domain is composed of It consists of about 60 tandem repeat domains, each of which has 156 amino acids.
  • the N-terminus of the MUC16 protein contains 56 SEA domains, and the MUC16 protein is truncated at the first or second SEA domain near the membrane end of the extracellular domain and releases free CA125 (Das, S. and S.K. Batra (2015). "Understanding the Unique Attributes of MUC16(CA125): Potential Implications in Targeted Therapy. "Cancer Res 75(22): 4669-4674, CA125 enters the circulation by being shed from the full-length MUC16 at the cell surface, Das , S., et al. (2015).
  • MUC16 membrane proximal ectodomain cleavage of MUC16 occurs in the acidifying Golgi/post-Golgi compartments" Sci Rep 5:9759).
  • CA125 indicators have also been found to increase in other tumors, such as breast cancer, pancreatic cancer, colorectal cancer and non-small cell lung cancer.
  • MUC16 promotes tumor invasion, metastasis, and suppresses immune responses. Based on this, MUC16 may be a potential target for cancer treatment.
  • a therapy targeting MUC16 has entered clinical research very early, the clinical results have failed to meet expectations (Aithal, A., et al.
  • immune checkpoint inhibitors have opened a new era of tumor immunotherapy, which can relieve immune suppression by blocking the binding of immune checkpoint molecules (such as PD-1 or CTLA4) to ligands, thereby restoring the function of T cells.
  • immune checkpoint inhibitor drugs have been approved for marketing in some indications, such as melanoma, non-small cell lung cancer, lymphoma, etc. (Pico de Coana, Y., et al. (2015). "Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system. "Trends Mol Med 21(8): 482-491) (Bu, X., et al. (2017).
  • T cell linker is a type of bispecific antibody, which can simultaneously bind to CD3 of T cells and tumor-associated antigen (TAA) of tumor cells (Labrijn, A.F., et al. (2019).
  • TAA tumor-associated antigen
  • the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof targeting human CD3, which has the following advantages:
  • the present invention also provides an anti-MUC16 antibody targeting human MUC16, which has one or more of the following advantages:
  • Target cells that bind to MUC16 such as human or monkey MUC16 with high affinity and express human MUC16;
  • the present invention provides a novel anti-GPRC5D antibody, and a bispecific antibody with CD3 antibody constructed using it.
  • the anti-GPRC5D antibody of the present invention has a high binding affinity to human GPRC5D and can recognize human and monkey (eg, cynomolgus) GPRC5D.
  • the anti-GPRC5D antibody can specifically bind to GPRC5D (such as human GPRC5D and/or monkey (such as cynomolgus monkey) GPRC5D) with high affinity, and the binding activity is significantly better than that of the control antibody GC5B596.
  • GPRC5D such as human GPRC5D and/or monkey (such as cynomolgus monkey) GPRC5D
  • the invention also provides CD3-based bispecific antibodies, such as bispecific antibodies that specifically bind CD3 and MUC16.
  • the anti-CD3 ⁇ MUC16 bispecific antibody of the present invention has one or more of the following characteristics:
  • the anti-CD3/GPRC5D bispecific antibody of the present invention has one or more of the following characteristics, so it has bright therapeutic prospects in GPRC5D-related indications:
  • the CD3 signaling pathway can only be activated in the presence of cells expressing GPRC5D, for example, the CD3 signaling pathway can only be activated in the presence of cells expressing GPRC5D;
  • CD3 signaling pathway There is less non-specific activation of the CD3 signaling pathway; for example, in cells that do not express GPRC5D (such as HEK293T cells), the signaling pathway is only activated at high concentrations, and it is concentration-dependent;
  • cytokine induction specificity for example, only induce cells to release cytokines in cells expressing GPRC5D (such as HEK293T-GPRC5D cells), such as interferon, such as IFN ⁇ ; tumor necrosis factors such as TNF ⁇ and/or leukocytes Interkines such as IL-6;
  • in vitro binding assays such as Fortebio
  • human and monkey such as cynomolgus
  • CD3 proteins that is, have cross-species activity
  • the affinity K D is between 1-1000 nM, for example, the binding affinity K D for monkeys (such as cynomolgus monkeys) is 1-100 nM between, such as between 10-100nM, such as between 50-100nM, such as the human binding affinity K D between 1-1000nM, such as between 1-100nM, such as between 10-100nM, For example between 50-100nM, such as between 100-1000nM, such as between 200-1000nM;
  • (10) have better tumor suppression effect, such as tumor suppression rate at 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% %, more than 95%, and can even make the tumor completely regress;
  • (11) Can bind to human and monkey (such as cynomolgus monkey) GPRC5D expressed on cells, and/or can bind to human and monkey (such as cynomolgus monkey) CD3 expressed on cells, and have species crossover for GPRC5D and/or CD3 active.
  • Figure 1 Results of Jurkat/NFAT-luc reporter gene system activation by humanized anti-CD3 antibodies Roche-CD3/017-Roche-CD3, DA023AH23L2 ( Figure 1A) and 017-2 ( Figure 1B).
  • FIG. 1 Binding of murine anti-GPRC5D antibodies to cells expressing human GPRC5D protein (top) and cynomolgus monkey GPRC5D protein (bottom).
  • Figure 3 Binding of humanized anti-GPRC5D antibody to engineered cells expressing human GPRC5D protein.
  • Figure 4 Schematic diagram of the bispecific molecular structure.
  • a and B Anti-CD3/anti-GPRC5D bispecific antibody binds to HEK293T cells stably expressing human GPRC5D protein; C: Anti-CD3/anti-GPRC5D bispecific antibody Bi-29H6-6 binds to stable expression of cynomolgus monkey GPRC5D HEK293T cell binding of protein; D-F: anti-CD3/GPRC5D bispecific antibody Bi-29H6-6 does not bind to cells expressing GPRC5A, GPRC5B or GPRC5C protein.
  • Anti-CD3/GPRC5D bispecific antibody can activate CD3 signaling pathway in the presence of cells expressing human GPRC5D.
  • Anti-CD3/GPRC5D bispecific antibody fails to activate CD3 signaling pathway in the presence of cells not expressing human GPRC5D.
  • Anti-CD3/GPRC5D bispecific antibody induces PBMC-specific killing of tumor cells expressing GPRC5D.
  • Figure 13A Anti-tumor effect of anti-CD3/anti-GPRC5D bispecific antibody in human CD3E transgenic mouse MC38-huGPRC5D model;
  • B Anti-CD3/anti-GPRC5D bispecific antibody in NCG mice transplanted with human PBMC and inoculated with NCI - Antitumor effect in H929 tumor model.
  • Figure 15 Flow cytometry detection results of the binding of murine anti-human MUC16 antibody 776.1 and its mutants to 293T cells expressing human Muc16 protein.
  • Figure 16 Flow cytometry detection results of the binding of mouse-derived anti-human MUC16 antibody 776.1 and its mutants to 293T cells expressing rhesus monkey MUC16 protein.
  • FIG. 19 The results of immunohistochemical detection of MUC16 and CD8 in tumor tissues.
  • Figure 20 Schematic diagram of the structure of a bispecific antibody molecule
  • FIG. 21 Anti-CD3/anti-MUC16 bispecific antibody binding to OVCAR3 cells.
  • Figure 22 Binding of anti-CD3/anti-MUC16 bispecific antibody to 293T/rhesus MUC16 cells.
  • FIG. 23 Binding of anti-CD3/anti-MUC16 bispecific antibody to Jurkat cells.
  • Anti-CD3/anti-MUC16 bispecific antibody activates Jurkat/NFAT-luc cells in the presence of OVCAR3 tumor cells.
  • Anti-CD3/anti-MUC16 bispecific antibody does not activate Jurkat/NFAT-luc cells in the presence of 293T cells.
  • FIG. 26 A: Anti-CD3/anti-MUC16 bispecific antibody specifically mediates T cell killing of tumor cells; B: Anti-CD3/anti-MUC16 bispecific antibody specifically mediates T cell killing activity of OVCAR3 tumor cells, incubated for 72 Hour
  • Figure 27 The results of cytokine release after activation of T cells by anti-CD3/anti-MUC16 bispecific antibody, in which the release of cytokines IFN ⁇ , TNF ⁇ and IL-6 factors were compared with OVCAR3 or 293T cells co-cultured respectively.
  • Figure 28A Results of tumor volume growth in each group after administration.
  • Fig. 28B The results of relative tumor volume growth in each group after administration.
  • Anti-CD3/anti-MUC16 bispecific antibody specifically mediates repeated killing of tumor cells by T cells.
  • FIG. 31 T cell activation, exhaustion and apoptosis detection in TDCC repeated killing assay.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • first and second When referring to “first” and “second” herein, it is only for distinguishing two structural domains or two chains, and does not indicate the positions of the two structural domains in any way.
  • CD3 refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR), the T cell adapter antigen T cell surface glycoprotein CD3, which is composed of the following four receptor chains Composed of homodimers or heterodimers formed by two chains of CD3- ⁇ , CD3- ⁇ , CD3- ⁇ and CD3- ⁇ .
  • Human CD3- ⁇ (hCD3 ⁇ ) comprises the amino acid sequence described in UniProtKB/Swiss-Prot: P07766.
  • Human CD3-delta (hCD3delta) comprises the amino acid sequence described in UniProtKB/Swiss-Prot: P04234.
  • the CD3 described in the present invention refers to CD3 from human or monkey (eg, cynomolgus monkey).
  • CD3-binding antibody or "anti-CD3 antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to a single CD3 subunit (e.g., ⁇ , ⁇ , ⁇ , or ⁇ ), as well as antibodies that specifically recognize Dimeric complexes of two CD3 subunits (eg, ⁇ / ⁇ , ⁇ / ⁇ , and ⁇ / ⁇ CD3 dimers) and antibodies and antigen-binding fragments thereof associated therewith.
  • Antibodies and antigen-binding fragments of the invention can bind to soluble CD3, bound CD3, and/or cell surface expressed CD3.
  • Soluble CD3 includes native CD3 protein as well as recombinant CD3 protein variants, eg, monomeric and dimeric CD3 structures that lack a transmembrane region or are otherwise not associated with a cell membrane.
  • the antigen-binding region in the anti-CD3 antibody or antigen-binding fragment thereof (such as ScFv) or bispecific antibody of the present invention that binds to CD3 may have a relatively high affinity for CD3 or CD3-expressing cells (such as T cells). low binding activity.
  • the binding affinity of the antibody to CD3 can be detected by flow cytometry detection or biofilm layer optical interference detection, such as the assay described in Example 6.5 or Example 10.2.
  • the binding affinity of the CD3-binding antigen-binding region to human or monkey (cynomolgus monkey) CD3 is between 1-1000 nM.
  • the anti-CD3 antibody or antigen-binding fragment thereof of the invention or the antigen-binding region in the bispecific antibody that binds to CD3 binds human and/or monkey (eg, cynomolgus) CD3 with lower binding affinity Human and/or monkey (eg cynomolgus) T cells can thereby be activated.
  • Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells may also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).
  • T lymphocytes such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells may also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).
  • GPRC5D refers to tumor-associated antigen G protein-coupled receptor family C group 5 member D (eg, human GPRC5D protein under accession number NP_061124.1 or cynomolgus monkey GPRC5D protein under XP_005570249.2).
  • the human GPRC5D protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 57, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% identical amino acid sequence, or consists of said sequence.
  • the cynomolgus monkey GPRC5D protein of the present invention comprises the amino acid sequence shown in SEQ ID NO: 58, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, An amino acid sequence that is 96%, 97%, 98% or 99% identical, or consists of said sequence.
  • the anti-GPRC5D antibody or its antigen-binding fragment (such as Fab) of the present invention or the antigen-binding region that binds to GPRC5D in the bispecific antibody has high-affinity binding activity to cells expressing human GPRC5D, for example, has a higher affinity than the control Higher binding affinity of antibodies (eg GC5B596).
  • the determination is by flow cytometry, eg, performing the assay described in 6.2.
  • the antigen-binding region in the anti-GPRC5D antibody or antigen-binding fragment (e.g., Fab) or bispecific antibody of the invention that binds to GPRC5D is cross-reactive to human and monkey (e.g., cynomolgus) GPRC5D , that is capable of binding human and monkey (eg cynomolgus) GPRC5D.
  • the terms “whole antibody”, “full-length antibody”, “complete antibody” and “whole antibody” are used interchangeably herein to refer to a naturally occurring antibody comprising at least two heavy (H) chains interconnected by disulfide bonds. and two light chain (L) glycoproteins.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs) interspersed with more conservative regions (framework regions (FRs)).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FR composition, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions.
  • the antibody heavy chain constant region HC of the present invention is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region.
  • the heavy chain constant region comprises a LALA mutation. In some embodiments, the heavy chain constant region comprises D265A and P329A mutations. In some embodiments, the heavy chain constant region comprises LALA mutations and D265A and P329A mutations. In some embodiments, the bispecific antibody molecule of the present invention The heavy chain constant region comprises a "knot button mutation.” In some preferred embodiments, the antibody heavy chain constant region HC of the present invention
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 45 or 48; or
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of said amino acid sequence.
  • the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 47 or 50; or
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) or consists of said amino acid sequence.
  • antibody fragment includes a portion of an intact antibody.
  • antibody fragments are antigen-binding fragments.
  • antigen-binding fragment is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) Binding antigen.
  • Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single chain Fv, diabody, single domain antibody (sdAb).
  • the Fab fragment is a monovalent fragment consisting of VL, VH, CL and CH1 domains, for example, Fab fragments can be obtained by papain digestion of complete antibodies. Furthermore, digestion of whole antibodies by pepsin below the disulfide bonds in the hinge region yields F(ab')2, a dimer of Fab', a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting F(ab')2 dimers to Fab' monomers.
  • a Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments see: Fundamental Immunology, edited by W.E. Paul, Raven Press, N.Y.
  • the Fv fragment consists of the VL and VH domains of a single arm of an antibody.
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, using recombinant methods they can be linked by a synthetic linker peptide that enables the production of the two domains as a single protein chain, described in The VL and VH regions pair in a single protein chain to form a single-chain Fv (scFv).
  • the antibody fragments can be obtained by chemical methods, recombinant DNA methods or protease digestion methods.
  • Fab fragment or “Fab” are used interchangeably herein to refer to an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, and a light chain variable domain consisting of two polypeptide chains.
  • Immunoglobulin fragments of variable domain VL and light chain constant domain CL wherein one polypeptide chain comprises VH and a constant region selected from CH1 and CL from N-terminus to C-terminus, and the other polypeptide chain runs from N-terminus to C-terminus
  • the VL end comprises a VL and another constant region selected from CL and CH1, wherein the VH and VL domains pair to form an antigen binding site.
  • a Fab chain comprising the heavy chain constant region CH1 is also referred to as "Fab heavy chain”; correspondingly, a Fab chain comprising the light chain constant region CL is also referred to as "Fab light chain”.
  • Target refers to a bound substance to which a binding molecule is directed.
  • Targets can be antigens, ligands or receptors.
  • antigen refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • epipe refers to the portion of an antigen that specifically interacts with an antibody molecule.
  • target binding region refers to the portion of a multispecific binding molecule, such as a bispecific binding molecule, that binds a particular target or antigen.
  • the target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the rest of the bispecific antibody molecule and may or may not bind its target as a separate entity.
  • the target binding region can also be a receptor or a ligand, or a domain of a receptor capable of binding a ligand.
  • the "target-binding region” is also referred to as "antigen-binding region”.
  • the antigen-binding region used in the bispecific antibody molecule of the present invention comprises a VH/VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the VL pair can be comprised in a single polypeptide chain (eg, in a scFv) or in two separate polypeptide chains (eg, in a Fab heavy chain and a Fab light chain, respectively).
  • the term “monospecific” antibody refers to an antibody that has one or more binding sites, each of which binds to the same epitope of the same antigen.
  • the term “multispecific” antibody refers to an antibody having at least two antigen binding sites, at least one of which binds a different epitope, e.g., the same antigen, relative to the remaining antigen binding sites Different epitopes on different antigens or different epitopes on different antigens.
  • the present invention provides antibodies against GPRC5D, antibodies against CD3, and bispecific antibodies against GPRC5D and CD3.
  • the invention also provides bispecific antibodies against CD3 and MUC16.
  • immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody.
  • IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons consisting of two light chains and two heavy chains that are disulfide bonded. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2 and CH3 ). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL).
  • VH heavy chain variable region
  • CL light chain constant domain
  • the heavy chains of immunoglobulins can be assigned to one of five classes, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), some of which can be further divided into subclasses Classes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1 ) and ⁇ 2 (IgA2).
  • the light chains of immunoglobulins can be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domains.
  • IgG immunoglobulins essentially consist of two Fab molecules and two dimerized Fc regions (Fc dimers) linked by an immunoglobulin hinge region.
  • variable region refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen.
  • the variable regions of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions.
  • FRs conserved framework regions
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”).
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment schemes, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S.
  • CDR or “CDR sequence” covers a CDR sequence determined in any of the above ways.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • residue positions in the variable region of an antibody including heavy chain variable region residues and light chain variable region residues
  • residue positions in the variable region of an antibody including heavy chain variable region residues and light chain variable region residues
  • the HCDRs and LCDRs in the antibodies of the invention are determined according to the Kabat protocol.
  • the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment schemes may be different. That is, the CDR sequences of the same antibody variable region defined under different assignment schemes are different.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment scheme rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Fc domain or “Fc region” or “Fc fragment” are used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a native immunoglobulin "Fc domain” comprises two or three constant domains, a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes;
  • the numbering of amino acid residues in the Fc region or in the heavy chain constant region is according to e.g. Edelman, G.M. et al., Proc. Natl. Acad.
  • Fc domain or "Fc region” or “Fc fragment” does not include the heavy chain variable region VH and the light chain variable region VL and the heavy chain constant region CH1 and the light chain constant region of an immunoglobulin CL, but in some cases may include a hinge region N-terminal to the heavy chain constant region, such as EPKSS or EPKSC.
  • the heavy chain constant region Fc suitable for use in the present invention is from an antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3 or IgG4, preferably from the constant region of IgG1.
  • the Fc region comprises mutations that reduce binding to Fc ⁇ receptors, such as a LALA mutation, a D265A mutation and/or a P329A mutation, preferably a LALA mutation and D265A and P329A mutations.
  • Fc comprises the amino acid sequence shown in SEQ ID NO: 46 or SEQ ID NO: 49, or comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said amino acid sequence.
  • Fc fragments dimerize to form Fc dimers.
  • the Fc fragment heterodimerizes into an Fc heterodimer.
  • the Fc fragment may contain mutations for heterodimerization, such as a junction button mutation.
  • effector functions of immunoglobulins include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (eg, B-cell receptors), and B-cell activation.
  • chimeric antibody is an antibody molecule in which (a) the constant region or part thereof is altered, replaced or exchanged such that the antigen binding site is of a different or altered class, effector function and/or species constant region or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) etc. that confers new properties on the chimeric antibody; Changes, substitutions, or exchanges of the variable regions of the For example, mouse antibodies can be modified by exchanging their constant regions with those from human immunoglobulins. Due to the exchange of human constant regions, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.
  • the constant region or part thereof is altered, replaced or exchanged such that the antigen binding site is of a different or altered class, effector function and/or species constant region or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) etc. that confers new properties on the chimeric antibody; Change
  • a “humanized antibody” is an antibody that retains the antigen-specific reactivity of a non-human antibody (eg, a mouse monoclonal antibody), while being less immunogenic when administered to humans as a therapeutic. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remainder of the antibodies with their human counterparts (i.e., substituting the constant and variable regions that do not participate in binding with the corresponding parts of human antibodies) .
  • the term "anti”, “bind” or “specifically binds” means that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of a binding site to bind a particular target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer Interferometry or MSD assay or Surface Plasmon Resonance (SPR) assay.
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ), which is the ratio of the dissociation rate constant and the association rate constant (K dis and K on , respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio Kinetic Binding Assay herein.
  • Percent identity (%) refers to after aligning a candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without taking into account any When conservative substitutions are taken as part of sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular amino acid sequence shown in this specification.
  • the invention contemplates variants of the antibody molecules of the invention having a substantial degree of identity, for example at least 80% identity, with respect to the antibody molecules and their sequences specifically disclosed herein , 85%, 90%, 95%, 97%, 98%, or 99% or higher. Such variants may contain conservative changes, or be conservatively modified variants.
  • “conservative changes” include substitutions, deletions or additions to the polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the substitution of an amino acid for a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the 8 groups of amino acids containing mutually conservative substitutions are listed below: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M ), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine (C), Methionine (M).
  • the term "conservative sequence change” is used to refer to an amino acid modification that does not significantly affect or alter the antigen-binding characteristics of interest of an antibody molecule or binding protein molecule of the invention comprising an amino acid sequence.
  • conservatively modified variants retain at least 80%, 85%, 90%, 95%, 98%, 99% or higher, eg 100-110% or higher binding affinity for the antigen of interest relative to the parent antibody or binding protein.
  • knock-in-hole mutation or “knob-in-hole” mutation or “knob-in-hole” is used herein to refer to the introduction of mutations in the first Fc polypeptide and the second Fc polypeptide, respectively, using the "knob-in-hole” technique to A protrusion ("knob") and a complementary cavity (“hole”) are formed at the interface of the first Fc polypeptide and at the interface of the second Fc polypeptide. It is known in the art that "knot-in-button” technology can engineer the interface between different chains of an antibody molecule to facilitate proper association of the chains of the antibody molecule.
  • the technique involves introducing a "bump/knot” at the interface of one chain and a corresponding "cavity/button” at the interface of the other chain it is to mate with, so that the bump can fit into the cavity.
  • a preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain with which it is to be paired.
  • the bulge can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains such as tyrosine or tryptophan. Create the same or similar bulge at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired by replacing large amino acid side chains with smaller ones (e.g.
  • Another optional interface is the above-mentioned Fab fragment comprising the CL domain of the light chain and the CH1 domain of the heavy chain, which promotes correct alignment between the two chains of the Fab fragment by constructing a bulge-cavity interaction. Heterodimerization.
  • Single-chain variable fragment or “scFv” is used herein to refer to a single-chain antibody fragment comprising a heavy chain variable domain VH and a light chain variable domain VL connected by a linker, wherein VH and VL are paired to form Antigen binding site.
  • antibody constant regions or antibody constant domains can be selected according to the intended function of the antibody molecule.
  • the constant region may be an IgA, IgD, IgE, IgG or IgM region, especially an immunoglobulin constant domain of human IgG, for example, the constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably the constant structure of human IgG1 area.
  • a Fab fragment of an antibody may comprise the CH and CL constant regions from IgGl.
  • the Fc region of an antibody may comprise CH2 and CH3 domains from IgGl. Immunoglobulin constant regions can have native or variant sequences.
  • linker refers to any molecule that enables the direct linking of different parts of a bispecific binding molecule.
  • linkers that establish covalent linkages between different molecular moieties include peptide linkers and non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol copolymer.
  • the linker is a peptide linker (also known as a "linker peptide”), which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and/or serine (S) alone or in combination.
  • a peptide linker can link a first target-binding region of a binding molecule to a second target-binding region.
  • a peptide linker can also join one part of an antibody to another part of an antibody, such as joining a light chain variable region to a heavy chain variable region.
  • the peptide linker is of a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the connecting peptide is 5-50 amino acids in length, eg, 10, 15, 20, 25, 30 amino acids in length.
  • the connecting peptide comprises the amino acid sequence (GS)n, (GGS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, wherein n is an integer equal to or greater than 1 , for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • the connecting peptide is a hinge region or portion of a hinge region from an immunoglobulin, including a native hinge region or portion thereof, or a mutated hinge region or portion thereof.
  • the connecting peptide is, for example, the hinge region or part thereof (eg EPKSC) or a mutated hinge region or part thereof of an immunoglobulin (eg IgG, eg IgGl, IgG2, IgG3 or IgG4), eg EPKSS.
  • suitable flexible linker peptides can be rationally designed using computer programs to model the three-dimensional structures of proteins and peptides, or by phage display methods.
  • half effective concentration refers to the concentration of drug, antibody or poison that induces 50% of the response between baseline and maximum after a specified exposure time.
  • host cell refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom.
  • a host cell is any type of cellular system that can be used to produce an antibody molecule of the invention, including eukaryotic cells, eg, mammalian cells, insect cells, yeast cells; and prokaryotic cells, eg, E. coli cells.
  • Host cells include cultured cells as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses) that incorporate recombinant polynucleotides. virus and adeno-associated virus).
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rodents). mouse).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rodents.
  • rodents e.g., mice and rodents.
  • an individual is a human being.
  • treating refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical indications of a disease, alleviating symptoms, or arresting or inhibiting the further development of a disease, condition, or disorder.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • prevention refers to the administration of a drug prior to the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.
  • therapeutic agent encompasses any substance that is effective in the prevention or treatment of tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants ).
  • cytotoxic agent is used in the present invention to refer to a substance that inhibits or prevents cell function and/or causes cell death or destruction.
  • “Chemotherapeutic agents” include chemical compounds useful in the treatment of cancer or disorders of the immune system.
  • small molecule drug refers to low molecular weight organic compounds capable of modulating biological processes.
  • Small molecules are defined as molecules having a molecular weight of less than 10 kD, usually less than 2 kD and preferably less than 1 kD.
  • Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.
  • the term "immunomodulator” refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response.
  • the immune response can be a humoral or cellular response.
  • Immunomodulators include immunosuppressants.
  • the immunomodulators of the invention include immune checkpoint inhibitors or immune checkpoint agonists.
  • an effective amount refers to such an amount or dose of the antibody or fragment or composition or combination of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, relative to an untreated subject %, 80%, 85%, 90% or even 100%.
  • prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • anti-tumor effect or “tumor suppression effect” or “tumor suppression effect” refers to a biological effect that can be exhibited by various means, including but not limited to, for example, reduction in tumor volume, reduction in tumor cell number, reduction in tumor cell proliferation, or Tumor cell survival is reduced.
  • pharmaceutical excipient refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., which are administered together with the active substance.
  • adjuvant such as Freund's adjuvant (complete and incomplete)
  • excipient carrier or stabilizer, etc.
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
  • non-fixed combination means that the active ingredients (e.g., (i) the immunoconjugate of the invention, and (ii) the other therapeutic agent) are combined as separate entities simultaneously, with no particular time limit, or at the same or different times. Interval, sequential administration to the patient, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient.
  • fixed combination means that two or more active agents are administered to a patient simultaneously as a single entity.
  • Dosages and/or intervals of two or more active agents are preferably selected such that the combined use of the parts produces a greater effect in treating a disease or condition than can be achieved by any one component alone.
  • the components may each be in the form of separate formulations, which may be the same or different.
  • combination therapy refers to the administration of two or more therapeutic agents or treatment modalities, such as radiation therapy or surgery, to treat a disease described herein.
  • administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, eg, in a single capsule with fixed ratios of the active ingredients.
  • administration includes co-administration for each active ingredient in multiple or in separate containers (eg tablets, capsules, powders and liquids). Powders and/or liquids can be reconstituted or diluted to the desired dosage before administration.
  • administration also includes using each type of therapeutic agent in a sequential manner at about the same time or at different times. In either case, the treatment regimen will provide for the beneficial effect of the drug combination in treating the disorders or conditions described herein.
  • tissue sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of tissue or cell samples can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as tears, vitreous humor, cerebrospinal fluid , amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • the tissue sample is tumor tissue.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • One aspect of the present invention provides a humanized CD3 antibody, which has better binding affinity to CD3, and thus is more suitable for constructing the antigen-binding region in the bispecific antibody molecule.
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention binds CD3 (eg, human CD3 or monkey CD3 such as cynomolgus CD3) with a desired affinity.
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention is capable of binding both human CD3 and monkey CD3, eg, cynomolgus CD3.
  • the affinity of the antibody is determined by biofilm thin layer interferometry or surface plasmon resonance.
  • an anti-CD3 antibody of the invention binds to human CD3 or monkey CD3, eg, cynomolgus CD3, with an equilibrium dissociation constant ( KD ) between about 1 nM-1000 nM. In some embodiments, an anti-CD3 antibody of the invention binds to monkey CD3, eg, cynomolgus CD3, with a KD between about 10 nM-100 nM, or 20 nM-100 nM, or 50 nM-100 nM. In some embodiments, an anti-CD3 antibody of the invention binds human CD3 with a KD of between about 100-1000 nM (eg, between about 200, 300, 400, or 500-1000 nM).
  • KD equilibrium dissociation constant
  • an antibody or antigen-binding fragment thereof of the invention binds to CD3 on the surface of an effector cell.
  • an antibody or antigen-binding fragment thereof of the invention is capable of activating effector cells.
  • the effector cells are T cells.
  • the binding is detected using flow cytometry.
  • CD3 antibody activation is detected using a reporter gene detection system (eg, Jurkat/NFAT-luc reporter gene system).
  • the antibodies or antigen-binding fragments thereof of the invention are capable of activating effector cells to induce tumor cell killing.
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region.
  • HCDRs complementarity determining regions
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region.
  • LCDRs complementarity determining regions
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region.
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH). In some aspects, an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VH). In some aspects, an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs), LCDR1, LCDR2 and LCDR3, from the light chain variable region.
  • CDRs complementarity determining regions
  • the anti-CD3 antibody or antigen-binding fragment thereof of the invention further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the invention further comprises an antibody light chain constant region LC. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region HC and a light chain constant region LC.
  • the anti-CD3 antibody heavy chain variable region VH of the present invention is anti-CD3 antibody heavy chain variable region VH of the present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 3;
  • amino acid sequence (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3 , more preferably amino acid conservative substitutions), the amino acid sequence consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.
  • the light chain variable region VL of the anti-CD3 antibody of the present invention is the light chain variable region VL of the anti-CD3 antibody of the present invention.
  • (i) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 4 or consists of said amino acid sequence;
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4;
  • amino acid sequence (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4 , more preferably amino acid conservative substitutions), the amino acid sequence consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region.
  • the three complementarity determining regions (HCDRs) of the anti-CD3 antibody of the present invention are from the heavy chain variable region, HCDR1, HCDR2 and HCDR3 are selected from
  • the three complementarity determining regions (LCDRs) from the light chain variable region of the anti-CD3 antibody of the present invention, LCDR1, LCDR2 and LCDR3 are selected from
  • HCDR1 comprises the amino acid sequence of SEQ ID NO: 5, or consists of said amino acid sequence
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, or consists of said amino acid sequence
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 7, or Consists of said amino acid sequence
  • LCDR1 comprises the amino acid sequence of SEQ ID NO: 8, or consists of said amino acid sequence
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 9, or consists of said amino acid sequence
  • LCDR3 comprises The amino acid sequence of SEQ ID NO: 10, or consists of said amino acid sequence.
  • the anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises VH and VL, wherein
  • the VH contains the amino acid sequence shown in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith
  • the amino acid sequence is or consists of the amino acid sequence
  • the VL contains the amino acid sequence shown in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical or consist of said amino acid sequences.
  • the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 3, and as shown in Three complementarity-determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO:4.
  • the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises: HCDR1 as shown in SEQ ID NO: 5, HCDR2 as shown in SEQ ID NO: 6, HCDR2 as shown in SEQ ID NO: 7 HCDR3 as shown; LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9 and LCDR3 as shown in SEQ ID NO:10.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • an anti-CD3 antibody or antigen-binding fragment thereof of the invention has one or more of the following properties:
  • the anti-CD3 antibody of the invention is an antibody in IgG1 format or an antibody in IgG2 format or an antibody in IgG3 format or an antibody in IgG4 format, eg, is an antibody in IgG1 format.
  • the light chain constant region of an anti-CD3 antibody of the invention is a kappa or lamda light chain constant region, eg, a lamda light chain constant region.
  • the anti-CD3 antibody is a monoclonal antibody.
  • anti-CD3 antibodies are humanized.
  • the anti-CD3 antibody of the invention also encompasses antibody fragments thereof (e.g. antigen-binding fragments), preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibodies (e.g. scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • antibody fragments thereof e.g. antigen-binding fragments
  • Fab' fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibodies (e.g. scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • the scFv comprises the amino acid sequence set forth in SEQ ID NO: 11, or comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , an amino acid sequence that is 98% or 99% identical, or consists of said amino acid sequence.
  • the anti-CD3 antibody of the present invention is a single chain antibody comprising an anti-CD3 antibody fragment scFv and a heavy chain constant region Fc (optionally the Fc region comprises a hinge region), or consisting of scFv and a heavy chain constant region. region Fc (optionally said Fc region comprises a hinge region).
  • the anti-CD3 single chain antibody of the present invention comprises the amino acid sequence shown in SEQ ID NO: 13, or comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98% or 99% identical amino acid sequences, or consist of said amino acid sequences.
  • the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof targeting human CD3, which has the following advantages:
  • the anti-CD3 antibody or antigen-binding fragment thereof is humanized.
  • said anti-CD3 antibody or antigen-binding fragment thereof comprises:
  • VH CD3 The heavy chain variable region (VH CD3 ) whose sequence is as described in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with SEQ ID NO: 3 , 97%, 98%, 99% or more sequence identity and heavy chain variable regions with identical CDRs; and
  • VL CD3 The light chain variable region (VL CD3 ) whose sequence is as described in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of SEQ ID NO: 4 , 97%, 98%, 99% or more sequence identity and light chain variable regions having identical CDRs.
  • said anti-CD3 antibody or antigen-binding fragment thereof comprises:
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 1 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Heavy chains with 98%, 99% or more sequence identity and identical CDRs; and
  • a light chain (L) whose sequence is as described in SEQ ID NO: 2 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Light chains with 98%, 99% or more sequence identity and identical CDRs.
  • the antigen-binding fragment of the anti-CD3 antibody is Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; linear antibody; single chain antibody (eg scFv).
  • the antigen-binding fragment of the anti-CD3 antibody is scFv, which comprises the sequence shown in SEQ ID NO: 11 or has at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and sequences having identical CDRs.
  • the anti-CD3 antibody comprises a scFv and an Fc region comprising a sequence as shown in SEQ ID NO: 13 or having at least 90%, 91%, 92%, 93% of SEQ ID NO: 13 , 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and sequences having identical CDRs.
  • One aspect of the present invention provides a GPRC5D antibody, which has higher binding affinity to GPRC5D, for example, the GPRC5D antibody of the present invention is more suitable for constructing the antigen-binding region in bispecific antibody molecules.
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention binds GPRC5D (e.g., human GPRC5D or monkey GPRC5D, e.g., cynomolgus GPRC5D) with higher affinity, e.g., than a control antibody such as GC5B596.
  • GPRC5D e.g., human GPRC5D or monkey GPRC5D, e.g., cynomolgus GPRC5D
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention is capable of binding both human GPRC5D and monkey GPRC5D, eg, cynomolgus monkey GPRC5D.
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention binds to GPRC5D expressed by a cell.
  • the affinity of the anti-GPRC5D antibody to cell-expressed GPRC5D is determined by flow cytometry.
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region.
  • HCDRs complementarity determining regions
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region.
  • LCDRs complementarity determining regions
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region.
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH). In some aspects, an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VH). In some aspects, an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs), LCDR1, LCDR2 and LCDR3, from the light chain variable region.
  • CDRs complementarity determining regions
  • the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain constant region LC. In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region HC and a light chain constant region LC.
  • the heavy chain variable region of an anti-GPRC5D antibody of the invention is provided.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; or
  • amino acid sequence of 5, 4, 3, 2, 1) of amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) consists of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR region.
  • the light chain variable region of an anti-GPRC5D antibody of the invention is provided.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; or
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) of 1) consists of said amino acid sequence, preferably, said amino acid change does not occur in the CDR region.
  • the anti-GPRC5D antibody of the present invention has three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3 are selected from
  • CDRs are determined by the Kabat protocol.
  • the anti-GPRC5D antibody of the present invention has three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3 are selected from
  • CDRs are determined by the Kabat protocol.
  • HCDR1 comprises the amino acid sequence of SEQ ID NO: 64, 74 or 84, or consists of said amino acid sequence, or HCDR1 comprises, compared with the amino acid sequence of SEQ ID NO: 64, 74 or 84, has one, Two or three altered (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequences.
  • HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 65, 75, 85, 39 or 41, or HCDR2 comprises the amino acid sequence of SEQ ID NO: 65, 75, 85, 39 or 41 An amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of the amino acid sequence.
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 66, 76 or 86, or consists of said amino acid sequence, or HCDR3 comprises, compared with the amino acid sequence of SEQ ID NO: 66, 76 or 86, has one, Two or three altered (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequences.
  • LCDR1 comprises the amino acid sequence of SEQ ID NO: 67, 77 or 87, or consists of said amino acid sequence, or LCDR1 comprises, compared with the amino acid sequence of SEQ ID NO: 67, 77 or 87, has one, Two or three altered (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequences.
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 68 or 78, or consists of said amino acid sequence, or LCDR2 comprises one, two or three amino acid sequences compared with the amino acid sequence of SEQ ID NO: 68, 78 Amino acid sequence of a change (preferably amino acid substitution, preferably conservative substitution).
  • LCDR3 comprises the amino acid sequence of SEQ ID NO: 69, 79 or 88, or consists of said amino acid sequence, or LCDR3 comprises, compared with the amino acid sequence of SEQ ID NO: 69, 79 or 88, has one, Two or three altered (preferably amino acid substitutions, preferably conservative substitutions) amino acid sequences.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises VH and VL, wherein
  • said VH contains the amino acid sequence shown in SEQ ID NO: 62 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof
  • said VL contains the amino acid sequence shown in SEQ ID NO: 63 or has at least 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical or consist of said amino acid sequences;
  • said VH contains the amino acid sequence shown in SEQ ID NO: 72 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof The amino acid sequence of identity or consists of said amino acid sequence, and said VL contains the amino acid sequence shown in SEQ ID NO: 73 or has at least 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical or consist of said amino acid sequences;
  • said VH contains the amino acid sequence shown in SEQ ID NO: 82 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof
  • An amino acid sequence of identity or VH consisting of said amino acid sequence and said VL contains the amino acid sequence shown in SEQ ID NO: 83 or has at least 90%, 91%, 92%, 93%, 94%, 95% thereof %, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences;
  • said VH contains the amino acid sequence shown in SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40 or has at least 90%, 91%, 92%, 93%, 94%, 95% thereof , 96%, 97%, 98% or 99% identical amino acid sequence or consists of said amino acid sequence
  • said VL contains or has at least 90% of the amino acid sequence shown in SEQ ID NO: 17, 19 or 21 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consists of said amino acid sequence.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention comprises:
  • HCDR1 as shown in SEQ ID NO: 64, HCDR2 as shown in SEQ ID NO: 65, 39 or 41, HCDR3 as shown in SEQ ID NO: 66; LCDR1 as shown in SEQ ID NO: 67 , LCDR2 as shown in SEQ ID NO: 68 and LCDR3 as shown in SEQ ID NO: 69;
  • HCDR1 as shown in SEQ ID NO: 74, HCDR2 as shown in SEQ ID NO: 75, HCDR3 as shown in SEQ ID NO: 76; LCDR1 as shown in SEQ ID NO: 77, as shown in SEQ ID LCDR2 shown in NO: 78 and LCDR3 shown in SEQ ID NO: 79; or
  • HCDR1 as shown in SEQ ID NO: 84, HCDR2 as shown in SEQ ID NO: 85, HCDR3 as shown in SEQ ID NO: 86; LCDR1 as shown in SEQ ID NO: 87, as shown in SEQ ID LCDR2 shown in NO:78 and LCDR3 shown in SEQ ID NO:88.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention comprises VH and VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below:
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a heavy chain. In some embodiments of the invention, an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a light chain. In some embodiments of the invention, an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain.
  • the heavy chain of the anti-GPRC5D antibody comprises the amino acid sequence of SEQ ID NO: 60, 70, 80, 14, 22, 24, 26 or 28, or comprises at least 85% of said amino acid sequence, Amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consist of said amino acid sequences.
  • the light chain of the anti-GPRC5D antibody comprises the amino acid sequence of SEQ ID NO: 61, 71, 81, 15, 18 or 20, or comprises at least 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consist of said amino acid sequences.
  • the anti-GPRC5D antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 14, 22, 24, 26 or 28, or is related to the amino acid sequence Amino acid sequences having or consisting of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and the The light chain comprises the amino acid sequence shown in SEQ ID NO: 15, 18 or 20, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence , 97%, 98% or 99% identical amino acid sequence, or consists of said amino acid sequence.
  • the anti-GPRC5D antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise the amino acid sequence shown in the following SEQ ID NO, or have at least 85%, 90% of the amino acid sequence %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consisting of said amino acid sequences:
  • the amino acid changes of the anti-GPRC5D antibodies described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid changes of the anti-GPRC5D antibody of the present invention are changes that eliminate post-translational modifications, such as those present in the CDRs of the anti-GPRC5D antibody, such as the HCDR2 of the anti-GPRC5D antibody, which contains N55S and/or or G56E substitution.
  • an anti-GPRC5D antibody or antigen-binding fragment thereof of the invention has one or more of the following properties:
  • an anti-GPRC5D antibody of the invention is an IgG1 antibody or an IgG2 antibody or an IgG3 antibody or an IgG4 antibody, eg, is an IgG1 antibody.
  • the light chain constant region of an anti-GPRC5D antibody of the invention is a lambda or kappa light chain constant region, eg, a kappa light chain constant region.
  • the anti-GPRC5D antibody is a monoclonal antibody.
  • the anti-GPRC5D antibody is humanized.
  • the anti-GPRC5D antibody is a chimeric antibody.
  • the anti-GPRC5D antibody of the present invention also encompasses antibody fragments thereof (eg, antigen-binding fragments), preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibodies (eg, scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • antibody fragments thereof eg, antigen-binding fragments
  • Fab' fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibodies (eg, scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • the present invention provides an anti-MUC16 antibody targeting human MUC16, which has one or more of the following advantages:
  • Target cells that bind to MUC16 such as human or monkey MUC16 with high affinity and express human MUC16;
  • the anti-MUC16 antibody or antigen-binding fragment thereof is humanized.
  • an anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region.
  • HCDRs complementarity determining regions
  • an anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region.
  • LCDRs complementarity determining regions
  • an anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises 3 complementarity determining regions (HCDRs) from the heavy chain variable region and 3 complementarity determining regions (LCDRs) from the light chain variable region.
  • HCDRs complementarity determining regions
  • LCDRs complementarity determining regions
  • an anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH). In some aspects, an anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (VH). In some aspects, an anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs), LCDR1, LCDR2 and LCDR3, from the light chain variable region.
  • CDRs complementarity determining regions
  • the anti-MUC16 antibody or antigen-binding fragment thereof of the present invention further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain constant region LC. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region HC and a light chain constant region LC.
  • the heavy chain variable region VH of the anti-MUC16 antibody of the present invention is the heavy chain variable region VH of the anti-MUC16 antibody of the present invention.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 92 or 113; or
  • amino acid changes compared to the amino acid sequence of SEQ ID NO: 92 or 113 (preferably The amino acid sequence of an amino acid substitution, more preferably a conservative amino acid substitution) consists of said amino acid sequence, preferably, said amino acid change does not occur in a CDR region.
  • the light chain variable region VL of the anti-MUC16 antibody of the present invention is the light chain variable region VL of the anti-MUC16 antibody of the present invention.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 96, 100, 102 or 114; or
  • amino acid sequence of the amino acid change (preferably amino acid substitution, more preferably amino acid conservative substitution) consists of said amino acid sequence, preferably, said amino acid change does not occur in the CDR region.
  • the three complementarity determining regions (HCDRs) from the heavy chain variable region of the anti-MUC16 antibody of the present invention, HCDR1, HCDR2 and HCDR3 are selected from
  • the three complementarity determining regions (LCDRs) of the anti-MUC16 antibody of the present invention are from the light chain variable region, LCDR1, LCDR2 and LCDR3 are selected from
  • HCDR1 comprises the amino acid sequence of SEQ ID NO: 93, or consists of said amino acid sequence
  • HCDR2 comprises the amino acid sequence of SEQ ID NO: 94, or consists of said amino acid sequence
  • HCDR3 comprises the amino acid sequence of SEQ ID NO: 95, or Consists of said amino acid sequence
  • LCDR1 comprises the amino acid sequence of SEQ ID NO: 97, 101 or 103, or consists of said amino acid sequence
  • LCDR2 comprises the amino acid sequence of SEQ ID NO: 98, or consists of said amino acid sequence
  • / or LCDR3 comprises the amino acid sequence of SEQ ID NO: 99, or consists of said amino acid sequence.
  • the anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises VH and VL, wherein
  • the VH contains the amino acid sequence shown in SEQ ID NO: 92 or 113 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto or consist of said amino acid sequence, and said VL contains or has at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 96, 100, 102 or 114 %, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consist of said amino acid sequences.
  • the anti-MUC16 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 92 or 113, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 96, 100, 102 or 114.
  • the anti-MUC16 antibody or antigen-binding fragment thereof of the present invention comprises: HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94, HCDR2 as shown in SEQ ID NO: 95 HCDR3 as shown; LCDR1 as shown in SEQ ID NO:97, 101 or 103, LCDR2 as shown in SEQ ID NO:98 and LCDR3 as SEQ ID NO:99.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein occur in regions outside the CDRs (eg, in FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • an anti-MUC16 antibody or antigen-binding fragment thereof of the invention has one or more of the following properties:
  • an anti-MUC16 antibody of the invention is an antibody in IgG1 format or an antibody in IgG2 format or an antibody in IgG3 format or an antibody in IgG4 format, eg, is an antibody in IgG1 format.
  • the light chain constant region of an anti-MUC16 antibody of the invention is a kappa or lamda light chain constant region, eg, a lamda light chain constant region.
  • the anti-MUC16 antibody is a monoclonal antibody.
  • the anti-MUC16 antibody is humanized.
  • the anti-MUC16 antibody of the present invention also encompasses antibody fragments thereof (such as antigen-binding fragments), preferably antibody fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibodies (such as scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • antibody fragments thereof such as antigen-binding fragments
  • Fab' fragments selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibodies (such as scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • said anti-MUC16 antibody or antigen-binding fragment thereof comprises VH and VL, wherein:
  • the VH comprises HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, and the VL comprises HCDR3 as shown in SEQ ID NO: 97 LCDR1 as shown, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99; or
  • the VH comprises HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, and the VL comprises HCDR3 as shown in SEQ ID NO: 101 LCDR1 as shown, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99; or
  • the VH comprises HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, and the VL comprises HCDR3 as shown in SEQ ID NO: 103 LCDR1 as shown, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99; or
  • the anti-MUC16 antibody or antigen-binding fragment thereof comprises:
  • VH whose sequence is as described in SEQ ID NO: 92 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 92 VH with % or more sequence identity and identical CDRs; and a VL whose sequence is as set forth in SEQ ID NO: 96 or at least 90%, 91%, 92%, 93%, 94%, VLs with 95%, 96%, 97%, 98%, 99% or more sequence identity and identical CDRs; or
  • VH whose sequence is as described in SEQ ID NO: 92 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 92 % or more sequence identity and VH with identical CDR; VLs with 95%, 96%, 97%, 98%, 99% or more sequence identity and identical CDRs; or
  • VH whose sequence is as described in SEQ ID NO: 92 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 92 % or more sequence identity and VH with identical CDR; VLs with 95%, 96%, 97%, 98%, 99% or more sequence identity and identical CDRs; or
  • VH whose sequence is as described in SEQ ID NO: 113 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 113 VH with % or more sequence identity and identical CDRs; and a VL whose sequence is as set forth in SEQ ID NO: 114 or at least 90%, 91%, 92%, 93%, 94%, VLs of 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs.
  • said anti-MUC16 antibody or antigen-binding fragment thereof comprises:
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 107 light chain (L) or with SEQ ID NO: 107 have at least 90%, 91%, Light chains with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs; or
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 108 light chain (L) or with SEQ ID NO: 108 have at least 90%, 91%, Light chains with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs; or
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 109 light chain (L) or with SEQ ID NO: 109 have at least 90%, 91%, Light chains with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs; or
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 115 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 116 light chain (L) or with SEQ ID NO: 116 have at least 90%, 91%, Light chains of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs.
  • the antibodies comprise amino acid changes.
  • the amino acid changes described herein include amino acid substitutions, insertions or deletions.
  • the amino acid changes described herein occur in regions outside the CDRs (e.g. in FRs). More preferably, the amino acid changes of the present invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.
  • the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • an antibody of the invention may be a cysteine-engineered antibody, such as a "thiomAb,” in which one or more residues of the antibody are replaced with a cysteine residue.
  • an anti-CD3 antibody of the invention is a bispecific or multispecific antibody, e.g., comprising a first binding specificity for CD3 and a molecule directed against one or more (e.g., a tumor-associated antigen, e.g., GPRC5D or other binding specificities of MUC16).
  • a tumor-associated antigen e.g., GPRC5D or other binding specificities of MUC16.
  • an anti-GPRC5D antibody of the invention is a bispecific or multispecific antibody comprising, for example, a first binding specificity for GPRC5D and an additional binding specificity for one or more molecules (e.g., CD3) specificity.
  • an anti-MUC16 antibody of the invention is a bispecific or multispecific antibody comprising, for example, a first binding specificity for MUC16 and an additional binding specificity for one or more molecules (e.g., CD3). specificity.
  • one aspect of the invention relates to a bispecific antibody comprising
  • a first antigen-binding region and a second antigen-binding region wherein the first antigen-binding region specifically binds a tumor-associated antigen, and/or the second antigen-binding region specifically binds CD3.
  • tumor-associated antigen refers to an antigen associated with a tumor, such as a protein highly expressed in a tumor, such as GPRC5D or MUC16.
  • one aspect of the invention relates to a bispecific antibody comprising
  • a first antigen-binding region and a second antigen-binding region wherein the first antigen-binding region specifically binds to GPRC5D or MUC16, and/or the second antigen-binding region specifically binds to CD3.
  • the first antigen binding region is from an anti-GPRC5D antibody described herein, eg, is a Fab fragment of an anti-GPRC5D antibody. In some embodiments, the first antigen binding region is from an anti-MUC16 antibody described herein, eg, is a Fab fragment of an anti-MUC16 antibody.
  • the second antigen binding region is from an anti-CD3 antibody, such as an SP34 antibody or a humanized antibody thereof, such as an anti-CD3 humanized antibody described herein, such as a scFv fragment of an anti-CD3 antibody.
  • an anti-CD3 antibody such as an SP34 antibody or a humanized antibody thereof, such as an anti-CD3 humanized antibody described herein, such as a scFv fragment of an anti-CD3 antibody.
  • the first antigen-binding region of the bispecific antibody applicable to the present invention may comprise or consist of the anti-GPRC5D or MUC16 full-length antibody or its antigen-binding fragment of the present invention, as long as it can specifically bind to GPRC5D or MUC16, Including but not limited to, for example, full-length antibodies, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies that specifically bind to GPRC5D.
  • the second antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of an anti-CD3 full-length antibody or an antigen-binding fragment thereof, as long as it can specifically bind to CD3, including but not limited to, for example, a specific Full-length antibody, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy chain antibody that binds to CD3, etc.
  • the bispecific antibodies of the invention are IgG-like bispecific antibodies.
  • An "IgG-like bispecific antibody" as described herein refers to a bispecific antibody comprising an Fc dimer.
  • the bispecific antibodies of the invention comprise Fc dimers.
  • the bispecific antibodies of the invention are IgG-like bispecific antibodies comprising a Fab fragment as one antigen-binding region and a scFv fragment as the other antigen-binding region.
  • the IgG-like bispecific antibody comprises a Fab fragment specifically binding to GPRC5D or MUC16 as the first antigen-binding region, and comprises a scFv specifically binding to CD3 as the second antigen-binding region.
  • the present invention provides a bispecific antibody comprising one or two Fab fragments that specifically bind a first antigen, an scFv that specifically binds a second antigen, and an Fc heterodimer, wherein
  • the Fab fragment specifically binding to the first antigen is fused to the N-terminus of the scFv fragment specifically binding to the second antigen (such as the N-terminus of VH or VL) at the C-terminus of CH1 of the Fab heavy chain, and the scFv fragment
  • the C-terminus e.g., the C-terminus of VL or VH
  • the CH2 or hinge region of one of the Fc regions of the Fc heterodimer e.g., a knot-containing Fc region or a button-containing Fc region
  • the first Fab fragment that specifically binds the first antigen is fused to one of the Fc regions of the Fc heterodimer (for example, a button-containing Fc region or a knot-containing Fc region) at the C-terminus of CH1 of the Fab heavy chain CH2 or hinge region; a second Fab fragment that specifically binds the first antigen is fused to the C-terminus of CH1 of the Fab heavy chain to the N-terminus of a scFv fragment that specifically binds the second antigen (e.g., the N-terminus of VH or VL), and the The C-terminus of the scFv fragment (e.g., the C-terminus of VL or VH) is fused to the CH2 or hinge region of another Fc region (e.g., a knot-containing Fc region or a knot-containing Fc region) of an Fc heterodimer; for example, as shown in FIG. 4B or the structure shown in Figure 20A;
  • scFv fragment that specifically binds a second antigen (e.g., the C-terminus of a VL or VH) fused to another Fc region of an Fc heterodimer (e.g., a knot-containing Fc region or a buckle-containing Fc region) CH2 or hinge region; for example, the structure shown in Figure 4C or Figure 20B; or
  • the first Fab fragment that specifically binds the first antigen is fused to the N-terminal of the VH of the second Fab fragment that specifically binds the first antigen at the C-terminus of CH1 of the Fab heavy chain, and the second Fab fragment is at The C-terminus of CH1 of the Fab heavy chain is fused to the CH2 or hinge region of one of the Fc regions of the Fc heterodimer (e.g., a knot-containing Fc region or a button-containing Fc region);
  • the C-terminus e.g., the C-terminus of VL or VH
  • another Fc region e.g., a button-containing Fc region or a knot-containing Fc region
  • fusion comprises direct fusion or fusion via a linker.
  • the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising a Fab fragment that specifically binds a first antigen, an scFv that specifically binds a second antigen and Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • the first Fc region constitutes the first heavy chain
  • the C-terminal of the CH1 of the Fab fragment is fused to the N-terminal of the scFv fragment, and the C-terminal of the scFv fragment is fused to the CH2 or hinge region of the second Fc region to form a second heavy chain;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind a first antigen, one scFv that specifically binds a second antigen, and Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • CH1 of the first Fab fragment is fused to CH2 comprising the first Fc region or the hinge region to form the first heavy chain;
  • the C-terminus of CH1 of the second Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the scFv fragment is fused to CH2 or the hinge region of the second Fc region to form a second heavy chain;
  • VL-CL of the first and second Fab fragments respectively constitute two light chains
  • first Fab and second Fab fragments are the same or different.
  • the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising a Fab fragment that specifically binds a first antigen, an scFv that specifically binds a second antigen, and an Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the first Fc region to form the first heavy chain;
  • the C-terminus of the scFv fragment is fused to the CH2 or hinge region comprising the second Fc region to form the second heavy chain;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind a first antigen, one scFv that specifically binds a second antigen, and Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • the C-terminal of the CH1 of the first Fab fragment is fused to the N-terminal of the VH of the second Fab fragment, and the C-terminal of the CH1 of the second Fab fragment is fused to the CH2 of the first Fc region or the hinge region to form the first heavy chain;
  • the C-terminus of the scFv fragment is fused to the CH2 or hinge region of the second Fc region, constituting the second heavy chain;
  • VL-CL of the first and second Fab fragments respectively constitute two light chains
  • first Fab and second Fab fragments are the same or different.
  • the first antigen is selected from GPRC5D or MUC16 (eg, human GPRC5D or human MUC16).
  • the second antigen is CD3.
  • the first Fc region comprises a knot mutation and the second Fc region comprises a button mutation. In some embodiments, the first Fc region comprises a button mutation and the second Fc region comprises a knot mutation.
  • the C-terminus of the scFv fragment is the C-terminus of the VL of the ScFv fragment.
  • the N-terminus of the scFv fragment is the N-terminus of the VH of the ScFv fragment.
  • the Fab fragment that is one of the antigen binding regions of the bispecific antibody consists of two polypeptide chains comprising antibody VH, CH1, VL and CL domains, wherein the VH is paired with VL and the CH1 is paired with The CLs pair to form the antigen-binding region.
  • one chain comprises VH and CH1 from N-terminus to C-terminus (i.e., VH-CH1) and the other chain comprises VL and CL from N-terminus to C-terminus (i.e., VL-CL) .
  • the Fab in the bispecific antibody, can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the VH-containing chain or fused to the Fc domain of the antibody via another antigen-binding region such as a scFv fragment. N-terminal to a domain where the Fc domain may or may not contain a hinge region (eg EPKSS or EPKSC).
  • the Fab comprises a VH-CH1 chain and a VL-CL chain, and is fused to an antibody Fc domain through the C-terminus of CH1 of the VH-CH1 chain, wherein the Fc domain may or may not contain a hinge region (such as EPKSS or EPKSC).
  • a Fab chain linked to an Fc dimer is also referred to as a Fab heavy chain, while a Fab chain not linked to an Fc dimer is also referred to as a Fab light chain.
  • the fusion is direct, or through a linker.
  • the Fab fragment comprised in the bispecific antibody of the invention specifically binds GPRC5D.
  • the Fab fragment contained in the bispecific antibody of the present invention is from the anti-GPRC5D antibody of the present invention, which comprises the heavy chain variable region VH and the light chain variable region VL of the anti-GPRC5D antibody of the present invention.
  • the Fab heavy chain comprises VH and CH1, wherein VH is the VH of an anti-GPRC5D antibody of the invention.
  • the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 44; or
  • amino acid sequence comprising one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 44
  • the amino acid sequence that is altered is or consists of said amino acid sequence.
  • the Fab light chain comprises VL-CL, wherein VL is the VL of an anti-GPRC5D antibody of the invention.
  • the CL is a light chain constant region from an antibody kappa or lamda light chain, preferably a light chain constant region from a kappa light chain.
  • the CL is the light chain constant region of an anti-GPRC5D antibody of the invention.
  • the Fab light chain is the light chain of an anti-GPRC5D antibody of the invention.
  • the Fab fragment comprised in the bispecific antibody of the invention specifically binds MUC16.
  • the Fab fragment contained in the bispecific antibody of the present invention is from the anti-MUC16 antibody of the present invention, which comprises the heavy chain variable region VH and the light chain variable region VL of the anti-MUC16 antibody of the present invention.
  • the Fab heavy chain comprises VH and CH1, wherein VH is the VH of an anti-MUC16 antibody of the invention.
  • the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 44; or
  • amino acid sequence comprising one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acids compared with the amino acid sequence selected from SEQ ID NO: 44
  • the amino acid sequence that is altered is or consists of said amino acid sequence.
  • the Fab light chain comprises VL-CL, wherein VL is the VL of an anti-MUC16 antibody of the invention.
  • the CL is a light chain constant region from an antibody kappa or lamda light chain, preferably a light chain constant region from a kappa light chain.
  • the CL is the light chain constant region of an anti-MUC16 antibody of the invention.
  • the Fab light chain is the light chain of an anti-MUC16 antibody of the invention.
  • the scFv fragment that is one of the binding regions of a bispecific antibody consists of one polypeptide chain comprising antibody VH and VL domains, wherein the VH and VL domains are linked (e.g., via a linker) to pair to form an antigen binding site point.
  • the scFv is in trans configuration, comprising from N-terminus to C-terminus: VH, linker, and VL (VH-linker-VL).
  • the scFv is in a cis configuration comprising, from N-terminus to C-terminus: VL, linker, and VH (VL-linker-VH).
  • the scFv antigen binding site comprised in the antibody molecule of the present invention is a disulfide bond stabilized scFv.
  • the scFv in the bispecific antibody, can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain. In some embodiments, in the bispecific antibody, the scFv is fused at the N-terminus of the chain to the C-terminus of another antigen binding region (eg, a Fab heavy chain) and at the C-terminus of the chain to the antibody Fc domain the N-terminus.
  • another antigen binding region eg, a Fab heavy chain
  • the scFv in the bispecific antibody, may be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the VL-containing chain. In some embodiments, in the bispecific antibody, the scFv is fused at the N-terminus of the VH chain to the C-terminus of another antigen-binding region (e.g., a Fab heavy chain) and at the C-terminus of the VL chain to the Fc of the antibody. N-terminus of the domain.
  • another antigen-binding region e.g., a Fab heavy chain
  • the scFv in the bispecific antibody, may be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the VH-containing chain. In some embodiments, in the bispecific antibody, the scFv is fused at the N-terminus of the VL chain to the C-terminus of another antigen-binding region (e.g., a Fab heavy chain), and at the C-terminus of the VH chain to the Fc of the antibody. N-terminus of the domain.
  • another antigen-binding region e.g., a Fab heavy chain
  • the scFv comprised in the bispecific antibody of the invention specifically binds CD3.
  • the scFv contained in the bispecific antibody of the present invention is from the anti-CD3 antibody of the present invention, which comprises the heavy chain variable region VH and the light chain variable region VL of the anti-CD3 antibody of the present invention.
  • the two Fc regions in the bispecific antibody of the invention dimerize to form a dimeric Fc.
  • the two Fc regions heterodimerize to form a heterodimeric Fc.
  • first and second Fc regions are the same. In other embodiments, the first and second Fc regions are different, pair and heterodimerize.
  • An Fc region fragment suitable for use in an antibody molecule of the invention may be any antibody Fc region.
  • Fc regions can include native sequence Fc regions and variant Fc regions.
  • Native sequence Fc domains encompass the naturally occurring Fc sequences of various immunoglobulins, such as the Fc regions of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389/fimmu.2014.00520.).
  • the Fc region of an antibody of the invention may comprise two or three constant domains, namely a CH2 domain, a CH3 domain and optionally a CH4 domain.
  • the Fc region of an antibody may also have an IgG hinge region or a part of an IgG hinge region, eg, an IgG1 hinge region or a part of an IgG1 hinge region, at the N-terminus. Mutations may be contained in the hinge region.
  • the hinge region can be EPKSS or EPKSC.
  • the Fc region of the antibody of the present invention includes from N-terminus to C-terminus: CH2-CH3, or from N-terminus to C-terminus: hinge region-CH2-CH3.
  • the Fc region suitable for use in an antibody or bispecific antibody of the invention is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 or human IgG4 Fc.
  • the Fc region comprises or consists of the amino acid sequence SEQ ID NO: 49 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity .
  • the Fc region in an antibody or bispecific antibody of the invention can be mutated to obtain desired properties. Mutations to the Fc region are known in the art.
  • the Fc region is modified in properties characteristic of the effector function of the Fc region (eg, the complement activation function of the Fc region). In one embodiment, said effector function has been reduced or eliminated relative to the wild isotype Fc region. In one embodiment, effector function is reduced or eliminated by a method selected from the group consisting of using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.
  • the Fc region has reduced effector functions mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector functions, for example comprising mutations to achieve the above functions.
  • the antibody molecule of the invention may also comprise modifications in the Fc domain that alter the binding affinity for one or more Fc receptors.
  • the Fc receptor is an Fc gamma receptor, in particular a human Fc gamma receptor.
  • the Fc region comprises mutations that reduce binding to Fc ⁇ receptors.
  • the Fc region used in the invention has one or more of the L234A/L235A mutation, the D265A mutation, or the P329A mutation that reduces binding to the Fc ⁇ receptor.
  • the Fc region used in the invention has a L234A/L235A mutation, a D265A mutation, and a P329A mutation that reduce binding to Fc ⁇ receptors.
  • the Fc fragment may have mutations that result in increased serum half-life, such as mutations that improve binding of the Fc fragment to FcRn.
  • the Fc region comprising a mutation that reduces binding to an Fc ⁇ receptor comprises or is at least 90% identical to the amino acid sequence of SEQ ID NO: 46, e.g., 95%, 96%, 97%, 99% or higher The amino acid sequence identical to or consisting of it.
  • the Fc region comprises an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 46 and comprises L234A/L235A mutation, D265A mutation, and P329A mutation.
  • the Fc region comprised by the bispecific antibody of the present invention may contain mutations that favor heterodimerization.
  • mutations are introduced in the CH3 region of both Fc regions.
  • the CH3 region of the first Fc region and the CH3 region of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to associate with
  • the complementary engineered other CH3 domains heterodimerize such that the first and second CH3 domains heterodimerize without forming homodimers between the two first CH3 domains or the two second CH3 domains).
  • knob mutations and hole mutations are respectively introduced into the Fc region of the first monomer and the Fc region of the second monomer.
  • This technique is described, for example, in Merchant, A.M., et al. (1998). "An efficient route to human bispecific IgG.” Nat Biotechnol 16(7):677-681.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knot mutation); while in the CH3 region of another Fc region
  • the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (deduction mutation)
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the Leucine residues were replaced with alanine residues (L368A) (numbering according to EU index).
  • the knot mutation comprises or consists of the following: the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine at position 354 residue is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with cysteine residue replacement); and in the CH3 region of another Fc region, the buckle mutation comprises or consists of the following: the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally at position 366 The threonine residue at position 368 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index), optionally a tyrosine residue at position 349 Amin
  • one Fc region comprises amino acid substitutions S354C and T366W (knot mutation) and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A and Y407V (deduction mutation) (numbering according to the EU index).
  • the bispecific antibody of the invention comprises two Fc regions that are heterodimerized, wherein
  • one Fc-region polypeptide comprises mutation T366W and the other Fc-region polypeptide comprises mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises mutations T366W and Y349C and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and S354C, or
  • one Fc-region polypeptide comprises mutations T366W and S354C, and the other Fc-region polypeptide comprises mutations T366S, L368A, Y407V and Y349C;
  • the Fc region further comprises a mutation that reduces binding to Fc ⁇ receptors, for example, one or more of L234A/L235A mutation, D265A mutation, or P329A mutation; for example, L234A/L235A mutation, D265A mutation, and P329A mutation.
  • a mutation that reduces binding to Fc ⁇ receptors for example, one or more of L234A/L235A mutation, D265A mutation, or P329A mutation; for example, L234A/L235A mutation, D265A mutation, and P329A mutation.
  • the Fc region further comprises other mutations that facilitate heterodimer purification.
  • the H435R mutation Eric J. Smith, Scientific Reports
  • the Fc region with a Hole mutation can be introduced into one of the Fc regions of the heterodimer (e.g., the Fc region with a Hole mutation) to facilitate Protein A was used to purify heterodimers.
  • mutations such as C220S can also be introduced in the hinge region to facilitate the formation of heterodimers.
  • the two Fc regions of the bispecific antibody of the invention are heterodimerized, wherein
  • the first Fc region comprises a junction mutation comprising the amino acid sequence of SEQ ID NO: 43 or 89 or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity or Consisting thereof; in some embodiments, the Fc region comprises amino acids having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 43 or 89 sequence and comprises a L234A/L235A mutation, a D265A mutation, and a P329A mutation and a junction mutation (eg, S354C and T366W); in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC;
  • the second Fc region comprises a button mutation comprising an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to the amino acid sequence SEQ ID NO: 30, 42 or 59 therewith or consisting of it.
  • the Fc region comprises an amino acid sequence that is at least 90% identical, such as 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 30, 42 or 59 and comprising L234A/L235A mutations, D265A mutations and P329A mutations and junction mutations; in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC.
  • the present invention provides a bispecific antibody comprising one or two Fab fragments of an anti-GPRC5D antibody, a scFv and an Fc heterodimer of an anti-CD3 antibody, wherein
  • the Fab fragment of anti-GPRC5D is fused to the N-terminal of the VH of the anti-CD3 antibody scFv fragment at the C-terminal of CH1 of the Fab heavy chain, and the C-terminal of the VL of the scFv fragment is fused to the Fc region of the Fc heterodimer a CH2 or hinge region (e.g. comprising an Fc region of the junction), such as the structure shown in Figure 4A;
  • the Fab fragment of the first GPRC5D is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions of the Fc heterodimer (for example, the Fc region comprising the buckle); the second anti-GPRC5D Fab
  • the fragment is fused to the N-terminus of the VH of the anti-CD3 antibody scFv fragment at the C-terminus of CH1 of the Fab heavy chain, and the C-terminus of the VL of the scFv fragment is fused to another Fc region of an Fc heterodimer (e.g., an Fc region comprising a knot). region) CH2 or hinge region; for example, the structure shown in Figure 4B;
  • the Fab fragment of GPRC5D is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions of the Fc heterodimer (for example, the Fc region comprising the button); the VL of the scFv fragment of the anti-CD3 antibody fused to the CH2 or hinge region of another Fc region of an Fc heterodimer (eg, the Fc region comprising the junction); for example, the structure shown in Figure 4C.
  • fusion comprises direct fusion or fusion via a linker.
  • the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising a Fab fragment of an anti-GPRC5D antibody, a scFv and an Fc heterodimer of an anti-CD3 antibody, in
  • the Fab fragment comprises VH-CH1 and VL-CL, the scFv comprises VH-linker-VL,
  • the Fc region comprising the buckle mutation constitutes the first heavy chain
  • the C-terminus of CH1 of the Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to CH2 or the hinge region of the Fc region comprising a junction mutation to form a second heavy chain;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 30, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95% identical to said amino acid sequence , 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 31, 34 or 35, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody, comprising two Fab fragments of an anti-GPRC5D antibody, a scFv and an Fc heterodimer of an anti-CD3 antibody, in
  • the Fab fragment comprises VH-CH1 and VL-CL, the scFv comprises VH-linker-VL,
  • the C-terminus of CH1 of the first Fab fragment is fused to the CH2 or hinge region of the Fc region comprising a buckle mutation to form the first heavy chain;
  • the C-terminus of CH1 of the second Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of the Fc region comprising the junction mutation, constituting the second heavy chain;
  • VL-CL of the first and second Fab fragments respectively constitute two light chains
  • first Fab and second Fab fragments are the same or different.
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 32, 33 or 36, or has at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence Amino acid sequences that are %, 95%, 96%, 97%, 98% or 99% identical, or consist of said sequences;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 31, 34 or 35, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95% of the amino acid sequence , 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said sequence; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 33, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95% identical to said amino acid sequence , 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 31, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 32, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95% identical to said amino acid sequence , 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 34, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence %, 98% or 99% identical amino acid sequences, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 36, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95% identical to said amino acid sequence , 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody, comprising a Fab fragment of an anti-GPRC5D antibody, a scFv and an Fc heterodimer of an anti-CD3 antibody, wherein
  • the Fab fragment comprises VH-CH1 and VL-CL, the scFv comprises VH-linker-VL,
  • the C-terminus of CH1 of the Fab fragment is fused to CH2 or the hinge region of the Fc region comprising a buckle mutation to form the first heavy chain;
  • the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of the Fc region comprising the junction mutation, constituting the second heavy chain;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 32, 33 or 36, or has at least 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence Amino acid sequences that are %, 95%, 96%, 97%, 98% or 99% identical, or consist of said sequences;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 37, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the present invention provides a bispecific antibody targeting both human CD3 and human MUC16 (anti-CD3 ⁇ MUC16 bispecific antibody), which has the following advantages:
  • the present invention provides an anti-CD3 ⁇ MUC16 bispecific antibody comprising:
  • the present invention provides an anti-CD3 ⁇ MUC16 bispecific antibody comprising:
  • VH CD3 1 heavy chain 1 with the structure VH-CH1-Fc, 1 heavy chain 2 with the structure VH-CH1-scFv-Fc, and 2 light chains with the structure VL-CL, of which 2 light chains Pair with the VH-CH1 structures of heavy chains 1 and 2 respectively to form two antigen recognition sites that recognize MUC16 molecules, and the scFv forms an antigen recognition site that recognizes CD3 molecules, wherein the VH that recognizes MUC16 molecules contains as shown in SEQ ID NO: 93
  • the HCDR1 shown in SEQ ID NO: 94, the HCDR2 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule includes LCDR1 shown in SEQ ID NO: 97, shown in SEQ ID NO: 98
  • the LCDR2 shown in SEQ ID NO:99 is LCDR3; the scFv that recognizes CD3 molecules comprises VH that recognizes CD3 (in order to be distinguished from the VH that recognizes MUC16,
  • VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-Fc
  • 1 heavy chain 2 with the structure of VH-CH1-scFv-Fc
  • 2 light chains with the structure of VL-CL.
  • the VH-CH1 structures of chains 1 and 2 are paired to form two antigen recognition sites that recognize MUC16 molecules
  • scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains HCDR1 as shown in SEQ ID NO:93 , HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95
  • the VL recognizing the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO: 101, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecules comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises HC
  • VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-Fc
  • 1 heavy chain 2 with the structure of VH-CH1-scFv-Fc
  • 2 light chains with the structure of VL-CL.
  • the VH-CH1 structures of chains 1 and 2 are paired to form two antigen recognition sites that recognize MUC16 molecules
  • scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains HCDR1 as shown in SEQ ID NO:93 , HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95
  • the VL that recognizes the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO: 103, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecules comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises
  • the pairing forms an antigen recognition site for recognizing the MUC16 molecule
  • scFv forms an antigen recognition site for recognizing the CD3 molecule
  • the VH for recognizing the MUC16 molecule contains HCDR1 as shown in SEQ ID NO: 93, and HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO:95
  • the VL recognizing the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO:101, LCDR2 as shown in SEQ ID NO:98 and LCDR3 as shown in SEQ ID NO:99
  • the scFv recognizing CD3 molecule comprises VH CD3 and VL CD3 , wherein said VH CD3 comprises HCDR1 as shown in SEQ
  • the pairing forms an antigen recognition site for recognizing the MUC16 molecule
  • scFv forms an antigen recognition site for recognizing the CD3 molecule
  • the VH for recognizing the MUC16 molecule contains HCDR1 as shown in SEQ ID NO: 93, and HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO:95
  • the VL recognizing the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO:103, LCDR2 as shown in SEQ ID NO:98 and LCDR3 as shown in SEQ ID NO:99
  • the scFv recognizing CD3 molecule comprises VH CD3 and VL CD3 , wherein said VH CD3 comprises HCDR1 as shown in SEQ
  • VH-CH1-VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-VH-CH1-Fc
  • 1 heavy chain 2 with the structure of scFv-Fc 2 light chains with the structure of VL-CL, of which 2 light chains Pair with the two VH-CH1 structures in heavy chain 1 to form two antigen recognition sites that recognize MUC16 molecules
  • the scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains such as SEQ ID NO: 93 HCDR1 as shown, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, recognize the VL of the MUC16 molecule comprising LCDR1 as shown in SEQ ID NO: 97, as shown in SEQ ID NO: 98 LCDR2 as shown and LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecules comprises VH CD3 and VL CD3
  • VH-CH1-VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-VH-CH1-Fc
  • 1 heavy chain 2 with the structure of scFv-Fc 2 light chains with the structure of VL-CL
  • the 2 light chains are respectively related to the heavy
  • the two VH-CH1 structures in chain 1 are paired to form two antigen recognition sites that recognize MUC16 molecules
  • the scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains as shown in SEQ ID NO: 93 HCDR1, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95
  • the VL of the MUC16 molecule comprising LCDR1 as shown in SEQ ID NO: 101, as shown in SEQ ID NO: 98 LCDR2 and LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecule comprises VH CD3 and
  • VH-CH1-VH-CH1-Fc 1 heavy chain 2 with the structure of scFv-Fc, and 2 light chains with the structure of VL-CL, and the 2 light chains are respectively related to the heavy
  • the two VH-CH1 structures in chain 1 are paired to form two antigen recognition sites that recognize MUC16 molecules, and the scFv forms an antigen recognition site that recognizes CD3 molecules, wherein the VH that recognizes MUC16 molecules contains as shown in SEQ ID NO: 93 HCDR1, HCDR2 as shown in SEQ ID NO:94 and HCDR3 as shown in SEQ ID NO:95, recognizes the VL of the MUC16 molecule comprising LCDR1 as shown in SEQ ID NO:103, as shown in SEQ ID NO:98 LCDR2 and LCDR3 as shown in SEQ ID NO: 99; the scFv recognizing CD3 molecule comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises HCDR
  • the present invention provides an anti-CD3 ⁇ MUC16 bispecific antibody comprising:
  • VH for recognizing the MUC16 molecule comprises a sequence selected from SEQ ID NO:
  • the sequence of 92 or 113, the VL recognizing the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102 or 114;
  • the scFv recognizing the CD3 molecule comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises SEQ ID NO: 3 and VL CD3 comprises the sequence shown in SEQ ID NO:4.
  • the two antigen recognition sites that recognize the MUC16 molecule included in the above bispecific antibody may have the same VH/VL sequence, or different VH/VL sequences.
  • the present invention provides an anti-CD3 ⁇ MUC16 bispecific antibody comprising:
  • VH-CH1-Fc 1 heavy chain 1 with the structure VH-CH1-Fc
  • 1 heavy chain 2 with the structure VH-CH1-scFv-Fc
  • 2 light chains with the structure VL-CL, of which 2 light chains Pair with the VH-CH1 structures of heavy chains 1 and 2 to form two antigen recognition sites that recognize MUC16 molecules
  • the scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains the sequence of SEQ ID NO: 113 Or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 113 and having the same CDRs Sequence
  • the VL recognizing the MUC16 molecule comprises a sequence selected from SEQ ID NO: 114 or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of SEQ ID NO: 114 , 98%,
  • VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-Fc
  • 1 heavy chain 2 with the structure of scFv-Fc
  • 1 light chain with the structure of VL-CL wherein the light chain and the VH of the heavy chain 1 - CH1 structural pairing forms an antigen recognition site for recognizing MUC16 molecules
  • scFv forms an antigen recognition site for recognizing CD3 molecules
  • the VH for recognizing MUC16 molecules contains a sequence selected from SEQ ID NO: 113 or contains the sequence with SEQ ID NO: 113
  • the VL recognizing the MUC16 molecule comprises selected A sequence from or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of SEQ ID NO: 114 Sequence identity and a
  • VH for recognizing the MUC16 molecule comprises a sequence selected from SEQ ID NO: The sequence of 113 or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 113 and having The sequence of the same CDR, the VL recognizing the MUC16 molecule comprises a sequence selected from SEQ ID NO: 114 or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of SEQ ID NO:
  • an anti-CD3 ⁇ MUC16 bispecific antibody as provided above in the present invention wherein the Fc region can be selected from the natural Fc of any IgG, IgA, IgM antibody known in the prior art Regions, such as consensus or germline IgG Fc regions.
  • the IgG Fc region may be from the isotype human IgGl, IgG2, IgG3 or IgG4.
  • the IgG Fc region is the Fc sequence of human IgG1.
  • the Fc region comprises mutations, eg, mutations that increase the stability of the bispecific antibody dimer (eg, a knob-in-hole structure), mutations that decrease or increase effector function.
  • the scFv of the anti-CD3 ⁇ MUC16 bispecific antibody of the present invention is connected to Fc or CH1 through a linker.
  • the linker can be any general flexible sequence known in the art or obtained in the future, usually a short GGSGG; GSGGG; SGGGG; GGTGS; GTSPGG; GNGGGS; G4S-GGSGG-G4S-SGGGG; GGG; DGGGS; TGEKP; GGRR; EGKSSGSGSESKVD; DGGGSERP; GSTSGSGKPGSGEGSTKG et al.
  • the linker is (G4S)n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9. In a specific embodiment, the linker is selected from (G4S)3.
  • the present invention provides an anti-CD3 ⁇ MUC16 bispecific antibody comprising:
  • heavy chain 1 with the structure of VH-CH1-VH-CH1-Fc
  • heavy chain 2 with the structure of scFv-Fc
  • 2 light chains with the structure of VL-CL, of which 2 light chains Pair with the two VH-CH1 structures in heavy chain 1 to form two antigen recognition sites that recognize MUC16 molecules, and the scFv forms an antigen recognition site that recognizes CD3 molecules
  • heavy chain 1 contains SEQ ID NO: 119 Sequences
  • the heavy chain 2 comprises the sequence set forth in SEQ ID NO: 117
  • the light chain comprises the sequence set forth in SEQ ID NO: 116.
  • the invention provides a nucleic acid encoding any of the above anti-CD3 antibodies or anti-GPRC5D antibodies or MUC16 or bispecific antibodies or any chain thereof.
  • the nucleic acid of the present invention comprises a code selected from SEQ ID NO: 1, 2, 3, 4, 11, 13, 14-29, 31-38, 40, 60-63, 70-73, 80-83, 92, 96, 100, 102, 106-109, 113-119 or 121 in any nucleic acid sequence shown in the amino acid sequence, or encoding and selected from SEQ ID NO: 1, 2, 3, 4, 11, 13, 14-29 , 31-38, 40, 60-63, 70-73, 80-83, 92, 96, 100, 102, 106-109, 113-119 or 121 have at least 85%, A nucleic acid having an amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • each antibody or polypeptide amino acid sequence may be encoded by multiple nucleic acid sequences due to codon degeneracy.
  • Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, eg, by de novo solid-phase DNA synthesis, or by PCR amplification.
  • the invention provides nucleic acids encoding any of the above antibodies or any antibody chains.
  • the polypeptide encoded by the nucleic acid is capable of displaying human CD3 and/or GPRC5D and/or MUC16 antigen (and/or monkey (eg rhesus or cynomolgus)) binding capacity when expressed from a suitable expression vector.
  • the invention provides a nucleic acid encoding any of the above bispecific antibodies.
  • the polypeptide encoded by the nucleic acid When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of displaying human or monkey (eg, rhesus or cynomolgus) CD3 and another antigen (eg, human or monkey (eg, rhesus or cynomolgus) CD3). ) binding ability of GPRC5D or MUC16 antigen).
  • the nucleic acids encoding each chain of the bispecific antibody may be in the same vector or in different vectors.
  • the nucleic acids encoding each chain of the bispecific antibody can be introduced into the same or different host cells for expression.
  • the production method of the bispecific antibody of the present invention comprises the steps of: cultivating a host cell comprising a nucleic acid encoding each chain of said molecule under conditions suitable for expressing each chain of said molecule, Production of bispecific antibodies of the invention.
  • a vector comprising said nucleic acid is provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • YACs yeast artificial chromosomes
  • the vector is eg a pcDNA vector, eg pcDNA3.1.
  • a host cell comprising said nucleic acid or said vector is provided, eg for cloning or expressing a vector encoding an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells such as CHO cells (e.g. CHO-S, such as ExpiCHO-S) or 293 cells (e.g. 293F or HEK293 cells)) or suitable for the production of antibodies or fragments thereof of other cells.
  • the host cell is prokaryotic, such as a bacterium, such as E. coli.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains in which glycosylation pathways have been "humanized” result in the production of antibodies with partially or fully human glycosylation patterns.
  • Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted for growth in suspension can be used.
  • useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7); the human embryonic kidney line (HEK293, 293F or 293T cells), and the like.
  • Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as YO, NSO and Sp2/0. Suitable mammalian host cell lines for antibody production are known in the art.
  • a method for preparing an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the present invention comprises, in a suitable anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or Under the condition that the bispecific antibody or its chain is expressed, the nucleic acid or A host cell comprising an expression vector of said nucleic acid, as provided above, and optionally recovering said anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody from said host cell (or host cell culture medium) Sexual antibodies.
  • the polynucleotide encoding the polypeptide chain of the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention can be inserted into one or more vectors for further cloning and/or expression in host cells.
  • Expression vectors can be constructed using methods well known to those skilled in the art. Once an expression vector comprising one or more nucleic acid molecules of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.
  • the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody prepared as described herein can be obtained by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, Purification by size exclusion chromatography, etc.
  • the actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art.
  • the purity of antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • anti-CD3 antibodies or anti-GPRC5D antibodies or anti-MUC16 antibodies or bispecific antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biology by a variety of assays known in the art active.
  • the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention is tested for its target (e.g. antigen, e.g. free antigen or antigen expressed on a cell) binding activity, e.g. by known methods Such as biofilm thin layer interferometry, ELISA, flow cytometry and so on. Binding to CD3 and/or GPRC5D and/or anti-MUC16 antibody (or CD3 and/or GPRC5D and/or MUC16 expressed on cells) can be assayed using methods known in the art, exemplary methods are disclosed herein.
  • target e.g. antigen, e.g. free antigen or antigen expressed on a cell
  • binding activity e.g. by known methods
  • binding activity e.g. by known methods
  • radioimmunoassay or biofilm thin layer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS) is used to measure.
  • RIA radioimmunoassay
  • BLI biofilm thin layer interferometry
  • ECL electrochemiluminescence
  • SPR surface plasmon resonance
  • FACS flow cytometry
  • the present invention also provides assays for identifying the biological activity of an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody.
  • the biological activity is selected from the properties of the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the invention.
  • the binding activity of the antibody molecules of the present invention to cells expressing CD3 and/or GPRC5D and/or MUC16 methods known in the art can be used, such as fluorescent reporter molecules and flow cytometry, or the methods disclosed in the examples herein. Exemplary methods are used to determine, for example, the binding of the antibody molecule of the present invention to CD3 and/or GPRC5D and/or MUC16 expressed on cells by the method shown in Example 5 or 6 or 7 or 8 or 10.
  • the activation activity of the antibody molecules of the present invention on T cells can be determined by methods known in the art, such as T cell activation assay systems, such as the NFAT-luc reporter gene system such as the Jurkat/NFAT-luc reporter gene system, for example By implementing the methods shown in 1 or 6 or 10 or 12 or 13 or 14, by detecting the CD3 signaling pathway in T cells or by detecting cytokines (such as interferons, such as IFN ⁇ ; tumor necrosis factors such as TNF ⁇ and /or the release of interleukins such as IL-6).
  • T cell activation assay systems such as the NFAT-luc reporter gene system such as the Jurkat/NFAT-luc reporter gene system
  • cytokines such as interferons, such as IFN ⁇ ; tumor necrosis factors such as TNF ⁇ and /or the release of interleukins such as IL-6).
  • the tumor inhibitory activity or structural safety of the antibody molecules of the present invention can be determined by methods known in the art, such as conducting tumor suppression experiments on mouse tumor models, such as by the methods shown in Example 7 or 11 .
  • Cells for use in any of the above in vitro assays are primary cells or cell lines, including those that naturally express or overexpress CD3 (e.g., human or monkey (e.g., cynomolgus monkey)) or GPRC5D (e.g., human or monkey (e.g., cynomolgus monkey) GPRC5D ) or MUC16 (e.g. human or monkey (e.g. cynomolgus) MUC16), e.g. cells overexpressing CD3 or GPRC5D or MUC16, e.g. T cells or 293 cells or CHO cells or Jurkat cells or myeloma cells e.g. HEK293 or CHO-K1 or Jurkat/NFAT-Luc cells or NCI-H929.
  • CD3 e.g., human or monkey (e.g., cynomolgus monkey)
  • GPRC5D e.g., human or monkey (e
  • Immunoconjugates pharmaceutical compositions, pharmaceutical combination products and kits of anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention
  • the invention provides an immunoconjugate comprising any of the anti-CD3 antibodies or anti-GPRC5D antibodies or anti-MUC16 antibodies or bispecific antibodies described herein.
  • the immunoconjugate comprises one or more additional therapeutic agents (eg, cytotoxins or small molecule compounds) or markers.
  • the present invention provides a composition or a medicament or a preparation comprising any anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody described herein, preferably the composition is a pharmaceutical composition.
  • the composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition, comprises a combination of an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the invention, and one or more other therapeutic agents.
  • composition or drug or preparation of the present invention may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • compositions or medicaments or formulations of the invention can be in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injections or eye drops), powders or suspensions, liposomes, and suppositories.
  • liquid solutions eg, injections or eye drops
  • powders or suspensions e.g., liposomes
  • suppositories e.g., suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use.
  • An anti-CD3 antibody comprising an anti-CD3 antibody described herein can be prepared by mixing an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the invention having the desired purity with one or more optional pharmaceutical excipients Or anti-GPRC5D antibody or bispecific antibody drug or preparation, for example in the form of lyophilized preparation or aqueous solution.
  • compositions or medicaments or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.
  • the present invention also provides a pharmaceutical combination or a pharmaceutical combination product, which comprises the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention, and one or more other therapeutic agents.
  • the present invention also provides a complete kit containing the drug combination, for example, the complete kit contains in the same package:
  • a first container containing a pharmaceutical composition comprising an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the invention
  • the other therapeutic agent is, for example, a chemotherapeutic agent, cytokine, cytotoxic agent, other antibody, small molecule drug, or immunomodulatory agent (eg, immunosuppressant).
  • One aspect of the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention, or an immunoconjugate comprising the same compound, composition or drug or preparation.
  • the invention provides methods for specifically activating T cells in a subject (eg, thereby enhancing, stimulating or increasing an immune response in the subject), comprising administering to the subject a bispecific T cell of the invention Antibodies, or immunoconjugates, compositions, or medicaments or formulations comprising the same.
  • the disease is eg a tumor, eg cancer.
  • Cancer can be early, middle or advanced or metastatic.
  • the cancer can be a solid tumor or a hematological tumor.
  • the cancer is ovarian cancer, myeloma, colon cancer, rectal cancer, or colorectal cancer.
  • the ovarian cancer is a cancer associated with abnormal proliferation of ovarian cells comprising a heterogeneous set of lesions, most commonly those lesions derived from epithelial cell types.
  • the ovarian cancer is e.g. ovarian cancer, especially serous ovarian cancer, e.g. ovarian serous cystadenocarcinoma, ovarian serous adenocarcinoma, ovarian cancer mucinous adenocarcinoma, endometrioid adenocarcinoma, ovarian cancer clear cell adenocarcinoma.
  • the disease treatment will benefit from activation of the CD3 signaling pathway and/or activation of T cells.
  • the cancer is a cancer characterized by elevated protein levels and/or nucleic acid levels (e.g., increased expression) of GPRC5D, e.g., elevated GPRC5D protein in tumor cells of the cancer Levels and/or nucleic acid levels (e.g., increased expression), e.g., compared to protein levels and/or nucleic acid levels of GPRC5D in the same tissue of a healthy subject, or compared to GPRC5D in healthy tissues adjacent to a patient's tumor tissue Protein level and/or nucleic acid level.
  • elevated protein levels and/or nucleic acid levels e.g., increased expression
  • levels and/or nucleic acid levels e.g., increased expression
  • the cancer is a cancer characterized by elevated protein levels and/or nucleic acid levels (e.g., increased expression) of MUC16, e.g., elevated MUC16 protein in tumor cells of the cancer Levels and/or nucleic acid levels (e.g., increased expression), e.g., compared to protein levels and/or nucleic acid levels of MUC16 in the same tissue of a healthy subject, or compared to MUC16 in healthy tissues adjacent to a patient's tumor tissue Protein level and/or nucleic acid level.
  • elevated protein levels and/or nucleic acid levels e.g., increased expression
  • MUC16 protein in tumor cells of the cancer
  • nucleic acid levels e.g., increased expression
  • the patient or subject can be a mammal, eg, a primate, preferably a higher primate, eg, a human (eg, a patient suffering from or at risk of developing a cancer described herein).
  • the subject receives or has received other treatments, such as chemotherapy treatment and/or radiation therapy.
  • the anti-MUC16 antibodies disclosed herein do not bind to free circulating CA125 molecules, but only bind to cell membrane-bound MUC16 molecules expressed on the cell surface. It is known that the MUC16 molecule is a molecule specifically expressed by ovarian cancer, so a therapeutically effective amount of anti-MUC16 antibody or anti-CD3 ⁇ MUC16 bispecific antibody can specifically bind to ovarian cancer cells expressing MUC16 molecule, which helps to improve the immune system’s response to The killing of cancer cells, so as to achieve the purpose of treatment.
  • the present invention provides the use of an anti-MUC16 antibody or an anti-CD3 ⁇ MUC16 bispecific antibody in the preparation of a medicament for treating tumors (such as ovarian cancer).
  • the present invention relates to a method for preventing or treating a tumor (such as ovarian cancer) in a subject, the method comprising administering to the subject an effective amount of an anti-MUC16 antibody or an anti-CD3 ⁇ MUC16 bismuth disclosed herein.
  • a tumor such as ovarian cancer
  • Specific antibodies or pharmaceutical compositions comprising same.
  • the anti-GPRC5D antibody or anti-GPRC5D ⁇ CD3 bispecific antibody disclosed herein can be used to treat cancer such as myeloma, colon cancer, rectal cancer or colorectal cancer. Therefore, the present invention provides the use of an anti-MUC16 antibody or an anti-GPRC5D ⁇ CD3 bispecific antibody in the preparation of a medicament for treating cancer such as myeloma, colon cancer, rectal cancer or colorectal cancer.
  • the present invention relates to a method of preventing or treating a tumor (e.g. cancer such as myeloma, colon cancer, rectal cancer or colorectal cancer) in a subject, said method comprising administering to said subject an effective amount of An anti-GPRC5D antibody or an anti-CD3 ⁇ GPRC5D bispecific antibody disclosed herein or a pharmaceutical composition comprising the same.
  • a tumor e.g. cancer such as myeloma, colon cancer, rectal cancer or colorectal cancer
  • a pharmaceutical composition comprising the same.
  • the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention can be administered by any suitable method , including parenteral, intrapulmonary and intranasal administration, and, if required for local treatment, intralesional administration.
  • Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion.
  • Administration may be by any suitable route, for example by injection, eg intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
  • Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention (and immunoconjugates, compositions, pharmaceutical compositions, preparations, combination products, etc.) Dosage (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether administered for prophylactic or therapeutic purposes, previously treatment, the patient's clinical history and response to the antibodies, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient in one treatment or over a series of treatments.
  • the present invention provides the use of the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the present invention or an immunoconjugate or composition or combination product comprising the same in the production or preparation of a drug, so
  • the above-mentioned drugs are used for the purposes described herein, for example, for the prevention or treatment of the related diseases or conditions mentioned herein.
  • the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody can also be combined with one or more Other therapies such as therapeutic modalities and/or other therapeutic agents are administered in combination for the uses described herein, such as for the prevention and/or treatment of the associated diseases or conditions mentioned herein.
  • the treatment modality is, for example, surgery or radiation therapy.
  • the other therapeutic agent is, for example, a chemotherapeutic agent, cytokine, cytotoxic agent, other antibody, small molecule drug, or immunomodulatory agent (eg, immunosuppressant).
  • the present invention also relates to methods for diagnosis and detection (eg for diagnostic or non-diagnostic purposes) of anti-CD3 antibodies or anti-GPRC5D antibodies or MUC16 or bispecific antibodies and methods for diagnosis and detection comprising the same. combination.
  • the anti-GPRC5D antibodies provided herein can be used to detect the presence of GPRC5D in a biological sample.
  • the bispecific antibodies provided herein can be used to detect the presence of CD3 and/or GPRC5D in a biological sample.
  • the anti-MUC16 antibodies provided herein can be used to detect the presence of MUC16 in a biological sample.
  • bispecific antibodies provided herein can be used to detect the presence of CD3 and/or MUC16 in a biological sample.
  • bispecific antibodies provided herein can be used to detect the presence of a first antigen and/or a second antigen in a biological sample. In certain embodiments, bispecific antibodies provided herein can be used to detect the presence of a first antigen and/or a second antigen in a biological sample.
  • the term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg, RT-PCR).
  • the biological sample is a bodily fluid, such as blood, serum or plasma.
  • the method comprises contacting a biological sample with an anti-GPRC5D antibody or bispecific antibody as described herein under conditions that allow it to bind to GPRC5D, and detecting the binding of the anti-GPRC5D antibody or bispecific antibody to GPRC5D. Whether a complex is formed between the antibody and GPRC5D. Complex formation indicates the presence of GPRC5D.
  • the method can be an in vitro or in vivo method.
  • the method comprises contacting a biological sample with an anti-MUC16 antibody or bispecific antibody as described herein under conditions that allow it to bind to MUC16, and detecting the binding of the anti-MUC16 antibody or bispecific antibody to MUC16. Whether a complex is formed between the antibody and MUC16. Complex formation indicates the presence of MUC16.
  • the method can be an in vitro or in vivo method.
  • a labeled anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody is provided.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are detected indirectly, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.
  • the label is, for example, a label such as biotin or hFc.
  • the sample is obtained prior to treatment with an anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the invention. In some embodiments, the sample is obtained prior to other therapy. In some embodiments, the sample is obtained during or after treatment with the other therapy.
  • GPRC5D or MUC16 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.
  • the first antigen and/or the second antigen are detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.
  • the invention relates to the following specific embodiments:
  • a bispecific antibody comprising
  • first antigen-binding region specifically binds a tumor-associated antigen
  • second antigen-binding region specifically binds CD3
  • the second antigen-binding region is a scFv of an anti-CD3 antibody
  • G4S amino acid sequence
  • VH comprised by the scFv comprises 3 complementarity determining regions (HCDRs), HCDR1, HCDR2 and HCDR3, from the heavy chain variable region
  • VL comprised by the scFv comprises 3 a complementarity determining region (LCDR) from the light chain variable region, LCDR1, LCDR2 and LCDR3, where
  • HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 3, and LCDR1, LCDR2 and LCDR3 are selected from the group as shown in SEQ ID NO: 4 the three complementarity-determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown; or
  • HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 6
  • HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 7
  • LCDR1 consists of the amino acid sequence shown in SEQ ID NO : consists of the amino acid sequence shown in 8
  • LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 9
  • LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 10.
  • the VH contains the amino acid sequence shown in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith an amino acid sequence or consisting of said amino acid sequence,
  • VL contains the amino acid sequence shown in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof
  • bispecific antibody of embodiment 4, wherein said Fab comprises CH1, wherein said CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • CH1 comprises (i) comprising at least 85%, 90%, 91%, 92%, 93%, Amino acid sequences that are 94%, 95%, 96%, 97%, 98% or 99% identical or consist of said amino acid sequences; or
  • bispecific antibody according to any one of embodiments 1-6, wherein said bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two constituents of said Fc dimer The Fc regions are the same or different.
  • one or both of the Fc regions comprises a mutation that reduces binding to Fc ⁇ receptors, for example comprising one or more of the L234A/L235A mutation, the D265A mutation or the P329A mutation One, for example comprising L234A/L235A mutation, D265A mutation and P329A mutation.
  • one Fc-region polypeptide comprises the knot mutation T366W and the other Fc-region polypeptide comprises the buckle mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises the knot mutations T366W and Y349C and the other Fc-region polypeptide comprises the buckle mutations T366S, L368A, Y407V and S354C, or
  • one Fc-region polypeptide comprises the knot mutations T366W and S354C, and the other Fc-region polypeptide comprises the buckle mutations T366S, L368A, Y407V and Y349C;
  • the Fc region further comprises a mutation that reduces binding to Fc ⁇ receptors, for example, one or more of L234A/L235A mutation, D265A mutation, or P329A mutation; for example, L234A/L235A mutation, D265A mutation, and P329A mutation.
  • a mutation that reduces binding to Fc ⁇ receptors for example, one or more of L234A/L235A mutation, D265A mutation, or P329A mutation; for example, L234A/L235A mutation, D265A mutation, and P329A mutation.
  • the first Fc region contains a junction mutation, which
  • a) comprises or consists of the amino acid sequence SEQ ID NO: 43 or 89; or
  • SEQ ID NO: 43 or 89 comprising at least 90% identity to SEQ ID NO: 43 or 89, for example 95%, 96%, 97%, 99% or higher identity and comprising L234A/L235A mutations, D265A mutations and P329A mutations and junctions Amino acid sequences of or consisting of mutations (eg S354C and T366W); and/or
  • the second Fc region comprises a button mutation, which
  • a) comprises or consists of the amino acid sequence SEQ ID NO: 30, 42 or 59; or
  • b) comprising at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity to SEQ ID NO: 30, 42 or 59 and comprising L234A/L235A mutations, D265A mutations and P329A mutations and button mutations (eg Y349C, T366S, L368A and Y407V) or consist of the amino acid sequence.
  • bispecific antibody according to any one of embodiments 1-13, which is an IgG-like bispecific antibody comprising a Fab fragment that specifically binds a first antigen, an scFv that specifically binds a second antigen, and Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • the Fc region comprising the buckle mutation constitutes the first heavy chain
  • the C-terminus of CH1 of the Fab fragment is fused to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is fused to CH2 or the hinge region of the Fc region comprising a junction mutation to form a second heavy chain;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • bispecific antibody according to any one of embodiments 1-13, which is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind a first antigen, one scFv that specifically binds a second antigen and Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • the C-terminus of CH1 of the first Fab fragment is fused to the CH2 or hinge region of the Fc region comprising a buckle mutation to form the first heavy chain;
  • the C-terminus of CH1 of the second Fab fragment is fused to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is fused to CH2 or the hinge region of the Fc region comprising the junction mutation, constituting the second heavy chain;
  • VL-CL of the first and second Fab fragments respectively constitute two light chains
  • first Fab and second Fab fragments are the same or different.
  • bispecific antibody according to any one of embodiments 1-13, which is an IgG-like bispecific antibody comprising a Fab fragment that specifically binds a first antigen, an scFv that specifically binds a second antigen and Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • the C-terminus of CH1 of the Fab fragment is fused to CH2 or the hinge region of the Fc region comprising a buckle mutation to form the first heavy chain;
  • the C-terminus of the scFv fragment is fused to the CH2 or hinge region of the Fc region comprising the junction mutation, constituting the second heavy chain;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • bispecific antibody according to any one of embodiments 1-13, which is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind a first antigen, one scFv that specifically binds a second antigen and Fc heterodimer, where
  • the Fab fragment comprises VH-CH1 and VL-CL
  • the scFv comprises VH-linker-VL or VL-linker-VH
  • the C-terminus of CH1 of the first Fab fragment is fused to the N-terminus of the VH of the second Fab fragment, and the C-terminus of CH1 of the second Fab fragment is fused to CH2 or the hinge region of the Fc region comprising a buckle mutation to constitute the first heavy chain;
  • the C-terminus of the scFv fragment is fused to the CH2 or hinge region of the Fc region comprising the junction mutation, constituting the second heavy chain;
  • VL-CL of the first and second Fab fragments respectively constitute two light chains
  • first Fab and second Fab fragments are the same or different.
  • the first antigen is a tumor-associated antigen, eg selected from GPRC5D or MUC16 (eg human GPRC5D or human MUC16).
  • the first antigen is human GPRC5D
  • the first antigen-binding region comprises 3 CDRs, HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH, and 3 CDRs of the light chain variable region VL CDR, LCDR1, LCDR2 and LCDR3, where
  • the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in any one of SEQ ID NO: 62, 38 or 40; and the LCDR1, LCDR2 and LCDR3 are three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 63;
  • the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 72; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in 73; or
  • the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 82; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: The three complementarity determining regions LCDR1, LCDR2 and LCDR3 included in VL shown in 83 .
  • HCDR1 as shown in SEQ ID NO: 64, HCDR2 as shown in SEQ ID NO: 65, 39 or 41, HCDR3 as shown in SEQ ID NO: 66; LCDR1 as shown in SEQ ID NO: 67 , LCDR2 as shown in SEQ ID NO: 68 and LCDR3 as shown in SEQ ID NO: 69;
  • HCDR1 as shown in SEQ ID NO: 74, HCDR2 as shown in SEQ ID NO: 75, HCDR3 as shown in SEQ ID NO: 76; LCDR1 as shown in SEQ ID NO: 77, as shown in SEQ ID LCDR2 shown in NO: 78 and LCDR3 shown in SEQ ID NO: 79; or
  • HCDR1 as shown in SEQ ID NO: 84, HCDR2 as shown in SEQ ID NO: 85, HCDR3 as shown in SEQ ID NO: 86; LCDR1 as shown in SEQ ID NO: 87, as shown in SEQ ID LCDR2 shown in NO:78 and LCDR3 shown in SEQ ID NO:88.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof % or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 63 or has at least 90%, 91%, 92%, 93% thereof %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 72 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof % or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73 or has at least 90%, 91%, 92%, 93% thereof %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 82 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof % or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83 or has at least 90%, 91%, 92%, 93% thereof %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consist of said amino acid sequences; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40 or has at least 90%, 91%, 92%, 93%, 94% thereof %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises SEQ ID NO: 17, 19 or 21 shown
  • the amino acid sequence is or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise the amino acid sequences shown below or consisting of said amino acid sequence:
  • the first antigen is human MUC16
  • the first antigen-binding region comprises 3 CDRs, HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH, and 3 CDRs of the light chain variable region VL CDR, LCDR1, LCDR2 and LCDR3, where
  • the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 92 or 113; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: Three complementarity determining regions LCDR1 , LCDR2 and LCDR contained in VL indicated by 96 , 100 , 102 or 114 .
  • HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94, HCDR3 as shown in SEQ ID NO: 95; LCDR1 as shown in SEQ ID NO: 97, 101 or 103, as shown in SEQ ID NO: LCDR2 as shown in 98 and LCDR3 as SEQ ID NO:99.
  • amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 92 or 113 or consisting of
  • the amino acid sequence consists of; or comprises the amino acid sequence of SEQ ID NO: 92 or 113 or consists of the amino acid sequence.
  • the first antigen binding region comprises a light chain variable region VL
  • said light chain variable region comprises the same sequence as SEQ ID NO: 96, 100, 102 or 114 has or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence; or Comprising or consisting of the amino acid sequence of SEQ ID NO: 96, 100, 102 or 114.
  • the bispecific antibody according to any one of embodiments 25-28, wherein the first antigen binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 92 or 113 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof or an amino acid sequence of 99% identity or consists of said amino acid sequence; and said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 96, 100, 102 or 114 or at least 90%, 91% thereof , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consists of said amino acid sequence.
  • the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises the amino acids shown in SEQ ID NO: 92 sequence or consists of said amino acid sequence, and said VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 96, 100 or 102; or
  • the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 113, and the VL comprises SEQ ID NO: The amino acid sequence shown in 114 or consists of it.
  • An expression vector comprising the nucleic acid molecule according to embodiment 31, preferably, the expression vector is pCDNA, such as pCDNA3.1.
  • a host cell comprising the nucleic acid molecule of embodiment 31 or the expression vector of embodiment 32, preferably, said host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells Or 293T cells or CHO-S cells.
  • a method of preparing the bispecific antibody of any one of embodiments 1-30 comprising culturing the nucleic acid molecule of embodiment 31 or performing A host cell expressing the vector of Scheme 32, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • An immunoconjugate comprising the bispecific antibody of any one of embodiments 1-30.
  • a pharmaceutical composition or medicament or formulation comprising the bispecific antibody of any one of embodiments 1-30, or the immunoconjugate of embodiment 35, and optionally a pharmaceutically acceptable excipient.
  • a pharmaceutical combination comprising the bispecific antibody of any one of embodiments 1-30, or the immunoconjugate of embodiment 35, and one or more other therapeutic agents (e.g. chemotherapeutics, cellular factors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators).
  • therapeutic agents e.g. chemotherapeutics, cellular factors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators.
  • a method of preventing or treating cancer in a subject comprising administering to the subject an effective amount of the bispecific antibody of any one of embodiments 1-30, or the immunoconjugate of embodiment 35, Or the pharmaceutical composition or preparation of embodiment 36; or the pharmaceutical combination product of embodiment 37.
  • the cancer is a solid tumor or a hematological tumor, such as ovarian cancer (such as serous ovarian cancer, such as ovarian serous cystadenocarcinoma, ovarian serous adenocarcinoma, ovarian cancer Carcinoma mucinous adenocarcinoma, endometrioid adenocarcinoma, ovarian cancer (clear cell adenocarcinoma), myeloma, colon cancer, rectal cancer, or colorectal cancer.
  • ovarian cancer such as serous ovarian cancer, such as ovarian serous cystadenocarcinoma, ovarian serous adenocarcinoma, ovarian cancer Carcinoma mucinous adenocarcinoma, endometrioid adenocarcinoma, ovarian cancer (clear cell adenocarcinoma), myeloma, colon cancer, rectal cancer, or colorectal cancer
  • a method of detecting the presence of a first antigen in a biological sample comprising
  • the invention relates to the following specific embodiments:
  • An anti-CD3 antibody or an antigen-binding fragment thereof specifically binding to human CD3, comprising:
  • VH CD3 The heavy chain variable region (VH CD3 ) whose sequence is as described in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% with SEQ ID NO: 3 , 97%, 98%, 99% or more sequence identity and heavy chain variable regions with identical CDRs; and
  • VL CD3 The light chain variable region (VL CD3 ) whose sequence is as described in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of SEQ ID NO: 4 , 97%, 98%, 99% or more sequence identity and light chain variable regions having identical CDRs.
  • anti-CD3 antibody or antigen-binding fragment thereof of embodiment 1 comprising:
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 1 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Heavy chains with 98%, 99% or more sequence identity and identical CDRs; and
  • a light chain (L) whose sequence is as described in SEQ ID NO: 2 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Light chains with 98%, 99% or more sequence identity and identical CDRs.
  • An anti-MUC16 antibody or an antigen-binding fragment thereof that specifically binds to human MUC16 comprising VH and VL, wherein:
  • the VH comprises HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, and the VL comprises HCDR3 as shown in SEQ ID NO: 97 LCDR1 as shown, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99; or
  • the VH comprises HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, and the VL comprises HCDR3 as shown in SEQ ID NO: 101 LCDR1 as shown, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99; or
  • the VH comprises HCDR1 as shown in SEQ ID NO: 93, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, and the VL comprises HCDR3 as shown in SEQ ID NO: 103 LCDR1 as shown, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99; or
  • anti-MUC16 antibody or antigen-binding fragment thereof that specifically binds to human MUC16 comprising:
  • VH whose sequence is as described in SEQ ID NO: 92 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 92 % or more sequence identity and VH with identical CDR; VLs with 95%, 96%, 97%, 98%, 99% or more sequence identity and identical CDRs; or
  • VH whose sequence is as described in SEQ ID NO: 92 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 92 % or more sequence identity and VH with identical CDR; VLs with 95%, 96%, 97%, 98%, 99% or more sequence identity and identical CDRs; or
  • VH whose sequence is as described in SEQ ID NO: 92 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 92 % or more sequence identity and VH with identical CDR; VLs with 95%, 96%, 97%, 98%, 99% or more sequence identity and identical CDRs; or
  • VH whose sequence is as described in SEQ ID NO: 113 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO: 113 VH with % or more sequence identity and identical CDRs; and a VL whose sequence is as set forth in SEQ ID NO: 114 or at least 90%, 91%, 92%, 93%, 94%, VLs of 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs.
  • anti-MUC16 antibody or antigen-binding fragment thereof according to embodiment 3 or 4 comprising:
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 107 light chain (L) or with SEQ ID NO: 107 have at least 90%, 91%, Light chains with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs; or
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 108 light chain (L) or with SEQ ID NO: 108 have at least 90%, 91%, Light chains with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs; or
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 106 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 109 light chain (L) or with SEQ ID NO: 109 have at least 90%, 91%, Light chains with 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs; or
  • the heavy chain (H) whose sequence is shown in SEQ ID NO: 115 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and heavy chain with the same CDR, and sequence as described in SEQ ID NO: 116 light chain (L) or with SEQ ID NO: 116 have at least 90%, 91%, Light chains of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and having identical CDRs.
  • scFv comprises a sequence as shown in SEQ ID NO: 11 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity and sequences having identical CDRs.
  • An anti-CD3 ⁇ MUC16 bispecific antibody comprising:
  • VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-Fc
  • 1 heavy chain 2 with the structure of VH-CH1-scFv-Fc
  • 2 light chains with the structure of VL-CL.
  • the VH-CH1 structures of chains 1 and 2 are paired to form two antigen recognition sites that recognize MUC16 molecules
  • scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains HCDR1 as shown in SEQ ID NO:93 , HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95
  • the VL recognizing the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO: 101, LCDR2 as shown in SEQ ID NO: 98 and LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecules comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises HC
  • VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-Fc
  • 1 heavy chain 2 with the structure of VH-CH1-scFv-Fc
  • 2 light chains with the structure of VL-CL.
  • the VH-CH1 structures of chains 1 and 2 are paired to form two antigen recognition sites that recognize MUC16 molecules
  • scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains HCDR1 as shown in SEQ ID NO:93 , HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95
  • the VL recognizing the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO: 103, LCDR as shown in SEQ ID NO: 98 and 2 LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecule comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises
  • the pairing forms an antigen recognition site for recognizing the MUC16 molecule
  • scFv forms an antigen recognition site for recognizing the CD3 molecule
  • the VH for recognizing the MUC16 molecule contains HCDR1 as shown in SEQ ID NO: 93, and HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO:95
  • the VL recognizing the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO:101, LCDR2 as shown in SEQ ID NO:98 and LCDR3 as shown in SEQ ID NO:99
  • the scFv recognizing CD3 molecule comprises VH CD3 and VL CD3 , wherein said VH CD3 comprises HCDR1 as shown in SEQ
  • the pairing forms an antigen recognition site for recognizing the MUC16 molecule
  • scFv forms an antigen recognition site for recognizing the CD3 molecule
  • the VH for recognizing the MUC16 molecule contains HCDR1 as shown in SEQ ID NO: 93, and HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO:95
  • the VL recognizing the MUC16 molecule comprises LCDR1 as shown in SEQ ID NO:103, LCDR2 as shown in SEQ ID NO:98 and LCDR3 as shown in SEQ ID NO:99
  • the scFv recognizing CD3 molecule comprises VH CD3 and VL CD3 , wherein said VH CD3 comprises HCDR1 as shown in SEQ
  • VH-CH1-VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-VH-CH1-Fc
  • 1 heavy chain 2 with the structure of scFv-Fc 2 light chains with the structure of VL-CL, of which 2 light chains Pair with the two VH-CH1 structures in heavy chain 1 to form two antigen recognition sites that recognize MUC16 molecules
  • the scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains such as SEQ ID NO: 93 HCDR1 as shown, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95, recognize the VL of the MUC16 molecule comprising LCDR1 as shown in SEQ ID NO: 97, as shown in SEQ ID NO: 98 LCDR2 as shown and LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecules comprises VH CD3 and VL CD3
  • VH-CH1-VH-CH1-Fc 1 heavy chain 1 with the structure of VH-CH1-VH-CH1-Fc
  • 1 heavy chain 2 with the structure of scFv-Fc 2 light chains with the structure of VL-CL
  • the 2 light chains are respectively related to the heavy
  • the two VH-CH1 structures in chain 1 are paired to form two antigen recognition sites that recognize MUC16 molecules
  • the scFv forms an antigen recognition site that recognizes CD3 molecules
  • the VH that recognizes MUC16 molecules contains as shown in SEQ ID NO: 93 HCDR1, HCDR2 as shown in SEQ ID NO: 94 and HCDR3 as shown in SEQ ID NO: 95
  • the VL of the MUC16 molecule comprising LCDR1 as shown in SEQ ID NO: 101, as shown in SEQ ID NO: 98 LCDR2 and LCDR3 as shown in SEQ ID NO: 99
  • the scFv recognizing CD3 molecule comprises VH CD3 and
  • VH-CH1-VH-CH1-Fc 1 heavy chain 2 with the structure of scFv-Fc, and 2 light chains with the structure of VL-CL, and the 2 light chains are respectively related to the heavy
  • the two VH-CH1 structures in chain 1 are paired to form two antigen recognition sites that recognize MUC16 molecules, and the scFv forms an antigen recognition site that recognizes CD3 molecules, wherein the VH that recognizes MUC16 molecules contains as shown in SEQ ID NO: 93 HCDR1, HCDR2 as shown in SEQ ID NO:94 and HCDR3 as shown in SEQ ID NO:95, recognizes the VL of the MUC16 molecule comprising LCDR1 as shown in SEQ ID NO:103, as shown in SEQ ID NO:98 LCDR2 and LCDR3 as shown in SEQ ID NO: 99; the scFv recognizing CD3 molecule comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises HCDR
  • VH for recognizing the MUC16 molecule comprises a sequence selected from SEQ ID NO:
  • the sequence of 92 or 113, the VL recognizing the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102 or 114;
  • the scFv recognizing the CD3 molecule comprises VH CD3 and VL CD3 , wherein the VH CD3 comprises SEQ ID NO: 3 and VL CD3 comprises the sequence shown in SEQ ID NO:4.
  • anti-CD3 ⁇ MUC16 bispecific antibody comprising:
  • the anti-CD3 ⁇ MUC16 bispecific antibody according to any one of embodiments 8-11, wherein the Fc region is selected from an Fc region of an antibody of the IgG class, for example from an isotype IgG1, IgG2, IgG3 or IgG4
  • the Fc region preferably the Fc region comprises mutations, such as mutations that increase the stability of the bispecific antibody dimer (eg, a knob-in-hole structure), mutations that reduce or increase effector function.
  • heavy chain 1 with the structure of VH-CH1-VH-CH1-Fc
  • heavy chain 2 with the structure of scFv-Fc
  • 2 light chains with the structure of VL-CL, of which 2 light chains Pair with the two VH-CH1 structures in heavy chain 1 to form two antigen recognition sites that recognize MUC16 molecules, and the scFv forms an antigen recognition site that recognizes CD3 molecules
  • heavy chain 1 contains SEQ ID NO: 119 Sequences
  • the heavy chain 2 comprises the sequence set forth in SEQ ID NO: 117
  • the light chain comprises the sequence set forth in SEQ ID NO: 116.
  • An anti-CD3 antibody or an antigen-binding fragment thereof encoding as described in any one of embodiments 1-2 or 6-7, encoding an anti-MUC16 antibody or an antigen-binding fragment thereof as described in any one of embodiments 3-6
  • a host cell comprising the nucleic acid of embodiment 15 or the vector of embodiment 16.
  • a pharmaceutical composition comprising the anti-CD3 antibody or antigen-binding fragment thereof as described in any one of embodiments 1-2 or 6-7, the anti-CD3 antibody as described in any one of embodiments 3-6 A MUC16 antibody or an antigen-binding fragment thereof, or the anti-CD3 ⁇ MUC16 bispecific antibody according to any one of embodiments 8-13, and a pharmaceutically acceptable carrier.
  • composition according to embodiment 19 which further comprises other therapeutic agents, preferably, said other therapeutic agents are selected from chemotherapeutic agents and cytotoxic agents.
  • the anti-CD3 antibody or antigen-binding fragment thereof according to any one of embodiments 1-2 or 6-7, the anti-MUC16 antibody or antigen-binding fragment thereof according to any one of embodiments 3-6, Or the anti-CD3 ⁇ MUC16 bispecific antibody as described in any one of Embodiments 8-13 or the pharmaceutical composition as described in any one of Embodiments 19-20 is used in the preparation for treatment, prevention and/or diagnosis Use in a medicine for ovarian cancer.
  • a method for treating, preventing and/or diagnosing ovarian cancer comprising combining a therapeutically effective amount or a diagnostically effective amount of the anti-CD3 antibody or its antigen as described in any one of embodiments 1-2 or 6-7 Fragment, anti-MUC16 antibody or antigen-binding fragment thereof as described in any one of embodiments 3-6, or anti-CD3 ⁇ MUC16 bispecific antibody as described in any one of embodiments 8-13, or as described in embodiments
  • the pharmaceutical composition of any one of regimens 19-20 is administered to a subject in need thereof.
  • ovarian cancer is ovarian cancer, such as serous ovarian cancer, such as ovarian serous cystadenocarcinoma, ovarian cancer serous adenocarcinoma, ovarian cancer mucinous Adenocarcinoma, endometrioid adenocarcinoma, ovarian cancer clear cell adenocarcinoma.
  • serous ovarian cancer such as ovarian serous cystadenocarcinoma, ovarian cancer serous adenocarcinoma, ovarian cancer mucinous Adenocarcinoma, endometrioid adenocarcinoma, ovarian cancer clear cell adenocarcinoma.
  • the invention relates to the following specific embodiments:
  • An anti-GPRC5D antibody or an antigen-binding fragment thereof comprising 3 CDRs of the heavy chain variable region VH, HCDR1, HCDR2 and HCDR3, and 3 CDRs of the light chain variable region VL, LCDR1, LCDR2 and LCDR3, wherein
  • the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in any one of SEQ ID NO: 62, 38 or 40; and the LCDR1, LCDR2 and LCDR3 are three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 63;
  • the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 72; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in 73; or
  • the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 82; and the LCDR1, LCDR2 and LCDR3 are as SEQ ID NO: The three complementarity determining regions LCDR1, LCDR2 and LCDR3 included in VL shown in 83 .
  • HCDR1 as shown in SEQ ID NO: 64, HCDR2 as shown in SEQ ID NO: 65, 39 or 41, HCDR3 as shown in SEQ ID NO: 66; LCDR1 as shown in SEQ ID NO: 67 , LCDR2 as shown in SEQ ID NO: 68 and LCDR3 as shown in SEQ ID NO: 69;
  • HCDR1 as shown in SEQ ID NO: 74, HCDR2 as shown in SEQ ID NO: 75, HCDR3 as shown in SEQ ID NO: 76; LCDR1 as shown in SEQ ID NO: 77, as shown in SEQ ID LCDR2 shown in NO: 78 and LCDR3 shown in SEQ ID NO: 79; or
  • HCDR1 as shown in SEQ ID NO: 84, HCDR2 as shown in SEQ ID NO: 85, HCDR3 as shown in SEQ ID NO: 86; LCDR1 as shown in SEQ ID NO: 87, as shown in SEQ ID LCDR2 shown in NO:78 and LCDR3 shown in SEQ ID NO:88.
  • (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof % or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 63 or has at least 90%, 91%, 92%, 93% thereof %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 72 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof % or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 73 or has at least 90%, 91%, 92%, 93% thereof %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consisting of said amino acid sequences;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 82 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% thereof % or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83 or has at least 90%, 91%, 92%, 93% thereof %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences or consist of said amino acid sequences; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40 or has at least 90%, 91%, 92%, 93%, 94% thereof %, 95%, 96%, 97%, 98% or 99% identical amino acid sequence or consists of said amino acid sequence; and said light chain variable region comprises SEQ ID NO: 17, 19 or 21 shown
  • the amino acid sequence is or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • antibody or antigen-binding fragment thereof according to any one of embodiments 1-6, which further comprises a heavy chain constant region HC, for example, said antibody heavy chain constant region HC is the heavy chain of IgG1, IgG2, IgG3 or IgG4 Chain constant region, preferably the heavy chain constant region of IgG1.
  • the heavy chain constant region comprises a mutation that reduces binding to Fc ⁇ receptors, such as a LALA mutation, a D265A mutation and/or a P329A mutation, preferably a LALA mutation and D265A and P329A mutations.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 60, or comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said amino acid sequence; and said light chain comprises the amino acid sequence of SEQ ID NO: 61, or comprises at least 85%, 90% of said amino acid sequence %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consist of said amino acid sequences;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 70, or comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, An amino acid sequence of 97%, 98% or 99% identity, or consists of said amino acid sequence; and said light chain comprises the amino acid sequence of SEQ ID NO: 71, or comprises at least 85%, 90% of said amino acid sequence %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consist of said amino acid sequences;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 80, or comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said amino acid sequence; and said light chain comprises the amino acid sequence of SEQ ID NO: 81, or comprises at least 85%, 90% of said amino acid sequence %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences, or consist of said amino acid sequences;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 14, 22, 24, 26 or 28, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said amino acid sequence
  • said light chain comprises the amino acid shown in SEQ ID NO: 15, 18 or 20 sequence, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or by said amino acid sequence composition;
  • the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 14 or 24, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said amino acid sequence
  • said light chain comprises the amino acid sequence shown in SEQ ID NO: 15, or has at least Amino acid sequences that are 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consist of said amino acid sequences;
  • (vi) comprising the amino acid sequence shown in the following SEQ ID NO: 14 or 26, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of said amino acid sequence, and said light chain comprises the amino acid sequence shown in SEQ ID NO: 18, or has at least 85%, Amino acid sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, or consist of said amino acid sequences;
  • (vii) comprising the amino acid sequence shown in the following SEQ ID NO: 14, 22, 24, 26 or 28, or having at least 85%, 90%, 91%, 92%, 93%, 94%, Amino acid sequence of 95%, 96%, 97%, 98% or 99% identity, or consists of said amino acid sequence, and said light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or with said amino acid sequence Amino acid sequences having or consisting of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • antigen-binding fragment thereof according to any one of embodiments 1-17, wherein said antigen-binding fragment is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single chain antibody (eg scFv), (Fab') 2 , single domain antibody such as VHH, dAb (domain antibody) or linear antibody.
  • the antibody or antigen-binding fragment thereof of embodiment 19, which is a bispecific antibody comprising a first binding specificity for GPRC5D and a second binding specificity for CD3.
  • the bispecific antibody of embodiment 20, comprising a first antigen binding region and a second antigen binding region,
  • the first antigen-binding region specifically binds to GPRC5D, and comprises VH and VL, wherein
  • VH comprises HCDR1, HCDR2, HCDR3 as defined in embodiment 1 or 2 and said VL comprises LCDR1, LCDR2 and LCDR3 as defined in embodiment 1 or 2; or
  • VH and VL are VH and VL as defined in any one of embodiments 3-6;
  • the second antigen binding region specifically binds CD3.
  • said second antigen binding region comprises VH and VL, said VH comprising three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2 and HCDR3, and the VL comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3, wherein
  • HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in any one of SEQ ID NO: 3, and LCDR1, LCDR2 and LCDR3 are selected from such as SEQ ID NO: The three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in any one of 4;
  • HCDR1 consists of the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 consists of the amino acid sequence shown in SEQ ID NO: 6
  • HCDR3 consists of the amino acid sequence shown in SEQ ID NO: 7
  • LCDR1 consists of the amino acid sequence shown in SEQ ID NO : consists of the amino acid sequence shown in 8
  • LCDR2 consists of the amino acid sequence shown in SEQ ID NO: 9
  • LCDR3 consists of the amino acid sequence shown in SEQ ID NO: 10.
  • the VH contains the amino acid sequence shown in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith an amino acid sequence or consisting of said amino acid sequence,
  • VL contains the amino acid sequence shown in SEQ ID NO: 4 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereof
  • bispecific antibody of embodiment 24, said Fab comprising CH1, wherein said CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.
  • CH1 comprises (i) comprising at least 85%, 90%, 91%, 92%, 93%, Amino acid sequences that are 94%, 95%, 96%, 97%, 98% or 99% identical or consist of said amino acid sequences; or
  • bispecific antibody of embodiment 29, wherein one or both of the Fc regions comprises a mutation that reduces binding to Fc ⁇ receptors, for example comprising one or more of the L234A/L235A mutation, the D265A mutation or the P329A mutation One, for example comprising L234A/L235A mutation, D265A mutation and P329A mutation.
  • bispecific antibody of embodiment 30, wherein one or both of said Fc regions comprises or has at least 90% identity, such as 95%, to the amino acid sequence shown in SEQ ID NO: 46 or 49 , 96%, 97%, 99% or more identical amino acid sequences or consist thereof.
  • one Fc-region polypeptide comprises the knot mutation T366W and the other Fc-region polypeptide comprises the buckle mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises the knot mutations T366W and Y349C and the other Fc-region polypeptide comprises the buckle mutations T366S, L368A, Y407V and S354C, or
  • one Fc-region polypeptide comprises the knot mutations T366W and S354C, and the other Fc-region polypeptide comprises the buckle mutations T366S, L368A, Y407V and Y349C;
  • the Fc region further comprises a mutation that reduces binding to Fc ⁇ receptors, for example, one or more of L234A/L235A mutation, D265A mutation, or P329A mutation; for example, L234A/L235A mutation, D265A mutation, and P329A mutation.
  • a mutation that reduces binding to Fc ⁇ receptors for example, one or more of L234A/L235A mutation, D265A mutation, or P329A mutation; for example, L234A/L235A mutation, D265A mutation, and P329A mutation.
  • the first Fc region contains a junction mutation, which
  • a) comprises or consists of the amino acid sequence SEQ ID NO: 43 or 89; or
  • SEQ ID NO: 43 or 89 comprising at least 90% identity to SEQ ID NO: 43 or 89, for example 95%, 96%, 97%, 99% or higher identity and comprising L234A/L235A mutations, D265A mutations and P329A mutations and junctions Amino acid sequences of or consisting of mutations (eg S354C and T366W); and/or
  • the second Fc region comprises a button mutation, which
  • a) comprises or consists of the amino acid sequence SEQ ID NO: 30, 42 or 59; or
  • b) comprising at least 90% identity, such as 95%, 96%, 97%, 99% or higher identity to SEQ ID NO: 30, 42 or 59 and comprising L234A/L235A mutations, D265A mutations and P329A mutations and button mutations (eg Y349C, T366S, L368A and Y407V) or consist of the amino acid sequence.
  • the bispecific antibody according to any one of embodiments 21-35 comprising a first antigen-binding region, a second antigen-binding region and an Fc dimer, wherein the first antigen-binding region is a Fab fragment that specifically binds GPRC5D , the second antigen-binding region is a scFv that specifically binds to CD3.
  • bispecific antibody of embodiment 36 comprising a first antigen-binding domain Fab, a second antigen-binding domain scFv and Fc dimers, wherein said Fab comprises VH-CH1 and VL-CL, said scFv comprising a VH-linker-VL, one Fc region in the Fc dimer comprising a button mutation and the other comprising a knot mutation; wherein
  • the Fc region comprising the buckle mutation constitutes the first heavy chain
  • the C-terminus of CH1 of the Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of the Fc region containing the junction mutation, constituting the second heavy chain ;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 30, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of said amino acid sequence , an amino acid sequence that is 98% or 99% identical, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 31, 34 or 35, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, Amino acid sequences that are 96%, 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the bispecific antibody of embodiment 36 comprising two first antigen binding domains Fab, one second antigen binding domain scFv and an Fc dimer, wherein said Fab comprises VH-CH1 and VL-CL, said The scFv comprises a VH-linker-VL, one Fc region in the Fc dimer comprises a button mutation and the other a knot mutation;
  • the C-terminus of CH1 of the first Fab fragment is fused to the CH2 or hinge region of the Fc region comprising a buckle mutation to form the first heavy chain;
  • the C-terminus of CH1 of the second Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of the Fc region comprising the junction mutation, constituting the second heavy chain ;
  • VL-CL of the first and second Fab fragments respectively constitute two light chains
  • first Fab and second Fab fragments are the same or different.
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 33, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of said amino acid sequence , 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 31, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence Amino acid sequences that are % or 99% identical, or consist of said sequences;
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 32, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of said amino acid sequence , 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 34, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consisting of said sequences; or
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 36, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence , 97%, 98% or 99% identical amino acid sequence, or consists of said sequence;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • the bispecific antibody of claim 36 it comprises a first antigen-binding region Fab, a second antigen-binding region scFv and Fc dimer, wherein said Fab comprises VH-CH1 and VL-CL, said scFv Comprising VH-linker-VL, one Fc region in the Fc dimer contains a button mutation and the other a knot mutation;
  • the C-terminus of CH1 of the Fab fragment is fused to CH2 or the hinge region of the Fc region comprising a buckle mutation to form the first heavy chain;
  • the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of the Fc region comprising the junction mutation, constituting the second heavy chain;
  • the VL-CL of the Fab fragment constitutes the light chain.
  • the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 32, 33 or 36, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence %, 97%, 98% or 99% identical amino acid sequences, or consist of said sequences;
  • the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 37, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences that are 97%, 98% or 99% identical, or consist of said sequences; and/or
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 20, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of said amino acid sequence % or 99% identical amino acid sequences, or consist of said sequences.
  • An expression vector comprising the nucleic acid molecule according to embodiment 43, preferably, the expression vector is pCDNA, such as pCDNA3.1.
  • a host cell comprising the nucleic acid molecule of embodiment 43 or the expression vector of embodiment 44, preferably, said host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells Or 293T cells or CHO-S cells.
  • An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-20 or the bispecific antibody of any one of embodiments 21-42.
  • a pharmaceutical composition or medicament or formulation comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-20 or the bispecific antibody of any one of embodiments 21-42, or The immunoconjugate of embodiment 47 and optionally a pharmaceutically acceptable excipient.
  • a pharmaceutical combination comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1-20 or the bispecific antibody of any one of embodiments 21-42, or the antibody of any one of embodiments 47.
  • Immunoconjugates and one or more additional therapeutic agents (eg, chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).
  • a method of preventing or treating cancer in a subject comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 1-20 or any of embodiments 21-42.
  • a method of detecting the presence of GPRC5D in a biological sample comprising
  • the SP34 antibody is a murine antibody that specifically binds to the CD3 epsilon subunit (CD3 ⁇ ), and it has cross-species activity with humans and monkeys (Pessano, S., et al. (1985). "The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits. "EMBO J 4(2): 337-344.). In this example, antibody SP34 was used as a candidate molecule for the construction of humanization.
  • Mouse anti-SP34 was humanized by CDR grafting technology.
  • the human germline gene sequence IGHV3-73*01 with high homology to the SP34 heavy chain variable region and light chain variable region was respectively obtained and IGLV7-46*01, which were used as the framework of the variable region of the humanized antibody; respectively, the CDRs of the mouse anti-SP34 were grafted into the framework of the corresponding heavy and light chain variable region of the humanized antibody to form humanized anti- CD3 Antibody Variable Region.
  • the sequence of the humanized control antibody comes from the patent US 2016/0075785A1, its heavy chain amino acid sequence is SEQ ID NO: 51, and its light chain is SEQ ID NO: 52.
  • its heavy chain amino acid sequence is SEQ ID NO: 51
  • its light chain is SEQ ID NO: 52.
  • sequence listing please refer to the sequence listing .
  • Table 1 and the amino acid sequence of the variable region of the control antibody Roche-CD3 were sent to General Biology System (Anhui) Co., Ltd. (General Biology System (Anhui) Co., Ltd.) for codon optimization and gene synthesis.
  • the genes encoding the VH region and VL region of the Roche-CD3 antibody were sequentially inserted into the gene encoding the human IgG1 heavy chain constant region (amino acid sequence SEQ ID NO: 45) and the lambda light chain constant region (amino acid sequence SEQ ID NO: 47) encoding Gene expression vector pcDNA3.1(+) to obtain the plasmid expressing the full-length heavy chain hu34H1 (SEQ ID NO: 1) of the anti-CD3 humanized antibody 017-2 and expressing the full-length light chain hu34L2 (SEQ ID NO: 2) Plasmids.
  • the synthesized VH region (hu34H1) and VL region (hu34L2) are connected by (G4S)3 peptide SEQ ID NO: 12 to form a single-chain antibody 23L2 (23L2ScFv) (SEQ ID NO: 11), which is inserted into a human IgG1 heavy chain constant A plasmid expressing DA023AH23L2 (scFv-Fc) (SEQ ID NO: 13) was obtained from the expression vector pcDNA3.1(+) of the gene encoding the Fc region (amino acid sequence SEQ ID NO: 46).
  • the heavy chain constant regions used all contained mutations at the L234A, L235A, D265A and P329A (encoded according to Eu) sites to eliminate effector functions.
  • the single-chain antibody (23L2) is named 23L2ScFv, and its amino acid sequence is shown in SEQ ID NO: 11.
  • the synthetic anti-CD3 single-chain antibody comprising the Fc domain of the constant region is named DA023AH23L2 (VH-connecting peptide-VL-Fc), and its sequence is SEQ ID NO: 13, wherein the amino acid sequence of VH-connecting peptide-VL is SEQ ID NO: 11, the amino acid sequence of the Fc region is SEQ ID NO: 46.
  • the plasmids obtained above were co-transfected into ExpiCHO-S cells to express control antibodies Roche-CD3, 017-2 and DA023AH23L2 using the ExpiCHO TM Expression System (ThermoFisher, Cat#A29133). Cells were cultured for 10-12 days after transfection. When the cell survival rate dropped to 60% to 70%, the supernatant was collected, and the protein expressed in the supernatant was purified using MabSelect Sure protein A affinity chromatography system (GE healthcare). Antibody. The purified antibody was concentrated, sterile filtered, and the purity of the antibody protein was tested by SDS-PAGE and molecular exclusion. The results showed that the purity of the antibody met the requirements and could be used in the next experiment.
  • NFAT-luc luciferase reporter gene system
  • the reporter gene element in NFAT-luc2P was transfected into Jurkat (Cell Bank of Type Culture Collection Committee, Chinese Academy of Sciences) by Gene Pul Ser X Cell TM (BIO-RAD) electroporation , Cat# SCSP-513) cells, and then screened with hygromycin B (Invitrogen, Cat#10687010) to obtain NFAT-luc2P polyclonal cell lines, polyclonal cells obtained stable and high expression NFAT-luc2P monoclonal after limiting dilution Cell line Jurkat/NFAT-luc.
  • test buffer 99% 1640 medium + 1% FBS
  • test buffer 99% 1640 medium + 1% FBS
  • cell density 5 ⁇ 10 5 /mL.
  • Cells were seeded in 384-well plates (Corning, Cat#3570), 40 ⁇ l per well.
  • humanized anti-CD3 antibodies DA023AH23L2 and Roche-CD3 final concentration: initial 20 nM, 3-fold dilution, 8 gradients) were added in serial dilutions, 10 ⁇ l per well. Place the 384-well plate in a 37°C incubator.
  • Human GPRC5D protein (NP_061124.1 (NCBI sequence number), sp
  • CHO-K1 ACTT, Cat#CCL-61
  • HEK293T Cell Bank of Type Culture Collection Committee, Chinese Academy of Sciences, respectively, Cat#SCSP-502
  • the gene sequence of cynomolgus monkey GPRC5D protein (XP_005570249.2 (NCBI sequence number), SEQ ID NO: 58) was codon optimized and gene synthesized by General Biosystems (Anhui) Co., Ltd. Using Lipofectamine TM 2000, the gene sequence encoding cynomolgus monkey GPRC5D protein was transfected into HEK293T cells.
  • the transfected cells were screened with 0.3 ⁇ g/mL puromycin (Gibco, Cat#A1113802) and monoclonal plated to obtain CHO-K1 (CHO-K1-HuGPRC5D) and HEK293T engineered monoclonal cells highly expressing human GPRC5D (HEK293T-HuGPRC5D) and HEK293T polyclonal cells (HEK293T-CynoGPRC5D) highly expressing cynomolgus monkey GPRC5D (Table 2).
  • the amino acid sequences encoding human GPRC5D and cynomolgus monkey GPRC5D proteins are shown in the sequence listing.
  • the protein cell line Promoter resistance CHO-K1-HuGPRC5D cell line HuGPRC5D CHO-K1 CMV Puromycin HEK293T-HuGPRC5D cell line HuGPRC5D HEK293T CMV Puromycin HEK293T-CynoGPRC5D CynoGPRC5D HEK293T CMV Puromycin
  • mice The CHO-K1-HuGPRC5D cell line (Table 2) was used to immunize 5 BALB/c mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.). Mice were injected biweekly with four alternating injections (Table 3). Venous blood was collected from the mice 7 days after the first immunization and 7 days after the second booster immunization, and the mouse immune serum was analyzed by flow cytometry using the HEK293T-HuGPRC5D cell line and the HEK293T-CynoGPRC5D cell line (Table 2). For FACS assays, two mice were selected for animal fusion. Four days after the last booster, the spleens of the mice were harvested, and lymphocytes isolated from the spleens were used for fusion to generate hybridomas.
  • Tissue culture supernatants from 249 hybridomas were tested for primary screening by FACS assay in which 31 clones binding to HEK293T-HuGPRC5D cell line or HEK293T-CynoGPRC5D cell line were selected for the next round of confirmatory testing . Positive cultures were retested with screened antigen to confirm secretion.
  • Selected hybridoma cell lines (parental clones) were subcloned to ensure monoclonality. Subcloning was performed by replating the parental clones using the one-step cloning system. Twenty-four subclones were transferred to 96-well culture plates. The subclones were screened by FACS method, and antibodies binding to HuGPRC5D and CynoGPRC5D were screened.
  • the light chain and heavy chain variable region sequences of the murine anti-GPRC5D antibody were obtained by polymerase chain reaction (PCR) amplification technique.
  • the total RNA of 24 antibodies was isolated with RNAprep pure Cell Kit (TIANGEN, Cat#DP430), and reverse transcribed into cDNA with RevertAid First Strand cDNA Synthesis Kit (Thermo, K1622).
  • the variable regions of the heavy and light chains were cloned by PCR, all PCRs being performed with high-fidelity polymerases.
  • the antibody heavy chain and light chain variable domain sequence fragments amplified by PCR were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing.
  • the amino acid sequences encoding the light chain variable domain and the heavy chain variable domain of the generated antibody were determined, and the Kabat light chain and heavy chain variable region sequences of the antibody are shown in Table 4.
  • Example 4 Characterization of GPRC5D antibodies obtained by hybridoma technology
  • the DNA sequences of the three murine antibodies in Table 4 were codon optimized and gene synthesized by General Biosystems (Anhui) Co., Ltd.
  • the genes encoding the VH region and VL region of the antibody were sequentially inserted into the expression vector pcDNA3.1 ( +) to obtain murine chimeric antibodies.
  • Humanized control antibody GPRC5D (clone number GC5B596, heavy chain amino acid sequence is SEQ ID NO: 55, light chain amino acid sequence is SEQ I DNO: 56) and CD3 (clone number CD3B219, heavy chain amino acid sequence is SEQ ID NO: 53.
  • the amino acid sequence of the light chain is SEQ I DNO: 54)
  • the sequence is from the patent WO2018017786A2.
  • the plasmids encoding the heavy and light chains of the anti-GPRC5D antibody were co-transfected into ExpiCHO-S cells to express the murine chimeric antibody and control antibodies GC5B596 and CD3B219 as described in Table 5, and were purified accordingly.
  • ExpiCHO TM expression system (ThermoFisher, Cat#A29133) was used to co-transfect ExpiCHO-S cells with the plasmids encoding the heavy and light chains of the anti-GPRC5D antibody described in Table 5 to express the anti-GPRC5D chimeric antibody, and similar expression control antibodies GC5B596 and CD3B219. Cells were cultured for 10-12 days after transfection.
  • the cell survival rate dropped to 60% to 70%, the supernatant was collected, and the protein expressed in the supernatant was purified using MabSelect Sure protein A affinity chromatography system (GE healthcare).
  • Antibody The purified antibody was concentrated, sterile filtered, and the purity of the antibody protein was detected by SDS-PAGE and molecular exclusion. The results showed that the purity of the antibody was >90%, which could be used in the next experiment.
  • Anti-GPRC5D chimeric antibody binds to cells expressing human GPRC5D protein or cynomolgus GPRC5D protein
  • the anti-GPRC5D chimeric antibody of the present invention can bind to human GPRC5D protein or cynomolgus GPRC5D protein stably expressed on the cell surface is determined by cell binding experiments.
  • the single cell suspensions of the two cells were obtained, and the medium was discarded after centrifugation at 400 ⁇ g at room temperature.
  • the obtained cell pellet was washed once with PBS and then resuspended with the anti-GPRC5D chimeric antibody shown in Table 5 and the control antibody GC5B596 (initial concentration was 133.3nM, 4-fold equivalent serial dilution, a total of 8 concentrations) diluted serially, at 4°C Incubate for 30 minutes.
  • Antibody 29H12C9 was humanized as described in Example 1.
  • the sequence of 29H12C9 was retrieved and compared in the IMGT database, and the human germline gene sequence IGHV1-46*01 with high homology to the heavy chain variable region of 29H12C9 was respectively obtained as the heavy chain variable region humanization framework.
  • the human germline gene sequence IGKV4-1*01 with high homology to the light chain variable region was used as the humanized framework for the light chain variable region, and the CDRs of the 29H12C9 heavy and light chain variable region were grafted into the corresponding humanized In frame, the variable regions of the humanized 29H12C9 antibody were formed.
  • variable region The amino acid sequence of the variable region was sent to General Biosystems (Anhui) Co., Ltd. for codon optimization and gene synthesis.
  • the genes encoding the VH region and VL region of the antibody were sequentially inserted into the expression vector pcDNA3 containing the gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 48) and the encoding gene for the kappa light chain constant region (SEQ ID NO: 50). 1(+) to obtain a plasmid expressing the full-length heavy chain of the anti-GPRC5D humanized antibody and a plasmid encoding the full-length light chain.
  • Table 6 lists the name, variable region sequence, and heavy and light chain sequences of each antibody.
  • Table 6 Names of humanized antibodies and SEQ ID NOs of corresponding sequences.
  • the 55th and 56th amino acids of the antibody CDR2 can also be mutated to obtain the following antibodies:
  • the combination of plasmids encoding the heavy and light chains (Table 6) was co-transfected into ExpiCHO-S cells as described in Example 4.1 to express the humanized anti-GPRC5D antibody, and was purified accordingly as described in Example 1.1.
  • the purified antibody was concentrated, sterile filtered, and the purity of the antibody was detected by SDS-PAGE and size exclusion chromatography (SEC).
  • the purity of the antibody was confirmed by size exclusion chromatography. Specifically, 20 ⁇ g of antibody samples were injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na 2 SO 4 (pH 7.0) as the running buffer. Run for 30 minutes. The collected effluent was measured by Agilent 1220 HPLC, and the data was analyzed using OpenLAB software. The results showed that the purity of the antibody was >90%, which could be used for the next experiment.
  • the anti-GPRC5D humanized antibody of the present invention can bind to the human GPRC5D protein stably expressed on the cell surface is judged by cell binding experiments.
  • a single cell suspension of HEK293T-huGPRC5D cells was obtained after enzymatic digestion of the HEK293T-HuGPRC5D cell line shown in Table 2, centrifuged at room temperature at 400 ⁇ g, and the culture medium was discarded. The cell pellet was washed once with PBS, and then resuspended with serially diluted anti-GPRC5D humanized antibody and mouse chimeric antibody 29H12C9 (initial concentration was 166.67nM, 4-fold equivalent serial dilution with a total of 8 concentration points), and incubated at 4°C for 30 minute.
  • the present invention constructed three bispecific antibodies with different asymmetric structures as shown in Figure 4, wherein the anti-GPRC5D antigen-binding region of each bispecific antibody was derived from the humanized GPRC5D antibody produced in Example 5 above, and the anti-GPRC5D antibody
  • the antigen-binding region of CD3 is derived from the humanized CD3 antibody produced in Example 1, and the constant region of the bispecific antibody contains a knob-in-hole (KIH) (Merchant, A.M., et al. (1998) .
  • bispecific antibodies are also referred to herein as “anti-CD3/GPRC5D bispecific antibodies” or “anti-CD3/anti-GPRC5D bispecific antibodies”, sometimes referred to simply as “bispecific antibody molecules", “double antibodies” .
  • the anti-GPRC5D portion of the anti-CD3/GPRC5D bispecific antibody targets GPRC5D-expressing cells, while the anti-CD3 portion activates T cells.
  • the double antibody simultaneously binds to GPRC5D on tumor cells and CD3 on T cells, helping to kill tumor cells through activated T cells.
  • the present invention uses standard construction methods to obtain three anti-CD3/GPRC5D bispecific antibodies with the structures shown in Figure 4, and their amino acid sequences are listed in Table 7 below.
  • the heavy chain of the bispecific antibody is named according to the sequence from the N-terminal to the C-terminal, for example, 23L2-Rknob (also known as Hu34Scfv-Rknob) indicates that the sequence includes the ScFv derived from 23L2 from the N-terminal to the C-terminal And the constant region containing the knot mutation; 29H6-Rhole means that the sequence from the N segment to the C-terminus includes the sequence derived from 29H6 and the constant region containing the button mutation.
  • the 55th asparagine (N) or the 56th glycine (G) of the 29H6 heavy chain were mutated to serine (S) or glutamic acid ( E), after the mutation, it is abbreviated as NS or GE (Kabat) in the molecule.
  • Table 7 Construction and sequence of the bispecific antibody molecule of the present invention
  • Each chain of each bispecific antibody molecule was constructed as follows:
  • 23L2-Rknob also known as Hu34Scfv-Rknob: the nucleotide sequence encoding anti-CD3 single-chain antibody 23L2 was cloned into the pcDNA3.1(+) vector with Fc (Knob), and expressed to obtain 23L2-Knob molecule.
  • 29H6-23L2-Rknob Using the humanized anti-GPRC5D antibody heavy chain coding sequence as a template to amplify the nucleotide sequence encoding the heavy chain variable region and the heavy chain constant region CH1 segment, and using overlapping PCR technology to combine the sequence with the coding sequence The complete nucleotide sequence of 23L2-Rknob was directly spliced (with no linking peptide between the two sequences), and the spliced nucleotide sequence was cloned into the expression vector pcDNA3.1(+) vector to obtain 29H6-23L2- Rknob molecules.
  • 29H6NS-23L2-Rknob Using the constructed 29H6-23L2-Knob as a template, the 55th asparagine (N) was mutated to serine (S) by overlapping PCR technique, and cloned into the expression vector pcDNA3.1(+ ) vector, the 29H6NS-23L2-Rknob molecule is obtained by expression.
  • 29H6GE-23L2-Rknob Using the constructed 29H6-23L2-Rknob as a template, the glycine (G) at position 56 was mutated to glutamic acid (E) by overlapping PCR technique, and cloned into the expression vector pcDNA3.1(+ ) vector, the 29H6GE-23L2-Rknob molecule is obtained by expression.
  • 29H6-Rhole The nucleotide sequence encoding the heavy chain variable region and heavy chain constant region CH1 segment of the humanized anti-GPRC5D antibody was cloned into the pcDNA3.1(+) vector with Fc (Hole), and expressed to obtain 29H6 -Rhole molecules.
  • 29H6NS-Rhole Using the constructed 29H6-Rhole as a template, the asparagine (N) at position 55 was mutated to serine (S) by overlapping PCR technology, and cloned into the expression vector pcDNA3.1(+), The expression obtained 29H6NS-Rhole molecule.
  • 29H6GE-Rhole Using the constructed 29H6-Hole as a template, the glycine (G) at position 56 was mutated to glutamic acid (E) by overlapping PCR technology, and cloned into the expression vector pcDNA3.1(+), The expression obtained 29H6GE-Rhole molecule.
  • 29HL3 The nucleotide sequence encoding the light chain of the humanized anti-29H6 antibody was cloned into the pcDNA3.1(+) vector, and the 29HL3 molecule was obtained by expression.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/CN2023/071314 2022-01-10 2023-01-09 一种抗体及其用途 Ceased WO2023131328A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2024541191A JP2025504795A (ja) 2022-01-10 2023-01-09 抗体及びその用途
CN202380016649.XA CN118510810A (zh) 2022-01-10 2023-01-09 一种抗体及其用途
US18/728,022 US20250092135A1 (en) 2022-01-10 2023-01-09 Antibody and use thereof
EP23737190.1A EP4464722A4 (en) 2022-01-10 2023-01-09 ANTIBODIES AND THEIR USE

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CNPCT/CN2022/071080 2022-01-10
CN2022071080 2022-01-10
CN202210352672 2022-04-01
CN202210352672.3 2022-04-01
CN202310005123.3 2023-01-03
CN202310005123 2023-01-03

Publications (2)

Publication Number Publication Date
WO2023131328A1 true WO2023131328A1 (zh) 2023-07-13
WO2023131328A9 WO2023131328A9 (zh) 2024-02-22

Family

ID=87073288

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/071314 Ceased WO2023131328A1 (zh) 2022-01-10 2023-01-09 一种抗体及其用途

Country Status (6)

Country Link
US (1) US20250092135A1 (https=)
EP (1) EP4464722A4 (https=)
JP (1) JP2025504795A (https=)
CN (1) CN118510810A (https=)
TW (1) TW202334234A (https=)
WO (1) WO2023131328A1 (https=)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117024596A (zh) * 2023-08-18 2023-11-10 镜像绮点(上海)细胞技术有限公司 肿瘤原代细胞特异性标记与活体成像
WO2025194478A1 (en) * 2024-03-22 2025-09-25 Biofront Ltd Antibodies binding to cd3 and gprc5d, and uses thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120005019A (zh) * 2023-11-14 2025-05-16 成都优赛诺生物科技有限公司 一种靶向gprc5d的单域抗体、嵌合抗原受体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7429382B2 (en) 2002-10-16 2008-09-30 Corixa Corporation Antibodies that bind cell-associated CA 125/O772P and methods of use thereof
US20160075785A1 (en) 2014-08-04 2016-03-17 Hoffmann-La Roche Inc. Bispecific t cell activating antigen binding molecules
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
US20180112001A1 (en) * 2016-09-23 2018-04-26 Regeneron Pharmaceuticals, Inc. Anti-MUC16 Antibodies, Antibody-Drug Conjugates, and Bispecific Antigen-Binding Molecules that Bind MUC16 and CD3, and Uses Thereof
US20190389966A1 (en) 2018-06-21 2019-12-26 Regeneron Pharmaceuticals, Inc. Methods for Treating Cancer with Bispecific Anti-CD3xMUC16 Antibodies and Anti-PD-1 Antibodies
CN113429482A (zh) * 2014-12-05 2021-09-24 纪念斯隆-凯特琳癌症中心 靶向g-蛋白偶联受体的抗体和使用方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016538275A (ja) * 2013-11-04 2016-12-08 グレンマーク ファーマシューティカルズ， エセ．アー． Ｔ細胞再標的化ヘテロ二量体免疫グロブリン（ｈｅｔｅｒｏ−ｄｉｍｅｒｉｃ ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ）の製造
CN114174338A (zh) * 2019-07-31 2022-03-11 豪夫迈·罗氏有限公司 与gprc5d结合的抗体

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7429382B2 (en) 2002-10-16 2008-09-30 Corixa Corporation Antibodies that bind cell-associated CA 125/O772P and methods of use thereof
US20160075785A1 (en) 2014-08-04 2016-03-17 Hoffmann-La Roche Inc. Bispecific t cell activating antigen binding molecules
CN113429482A (zh) * 2014-12-05 2021-09-24 纪念斯隆-凯特琳癌症中心 靶向g-蛋白偶联受体的抗体和使用方法
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
US20180037651A1 (en) * 2016-07-20 2018-02-08 Janssen Pharmaceutica Nv Anti- gprc5d antibodies, bispecific antigen binding molecules that bind gprc5d and cd3, and uses thereof
US20180112001A1 (en) * 2016-09-23 2018-04-26 Regeneron Pharmaceuticals, Inc. Anti-MUC16 Antibodies, Antibody-Drug Conjugates, and Bispecific Antigen-Binding Molecules that Bind MUC16 and CD3, and Uses Thereof
US20190389966A1 (en) 2018-06-21 2019-12-26 Regeneron Pharmaceuticals, Inc. Methods for Treating Cancer with Bispecific Anti-CD3xMUC16 Antibodies and Anti-PD-1 Antibodies

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
"Fundamental Immunology", 1993, RAVEN PRESS
AITHAL, A ET AL.: "MUC 16 as a novel target for cancer therapy", EXPERT OPIN THER TARGETS, vol. 22, no. 8, 2018, pages 675 - 686
AL-LAZIKANI ET AL.: "Standard conformations for the canonical structures of immunoglobulins", JOURNAL OF MOLECULAR BIOLOGY, vol. 273, 1997, pages 927 - 948, XP004461383, DOI: 10.1006/jmbi.1997.1354
ATAMANIUK, J. ET AL.: "Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma.", EUR J CLIN INVEST, vol. 42, no. 9, 2012, pages 953 - 960, XP055408192, DOI: 10.1111/j.1365-2362.2012.02679.x
BORELLA, F. ET AL.: "Immune Checkpoint Inhibitors in Epithelial Ovarian Cancer: An Overview on Efficacy and Future Perspectives", DIAGNOSTICS (BASEL, vol. 10, 2020, pages 3
BU, X. ET AL.: "Immune Checkpoint Blockade in Breast Cancer Therapy", ADV EXP MED BIOL, vol. 1026, 2017, pages 383 - 402
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 877 - 883
DAS, S. ET AL.: "Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifying Golgi/post-Golgi compartments", SCI REP, vol. 5, 2015, pages 9759
DAS, S.S. K. BATRA: "Understanding the Unique Attributes of MUC16 (CA125): Potential Implications in Targeted Therapy", CANCER RES, vol. 75, no. 22, 2015, pages 4669 - 4674, XP093057260, DOI: 10.1158/0008-5472.CAN-15-1050
EDELMAN, G.M. ET AL., PROC. NATL. ACAD. USA, vol. 63, 1969, pages 78 - 85
ELLERMAN, D.: "Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety", METHODS, vol. 154, 2019, pages 102 - 117, XP055712218, DOI: 10.1016/j.ymeth.2018.10.026
ERIC J. SMITH, SCIENTIFIC REPORTS, vol. 5, pages 17943
GOEBELER, M. E. AND R. C. BARGOU: "T cell-engaging therapies BiTEs and beyond", NAT REV CLIN ONCOL, vol. 17, no. 7, pages 418 - 434, XP037175965, DOI: 10.1038/s41571-020-0347-5
HARGADON, K. M. ET AL.: "Immune checkpoint blockade therapy for cancer: An overview of FDA-approved immune checkpoint inhibitors.", INT IMMUNOPHARMACOL, vol. 62, 2018, pages 29 - 39, XP055637409, DOI: 10.1016/j.intimp.2018.06.001
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
KODAMA, T. ET AL.: "Anti-GPRC5D/CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma.", MOL. CANCER THER., vol. 18, no. 9, 2019, pages 1555 - 1564, XP055743011, DOI: 10.1158/1535-7163.MCT-18-1216
LABRIJN, A. F. ET AL.: "Bispecific antibodies: a mechanistic review of the pipeline", NAT REV DRUG DISCOV, vol. 18, no. 8, 2019, pages 585 - 608
LORUSSO, D. ET AL.: "Emerging role of immune checkpoint inhibitors in the treatment of ovarian cancer.", EXPERT OPIN EMERG DRUGS, vol. 25, no. 4, 2020, pages 445 - 453
MERCHANT, A. M. ET AL.: "An efficient route to human bispecific IgG", NAT BIOTECHNOL, vol. 16, no. 7, 1998, pages 677 - 681, XP002141015, DOI: 10.1038/nbt0798-677
MERCHANT, A. M. ET AL.: "An efficient route to human bispecific IgG.", NAT BIOTECHNOL, vol. 16, no. 7, 1998, pages 677 - 681, XP002141015, DOI: 10.1038/nbt0798-677
PESSANO, S. ET AL.: "The T3/T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits", EMBO J, vol. 4, no. 2, 1985, pages 337 - 344, XP055348187
PICO DE COANA, Y ET AL.: "Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system.", TRENDS MOL MED, vol. 21, no. 8, 2015, pages 482 - 491
PILLARISETTI, K. ET AL.: "A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma.", BLOOD, vol. 135, no. 15, 2020, pages 1232 - 1243
See also references of EP4464722A4
SMITH, E. L. ET AL.: "GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.", SCI TRANSL MED, vol. 11, 2019, pages 485

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117024596A (zh) * 2023-08-18 2023-11-10 镜像绮点(上海)细胞技术有限公司 肿瘤原代细胞特异性标记与活体成像
CN117024596B (zh) * 2023-08-18 2024-02-27 镜像绮点(上海)细胞技术有限公司 肿瘤原代细胞特异性标记与活体成像
WO2025194478A1 (en) * 2024-03-22 2025-09-25 Biofront Ltd Antibodies binding to cd3 and gprc5d, and uses thereof

Also Published As

Publication number Publication date
US20250092135A1 (en) 2025-03-20
EP4464722A4 (en) 2026-03-04
WO2023131328A9 (zh) 2024-02-22
TW202334234A (zh) 2023-09-01
CN118510810A (zh) 2024-08-16
EP4464722A1 (en) 2024-11-20
JP2025504795A (ja) 2025-02-19

Similar Documents

Publication Publication Date Title
TWI830151B (zh) 抗GPRC5DxBCMAxCD3三特異性抗體及其用途
JP7287963B2 (ja) 抗ｔｉｇｉｔ抗体並びに治療剤及び診断剤としてのその使用
JP7554781B2 (ja) 抗ｃｅａｃａｍ５モノクローナル抗体およびその調製方法およびその使用
WO2022161314A1 (zh) 针对cd16a的单域抗体及其用途
JP2024520666A (ja) Ｔ細胞エンゲージャ分子及びその使用
WO2023131328A1 (zh) 一种抗体及其用途
WO2018176992A1 (zh) 新型双特异性抗体及其用途
KR20250027562A (ko) Dll3 및 cd3에 특이적으로 결합하는 항원 결합 분자 및 이의 의약적 용도
TW202313699A (zh) 新型抗ｓｉｒｐａ抗體
CN120958026A (zh) Fap/4-1bb/cd40结合分子及其医药用途
WO2023036215A1 (zh) 双特异性抗原结合分子及其应用
CN116135884A (zh) 抗tigit-抗pd-l1双特异性抗体、其药物组合物及用途
US20230146593A1 (en) Method for treatment and prophylaxis of crs in patients comprising a combination of bispecific antibodies binding to cds x cancer cell and tnf alpha or il-6 inhibitor
WO2024235317A1 (zh) 结合lilrb4和cd3的抗体及其用途
EP4644422A1 (en) Anti-nkg2a antibody and use thereof
TW202432596A (zh) 針對磷脂醯肌醇蛋白聚醣3的雙特異性抗體及其應用
WO2023125941A1 (zh) Tigit单域抗体以及基于其的双特异性抗体
WO2022148413A1 (zh) 特异性结合4-1bb的抗体及其抗原结合片段
CN118846036A (zh) 一种包含特异性结合cd3和muc16的双特异性抗体的药物组合
WO2025092742A1 (zh) 结合bcma的抗体以及包含其的多特异性抗体
WO2025113640A1 (zh) 结合lilrb1/2或pd1-lilrb1/2的抗体及其用途
WO2024094159A1 (zh) 靶向人源ror1的单域抗体
TW202521577A (zh) 抗ｏｘ４０ｌ抗體以及其用途
HK40104756A (zh) 抗tigit抗体及其作为治疗和诊断的用途
CN120659811A (zh) 双特异性抗原结合分子及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23737190

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2024541191

Country of ref document: JP

Ref document number: 202380016649.X

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 2023737190

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023737190

Country of ref document: EP

Effective date: 20240812

WWP Wipo information: published in national office

Ref document number: 18728022

Country of ref document: US