WO2024037627A1 - 一种双特异性抗体及其应用 - Google Patents

一种双特异性抗体及其应用 Download PDF

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Publication number
WO2024037627A1
WO2024037627A1 PCT/CN2023/113765 CN2023113765W WO2024037627A1 WO 2024037627 A1 WO2024037627 A1 WO 2024037627A1 CN 2023113765 W CN2023113765 W CN 2023113765W WO 2024037627 A1 WO2024037627 A1 WO 2024037627A1
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seq
sequence
antigen
antibody
polypeptide chain
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PCT/CN2023/113765
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English (en)
French (fr)
Inventor
周金花
周冲
吴崇兵
姜晓玲
殷刘松
杨化冰
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盛禾(中国)生物制药有限公司
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Publication of WO2024037627A1 publication Critical patent/WO2024037627A1/zh

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a bispecific antibody that combines tumor-associated antigen (TAA) and TNFR2 and its application.
  • TAA tumor-associated antigen
  • Monoclonal antibodies have been widely used to treat a variety of human diseases, including cancer, autoimmune diseases, infectious diseases, and cardiovascular diseases.
  • monoclonal antibodies including murine, fully human, and chimeric antibodies, which have been approved by the FDA for therapeutic use.
  • Most of these antibodies are monospecific antibodies that recognize a single epitope and can be selected to activate or inhibit the activity of the target molecule through this single epitope.
  • trastuzumab one of the best-selling anti-cancer protein therapeutics, blocks the growth of cancer cells by attaching itself to Her2 to prevent the attachment of human epidermal growth factor to Her2.
  • Zizumab also stimulates the body's own immune cells to destroy cancer cells.
  • Bispecific antibody refers to an antibody molecule that can bind two (or more) different antigenic epitopes at the same time. Compared with traditional monoclonal antibodies, bispecific antibodies have a unique mechanism of action: (1) Bispecific antibodies can simultaneously bind to two or more different antigen molecules or different epitopes of the same molecule, and combined drugs often does not have this effect. (2) Mediate cell-cell interactions. Bispecific antibodies can bind to two antigens on effector cells and target cells respectively, build a bridge between effector cells and target cells, and promote cell-cell interactions, such as mediating Guide immune cells to kill tumor cells. Therefore, bispecific antibodies have unique advantages that traditional monoclonal antibodies do not have.
  • Tumor necrosis factor receptor 2 also known as tumor necrosis factor receptor superfamily 1B (TNFRSF1B) and CD120b
  • TNFRSF1B tumor necrosis factor receptor superfamily 1B
  • CD120b CD120b
  • ECD extracellular domain
  • ICD intracellular domain
  • TNFR2 has been shown to enhance activation of effector T cells (Teff) and reduce regulatory T cell (Treg)-mediated suppression.
  • Activation of TNFR2 induces signaling through the mitogen-activated protein kinase (MAPK) signaling pathway, which coordinates the transcription of genes that promote evasion of apoptosis and cell proliferation through TRAF2/3 signaling and NF ⁇ B.
  • TNFR2 can be expressed not only on cancer cells and tumor-infiltrating Tregs, but also on effector Teff cells.
  • agonistic antibodies to TNFR2 can stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFN ⁇ and IL2; antagonistic antibodies to TNFR2 can block TNF-TNFR2 and inhibit Treg proliferation.
  • the present invention aims to develop a bispecific antibody that can not only specifically recognize tumor-related antigens, but also use TNFR2 agonistic antibodies to stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFN ⁇ and IL2. ; Or use TNFR2 antagonist antibodies to block TNF-TNFR2 and inhibit Treg proliferation.
  • the inventor has developed a bispecific antibody with good performance that can bind to tumor-associated antigen (TAA) and TNFR2.
  • the bispecific antibody of the present invention can target tumor-associated antigen (TAA) and can also utilize TNFR2 agonistic antibodies stimulate the proliferation of CD8+ T cells, activate CD8+ T cells, and release anti-tumor factors such as IFN ⁇ and IL2; or use TNFR2 antagonist antibodies to block TNF-TNFR2 and inhibit Treg proliferation.
  • the invention provides a bispecific antibody, the bispecific antibody comprising: (a) a first antibody that specifically binds a first antigen or an antigen-binding fragment thereof; and (b) a third antibody that specifically binds a second antigen.
  • TAA tumor-associated antigen
  • the first antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain; the second antibody or antigen-binding fragment thereof comprises a scFv or VHH.
  • the heavy chain variable region of one heavy chain of the first antibody forms an antigen-binding site with the light chain variable region of one light chain, and the heavy chain variable region of another heavy chain interacts with another light chain.
  • the light chain variable region of the chain forms the antigen-binding site.
  • the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more of the scFvs.
  • the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and a scFv linked to the N-terminus of a heavy chain of the first antibody or an antigen-binding fragment thereof.
  • the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and a scFv linked to the C-terminus of a heavy chain of the first antibody or an antigen-binding fragment thereof.
  • the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and two of the scFvs.
  • two of the scFvs are respectively linked to the N-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.
  • two of the scFvs are respectively linked to the C-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.
  • the bispecific antibody comprises a first antibody that specifically binds a tumor-associated antigen (TAA) and two scFvs that specifically bind TNFR2, and the two scFvs that specifically bind TNFR2 are respectively connected to the first antibody.
  • TAA tumor-associated antigen
  • the bispecific antibody comprises a first antibody that specifically binds a tumor-associated antigen (TAA) and two scFvs that specifically bind TNFR2, and the two scFvs that specifically bind TNFR2 are respectively connected to the first antibody.
  • TAA tumor-associated antigen
  • the C-termini of the two heavy chains of an antibody is respectively connected to the first antibody.
  • the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, for each of the polypeptide chains: (a) the first polypeptide chains each independently comprising the light chain of the first antibody or antigen-binding fragment thereof; and (b) the second polypeptide chain each independently comprising the heavy chain of the first antibody or antigen-binding fragment thereof and the scFv.
  • the two first polypeptide chains of the bispecific antibody are the same or different, and/or the two second polypeptide chains are the same or different.
  • the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one or more of the VHHs.
  • the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and one said VHH, said VHH being connected to the N-terminus of the heavy chain of said first antibody or antigen-binding fragment thereof.
  • the bispecific antibody comprises a first antibody or an antigen-binding fragment thereof and a VHH connected to the C-terminus of the heavy chain of the first antibody or an antigen-binding fragment thereof.
  • the bispecific antibody comprises a first antibody or antigen-binding fragment thereof and two of the VHHs.
  • two of the VHHs are respectively linked to the N-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.
  • two of the VHHs are respectively linked to the C-termini of the two heavy chains of the first antibody or antigen-binding fragment thereof.
  • the bispecific antibody comprises a first antibody that specifically binds a tumor-associated antigen (TAA) and two VHHs that specifically bind TNFR2, and the two VHHs that specifically bind TNFR2 are respectively connected to the first antibody.
  • TAA tumor-associated antigen
  • the bispecific antibody comprises a first antibody that specifically binds a tumor-associated antigen (TAA) and two VHHs that specifically bind TNFR2, and the two VHHs that specifically bind TNFR2 are respectively connected to the first antibody.
  • TAA tumor-associated antigen
  • the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, for each of the polypeptide chains: (a) the first polypeptide chains each independently comprising the light chain of the first antibody or antigen-binding fragment thereof; and (b) the second polypeptide chain each independently comprising the heavy chain of the first antibody or antigen-binding fragment thereof and the VHH.
  • the two first polypeptide chains of the bispecific antibody are the same or different, and/or the two second polypeptide chains are the same or different.
  • the tumor associated antigen is selected from GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2 , AFP, ALK, B7H3, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, ⁇ -catenin ( ⁇ -catenin), brc-ab1, BRCA1, BORIS, CA9, CA125, carbonic anhydrase IX, cysteine Caspase-8 (caspase-8), CALR, CCR5, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA(FOLH1), RAGE protein, cyclin-B1, CYP1B1, EGFR, EG
  • the heavy chain variable region and the light chain variable region of the scFv are linked by linker L1.
  • the scFv is linked to the N-terminus or C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof via linker L2.
  • the VHH is linked to the N-terminus or C-terminus of the heavy chain of the first antibody or antigen-binding fragment thereof via linker L2.
  • the linker L1 and linker L2 are the same or different. In some embodiments, the linker L1 and/or the linker L2 have an amino acid sequence shown as (G4S) x , where x is an integer selected from 1-6; preferably, the linker L1 and/or Linker L2 is (G4S) 2 , (G4S) 3 or (G4S) 4 .
  • the heavy chain of the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a heavy chain constant region
  • the light chain comprises a light chain variable region and a light chain constant region; preferably , the first antibody or its antigen-binding fragment is a full-length antibody.
  • the heavy chain of the first antibody or antigen-binding fragment thereof comprises a first Fc region and a second Fc region.
  • the first Fc region and the second Fc region are the same or different.
  • the first Fc region or second Fc region is selected from IgG, IgA, IgD, IgE, IgM, or variants thereof.
  • the first Fc region or second Fc region is selected from IgG1, IgG2, IgG3, IgG4, or variants thereof.
  • the first Fc region or second Fc region comprises one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.
  • the first antibody or antigen-binding fragment thereof specifically binds to B7H3, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 2, or is the same as SEQ ID NO: :2 has a sequence of at least 80% identity; HCDR2 is as set forth in SEQ ID NO:3, or is a sequence that has at least 80% identity as SEQ ID NO:3; HCDR3 is as set forth in SEQ ID NO:4, or is A sequence having at least 80% identity with SEQ ID NO:4; LCDR1 as set forth in SEQ ID NO:6, or a sequence having at least 80% identity with SEQ ID NO:6; LCDR2 as set forth in SEQ ID NO:7 is shown, or is a sequence with at least 80% identity with SEQ ID NO:7; LCDR3 is as shown in SEQ ID NO:8, or is a sequence with at least 80% identity with SEQ ID NO:8.
  • the first antibody or antigen-binding fragment thereof specifically binds to CD24, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 10, or is the same as SEQ ID NO.
  • :10 has a sequence of at least 80% identity
  • HCDR2 is as shown in SEQ ID NO:11, or is a sequence that has at least 80% identity as SEQ ID NO:11
  • HCDR3 is as shown in SEQ ID NO:12, or is A sequence that is at least 80% identical to SEQ ID NO:12
  • LCDR1 is as set forth in SEQ ID NO:14, or is a sequence that is at least 80% identical to SEQ ID NO:14
  • LCDR2 is as set forth in SEQ ID NO:15 is shown, or is a sequence having at least 80% identity with SEQ ID NO: 15
  • LCDR3 is as shown in SEQ ID NO: 16, or is a sequence having at least 80% identity with SEQ ID NO: 16.
  • the first antibody or antigen-binding fragment thereof specifically binds to Claudin 18.2, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 18, or is the same as SEQ ID NO: 18 ID NO:18 is a sequence with at least 80% identity; HCDR2 is as set forth in SEQ ID NO:19, or is a sequence with at least 80% identity as SEQ ID NO:19; HCDR3 is as set forth in SEQ ID NO:20, Or a sequence having at least 80% identity with SEQ ID NO:20; LCDR1 as shown in SEQ ID NO:22, or a sequence with at least 80% identity as SEQ ID NO:22; LCDR2 as SEQ ID NO: 23, or a sequence with at least 80% identity with SEQ ID NO:23; LCDR3 is as shown in SEQ ID NO:24, or a sequence with at least 80% identity with SEQ ID NO:24.
  • the first antibody or antigen-binding fragment thereof specifically binds to Claudin18.2, wherein the HCDR1 of the first antibody or antigen-binding fragment thereof is as set forth in SEQ ID NO: 26, or is the same as SEQ ID NO: 26.
  • ID NO:26 has a sequence with at least 80% identity
  • HCDR2 is as shown in SEQ ID NO:27, or is a sequence with at least 80% identity with SEQ ID NO:27
  • HCDR3 is as shown in SEQ ID NO:28, Or it is a sequence with at least 80% identity with SEQ ID NO:28
  • LCDR1 is as shown in SEQ ID NO:30, or it is a sequence with at least 80% identity with SEQ ID NO:30
  • LCDR2 is as shown in SEQ ID NO: 31, or a sequence having at least 80% identity with SEQ ID NO:31
  • LCDR3 is as SEQ ID NO:32, or a sequence having at least 80% identity with SEQ ID NO:32.
  • the scFv specifically binds TNFR2, and the HCDR1 of the scFv is as set forth in SEQ ID NO: 34, or is a sequence with at least 80% identity to SEQ ID NO: 34; HCDR2 is as set forth in SEQ ID NO: 34 :35, or a sequence having at least 80% identity with SEQ ID NO:35; HCDR3 is as set forth in SEQ ID NO:36, or a sequence with at least 80% identity with SEQ ID NO:36; and , LCDR1 is as shown in SEQ ID NO:38, or is a sequence that has at least 80% identity with SEQ ID NO:38; LCDR2 is as shown in SEQ ID NO:39, or is a sequence that has at least 80% identity with SEQ ID NO:39 A sequence of identity; LCDR3 is as set forth in SEQ ID NO:40, or is a sequence that is at least 80% identical to SEQ ID NO:40.
  • the VHH specifically binds TNFR2, and the HCDR1 of the VHH is as set forth in SEQ ID NO: 42, or is a sequence with at least 80% identity to SEQ ID NO: 42; HCDR2 is as shown in SEQ ID NO: 42 :43, or a sequence having at least 80% identity with SEQ ID NO:43; HCDR3 is as shown in SEQ ID NO:44, or a sequence with at least 80% identity with SEQ ID NO:44.
  • the first antibody or antigen-binding fragment thereof specifically binds B7H3, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 1, or Is a sequence that has at least 80% identity with SEQ ID NO:1; the light chain variable region VL is as shown in SEQ ID NO:5, or is a sequence that has at least 80% identity with SEQ ID NO:5.
  • the first antibody or antigen-binding fragment thereof specifically binds CD24, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 9, or Is a sequence that has at least 80% identity with SEQ ID NO:9; the light chain variable region VL is as shown in SEQ ID NO:13, or is a sequence that has at least 80% identity with SEQ ID NO:13.
  • the first antibody or antigen-binding fragment thereof specifically binds to Claudin 18.2, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 17 , or a sequence with at least 80% identity with SEQ ID NO:17; the light chain variable region VL is as shown in SEQ ID NO:21, or a sequence with at least 80% identity with SEQ ID NO:21.
  • the first antibody or antigen-binding fragment thereof specifically binds to Claudin 18.2, wherein the heavy chain variable region VH of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 25 , or a sequence with at least 80% identity with SEQ ID NO:25; the light chain variable region VL is as shown in SEQ ID NO:29, or a sequence with at least 80% identity with SEQ ID NO:29.
  • the scFv specifically binds TNFR2, and the scFv heavy chain variable region VH is as shown in SEQ ID NO: 33, or is a sequence with at least 80% identity to SEQ ID NO: 33; light
  • the chain variable region VL is as set forth in SEQ ID NO:37, or is a sequence having at least 80% identity with SEQ ID NO:37.
  • the VHH specifically binds TNFR2, and the VHH heavy chain variable region VH is set forth in SEQ ID NO: 41, or is a sequence that is at least 80% identical to SEQ ID NO: 41.
  • the first polypeptide chain of the bispecific antibody is selected from any amino acid sequence of SEQ ID NO:46-50, 59-63, 71-76, or is the same as SEQ ID NO:46 - any amino acid sequence of 50, 59-63, 71-76 has an amino acid sequence with at least 80% identity;
  • the second polypeptide chain of the bispecific antibody is selected from SEQ ID NO: 51-58, 64-70 , any amino acid sequence of 77-82, or an amino acid sequence having at least 80% identity with any amino acid sequence of SEQ ID NO: 51-58, 64-70, 77-82.
  • the bispecific antibody comprises:
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antibody according to any one of the above.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding the first polypeptide chain of the bispecific antibody of any one of the above.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding the second polypeptide chain of the bispecific antibody of any one of the above.
  • the present invention provides a multifunctional fusion protein comprising the bispecific antibody described in any one of the above.
  • the multifunctional fusion protein further comprises one or more third antibodies or antigen-binding portions thereof that specifically bind to other antigens.
  • the antigen that binds the third antibody or antigen-binding portion thereof is selected from a tumor associated antigen (TAA) or an immune checkpoint.
  • TAA tumor associated antigen
  • the antigen that binds the third antibody or antigen-binding portion thereof is selected from the group consisting of GPC3, CD19, CD20 (MS4A1), CD22, CD24, CD30, CD33, CD38, CD40, CD123, CD133, CD138, CDK4, CEA, Claudin18.2, AFP, ALK, BAGE protein, BCMA, BIRC5 (survivin), BIRC7, ⁇ -catenin ( ⁇ -catenin), brc-ab1, BRCA1, BORIS, CA9, CA125, carbonic anhydrase IX, Caspase-8 (caspase-8), CALR, CCR5, NA17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA -1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, cyclin-B1, CYP1B1,
  • the multifunctional fusion protein further comprises a cytokine.
  • the cytokine is selected from IL-1, IL-2, IL-2R ⁇ , IL-2R ⁇ , IL-3, IL-3R ⁇ , IL-4, IL-4R ⁇ , IL-5, IL- 5R ⁇ , IL-6, IL-6R ⁇ , IL-7, IL-7R ⁇ , IL-8, IL-9, IL-9R ⁇ , IL-10, IL-10R1, IL-10R2, IL-11, IL-11R ⁇ , IL-12, IL-12R ⁇ , IL-12R ⁇ 2, IL-12R ⁇ 1, IL-13, IL-13R ⁇ , IL-13R ⁇ 2, IL-14, IL-15, IL-15R ⁇ sushi, IL-16, IL-17, IL- 18.
  • IL-19 IL-20, IL-20R1, IL-20R2, IL-21, IL-21R ⁇ , IL-22, IL-23, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFN ⁇ , IFN ⁇ or GM-CSF.
  • the cancer is selected from human brain astroblastoma, human pharyngeal cancer, adrenal tumors, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer , brain and spinal cord cancer, metastatic brain tumors, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, renal chromophobe cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, desmoplastic Small round cell tumor, ependymoma, Ewing tumor, extraskeletal myxoid chondrosarcoma, osteofibrous dysplasia, osteofibrous dysplasia, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck
  • the present invention also provides the use of the bispecific antibody described in any one of the above or the multifunctional fusion protein described in any of the above in the preparation of drugs for the treatment of autoimmune diseases.
  • the autoimmune disease is selected from the group consisting of graft versus host disease, rheumatoid arthritis, Crohn's disease, multiple sclerosis, colitis, psoriasis, autoimmune uveitis, pemphigus Sores, epidermolysis bullosa, or type 1 diabetes.
  • the use is achieved by one or more of tumor immunotherapy, cell therapy, or gene therapy.
  • the present invention also provides a pharmaceutical composition, which contains the bispecific antibody described in any one of the above and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also provides a pharmaceutical composition, which contains the multifunctional fusion protein described in any one of the above and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also provides an antibody-drug conjugate, which contains the bispecific antibody described in any one of the above.
  • the conjugated drug is selected from the group consisting of cytotoxins, small molecule chemicals, or immunotoxins.
  • VH Antibody heavy chain variable region
  • VL Antibody light chain variable region
  • CDR Complementarity determining region in the immunoglobulin variable region
  • FR Antibody framework region, that is, the antibody variable region except CDR residues Amino acid residue
  • IgG Immunoglobulin G.
  • antibody refers to a natural immunoglobulin or an immunoglobulin prepared by partial or complete synthesis.
  • the antibody can be isolated from natural resources such as plasma or serum in which the antibody naturally occurs, or from the culture supernatant of hybridoma cells that produce the antibody, from animal immune serum, or from phage library screening. Alternatively, it may be partially or completely synthesized by using techniques such as genetic recombination.
  • Preferred antibodies include, for example, antibodies of immunoglobulin isotypes or subclasses of these isotypes.
  • Human immunoglobulins are known to include nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM.
  • isotypes the antibodies of the invention may include IgGl, IgG2, IgG3 and/or IgG4.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • a chimeric antibody you must first establish a hybridoma that secretes mouse-derived specific monoclonal antibodies, then clone the variable region gene from mouse hybridoma cells, and then clone the constant region gene of the human antibody as needed, and combine the mouse variable region
  • the gene is connected to the human constant region gene to form a chimeric base It is then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • partial antibodies are immunoglobulin molecules composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC).
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only the light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • bispecific antibody refers to a protein molecule capable of specifically binding to two target antigens or target antigen epitopes.
  • bispecific antigen-binding proteins including antibodies or antigen-binding fragments (such as Fab, scFv, etc.), “bispecific antibodies” and “bisantibodies” can be used interchangeably.
  • antigen-binding fragment of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen that the full-length antibody binds and/or competes with the full-length antibody for the antigen. Specific binding, which is also known as the "antigen-binding moiety".
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabody), linear antibody (linear antibody), Nanobody (such as technology from Ablynx), domain antibody (such as technology from Domantis), and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide .
  • CDR complementarity determining region
  • antibody drug conjugate refers to a binding protein (such as an antibody or antigen-binding fragment thereof) linked to one or more conjugated drugs (which may optionally be therapeutic or cytotoxic agents), Its structure usually consists of three parts: an antibody or antibody-based ligand, a drug part, and a linker that couples the antibody or antibody-based ligand to the drug.
  • ADCs typically have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drugs coupled to antibodies.
  • polypeptide refers to an amino acid chain of any length, regardless of modification (eg, phosphorylation or glycosylation).
  • polypeptide includes proteins and fragments thereof.
  • Polypeptides may be "exogenous,” meaning that they are “heterologous,” ie, foreign to the host cell utilized, such as a human polypeptide produced by a bacterial cell.
  • Polypeptides are disclosed herein as sequences of amino acid residues. Those sequences are written from left to right in amino terminus to carboxyl terminus direction. According to standard nomenclature, amino acid residue sequences are named by three-letter or single-letter codes.
  • scFv refers to a molecule comprising an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) joined by a linker.
  • VH antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeats of the GGGGS amino acid sequence or variants thereof, for example using 1 to 6 repeats of the GGGGS amino acid sequence or variants thereof.
  • VHH refers to a single antigen-binding polypeptide containing only one heavy chain variable domain (VHH), which is derived from the variable domain of a heavy chain molecule that naturally does not contain a light chain to distinguish it from four-chain immunoglobulins. Regular VH.
  • VHH molecules may be derived from antibodies produced in Camelidae species such as camels, llamas, vicu ⁇ as, dromedaries, alpacas and guanacos. Other species outside of the family Camelidae may produce heavy chain molecules that naturally lack light chains, and such VHHs are within the scope of this application.
  • host cell refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expressing a selected gene of interest.
  • the term includes parental cells The offspring, regardless of whether the offspring is identical in morphology or genetic composition to the original parent cells, as long as the offspring has the selected target gene.
  • Commonly used host cells include bacteria, yeast, mammalian cells, etc.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid operably linked thereto and are referred to herein as "expression vectors.”
  • pharmaceutically acceptable carrier includes any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water and emulsions, as well as various types of wetting agents and the like.
  • identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a control polypeptide sequence after aligning the sequences and introducing gaps where necessary to obtain maximum percent sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software, such as BLAST software or the FASTA package.
  • the term "at least 80% identity” means that the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the control polypeptide sequence is more than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
  • the term "specific" means that one of the molecules involved in specific binding does not show any significant binding to molecules other than one or more of the binding partner molecules. Additionally, the term is used when the domain containing the variable region of an antibody is specific for a particular epitope among multiple epitopes in the antigen. When the epitope bound by the antibody variable region-containing domain is comprised in several different antigens, the antigen-binding molecule comprising the antibody variable region-containing domain can bind to various antigens having the epitope.
  • tumor-associated antigen refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed entirely or as fragments on the surface of cancerous cells and which can be used to preferentially target Pharmacological agents target cancerous cells.
  • epitope refers to an antigenic determinant in an antigen, and refers to an antigenic site to which a domain of an antigen-binding molecule including an antibody variable region disclosed in this specification binds. Therefore, epitopes can be defined based on their structure. In addition, the epitope can also be defined based on the antigen-binding activity of the antigen-binding molecule that recognizes the epitope.
  • the antigen is a peptide or polypeptide
  • the epitope can be specified by the amino acid residues forming the epitope; when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.
  • positive control refers to natural or engineered cells or antibodies that can bind to or express the target protein.
  • the positive control referred to herein refers to a single-target positive control.
  • control refers to the use of the same species source, the same subtype, the same dose, the same immunoglobulin and subtype immunoglobulin, the same label, etc. as the experimental sample in the same experiment to eliminate the
  • the experimental background impact of non-specific binding samples on experimental values serves as a control to further illustrate the experimental effect.
  • Treg Treg cells
  • Regulatory T cells are sometimes referred to as suppressor T cells and are characterized by expression of the biomarkers CD4, FOXP3 and CD25, representing the regulation of the immune system and maintenance of tolerance to self-antigens T cell subsets that protect against autoimmune diseases.
  • Tregs are immunosuppressive and generally inhibit or downregulate the induction and proliferation of effector T (Teff) cells.
  • Tregs can develop in the thymus (so-called CD4+Foxp3+ "native" Tregs) or differentiate from naive CD4+ T cells in the periphery, for example, following exposure to TGF ⁇ or retinoic acid.
  • Teff Teff cell
  • effector T cell is a cell formed by proliferation and differentiation of T cells after receiving antigen stimulation. Effector T cells have the function of releasing lymphokines. In the process, a small number of T cells become memory T cells. Effector T cells contact target cells and trigger granule exocytosis. The released perforin forms small pores on the surface of target cells through polymerization, thereby mediating the killing effect. The target cell death process is similar to apoptosis. At the same time, effector T cells can also release immune active substances - lymphokines, such as interleukins, interferons, etc.
  • immune active substances - lymphokines such as interleukins, interferons, etc.
  • HCDR1 shown in SEQ ID NO: 2, 10, 18, 34, and 42 is divided based on the first cysteine after the heavy chain variable region. Starting from the fourth digit, the length of HCDR1 is generally 10-12, ending with the amino acid before tryptophan.
  • Figure 1 depicts an exemplary bispecific antibody, which includes a full-length antibody that specifically recognizes a tumor-associated antigen (TAA) and an scFv that specifically recognizes TNFR2, the scFv being connected to two ends of the full-length antibody through a linker. The C-terminus of the heavy chain.
  • TAA tumor-associated antigen
  • Figure 2 depicts an exemplary bispecific antibody, which includes a full-length antibody that specifically recognizes a tumor-associated antigen (TAA) and an scFv that specifically recognizes TNFR2, the scFv being connected to two ends of the full-length antibody through a linker. N-terminus of the heavy chain.
  • TAA tumor-associated antigen
  • Figure 3 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, the VHH being connected to two ends of the full-length antibody via a linker. The C-terminus of the heavy chain.
  • TAA tumor-associated antigen
  • Figure 4 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, the VHH being connected to two ends of the full-length antibody via a linker. N-terminus of the heavy chain.
  • TAA tumor-associated antigen
  • Figure 5 depicts an exemplary bispecific antibody, which includes a full-length antibody that specifically recognizes a tumor-associated antigen (TAA) and an scFv that specifically recognizes TNFR2, the scFv being connected to two ends of the full-length antibody through a linker. C-terminus of the light chain.
  • TAA tumor-associated antigen
  • Figure 6 depicts an exemplary bispecific antibody, which includes a full-length antibody that specifically recognizes a tumor-associated antigen (TAA) and an scFv that specifically recognizes TNFR2, the scFv being connected to two ends of the full-length antibody through a linker. N-terminus of the light chain.
  • TAA tumor-associated antigen
  • Figure 7 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, the VHH being connected to two ends of the full-length antibody via a linker. C-terminus of the light chain.
  • TAA tumor-associated antigen
  • Figure 8 depicts an exemplary bispecific antibody comprising a full-length antibody capable of specifically recognizing a tumor-associated antigen (TAA) and a VHH capable of specifically recognizing TNFR2, the VHH being connected to two ends of the full-length antibody via a linker. N-terminus of the light chain.
  • TAA tumor-associated antigen
  • Figure 9 shows the binding activity of antibodies TB-A1, TB-E, TB-B, and TB-F to TNFR2 protein.
  • Figure 10 shows the binding activity of antibodies TB-G, TB-D1, and TB-H to TNFR2 protein.
  • Figure 11 shows the binding activity of antibody TB-A2 to TNFR2 protein.
  • Figure 12 shows the binding activity of antibodies TB-C1, TB-C2, and TB-D2 to TNFR2 protein.
  • Figure 13 shows the binding activity of antibodies TB-A1, TB-E, TB-B, and TB-F to B7H3 protein.
  • Figure 14 shows the binding activity of antibodies TB-G, TB-D1, and TB-H to B7H3 protein.
  • Figure 15 shows the binding activity of antibodies TB-A2 and TB-C1 to B7H3 protein.
  • Figure 16 shows the binding activity of antibodies TB-C2 and TB-D2 to B7H3 protein.
  • Figure 17 shows the binding activities of both ends of antibodies TB-A1, TB-E, TB-B, and TB-F.
  • Figure 18 shows the binding activities of antibodies TB-G, TB-D1, and TB-H at both ends.
  • Figure 19 shows the binding activity of antibody TB-C1 at both ends.
  • Figure 20 shows the binding activities of antibodies TB-A1, TB-D1, and TB-C1 at both ends.
  • Figure 21 shows the anti-tumor growth curves of antibodies TB-C1 and TB-C2.
  • Figure 22 shows the binding activity of antibodies TD-A1, TD-E, TD-B, and TD-F to TNFR2 protein.
  • Figure 23 shows the binding activity of antibodies TD-C, TD-G, TD-D1, and TD-H to TNFR2 protein.
  • Figure 24 shows the binding activity of antibodies TD-A1 and TD-A2 to TNFR2 protein.
  • Figure 25 shows the binding activity of antibodies TD-D1 and TD-D2 to TNFR2 protein.
  • Figure 26 shows the ADCC activities of antibodies TD-A1 and TD-D1.
  • bispecific antibodies were constructed based on the 8 structures in Figure 1-8, named TB-A to TB-H in sequence; for TNFR2 and CD24, bispecific antibodies were constructed based on the 8 structures in Figure 1-8 specific antibodies, named TD-A to TD-H in sequence; against TNFR2 and Claudin18.2, bispecific antibodies were constructed according to the four structures in Figures 1, 2, 5, and 6, named TC-A, TC- B, TC-E and TC-F.
  • the bispecific antibody with the partial structure of this patent simultaneously constructs IgG1 subtype and IgG4 subtype.
  • Antibody TB -A1 is an antibody of IgG1 subtype directed against TNFR2 and B7H3 targets and constructed according to the structure of Figure 1.
  • Antibody TB-A2 is an antibody of IgG4 subtype directed against TNFR2 and B7H3 targets and constructed according to the structure of Figure 1.
  • the linker used in the bispecific antibody has 2 GGGGS repeats (i.e., GGGSGGGGS, hereinafter abbreviated as (G4S) 2 ) or 3 GGGGS repeats (ie, GGGGSGGGGSGGGGS, hereinafter abbreviated as (G4S) 3 ).
  • the CL used in this example is kappa ( ⁇ ) type, and the sequence is shown in SEQ ID NO: 45.
  • Each chain of the bispecific antibodies with different structures designed above was gene synthesized, and then molecular cloning technology was used to insert the antibody fragment into the PCDNA3.1 vector to construct a mammalian cell expression plasmid, and liposome transfection was used. Introduce the host cell strain CHO cells, use cell Fed-batch to obtain the fermentation supernatant, take the fermentation broth supernatant and perform a series of purification steps such as affinity chromatography and ion exchange chromatography, and finally purify the constructed antibody. The purified antibodies were tested for expression level, purity, SDS-PAGE, etc. to confirm the bispecific antibodies.
  • Dilute human-TNFR2-His (manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) to 0.3 ⁇ g/mL with coating solution (1 ⁇ PBS, pH7.4), and coat it into a 96-well enzyme plate. , 100 ⁇ L/well, overnight at 4°C. Pour off the coating solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 4 times with a plate washer, and pat dry on flat paper. Block with 3% skimmed milk powder, 300 ⁇ L/well, and incubate at 37°C for 1 hour. Pour off the blocking solution, wash 4 times with a plate washer, and pat dry on a flat paper.
  • the negative control is commercially available trastuzumab for injection (Herceptin).
  • the antibody sequence variable region of the positive control 1 consists of SEQ ID NO:83 and SEQ ID NO:84.
  • the antibody sequence variable region of positive control 2 consists of SEQ ID NO:41.
  • the positive control and antibody were diluted to 10 ⁇ g/mL with 3% skimmed milk powder, and diluted 3 times using this as the initial concentration. A total of 11 gradients were diluted. Another blank well was set and only the diluent was added. 100 ⁇ L/well, incubate at 37°C for 1 hour.
  • the negative control is commercially available trastuzumab for injection (Herceptin).
  • the antibody sequence variable region of the positive control consists of SEQ ID NO: 1 and SEQ ID NO: 5.
  • Added Constant region of human IgGl see SEQ ID NO:85 and SEQ ID NO:86).
  • Dilute human-B7H3Fc (Manufacturer: Acro, CAT: B73-H5253, LOT: 2053b-201XF1-UD) to 0.5 ⁇ g/mL with coating solution (1 ⁇ PBS, pH7.4), and coat it on the 96-well enzyme label plate, 100 ⁇ L/well, and incubate at 4°C overnight.
  • coating solution (1 ⁇ PBS, pH7.4)
  • Coffee off the coating solution wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 4 times with a plate washer, and pat dry on flat paper.
  • Block with 3% skimmed milk powder 300 ⁇ L/well, and incubate at 37°C for 1 hour.
  • Pour off the blocking solution wash 4 times with a plate washer, and pat dry on a flat paper.
  • a negative control was set up, and the negative control was commercially available trastuzumab for injection (Herceptin). Starting from 300nM, use this as the initial concentration to dilute 3 times, diluting a total of 8 gradients, setting up another blank well, and adding only diluent. 100 ⁇ L/well, incubate at 37°C for 1 hour. Discard the liquid in the wells, wash 4 times with a plate washer, and pat dry on flat paper.
  • Dilute human-TNFR2-His (Manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) to 0.5 ⁇ g/mL, add 100 ⁇ L to each well, incubate at room temperature for 1 hour, then wash the plate 3 times with PBST, and then add HRP-labeled His antibody was diluted 1:10000 with sample diluent, 100 ⁇ L was added to each well, and incubated at room temperature for 1 hour. Wash in plate washer 6 times and pat dry on flat paper. Add TMB chromogenic solution, 100 ⁇ L/well, wrap it in aluminum foil, and develop color for 8 minutes at 37°C in the dark.
  • Use coating solution (1 ⁇ PBS, pH7.4) to dilute human-TNFR2mFc (manufacturer: Kaika, CAT: TNF-HM3R2, LOT: 031202) to 0.5 ⁇ g/mL, and coat it into a 96-well enzyme plate. 100 ⁇ L/well, 4°C overnight. Pour off the coating solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 4 times with a plate washer, and pat dry on flat paper. Block with 3% skimmed milk powder, 300 ⁇ L/well, and incubate at 37°C for 1 hour. Pour off the blocking solution, wash 4 times with a plate washer, and pat dry on a flat paper.
  • Dilute human-B7H3His (manufacturer: Acro, CAT: TB73-H52E2, LOT: 2052-21AXF1-YS) protein to 0.5 ⁇ g/mL, add 100 ⁇ L to each well, incubate at room temperature for 1 hour, then wash the plate 3 times with PBST, and then HRP-labeled his antibody was diluted 1:10000 with sample diluent, 100 ⁇ L was added to each well, and incubated at room temperature for 1 h. Wash in plate washer 6 times and pat dry on flat paper. Add TMB chromogenic solution, 100 ⁇ L/well, wrap it in aluminum foil, and develop color for 8 minutes at 37°C in the dark.
  • 50 ⁇ L OKT3 diluent and 50 ⁇ L antibody diluent were used to set up irrelevant antibodies.
  • the irrelevant antibodies were human IgG1kappa Isotype control (HG1K, Sino Biological).
  • a negative control group was set up. Coated into a 96-well flat bottom plate, the total volume was 100 ⁇ L/well, and incubated at 4°C overnight. .
  • CD8+T cells were negatively separated from frozen PBMC (ORiCELLS, ID: PCH20210300033, separation efficiency is 30%), and the sorted CD8+T cells were tested for purity; CFSE labeled CD8+T cells at a concentration of 5 ⁇ M Cells were incubated at 37°C for 10 min, and complete medium was added to terminate staining. Wash the previously coated plate once with PBS, add CFSE-labeled or unlabeled T cells at 2 ⁇ 10 5 /well, 100 ⁇ L/well, and add Anti-CD28 with a final concentration of 1 ⁇ g/mL, 100 ⁇ L/well. The total experimental system is 200 ⁇ L/well.
  • Example 7 BLI method to detect the affinity of antibodies to TNFR2 protein (TNFR2+B7H3)
  • PBST 0.02% Tween 20, pH 7.4, 1*PBS
  • the AHC sensor was equilibrated with 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) as a buffer for 60 s
  • the antibody in the sample plate was solidified for 300 s
  • the secondary equilibration buffer was 180 s.
  • 100nM human-TNFR2-His manufactured by SINO, CAT: 10417-H08H, LOT: LC12SE2902
  • protein binds to the antibody for 300s, and then dissociates for 600s.
  • Example 8 BLI method to detect the affinity of antibodies to B7H3 protein (TNFR2+B7H3)
  • PBST 0.02% Tween 20, pH 7.4, 1*PBS
  • the AHC sensor was equilibrated with 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) as a buffer for 60 s, the antibody in the sample plate was solidified for 300 s, and the secondary equilibration buffer was 180 s.
  • 100nM human-B7H3His (manufacturer: Acro, CAT: TB73-H52E2, LOT: 2052-21AXF1-YS) protein binds to the antibody for 300s and then dissociates for 600s.
  • Example 9 Pharmacodynamic study of each candidate test substance in the mouse colon cancer cell line MC38-hB7H3 tumor model subcutaneously transplanted into hTNFR2 mice (TNFR2+B7H3)
  • mice Female C57BL/6-hTNFR2 mice aged 6-7 weeks were selected and inoculated subcutaneously with MC38-hB7H3 tumors. After the tumor volume reached approximately 100 ⁇ 50 mm, they were randomly divided into four groups. There are 5 mice in each group, and the groups include: (1) G1: PBS group; (2) G2: control antibody group, the sequence variable region consists of SEQ ID NO: 83 and SEQ ID NO: 84, with the addition of human IgG1 constant area (see SEQ ID NO:85 and SEQ ID NO: 86); (3) G3: Antibody TB-C1 group; (4) G4: Antibody TB-C2 group.
  • G1 PBS group
  • G2 control antibody group, the sequence variable region consists of SEQ ID NO: 83 and SEQ ID NO: 84, with the addition of human IgG1 constant area (see SEQ ID NO:85 and SEQ ID NO: 86); (3) G3: Antibody TB-C1 group; (4) G4: Antibody TB
  • the negative control group used PBS for intratumoral administration, and the samples from the remaining groups were administered intratumorally at 10 mg/kg.
  • the frequency of administration was 2 times a week for 4 consecutive weeks, with a total of 8 administrations; the tumor volume and body weight of the mice were measured every other day, and the tumor volume was calculated according to a ⁇ b 2 /2 (a is the long diameter, b is short diameter) calculation.
  • the experimental plan design is shown in Table 9.
  • Dilute human-TNFR2-His (manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) to 0.3 ⁇ g/mL with coating solution (1 ⁇ PBS, pH7.4), and coat it into a 96-well enzyme plate. , 100 ⁇ L/well, overnight at 4°C. Pour off the coating solution, wash the plate with 300 ⁇ L of 1 ⁇ PBST per well, wash 4 times with a plate washer, and pat dry on flat paper. Block with 3% skimmed milk powder, 300 ⁇ L/well, and incubate at 37°C for 1 hour. Pour off the blocking solution, wash 4 times with a plate washer, and pat dry on a flat paper.
  • the negative control is commercially available trastuzumab for injection (Herceptin).
  • the antibody sequence variable region of the positive control 1 consists of SEQ ID NO:83 and SEQ ID NO:84.
  • the antibody sequence variable region of positive control 2 consists of SEQ ID NO:41.
  • the positive control and antibody were diluted to 10 ⁇ g/mL with 3% skimmed milk powder, and diluted 3 times using this as the initial concentration. A total of 11 gradients were diluted. Another blank well was set and only the diluent was added. 100 ⁇ L/well, incubate at 37°C for 1 hour.
  • the antibody sequence variable region of the positive control consists of SEQ ID NO:9 and SEQ ID NO:13. Add the constant region of human IgG1 (see SEQ ID NO:85 and SEQ ID NO:86) and dilute it. Set the actual final concentration of the positive control and the antibody to be tested to 150 nM, 100 ⁇ L per well.
  • Tumor cells MCF-7 that highly express human CD24 purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu74) were washed twice, 1 ⁇ 10 5 /well, and added to a 96-well plate at 100 ⁇ L/tube.
  • the positive control and the antibody to be tested were added to the 96-well plate in sequence at 50 ⁇ L/well, and an equal volume of diluent was added to the blank control group and negative control group. After mixing, incubate the 96-well plate at 4°C for 1 hour. Add pre-cooled diluent and wash once, centrifuge (3000 rpm, 3 min), discard the supernatant, and harvest the cells. Take the fluorescent secondary antibody goat anti-human IgG Fc (manufacturer Biolegend, product number: 398004, batch number: B340957), and dilute it at a usage concentration of 1:500.
  • negative control 1 PBS group
  • the antibody sequence variable region of negative control 2 consists of SEQ ID NO:83 and SEQ ID NO:84, with the constant region of human IgG1 added (see SEQ ID NO:85 and SEQ ID NO:86);
  • the antibody sequence variable region of negative control 3 consists of SEQ ID NO:9 and SEQ ID NO:13, and the constant region of human IgG1 (SEQ ID NO:85 and SEQ ID NO:86) is added .
  • Tumor cells MCF-7 that highly express human CD24 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu74) were washed twice, 1 ⁇ 10 5 /well, and added to a 96-well plate at 100 ⁇ L/tube.
  • the negative control and the antibody to be tested were added to the 96-well plate in sequence at 50 ⁇ L/well, and an equal volume of diluent was added to the blank control group and the negative control group.
  • Human TNFR2 protein (SINO, CAT: 10417-H08H) was added into the 96-well plate at 50 ⁇ L/well. After mixing, the 96-well plate was incubated at 4°C for 1 hour.
  • Example 13 BLI method to detect the affinity of antibodies to TNFR2 protein (TNFR2+CD24)
  • the antibody sequence variable region of positive control 1 consists of SEQ ID NO:83 and SEQ ID NO:84, with the constant region of human IgG1 added (see SEQ ID NO:85 and SEQ ID NO:86);
  • the antibody sequence variable region of positive control 2 consists of SEQ ID NO:41.
  • the AHC sensor was equilibrated with 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) as a buffer for 60 s, the antibody in the sample plate was solidified for 300 s, and the secondary equilibration buffer was 180 s.
  • 100nM human-TNFR2-His (manufacturer: SINO, CAT: 10417-H08H, LOT: LC12SE2902) protein binds to the antibody for 300s, and then dissociates for 600s. After dissociation, use 10mM glycine (pH2.0) as the regeneration buffer and regenerate for 30 seconds. The sensor was regenerated with 10mM glycine (pH2.0).
  • the affinity results of the antibody molecules are shown in Table 12. The results show that the affinities of the TNFR2 end of the antibodies TD-A1, TD-C, TD-D1, TD-G and TD-H are all at the nanomolar or sub-nanomolar level.
  • the irrelevant antibodies are human IgG1kappa Isotype control (HG1K, Sino Biological). Set up a negative control group and a positive control group.
  • the antibody sequence variable region of the positive control consists of SEQ ID NO:83 and SEQ ID NO:84. Added Constant region of human IgGl (see SEQ ID NO:85 and SEQ ID NO:86).
  • IgG1 isotype control and positive control (antibody sequence variable region by SEQ ID NO:9 and SEQ ID NO:13, adding the constant region of human IgG1, see SEQ ID NO:85 and SEQ ID NO:86).
  • MCF-7 cells overexpressing human CD24 purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu74 were used as target cells.
  • RPMI1640 basic medium containing 5% FBS
  • the cells were incubated with 1 ⁇ 10 4 /well, 50 ⁇ L/well were spread on a 96-well plate; use RPMI1640 basic medium (containing 5% FBS) to dilute the antibody, with a starting concentration of 120nM, and then perform 5-fold gradient dilution, a total of 8 concentration gradients, 100 ⁇ L/well ; Resuspend NK158V cells and add 50 ⁇ L/well into the corresponding wells.
  • the effect-to-target ratio is 5:1.
  • M target cell maximum lysis well
  • ST target cell spontaneous release well
  • SE effector cell spontaneous release well
  • BV medium blank control well
  • BM medium blank control well
  • negative control 1 PBS group
  • negative control 2 antibody sequence consists of SEQ ID NO: 87 and SEQ ID NO: 88
  • negative control 3 antibody sequence consists of SEQ ID NO: 79 and SEQ ID NO :71 composition
  • irrelevant antibody human IgG1kappa Isotype control, manufacturer: Sino Biological, product number: HG1K, batch number: MA140C2803
  • dilute the negative control, irrelevant antibody and the antibody to be tested and the actual final concentration is set to 150nM, per well 100 ⁇ L.
  • Tumor cells CHOK1-18.2 (P6-4-1), which highly expresses human Claudin18.2 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu74), were washed twice, 1 ⁇ 10 5 /well, and added to 96 wells at 100 ⁇ L/tube. board. Add negative control 2, negative control 3, irrelevant antibody and test antibody to the 96-well plate in sequence at 50 ⁇ L/well, and add an equal volume of diluent to the blank control group and negative control group. Human TNFR2 protein (SINO, CAT: 10417-H08H) was added into the 96-well plate at 100 ⁇ L/well. After mixing, the 96-well plate was placed at 4°C and incubated for 1 hour.

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Abstract

本发明提供了一种双特异性抗体，其包含：(a)特异性结合第一抗原的第一抗体或其抗原结合片段；和(b)特异性结合第二抗原的第二抗体或其抗原结合片段；其中，所述第一抗原为肿瘤相关抗原(TAA)，并且所述第二抗原为TNFR2。

Description

一种双特异性抗体及其应用 技术领域
本发明属于生物医药领域，具体涉及一种结合肿瘤相关抗原(TAA)和TNFR2的双特异性抗体及其应用。
背景技术
单克隆抗体(mAb)已经广泛用于治疗多种人类疾病，包括癌症、自身免疫疾病、感染性疾病以及心血管疾病等。目前，存在超过80种单克隆抗体，包括鼠源、全人源化和嵌合抗体，它们已经被FDA批准用于治疗用途。这些抗体大多数是单特异性抗体，其识别单一表位并且可被选择以便通过此单一表位激活或抑制靶分子的活性。例如，曲妥珠单抗(trastuzumab)是最畅销的抗癌蛋白质治疗剂之一，通过将自己附着在Her2上来阻止人体表皮生长因子在Her2上的附着，从而阻断癌细胞的生长，曲妥珠单抗还可以刺激身体自身的免疫细胞去摧毁癌细胞。但是，许多生理反应需要有待触发的两种或更多种不同蛋白质或蛋白质亚基的交联或共接合。以异聚细胞-表面受体复合物的活化为例，对于这些受体复合物，活化通常通过配体与不同蛋白质上的多个结构域的相互作用而实现，由此造成一种或两种受体组分的接近相关的活化。
双特异性抗体(bispecific antibody，BsAb)是指能同时结合两个(或多个)不同抗原表位的抗体分子。与传统的单克隆抗体相比，双特异性抗体具有独特的作用机制：(1)双特异性抗体可以同时结合2个或多个不同的抗原分子或相同分子的不同表位，而联合用药往往不具备这种效应。(2)介导细胞间的相互作用，双特异性抗体可分别结合效应细胞和靶细胞上的两种抗原上，在效应细胞和靶细胞之间架起桥梁，促进细胞间的相互作用，例如介导免疫细胞对肿瘤细胞的杀伤。因此双特异性抗体具有传统单克隆抗体不具备的独特优势。
肿瘤坏死因子受体2(TNFR2)，也称为肿瘤坏死因子受体超家族1B(TNFRSF1B)和CD120b，是一种75-KDA型I型跨膜蛋白，属于肿瘤坏死因子受体超家族成员，其含有细胞外结构域(ECD，残留物1-257)和具有TRAF2结合结构域的细胞内结构域(ICD，残基288-461)。在正常的T细胞中，TNFA-TNFR2相互作用通过NFKB信号通路触发细胞存活信号。然而，在自身免疫T细胞中，TNFA-TNFR2相互作用通过Caspase途径触发凋亡信号。
目前，TNFR2已被证明能增强效应T细胞(Teff)的激活并减少调节T细胞(Treg)介导的抑制作用。TNFR2的活化通过有丝分裂原活化的蛋白激酶(MAPK)信号传导途径诱导信号传导，其协调通过TRAF2/3信号传导和NFκB介导的促进逃避细胞凋亡和细胞增殖的基因的转录。TNFR2不仅可以在癌细胞，浸润肿瘤的Treg上表达，还可以在效应Teff细胞上表达。研究表明，TNFR2的激动型抗体能够刺激CD8+T细胞的增殖，活化CD8+T细胞，释放IFNγ和IL2等抗肿瘤因子；TNFR2的拮抗型抗体则能够阻断TNF-TNFR2，抑制Treg增殖。
本发明旨在开发一种双特异性抗体，既能够特异性识别肿瘤相关抗原，又能够利用TNFR2激动型抗体刺激CD8+T细胞的增殖，活化CD8+T细胞，释放IFNγ和IL2等抗肿瘤因子；或利用TNFR2拮抗型抗体阻断TNF-TNFR2，抑制Treg增殖。
发明内容
在本申请中，发明人开发了具有良好性能的能够与肿瘤相关抗原(TAA)和TNFR2结合的双特异性抗体，本发明的双特异性抗体可以靶向肿瘤相关抗原(TAA)，同时可以利用TNFR2激动型抗体刺激CD8+T细胞的增殖，活化CD8+T细胞，释放IFNγ和IL2等抗肿瘤因子；或利用TNFR2拮抗型抗体阻断TNF-TNFR2，抑制Treg增殖。
本发明提供了一种双特异性抗体，所述双特异性抗体包含：(a)特异性结合第一抗原的第一抗体或其抗原结合片段；和(b)特异性结合第二抗原的第二抗体或其抗原结合片段；其中，所述第一抗原为肿瘤相关抗原(TAA)，并且所述第二抗原为TNFR2。
在一些实施方案中，所述第一抗体或其抗原结合片段包含重链和轻链；所述第二抗体或其抗原结合片段包含scFv或VHH。
在一些实施方案中，所述第一抗体的一条重链的重链可变区与一条轻链的轻链可变区形成抗原结合部位，另一条重链的重链可变区与另一条轻链的轻链可变区形成抗原结合部位。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段和一个或多个所述的scFv。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段和一个所述的scFv，所述scFv连接于所述第一抗体或其抗原结合片段的重链的N端。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段和一个所述的scFv，所述scFv连接于所述第一抗体或其抗原结合片段的重链的C端。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段和两个所述的scFv。
在一些实施方案中，两个所述scFv分别连接于所述第一抗体或其抗原结合片段的两条重链的N端。
在一些实施方案中，两个所述scFv分别连接于所述第一抗体或其抗原结合片段的两条重链的C端。
在一些实施方案中，所述双特异性抗体包含特异性结合肿瘤相关抗原(TAA)的第一抗体和两个特异性结合TNFR2的scFv，两个特异性结合TNFR2的scFv分别连接于所述第一抗体两条重链的N端。
在一些实施方案中，所述双特异性抗体包含特异性结合肿瘤相关抗原(TAA)的第一抗体和两个特异性结合TNFR2的scFv，两个特异性结合TNFR2的scFv分别连接于所述第一抗体两条重链的C端。
在一些实施方案中，所述双特异性抗体包含两条第一多肽链和两条第二多肽链，对于所述每条多肽链：(a)所述第一多肽链各自独立地包含所述第一抗体或其抗原结合片段的轻链；和(b)所述第二多肽链各自独立地包含所述第一抗体或其抗原结合片段的重链和所述scFv。
在一些实施方案中，所述双特异性抗体的两条第一多肽链相同或不同，和/或两条第二多肽链相同或不同。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段和一个或多个所述的VHH。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段 和一个所述的VHH，所述VHH连接于所述第一抗体或其抗原结合片段的重链的N端。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段和一个所述的VHH，所述VHH连接于所述第一抗体或其抗原结合片段的重链的C端。
在一些实施方案中，所述双特异性抗体包含一个第一抗体或其抗原结合片段和两个所述的VHH。
在一些实施方案中，两个所述VHH分别连接于所述第一抗体或其抗原结合片段的两条重链的N端。
在一些实施方案中，两个所述VHH分别连接于所述第一抗体或其抗原结合片段的两条重链的C端。
在一些实施方案中，所述双特异性抗体包含特异性结合肿瘤相关抗原(TAA)的第一抗体和两个特异性结合TNFR2的VHH，两个特异性结合TNFR2的VHH分别连接于所述第一抗体两条重链的N端。
在一些实施方案中，所述双特异性抗体包含特异性结合肿瘤相关抗原(TAA)的第一抗体和两个特异性结合TNFR2的VHH，两个特异性结合TNFR2的VHH分别连接于所述第一抗体两条重链的C端。
在一些实施方案中，所述双特异性抗体包含两条第一多肽链和两条第二多肽链，对于所述每条多肽链：(a)所述第一多肽链各自独立地包含所述第一抗体或其抗原结合片段的轻链；和(b)所述第二多肽链各自独立地包含所述第一抗体或其抗原结合片段的重链和所述VHH。
在一些实施方案中，所述双特异性抗体的两条第一多肽链相同或不同，和/或两条第二多肽链相同或不同。
在一些实施方案中，所述肿瘤相关抗原(TAA)选自GPC3、CD19、CD20(MS4A1)、CD22、CD24、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin18.2、AFP、ALK、B7H3、BAGE蛋白质、BCMA、BIRC5(存活素)、BIRC7、β-连环蛋白(β-catenin)、brc-ab1、BRCA1、BORIS、CA9、CA125、碳酸酐酶IX、半胱天冬酶-8(caspase-8)、CALR、CCR5、NA17、NKG2D、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、OX40、p15、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、PRAME、PSMA(FOLH1)、RAGE蛋白质、周期素-B1、CYP1B1、EGFR、EGFRvIII、ErbB2/Her2、ErbB3、ErbB4、ETV6-AML、EpCAM、EphA2、Fra-1、FOLR1、GAGE蛋白、GD2、GD3、GloboH、GM3、gp100、Her2、HLA/B-raf、HLA/k-ras、HLA/MAGE-A3、hTERT、IL13Rα2、LMP2、κ-Light、LeY、MAGE-1、MAGE-2、MAGE-3、MAGE-4、MAGE-6、MAGE-12、MART-1、间皮素、ML-IAP、MOv-γ、Muc1、Muc2、Muc3、Muc4、Muc5、Muc16、MUM1、Ras、RGS5、Rho、ROR1、SART-1、SART-3、STEAP1、STEAP2、TAG-72、TGF-β、TMPRSS2、汤-诺氏抗原、TRP-1、TRP-2、酪氨酸酶和尿溶蛋白-3、5T4。优选地，所述肿瘤相关抗原(TAA)选自B7H3、CD24或Claudin18.2。
在一些实施方案中，所述scFv的重链可变区与轻链可变区通过连接子L1连接。
在一些实施方案中，所述scFv通过连接子L2与所述第一抗体或其抗原结合片段的重链的N端或C端连接。
在一些实施方案中，所述VHH通过连接子L2与所述第一抗体或其抗原结合片段的重链的N端或C端连接。
在一些实施方案中，所述连接子L1和连接子L2相同或不同。在一些实施方案中，所述连接子L1和/或连接子L2具有如(G4S)x所示的氨基酸序列，x为选自1-6的整数；优选地，所述连接子L1和/或连接子L2为(G4S)2、(G4S)3或(G4S)4。
在一些实施方案中，所述第一抗体或其抗原结合片段的重链包含重链可变区和重链恒定区，并且所述轻链包含轻链可变区和轻链恒定区；优选地，所述第一抗体或其抗原结合片段为全长抗体。
在一些实施方案中，所述第一抗体或其抗原结合片段的重链包含第一Fc区和第二Fc区。在一些实施方案中，第一Fc区和第二Fc区是相同的或不同的。一些实施方案中，所述第一Fc区或第二Fc区选自IgG、IgA、IgD、IgE、IgM或其变体。在一些实施方案中，所述第一Fc区或第二Fc区选自IgG1、IgG2、IgG3、IgG4或其变体。在一些实施方案中，所述第一Fc区或第二Fc区包含一个或多个氨基酸突变，优选氨基酸置换、插入或缺失。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合B7H3，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:2所示，或为与SEQ ID NO:2具有至少80％同一性的序列；HCDR2如SEQ ID NO:3所示，或为与SEQ ID NO:3具有至少80％同一性的序列；HCDR3如SEQ ID NO:4所示，或为与SEQ ID NO:4具有至少80％同一性的序列；LCDR1如SEQ ID NO:6所示，或为与SEQ ID NO:6具有至少80％同一性的序列；LCDR2如SEQ ID NO:7所示，或为与SEQ ID NO:7具有至少80％同一性的序列；LCDR3如SEQ ID NO:8所示，或为与SEQ ID NO:8具有至少80％同一性的序列。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合CD24，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:10所示，或为与SEQ ID NO:10具有至少80％同一性的序列；HCDR2如SEQ ID NO:11所示，或为与SEQ ID NO:11具有至少80％同一性的序列；HCDR3如SEQ ID NO:12所示，或为与SEQ ID NO:12具有至少80％同一性的序列；LCDR1如SEQ ID NO:14所示，或为与SEQ ID NO:14具有至少80％同一性的序列；LCDR2如SEQ ID NO:15所示，或为与SEQ ID NO:15具有至少80％同一性的序列；LCDR3如SEQ ID NO:16所示，或为与SEQ ID NO:16具有至少80％同一性的序列。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:18所示，或为与SEQ ID NO:18具有至少80％同一性的序列；HCDR2如SEQ ID NO:19所示，或为与SEQ ID NO:19具有至少80％同一性的序列；HCDR3如SEQ ID NO:20所示，或为与SEQ ID NO:20具有至少80％同一性的序列；LCDR1如SEQ ID NO:22所示，或为与SEQ ID NO:22具有至少80％同一性的序列；LCDR2如SEQ ID NO:23所示，或为与SEQ ID NO:23具有至少80％同一性的序列；LCDR3如SEQ ID NO:24所示，或为与SEQ ID NO:24具有至少80％同一性的序列。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:26所示，或为与SEQ ID NO:26具有至少80％同一性的序列；HCDR2如SEQ ID NO:27所示，或为与SEQ ID NO:27具有至少80％同一性的序列；HCDR3如SEQ ID NO:28所示，或为与SEQ ID NO:28具有至少80％同一性的序列；LCDR1如SEQ ID NO:30所示，或为与SEQ ID NO:30具有至少80％同一性的序列；LCDR2如SEQ ID NO:31所示，或为与SEQ ID NO:31具有至少80％同一性的序列；LCDR3如SEQ  ID NO:32所示，或为与SEQ ID NO:32具有至少80％同一性的序列。
在一些实施方案中，所述scFv特异性结合TNFR2，所述scFv的HCDR1如SEQ ID NO:34所示，或为与SEQ ID NO:34具有至少80％同一性的序列；HCDR2如SEQ ID NO:35所示，或为与SEQ ID NO:35具有至少80％同一性的序列；HCDR3如SEQ ID NO:36所示，或为与SEQ ID NO:36具有至少80％同一性的序列；以及，LCDR1如SEQ ID NO:38所示，或为与SEQ ID NO:38具有至少80％同一性的序列；LCDR2如SEQ ID NO:39所示，或为与SEQ ID NO:39具有至少80％同一性的序列；LCDR3如SEQ ID NO:40所示，或为与SEQ ID NO:40具有至少80％同一性的序列。
在一些实施方案中，所述VHH特异性结合TNFR2，所述VHH的HCDR1如SEQ ID NO:42所示，或为与SEQ ID NO:42具有至少80％同一性的序列；HCDR2如SEQ ID NO:43所示，或为与SEQ ID NO:43具有至少80％同一性的序列；HCDR3如SEQ ID NO:44所示，或为与SEQ ID NO:44具有至少80％同一性的序列。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合B7H3，其中，所述第一抗体或其抗原结合片段的重链可变区VH如SEQ ID NO:1所示，或为与SEQ ID NO:1具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:5所示，或为与SEQ ID NO:5具有至少80％同一性的序列。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合CD24，其中，所述第一抗体或其抗原结合片段的重链可变区VH如SEQ ID NO:9所示，或为与SEQ ID NO:9具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:13所示，或为与SEQ ID NO:13具有至少80％同一性的序列。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片段的重链可变区VH如SEQ ID NO:17所示，或为与SEQ ID NO:17具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:21所示，或为与SEQ ID NO:21具有至少80％同一性的序列。
在一些实施方案中，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片段的重链可变区VH如SEQ ID NO:25所示，或为与SEQ ID NO:25具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:29所示，或为与SEQ ID NO:29具有至少80％同一性的序列。
在一些实施方案中，所述scFv特异性结合TNFR2，所述scFv重链可变区VH如SEQ ID NO:33所示，或为与SEQ ID NO:33具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:37所示，或为与SEQ ID NO:37具有至少80％同一性的序列。
在一些实施方案中，所述VHH特异性结合TNFR2，所述VHH重链可变区VH如SEQ ID NO:41所示，或为与SEQ ID NO:41具有至少80％同一性的序列。
在一些实施方案中，所述的双特异性抗体的第一多肽链选自SEQ ID NO:46-50、59-63、71-76的任一氨基酸序列，或为与SEQ ID NO:46-50、59-63、71-76的任一氨基酸序列具有至少80％同一性的氨基酸序列；所述双特异性抗体的第二多肽链选自SEQ ID NO:51-58、64-70、77-82的任一氨基酸序列，或为与SEQ ID NO:51-58、64-70、77-82的任一氨基酸序列具有至少80％同一性的氨基酸序列。
在一些实施方案中，所述的双特异性抗体包含：
(1)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:51所示的第二多 肽链；或
(2)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:56所示的第二多肽链；或
(3)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:53所示的第二多肽链；或
(4)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:54所示的第二多肽链；或
(5)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:57所示的第二多肽链；或
(6)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:55所示的第二多肽链；或
(7)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:58所示的第二多肽链；或
(8)如SEQ ID NO:47所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
(9)如SEQ ID NO:50所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
(10)如SEQ ID NO:48所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
(11)如SEQ ID NO:49所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
(12)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:64所示的第二多肽链；或
(13)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:69所示的第二多肽链；或
(14)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:66所示的第二多肽链；或
(15)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:67所示的第二多肽链；或
(16)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:68所示的第二多肽链；或
(17)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:70所示的第二多肽链；或
(18)如SEQ ID NO:60所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
(19)如SEQ ID NO:61所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
(20)如SEQ ID NO:62所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
(21)如SEQ ID NO:63所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
(22)如SEQ ID NO:71所示的第一多肽链，如SEQ ID NO:77所示的第二多肽链；或
(23)如SEQ ID NO:72所示的第一多肽链，如SEQ ID NO:78所示的第二 多肽链；或
(24)如SEQ ID NO:71所示的第一多肽链，如SEQ ID NO:81所示的第二多肽链；或
(25)如SEQ ID NO:72所示的第一多肽链，如SEQ ID NO:82所示的第二多肽链；或
(26)如SEQ ID NO:73所示的第一多肽链，如SEQ ID NO:79所示的第二多肽链；或
(27)如SEQ ID NO:74所示的第一多肽链，如SEQ ID NO:80所示的第二多肽链；或
(28)如SEQ ID NO:75所示的第一多肽链，如SEQ ID NO:79所示的第二多肽链；或
(29)如SEQ ID NO:76所示的第一多肽链，如SEQ ID NO:80所示的第二多肽链。
本发明提供一种分离的核酸分子，其包含编码上述任一项所述的双特异性抗体的核苷酸序列。优选地，所述分离的核酸分子包含编码上述任一项所述的双特异性抗体的第一多肽链的核苷酸序列。优选地，所述分离的核酸分子包含编码上述任一项所述的双特异性抗体的第二多肽链的核苷酸序列。
本发明提供一种多功能融合蛋白，其包含上述任一项所述的双特异性抗体。
在一些实施方案中，所述多功能融合蛋白还包含一个或多个与其他抗原特异性结合的第三抗体或其抗原结合部分。在一些实施方案中，所述结合第三抗体或其抗原结合部分的抗原选自肿瘤相关抗原(TAA)或免疫检查点。在一些实施方案中，所述结合第三抗体或其抗原结合部分的抗原选自GPC3、CD19、CD20(MS4A1)、CD22、CD24、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin18.2、AFP、ALK、BAGE蛋白质、BCMA、BIRC5(存活素)、BIRC7、β-连环蛋白(β-catenin)、brc-ab1、BRCA1、BORIS、CA9、CA125、碳酸酐酶IX、半胱天冬酶-8(caspase-8)、CALR、CCR5、NA17、NKG2D、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、OX40、p15、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、PRAME、PSMA(FOLH1)、RAGE蛋白质、周期素-B1、CYP1B1、EGFR、EGFRvIII、ErbB2/Her2、ErbB3、ErbB4、ETV6-AML、EpCAM、EphA2、Fra-1、FOLR1、GAGE蛋白、GD2、GD3、GloboH、GM3、gp100、Her2、HLA/B-raf、HLA/k-ras、HLA/MAGE-A3、hTERT、IL13Rα2、LMP2、κ-Light、LeY、MAGE-1、MAGE-2、MAGE-3、MAGE-4、MAGE-6、MAGE-12、MART-1、间皮素、ML-IAP、MOv-γ、Muc1、Muc2、Muc3、Muc4、Muc5、Muc16、MUM1、Ras、RGS5、Rho、ROR1、SART-1、SART-3、STEAP1、STEAP2、TAG-72、TGF-β、TMPRSS2、汤-诺氏抗原、TRP-1、TRP-2、酪氨酸酶和尿溶蛋白-3、5T4、PD-L1、CTLA4、PD-L2、PD-1、4-1BB、CD47、TIGIT、GITR、TIM3、ILT4、TREM2、LAG3、CD27或B7H4。
在一些实施方案中，所述多功能融合蛋白还包含细胞因子。在一些实施方案中，所述细胞因子选自IL-1、IL-2、IL-2Rα、IL-2Rβ、IL-3、IL-3Rα、IL-4、IL-4Rα、IL-5、IL-5Rα、IL-6、IL-6Rα、IL-7、IL-7Rα、IL-8、IL-9、IL-9Rα、IL-10、IL-10R1、IL-10R2、IL-11、IL-11Rα、IL-12、IL-12Rα、IL-12Rβ2、IL-12Rβ1、IL-13、IL-13Rα、IL-13Rα2、IL-14、IL-15、IL-15Rαsushi、IL-16、IL-17、IL-18、IL-19、IL-20、IL-20R1、IL-20R2、IL-21、IL-21Rα、IL-22、IL-23、IL-23R、IL-27R、IL-31R、TGF、VEGF、IFNγ、IFNα或GM-CSF。
本发明还提供上述任一项所述的双特异性抗体或上述任一项所述的多功能融合蛋白在制备治疗癌症的药物中的用途。在一些实施方案中，所述癌选自人脑星形胶质母细胞瘤、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、宫颈癌、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促结缔组织增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、乳头状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、前列腺癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌或子宫癌。
本发明还提供上述任一项所述的双特异性抗体或上述任一项所述的多功能融合蛋白在制备用于治疗自身免疫性疾病的药物中的用途。在一些实施方案中，所述自身免疫性疾病选自移植物抗宿主病、类风湿性关节炎、克罗恩病、多发性硬化症、结肠炎、牛皮癣、自身免疫性葡萄膜炎、天疱疮、大疱性表皮松解症或I型糖尿病。
在一些实施方案中，所述用途通过肿瘤免疫疗法、细胞疗法或基因疗法中的一种或多种来实现。
本发明还提供一种药物组合物，其包含上述任一项所述的双特异性抗体和药学上可接受的载体、稀释剂或赋形剂。
本发明还提供一种药物组合物，其包含上述任一项所述的多功能融合蛋白和药学上可接受的载体、稀释剂或赋形剂。
本发明还提供一种抗体药物偶联物，其包含上述任一项所述的双特异性抗体。
在一些实施方案中，所述偶联药物选自细胞毒素、小分子化学药物或免疫毒素。
缩写及术语定义
在本文中使用以下缩写。VH：抗体重链可变区；VL：抗体轻链可变区；CDR：免疫球蛋白可变区中的互补决定区；FR：抗体构架区，即抗体可变区中除CDR残基以外的氨基酸残基；IgG：免疫球蛋白G。
术语“抗体”是指天然的免疫球蛋白或者通过部分或完全合成而制备的免疫球蛋白。抗体可从天然存在该抗体的血浆或血清等的天然资源、或者产生抗体的杂交瘤细胞的培养上清中、动物免疫血清中、噬菌体文库筛选进行重建得到分离。备选地，可通过使用基因重组等的技术部分或完全地合成。优选的抗体包括，例如，免疫球蛋白的同种型或这些同种型的亚类的抗体。已知人免疫球蛋白包括IgGl、IgG2、IgG3、IgG4、IgAl、IgA2、IgD、IgE、IgM这9种类别(同种型)。在这些同种型中，本发明的抗体可以包括IgGl、IgG2、IgG3和/或IgG4。
术语“嵌合抗体”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基 因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
本文中所使用的部分抗体由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成，或者只有轻链恒定区(CL)。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。
术语“双特异性抗体”是指能够与两个目标抗原或目标抗原表位特异性结合的蛋白分子。在本发明中，包含抗体或抗原结合片段(例如Fab、scFv等)的“双特异性抗原结合蛋白”与“双特异性抗体”、“双抗”可以互换使用。
术语抗体的“抗原结合片段”是指抗体的多肽片段，例如全长抗体的多肽片段，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)、线性抗体(linear antibody)、纳米抗体(如技术来自Ablynx)、结构域抗体(如技术来自Domantis)、和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
术语“抗体药物偶联物”或“ADC”是指与一个或多个偶联药物(其可以任选地是治疗剂或细胞毒性剂)连接的结合蛋白(如抗体或其抗原结合片段)，其结构通常由三部分组成：抗体或抗体类配体、药物部分、以及将抗体或抗体类配体及药物偶联起来的连接子(linker)。ADC通常具有与抗体偶联的1、2、3、4、5、6、7、8、9或10个的药物。
术语“多肽”是指任何长度的氨基酸链，而与修饰(例如磷酸化或糖基化)无关。术语多肽包括蛋白质及其片段。多肽可以是“外源的”，意指它们是“异源的”，即是所利用的宿主细胞外来的，例如由细菌细胞产生的人多肽。本文将多肽公开为氨基酸残基序列。那些序列按氨基末端到羧基末端的方向从左到右书写。根据标准命名法，氨基酸残基序列以三字母或单字母代码命名。
术语“scFv”指包含通过接头连接的抗体重链可变结构域(VH)和抗体轻链可变结构域(VL)的分子。此类scFv分子可具有一般结构：NH2-VL-连接子-VH-COOH或NH2-VH-连接子-VL-COOH。合适的现有技术连接子由重复的GGGGS氨基酸序列或其变体组成，例如使用1-6个重复的GGGGS氨基酸序列或其变体。
术语“VHH”是指只包含一个重链可变区(VHH)的单一抗原结合多肽，其源自天然不含轻链的重链分子的可变结构域，以区别于四链免疫球蛋白的常规VH。这种VHH分子可以源自在骆驼科物种例如骆驼、美洲驼羊、骆马、单峰骆驼、羊驼和原驼中产生的抗体。骆驼科以外的其它物种可以产生天然缺乏轻链的重链分子，并且这样的VHH在本申请的范围内。术语“宿主细胞”指已经或者能够用核酸序列转化并从而表达所选的目的基因的细胞。该术语包括亲本细胞的 后代，无论该后代与原来的亲本细胞在形态或基因组成上是否相同，只要后代存在所选目的基因即可。常用的宿主细胞包括细菌、酵母、哺乳动物细胞等。
术语“载体”指能够增殖与其连接的另一种核酸的核酸分子。该术语包括作为自身复制型核酸结构的载体及并入接受其导入的宿主细胞的基因组中的载体。某些载体能够指导与其可操作连接的核酸的表达，本文称为“表达载体”。
术语“药学上可接受的载体”包括任何标准药物载体，诸如磷酸盐缓冲盐水溶液、水和乳液，以及各种类型的润湿剂等。
术语“同一性”定义为比对序列并在必要时引入缺口以获取最大百分比序列同一性后，候选序列中与对照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式进行，例如使用公众可得到的计算机软件，诸如BLAST软件或FASTA程序包。
术语“至少80％同一性”是指候选序列中与对照多肽序列中的氨基酸残基相同的氨基酸残基的百分率为80％以上，包括80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。
术语“特异性”表示参与特异性结合的分子之一不显示任何与不同于结合伙伴分子中的一个或数个的分子的显著结合。此外，在含抗体可变区的结构域对抗原中的多个表位中的特定表位具有特异性时，也使用该术语。当含抗体可变区的结构域所结合的表位被包含在数个不同抗原中时，包含含抗体可变区的结构域的抗原结合分子可以结合具有所述表位的各种抗原。
术语“肿瘤相关抗原”或“TAA”是指在癌性细胞的表面上完全或作为片段表达的分子(典型地是蛋白质、碳水化合物、脂质或它们的一些组合)，并且其可用于优先将药理学药剂靶向癌性细胞。
术语“表位”是指抗原中的抗原决定簇，并且是指在本说明书中公开的包含抗体可变区的抗原结合分子的结构域所结合的抗原位点。因此，可以根据其结构来定义表位。另外，也可以根据识别该表位的抗原结合分子中的抗原结合活性来定义该表位。当抗原是肽或多肽时，表位可以由形成表位的氨基酸残基指定；当表位是糖链时，表位可以通过其特定的糖链结构来确定。
术语“阳性对照”是指能够结合靶点蛋白或是表达靶点蛋白的天然或工程化细胞或抗体，本文所指的阳性对照是指单靶点阳性对照。
术语“阴性对照”是指在同一实验中，使用与实验样品相同种属来源、相同亚型、相同剂量、相同的免疫球蛋白及亚型的免疫球蛋白、相同标记等，用于消除实验中非特异结合样品对实验数值产生的实验背景影响，作为一种更加说明实验效果的对照。
术语“Treg”、“Treg细胞”或“调节性T细胞”有时被称为抑制性T细胞，其特征在于表达生物标志物CD4、FOXP3和CD25，代表调节免疫系统、维持对自身抗原的耐受性并预防自身免疫疾病的T细胞亚群。Treg是免疫抑制性的并且通常抑制或下调效应T(Teff)细胞的诱导和增殖。Treg可以在胸腺中发展(所谓的CD4+Foxp3+“天然”Treg)或从外周的幼稚CD4+T细胞中分化，例如，在暴露于TGFβ或视黄酸之后。
术语“Teff”、“Teff细胞”或“效应T细胞”是T细胞接受抗原刺激后，经过增殖，分化形成的细胞。效应T细胞具有释放淋巴因子的功能，在此过程中，有一小部分T细胞成为记忆T细胞。效应T细胞与靶细胞接触而激发颗粒胞吐， 所释放的穿孔素通过聚合作用而在靶细胞表面形成小孔，从而介导杀伤作用，其靶细胞死亡过程类似于细胞凋亡。同时，效应T细胞还能释放出免疫活性物质-淋巴因子，如白细胞介素，干扰素等。
本领域中有多种方法/系统来定义和描述CDR，这些系统和/或定义已经开发和精制多年，包括Kabat、Chothia、IMGT、AbM和Contact。Kabat是最常用的，基于序列变异性定义CDR；Chothia基于结构循环区域的位置基于序列变异性定义CDR；IMGT系统基于可变域结构内的序列变异性和位置定义CDR；AbM是基于牛津分子公司的AbM抗体建模软件进行定义，是Kabat和Chothia之间的折衷；Contact基于对复杂晶体结构的分析定义CDR，在多个方面与Chothia类似。除非特殊说明，否则本文使用Kabat定义CDR。特殊说明，本申请部分CDR采用定义标准进行划分，如SEQ ID NO:2、10、18、34、42所示的HCDR1，其划分以重链可变区第一个半胱氨酸后的第四位开始，HCDR1长度一般为10-12，截止到色氨酸前一个氨基酸。
附图说明
图1为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的scFv，所述scFv通过连接子连接到全长抗体的两条重链的C端。
图2为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的scFv，所述scFv通过连接子连接到全长抗体的两条重链的N端。
图3为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的VHH，所述VHH通过连接子连接到全长抗体的两条重链的C端。
图4为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的VHH，所述VHH通过连接子连接到全长抗体的两条重链的N端。
图5为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的scFv，所述scFv通过连接子连接到全长抗体的两条轻链的C端。
图6为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的scFv，所述scFv通过连接子连接到全长抗体的两条轻链的N端。
图7为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的VHH，所述VHH通过连接子连接到全长抗体的两条轻链的C端。
图8为描述示例性双特异性抗体，其包含能够特异性识别肿瘤相关抗原(TAA)的全长抗体和能够特异性识别TNFR2的VHH，所述VHH通过连接子连接到全长抗体的两条轻链的N端。
图9为抗体TB-A1、TB-E、TB-B、TB-F对TNFR2蛋白的结合活性。
图10为抗体TB-G、TB-D1、TB-H对TNFR2蛋白的结合活性。
图11为抗体TB-A2对TNFR2蛋白的结合活性。
图12为抗体TB-C1、TB-C2、TB-D2对TNFR2蛋白的结合活性。
图13为抗体TB-A1、TB-E、TB-B、TB-F对B7H3蛋白的结合活性。
图14为抗体TB-G、TB-D1、TB-H对B7H3蛋白的结合活性。
图15为抗体TB-A2、TB-C1对B7H3蛋白的结合活性。
图16为抗体TB-C2、TB-D2对B7H3蛋白的结合活性。
图17为抗体TB-A1、TB-E、TB-B、TB-F的两端结合活性。
图18为抗体TB-G、TB-D1、TB-H的两端结合活性。
图19为抗体TB-C1的两端结合活性。
图20为抗体TB-A1、TB-D1、TB-C1的两端结合活性。
图21为抗体TB-C1、TB-C2的抗肿瘤生长曲线。
图22为抗体TD-A1、TD-E、TD-B、TD-F对TNFR2蛋白的结合活性。
图23为抗体TD-C、TD-G、TD-D1、TD-H对TNFR2蛋白的结合活性。
图24为抗体TD-A1、TD-A2对TNFR2蛋白的结合活性。
图25为抗体TD-D1、TD-D2对TNFR2蛋白的结合活性。
图26为抗体TD-A1、TD-D1的ADCC活性。
具体实施方式
以下结合附图与具体实施例对本发明做进一步的描述，本发明的保护内容不局限于以下实施例。还应该理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求及其任何等同物为本发明的保护范围。
实施例1不同结构的双特异性抗体的制备
针对TNFR2和B7H3，根据图1-8的8种结构分别构建双特异性抗体，依次命名为TB-A至TB-H；针对TNFR2和CD24，根据图1-8的8种结构分别构建双特异性抗体，依次命名为TD-A至TD-H；针对TNFR2和Claudin18.2，根据图1、2、5、6的4种结构分别构建双特异性抗体，依次命名为TC-A、TC-B、TC-E和TC-F。本专利部分结构的双特异性抗体同时构建了IgG1亚型和IgG4亚型，抗体名称后带“1”的为IgG1亚型，抗体名称后带“2”的为IgG4亚型，例如：抗体TB-A1为针对TNFR2和B7H3靶点、根据图1结构构建的IgG1亚型的抗体，抗体TB-A2为针对TNFR2和B7H3靶点、根据图1结构构建的IgG4亚型的抗体。
抗B7H3抗体、抗CD24抗体、抗Claudin18.2抗体作为全长抗体时的序列以及TNFR2抗体作为scFv、VHH时的序列如表1所示。
表1序列表

TNFR2抗体的序列和B7H3抗体、CD24抗体、Claudin18.2抗体的序列通过连接子按照一定的顺序连接，可形成表2-4中的肽链。其中，在本实施例中，双特异性抗体中使用的连接子2个GGGGS重复(即GGGGSGGGGS，下文简写为(G4S)2)或3个GGGGS重复(即GGGGSGGGGSGGGGS，下文简写为(G4S)3)，本实施例中使用的CL为kappa(κ)型，序列如SEQ ID NO:45所示。
表2双特异性抗体(TNFR2+B7H3)肽链序列表
表3双特异性抗体(TNFR2+CD24)肽链序列表

表4双特异性抗体(TNFR2+Claudin18.2)肽链序列表
根据表2-4中的肽链组合，设计得到表5所示的双特异性抗体的氨基酸序列。
表5双特异性抗体序列

将上述设计的不同结构的双特异性抗体各条链进行基因合成，而后利用分子克隆技术，将抗体片段插入PCDNA3.1载体中，构建成哺乳动物细胞表达质粒，利用脂质体转染方式，导入宿主细胞株CHO细胞，利用细胞Fed-batch获得发酵上清液，取发酵液上清进行亲和层析、离子交换层析等一系列步骤的纯化，最终纯化得到构建的抗体。对纯化后的抗体检测表达量、纯度、SDS-PAGE等，确认双特异性抗体。
实施例2 ELISA检测抗体对TNFR2蛋白的结合活性(TNFR2+B7H3)
用包被液(1×PBS，pH7.4)将人-TNFR2-His(厂家：SINO，CAT：10417-H08H，LOT：LC12SE2902)稀释至0.3μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。设置阴性对照与阳性对照，阴性对照为市售所得注射用曲妥珠单抗(赫赛汀)，阳性对照1的抗体序列可变区由SEQ ID NO:83和SEQ ID NO:84组成，加入了人IgG1的恒定区(见SEQ ID NO:85和SEQ ID NO:86)；阳性对照2的抗体序列可变区由SEQ ID NO:41组成。将阳性对照和抗体用3％脱脂奶粉稀释为10μg/mL，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。用3％脱脂奶粉将山羊抗人IgG Fc按1:20000稀释，100μL/孔，37℃孵育1h。洗板机洗涤6次，平板纸上拍干。加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8min。加入终止液1M HCl终止显色反应，100μL/孔。在酶标仪上450nm处进行读数。抗体分子的ELISA结果分别如图9-12所示，结果显示抗体TB-A1至TB-H均能与人TNFR2蛋白结合。
实施例3 ELISA检测抗体对B7H3蛋白的结合活性(TNFR2+B7H3)
用包被液(1×PBS，pH7.4)将人-B7H3His(厂家：Acro，CAT：TB73-H52E2，LOT：2052-21AXF1-YS)稀释为0.3μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。设置阴性对照与阳性对照，阴性对照为市售所得注射用曲妥珠单抗(赫赛汀)，阳性对照的抗体序列可变区由SEQ ID NO:1和SEQ ID NO:5组成，加入了人IgG1的恒定区(见SEQ ID NO:85和SEQ ID NO:86)。将阳性对照和供试品用3％脱脂奶粉稀释为10μg/mL，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。用3％脱脂奶粉将山羊抗人IgG Fc按1:20000稀释，100μL/孔，37℃孵育1h。洗板机洗涤6次，平板纸上拍干。加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8min。加入终止液1M HCl终止显色反应，100μL/孔。在酶标仪上450nm处读数。抗体分子的ELISA结果分别如图13-16所示，结果显示，抗体TB-A1至TB-H均能与人B7H3蛋白结合。
实施例4构建抗体双靶点结合活性(TNFR2+B7H3)
用包被液(1×PBS，pH7.4)将人-B7H3Fc(厂家：Acro，CAT：B73-H5253，LOT：2053b-201XF1-UD)稀释为0.5μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。设置阴性对照，阴性对照为市售所得注射用曲妥珠单抗(赫赛汀)。300nM起始，以此为初始浓度进行3倍稀释，共稀释8个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。将人-TNFR2-His(厂家：SINO，CAT：10417-H08H，LOT：LC12SE2902)稀释至0.5μg/mL，每孔加入100μL，室温孵育1h，再用PBST洗板3次，而后将HRP标记的his抗体用样品稀释液按1:10000稀释，每孔加入100μL，室温孵育1h。洗板机洗涤6次，平板纸上拍干。加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8min。加入终止液1M HCl终止显色反应，100μL/孔。在酶标仪上450nm处读数。抗体分子的ELISA结果分别如图17-19所示，结果显示，抗体TB-A1至TB-H均具有两端结合活性。
实施例5构建抗体双靶点结合活性(TNFR2+B7H3)
用包被液(1×PBS，pH7.4)将人-TNFR2mFc(厂家：恺佧，CAT：TNF-HM3R2，LOT：031202)稀释为0.5μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。100nM起始，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。将人-B7H3His(厂家：Acro，CAT：TB73-H52E2，LOT：2052-21AXF1-YS)蛋白稀释至0.5μg/mL，每孔加入100μL，室温孵育1h，再用PBST洗板3次，而后将HRP标记的his抗体用样品稀释液按1:10000稀释，每孔加入100μL，室温孵育1h。洗板机洗涤6次，平板纸上拍干。加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8min。加入终止液1M HCl终止显色反应，100μL/孔。在酶标仪上450nm处读数。抗体分子的ELISA结果分别如图20所示，结果显示，抗体TB-A1、TB-D1和TB-C1均具有两端结合活性。
实施例6 CD8+T细胞激活实验(TNFR2+B7H3)
PBS稀释抗体至70nM(实验终浓度35nM，50μL/孔)；PBS稀释OKT3至10μg/mL(实验终浓度5μg/mL，50μL/孔)。50μLOKT3稀释液和50μL抗体稀释液，设置无关抗体，无关抗体为人IgG1kappa Isotype control(HG1K，Sino Biological)，设置阴性对照组，包被到96孔平底板中，总体积100μL/孔，4℃孵育过夜。第二天，从冻存PBMC(ORiCELLS，ID:PCH20210300033，分离效率为30％)中阴性分离CD8+T细胞，分选出的CD8+T细胞进行纯度检测；CFSE以5μM的浓度标记CD8+T细胞，37℃孵育10min，加入完全培养基终止染色。将前述用来包被的板用PBS清洗一次，以2×105/孔，100μL/孔加入CFSE标记或未标记的T细胞，加入终浓度为1μg/mL的Anti-CD28，100μL/孔。实验总体系为200μL/孔。37℃共孵育96h；离心后收集上清液，用于IFNγ、IL2细胞因子的检测，细胞使用PBS清洗细胞后进行流式检测。结果如表6所示，激动型抗体TB-A1、TB-B、TB-E、TB-F均对CD8+T细胞有激活作用。
表6 CD8+T细胞激活实验结果
实施例7 BLI方法检测抗体对TNFR2蛋白的亲和力(TNFR2+B7H3)
在使用前以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液浸润AHC传感器600s，除去传感器表面覆盖的蔗糖。AHC传感器以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液平衡60s，固化样品板中的抗体300s，二次平衡缓冲液180s。100nM的人-TNFR2-His(厂家：SINO，CAT：10417-H08H，LOT：LC12SE2902)蛋白与抗体结合300s，然后解离600s。解离后以10mM甘氨酸(pH2.0)作为再生缓冲液，再生30s。用10mM甘氨酸(pH2.0)再生传感器。抗体分子的亲和力结果如表7所示，结果显示，抗体TB-A1、TB-C1和TB-D1的TNFR2端的亲和力均为纳摩尔级别或者亚纳摩尔级别。
表7抗体对TNFR2蛋白的亲和力
实施例8 BLI方法检测抗体对B7H3蛋白的亲和力(TNFR2+B7H3)
在使用前以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液浸润AHC传感器600s，除去传感器表面覆盖的蔗糖。AHC传感器以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液平衡60s，固化样品板中的抗体300s，二次平衡缓冲液180s。100nM的人-B7H3His(厂家：Acro，CAT：TB73-H52E2，LOT：2052-21AXF1-YS)蛋白与抗体结合300s，然后解离600s。解离后以10mM甘氨酸(pH2.0)作为再生缓冲液，再生30s。用10mM甘氨酸(pH2.0)再生传感器。抗体分子的亲和力结果如表8所示，结果显示，抗体TB-A1、TB-C1和TB-D1均对B7H3蛋白有亲和力。
表8抗体对B7H3蛋白的亲和力
实施例9各候选受试物在hTNFR2小鼠皮下移植小鼠结肠癌细胞系MC38-hB7H3肿瘤模型中的药效学研究(TNFR2+B7H3)
选择6-7周龄的雌性C57BL/6-hTNFR2小鼠，分别皮下接种MC38-hB7H3肿瘤，待肿瘤体积约100±50mm3后随机分成四个小组。每组5只老鼠，分组包括：(1)G1：PBS组；(2)G2：对照抗体组，序列可变区由SEQ ID NO:83和SEQ ID NO:84组成，加入了人IgG1的恒定区(见SEQ ID NO:85和SEQ ID  NO:86)；(3)G3：抗体TB-C1组；(4)G4：抗体TB-C2组。阴性对照组采用PBS进行瘤内给药，其余组样品以10mg/kg进行瘤内给药。给药频次为每周2次，连续给药4周，共给药8次；隔天测量小鼠肿瘤体积与体重，肿瘤体积计算，按照a·b2/2(a为长径，b为短径)计算。实验方案设计见表9。
表9实验方案设计
结果如图21所示，从肿瘤生长曲线可以看出，抗体TB-C1和抗体TB-C2有明显的抗肿瘤作用。
实施例10 ELISA检测抗体对TNFR2蛋白的结合活性(TNFR2+CD24)
用包被液(1×PBS，pH7.4)将人-TNFR2-His(厂家：SINO，CAT：10417-H08H，LOT：LC12SE2902)稀释至0.3μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。设置阴性对照与阳性对照，阴性对照为市售所得注射用曲妥珠单抗(赫赛汀)，阳性对照1的抗体序列可变区由SEQ ID NO:83和SEQ ID NO:84组成，加入了人IgG1的恒定区(见SEQ ID NO:85和SEQ ID NO:86)；阳性对照2的抗体序列可变区由SEQ ID NO:41组成。将阳性对照和抗体用3％脱脂奶粉稀释为10μg/mL，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。用3％脱脂奶粉将山羊抗人IgG Fc按1:20000稀释，100μL/孔，37℃孵育1h。洗板机洗涤6次，平板纸上拍干。加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8min。加入终止液1M HCl终止显色反应，100μL/孔。在酶标仪上450nm处进行读数。抗体分子的ELISA结果分别如图22-25所示，结果显示抗体TD-A1至TD-H均能与人TNFR2蛋白结合。
实施例11流式细胞仪检测抗体对CD24蛋白的结合活性(TNFR2+CD24)
设置阳性对照，阳性对照的抗体序列可变区由SEQ ID NO:9和SEQ ID NO:13组成，加入人IgG1的恒定区(见SEQ ID NO:85和SEQ ID NO:86)，进行稀释，将阳性对照和待测抗体的实际终浓度设定为150nM，每孔100μL。将高表达人CD24的肿瘤细胞MCF-7(购自中科院细胞库，目录编号：TCHu74)清洗2遍，1×105/孔，以100μL/管加入96孔板中。阳性对照和待测抗体以50μL/孔依次加入96孔板中，空白对照组、阴性对照组加入等体积的稀释液。待混匀后，将96孔板置于4℃孵育1h。加入预冷的稀释液洗涤1次，离心(3000rpm，3min)，弃上清，收获细胞。取荧光二抗山羊抗人IgG Fc(厂家Biolegend，货号：398004，批号：B340957)，以1:500的使用浓度进行稀释。空白对照组加入100μL稀释液，其余样品组分别加入100μL荧光二抗稀释液。室温避光4℃孵育0.5h后，加入PBS清洗离心(3000rpm，3min)两次。弃去上清后，加入80μL的稀释液重悬。将各组的细胞依次进行流式检测，抗体分子的结果如表10所示，结果显示 抗体TD-A1至TD-H均能与高表达人CD24的MCF-7肿瘤细胞结合。
表10抗体TD-A1至TD-H对CD24蛋白的结合活性
实施例12构建抗体两端结合活性(TNFR2+CD24)
设置阴性对照组，阴性对照1(PBS组)，阴性对照2的抗体序列可变区由SEQ ID NO:83和SEQ ID NO:84组成，加入了人IgG1的恒定区(见SEQ ID NO:85和SEQ ID NO:86)；阴性对照3的抗体序列可变区由SEQ ID NO:9和SEQ ID NO:13组成，加入人IgG1的恒定区(SEQ ID NO:85和SEQ ID NO:86)。进行稀释，将阴性对照和待测抗体实际终浓度设定为150nM，每孔100μL。将高表达人CD24的肿瘤细胞MCF-7(购自中科院细胞库，目录编号：TCHu74)清洗2遍，1×105/孔，以100μL/管加入96孔板中。阴性对照和待测抗体以50μL/孔依次加入96孔板中，空白对照组、阴性对照组加入等体积的稀释液。将Human TNFR2蛋白(SINO，CAT：10417-H08H)以50μL/孔依次加入96孔板中，待混匀后，将96孔板置于4℃孵育1h。加入预冷的稀释液洗涤1次，离心(3000rpm，3min)，弃上清，收获细胞。取荧光二抗Anti-6X-his tag antibody(FITC)(厂家：abcam，货号：ab1206，批号：GR3420599-3)，以1:500的使用浓度进行稀释。空白对照组加入100μL稀释液，其余样品组分别加入100μL荧光二抗稀释液。室温避光4℃孵育0.5h后，加入PBS清洗离心(3000rpm，3min)两次。弃去上清后，加入80μL的稀释液重悬。将各组的细胞依次进行流式检测，抗体分子的结果如表11所示，结果显示：抗体TD-A1至TD-H均能同时结合TNFR2蛋白和高表达人CD24的MCF-7肿瘤细胞。
表11抗体两端结合活性
实施例13 BLI方法检测抗体对TNFR2蛋白的亲和力(TNFR2+CD24)
设置阳性对照组，阳性对照1的抗体序列可变区由SEQ ID NO:83和SEQ ID NO:84组成，加入了人IgG1的恒定区(见SEQ ID NO:85和SEQ ID NO:86)；阳性对照2的抗体序列可变区由SEQ ID NO:41组成。以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液浸润AHC传感器600s，除去传感器表面覆盖的蔗糖。AHC传感器以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液平衡60s，固化样品板中的抗体300s，二次平衡缓冲液180s。100nM的人-TNFR2-His(厂家：SINO，CAT：10417-H08H，LOT：LC12SE2902)蛋白与抗体结合300s，然后解离600s。解离后以10mM甘氨酸(pH2.0)作为再生缓冲液，再生30s。用10mM甘氨酸(pH2.0)再生传感器。抗体分子的亲和力结果如表12所示，结果显示，抗体TD-A1、TD-C、TD-D1、TD-G和TD-H的TNFR2端的亲和力均为纳摩尔级别或亚纳摩尔级别。
表12抗体对TNFR2蛋白的亲和力
实施例14 CD8+T细胞激活实验(TNFR2+CD24)
PBS稀释抗体至70nM(实验终浓度35nM，50μL/孔)；PBS稀释OKT3至10μg/mL(实验终浓度5μg/mL，50μL/孔)。设置无关抗体，无关抗体为人IgG1kappa Isotype control(HG1K，Sino Biological)，设置阴性对照组和阳性对照组，阳性对照的抗体序列可变区由SEQ ID NO:83和SEQ ID NO:84组成，加入了人IgG1的恒定区(见SEQ ID NO:85和SEQ ID NO:86)。50μLOKT3稀释液和50μL抗体稀释液，包被到96孔平底板中，总体积100μL/孔，4℃孵育过夜。第二天，从冻存PBMC(ORiCELLS，ID:PCH20210300033，分离效率为30％)中阴性分离CD8+T细胞，分选出的CD8+T细胞进行纯度检测；CFSE以5μM的浓度标记CD8+T细胞，37℃孵育10min，加入完全培养基终止染色。将前述用来包被的板用PBS清洗一次，以2×105/孔，100μL/孔加入CFSE标记或未标记的T细胞，加入终浓度为1μg/mL的抗-CD28，100μL/孔。实验总体系为200μL/孔。37℃共孵育96h；离心后收集上清液，用于IFNγ、IL2细胞因子的检测，细胞使用PBS清洗细胞后进行流式检测。结果如表13所示，抗体TD-A1对CD8+T细胞有激活作用。
表13 CD8+T细胞激活实验结果
实施例15抗体对MCF-7细胞的ADCC活性(TNFR2+CD24)
设置阴性对照(IgG1同型对照)和阳性对照(抗体序列可变区由SEQ ID  NO:9和SEQ ID NO:13组成，加入人IgG1的恒定区，见SEQ ID NO:85和SEQ ID NO:86)。使用过表达人CD24的MCF-7细胞(购自中科院细胞库，目录编号：TCHu74)作为靶细胞，1000rpm室温离心4分钟并使用RPMI1640基础培养基(含5％FBS)重悬后，以1×104/孔、50μL/孔铺于96孔板；使用RPMI1640基础培养基(含5％FBS)稀释抗体，起始浓度为120nM，后进行5倍梯度稀释，共8个浓度梯度，100μL/孔；重悬NK158V细胞，以50μL/孔加入对应孔中，效靶比为5:1。同时设置靶细胞最大裂解孔(M)、靶细胞自发释放孔(ST)、效应细胞自发释放孔(SE)、总体积校正空白孔(BV)和培养基空白对照孔(BM)。静置10分钟后，1000rpm室温离心4分钟，于5％CO2、37℃二氧化碳细胞培养箱中孵育4h。提前45分钟在M、BV孔加入20μL裂解液，混匀，孵育结束后1000rpm室温离心4分钟。吸取50μL上清至LDH分析板，加入50μL/孔分析缓冲液(assay buffer)溶解的底物，室温避光反应30分钟。加入50μL/孔终止液，静置10分钟，于490nm进行读数，计算细胞死亡率。
将抗体的浓度取对数后作为横坐标，选用Sigmoidaldose-response(VariableSlope)方式(GraphPadPrism软件，GraphPadSoftware，SanDiego，California)进行非线性回归，得到目标抗体对靶细胞的ADCC活性曲线。
由图26可知，阴性对照未显示对MCF-7细胞的杀伤，阳性对照、抗体TD-A1与抗体TD-D1显示对MCF-7细胞有明显的裂解死亡作用，且呈浓度依赖性。
实施例16抗体两端结合活性(TNFR2+Claudin18.2)
设置阴性对照组，阴性对照1(PBS组)，阴性对照2(抗体序列由SEQ ID NO:87和SEQ ID NO:88组成)、阴性对照3(抗体序列由SEQ ID NO:79和SEQ ID NO:71组成)、无关抗体(人IgG1kappa Isotype control，厂家：Sino Biological，货号：HG1K，批号：MA140C2803)，将阴性对照、无关抗体和待测抗体进行稀释，实际终浓度设定为150nM，每孔100μL。将高表达人Claudin18.2的肿瘤细胞CHOK1-18.2(P6-4-1)(购自中科院细胞库，目录编号：TCHu74)清洗2遍，1×105/孔，以100μL/管加入96孔板中。将阴性对照2、阴性对照3、无关抗体和待测抗体以50μL/孔依次加入96孔板中，空白对照组、阴性对照组加入等体积的稀释液。将Human TNFR2蛋白(SINO，CAT：10417-H08H)以100μL/孔依次加入96孔板中，待混匀后，将96孔板置于4℃孵育1h。加入预冷的稀释液洗涤1次，离心(3000rpm，3min)，弃上清。取荧光二抗Anti-6X-his tag antibody(FITC)(厂家：abcam，货号：ab1206，批号：GR3420599-3)，以1:500的使用浓度进行稀释。空白对照组加入100μL稀释液，其余样品组分别加入100μL荧光二抗稀释液。室温避光4℃孵育0.5h后，加入PBS清洗离心(3000rpm，3min)两次。弃去上清后，加入80μL的稀释液重悬。将各组的细胞依次进行流式检测。抗体分子的结果如表14所示，结果显示，所有抗体均能同时结合TNFR2蛋白和高表达人Claudin18.2的CHOK1-18.2肿瘤细胞。
表14抗体两端结合活性

本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。

Claims (51)

  1. 一种双特异性抗体，其特征在于，所述双特异性抗体包含：(a)特异性结合第一抗原的第一抗体或其抗原结合片段；和(b)特异性结合第二抗原的第二抗体或其抗原结合片段；所述第一抗原为肿瘤相关抗原(TAA)，并且所述第二抗原为TNFR2。
  2. 根据权利要求1所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段包含重链和轻链；和所述第二抗体或其抗原结合片段包含scFv或VHH。
  3. 根据权利要求1或2所述的双特异性抗体，其特征在于，所述第一抗体的一条重链的重链可变区与一条轻链的轻链可变区形成抗原结合部位，另一条重链的重链可变区与另一条轻链的轻链可变区形成抗原结合部位。
  4. 根据权利要求2或3所述的双特异性抗体，其特征在于，其包含一个第一抗体或其抗原结合片段和一个或多个所述的scFv或VHH。
  5. 根据权利要求4所述的双特异性抗体，其特征在于，其包含一个第一抗体或其抗原结合片段和一个所述的scFv或一个所述的VHH，所述scFv或VHH连接于所述第一抗体或其抗原结合片段的重链的N端或C端。
  6. 根据权利要求4所述的双特异性抗体，其特征在于，其包含一个第一抗体或其抗原结合片段和两个所述的scFv或两个所述的VHH。
  7. 根据权利要求6所述的双特异性抗体，其特征在于，两个所述的scFv或两个所述的VHH分别连接于所述第一抗体或其抗原结合片段的两条重链的N端或C端。
  8. 根据权利要求7所述的双特异性抗体，其中所述双特异性抗体包含两条第一多肽链和两条第二多肽链，其特征在于，对于所述每条多肽链：(a)所述第一多肽链各自独立地包含所述第一抗体或其抗原结合片段的轻链；和(b)所述第二多肽链各自独立地包含所述第一抗体或其抗原结合片段的重链和所述scFv或VHH。
  9. 根据权利要求8所述的双特异性抗体，其特征在于，两条第一多肽链相同或不同，和/或两条第二多肽链相同或不同。
  10. 根据权利要求1-9任一项所述的双特异性抗体，其特征在于，所述肿瘤相关抗原(TAA)选自GPC3、CD19、CD20(MS4A1)、CD22、CD24、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin18.2、AFP、ALK、B7H3、BAGE蛋白质、BCMA、BIRC5(存活素)、BIRC7、β-连环蛋白(β-catenin)、brc-ab1、BRCA1、BORIS、CA9、CA125、碳酸酐酶IX、半胱天冬酶-8(caspase-8)、CALR、CCR5、NA17、NKG2D、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、OX40、p15、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、PRAME、PSMA(FOLH1)、RAGE蛋白质、周期素-B1、CYP1B1、EGFR、EGFRvIII、ErbB2/Her2、ErbB3、ErbB4、ETV6-AML、EpCAM、EphA2、Fra-1、FOLR1、GAGE蛋白、GD2、GD3、GloboH、GM3、gp100、Her2、HLA/B-raf、HLA/k-ras、HLA/MAGE-A3、hTERT、IL13Rα2、LMP2、κ-Light、LeY、MAGE-1、MAGE-2、MAGE-3、MAGE-4、MAGE-6、MAGE-12、MART-1、间皮素、ML-IAP、MOv-γ、Muc1、Muc2、Muc3、Muc4、Muc5、Muc16、MUM1、Ras、RGS5、Rho、ROR1、SART-1、SART-3、STEAP1、STEAP2、TAG-72、TGF-β、TMPRSS2、汤-诺氏抗原、TRP-1、TRP-2、酪氨酸酶和尿溶蛋白-3、5T4。
  11. 根据权利要求10所述的双特异性抗体，其特征在于，所述肿瘤相关抗原(TAA)选自B7H3、CD24或Claudin18.2。
  12. 根据权利要求2-11任一项所述的双特异性抗体，其特征在于，所述scFv的重 链可变区与轻链可变区通过连接子L1连接。
  13. 根据权利要求2-12任一项所述的双特异性抗体，其特征在于，所述scFv或VHH通过连接子L2与所述第一抗体或其抗原结合片段的重链的N端或C端连接。
  14. 根据权利要求13所述的双特异性抗体，其特征在于，所述连接子L1和连接子L2相同或不同。
  15. 根据权利要求12-14任一项所述的双特异性抗体，其特征在于，所述连接子L1和/或连接子L2具有如(G4S)x所示的氨基酸序列，x为选自1-6的整数；优选地，所述连接子L1和/或连接子L2为(G4S)2、(G4S)3或(G4S)4。
  16. 根据权利要求1-15任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段的重链包含重链可变区和重链恒定区，并且所述轻链包含轻链可变区和轻链恒定区；优选地，所述第一抗体或其抗原结合片段为全长抗体。
  17. 根据权利要求16所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段的重链包含第一Fc区和第二Fc区。
  18. 根据权利要求17所述的双特异性抗体，其特征在于，第一Fc区和第二Fc区是相同的或不同的。
  19. 根据权利要求17或18所述的双特异性抗体，其特征在于，所述第一Fc区或第二Fc区选自IgG、IgA、IgD、IgE、IgM或其变体。
  20. 根据权利要求19所述的双特异性抗体，其特征在于，所述第一Fc区或第二Fc区选自IgG1、IgG2、IgG3、IgG4或其变体。
  21. 根据权利要求19或20所述的双特异性抗体，其特征在于，所述第一Fc区或第二Fc区包含一个或多个氨基酸突变，优选氨基酸置换、插入或缺失。
  22. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合B7H3，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:2所示，或为与SEQ ID NO:2具有至少80％同一性的序列；HCDR2如SEQ ID NO:3所示，或为与SEQ ID NO:3具有至少80％同一性的序列；HCDR3如SEQ ID NO:4所示，或为与SEQ ID NO:4具有至少80％同一性的序列；LCDR1如SEQ ID NO:6所示，或为与SEQ ID NO:6具有至少80％同一性的序列；LCDR2如SEQ ID NO:7所示，或为与SEQ ID NO:7具有至少80％同一性的序列；LCDR3如SEQ ID NO:8所示，或为与SEQ ID NO:8具有至少80％同一性的序列。
  23. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合CD24，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:10所示，或为与SEQ ID NO:10具有至少80％同一性的序列；HCDR2如SEQ ID NO:11所示，或为与SEQ ID NO:11具有至少80％同一性的序列；HCDR3如SEQ ID NO:12所示，或为与SEQ ID NO:12具有至少80％同一性的序列；LCDR1如SEQ ID NO:14所示，或为与SEQ ID NO:14具有至少80％同一性的序列；LCDR2如SEQ ID NO:15所示，或为与SEQ ID NO:15具有至少80％同一性的序列；LCDR3如SEQ ID NO:16所示，或为与SEQ ID NO:16具有至少80％同一性的序列。
  24. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:18所示，或为与SEQ ID NO:18具有至少80％同一性的序列；HCDR2如SEQ ID NO:19所示，或为与SEQ ID NO:19具有至少80％ 同一性的序列；HCDR3如SEQ ID NO:20所示，或为与SEQ ID NO:20具有至少80％同一性的序列；LCDR1如SEQ ID NO:22所示，或为与SEQ ID NO:22具有至少80％同一性的序列；LCDR2如SEQ ID NO:23所示，或为与SEQ ID NO:23具有至少80％同一性的序列；LCDR3如SEQ ID NO:24所示，或为与SEQ ID NO:24具有至少80％同一性的序列。
  25. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片段的HCDR1如SEQ ID NO:26所示，或为与SEQ ID NO:26具有至少80％同一性的序列；HCDR2如SEQ ID NO:27所示，或为与SEQ ID NO:27具有至少80％同一性的序列；HCDR3如SEQ ID NO:28所示，或为与SEQ ID NO:28具有至少80％同一性的序列；LCDR1如SEQ ID NO:30所示，或为与SEQ ID NO:30具有至少80％同一性的序列；LCDR2如SEQ ID NO:31所示，或为与SEQ ID NO:31具有至少80％同一性的序列；LCDR3如SEQ ID NO:32所示，或为与SEQ ID NO:32具有至少80％同一性的序列。
  26. 根据权利要求22-25任一项所述的双特异性抗体，其特征在于，所述scFv特异性结合TNFR2，所述scFv的HCDR1如SEQ ID NO:34所示，或为与SEQ ID NO:34具有至少80％同一性的序列；HCDR2如SEQ ID NO:35所示，或为与SEQ ID NO:35具有至少80％同一性的序列；HCDR3如SEQ ID NO:36所示，或为与SEQ ID NO:36具有至少80％同一性的序列；以及，LCDR1如SEQ ID NO:38所示，或为与SEQ ID NO:38具有至少80％同一性的序列；LCDR2如SEQ ID NO:39所示，或为与SEQ ID NO:39具有至少80％同一性的序列；LCDR3如SEQ ID NO:40所示，或为与SEQ ID NO:40具有至少80％同一性的序列。
  27. 根据权利要求22-25任一项所述的双特异性抗体，其特征在于，所述VHH特异性结合TNFR2，所述VHH的HCDR1如SEQ ID NO:42所示，或为与SEQ ID NO:42具有至少80％同一性的序列；HCDR2如SEQ ID NO:43所示，或为与SEQ ID NO:43具有至少80％同一性的序列；HCDR3如SEQ ID NO:44所示，或为与SEQ ID NO:44具有至少80％同一性的序列。
  28. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合B7H3，其中，所述第一抗体或其抗原结合片段的重链可变区VH如SEQ ID NO:1所示，或为与SEQ ID NO:1具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:5所示，或为与SEQ ID NO:5具有至少80％同一性的序列。
  29. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合CD24，其中，所述第一抗体或其抗原结合片段的重链可变区VH如SEQ ID NO:9所示，或为与SEQ ID NO:9具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:13所示，或为与SEQ ID NO:13具有至少80％同一性的序列。
  30. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片段的重链可变区VH如SEQ ID NO:17所示，或为与SEQ ID NO:17具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:21所示，或为与SEQ ID NO:21具有至少80％同一性的序列。
  31. 根据权利要求1-21任一项所述的双特异性抗体，其特征在于，所述第一抗体或其抗原结合片段特异性结合Claudin18.2，其中，所述第一抗体或其抗原结合片 段的重链可变区VH如SEQ ID NO:25所示，或为与SEQ ID NO:25具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:29所示，或为与SEQ ID NO:29具有至少80％同一性的序列。
  32. 根据权利要求28-31任一项所述的双特异性抗体，其特征在于，所述scFv特异性结合TNFR2，所述scFv重链可变区VH如SEQ ID NO:33所示，或为与SEQ ID NO:33具有至少80％同一性的序列；轻链可变区VL如SEQ ID NO:37所示，或为与SEQ ID NO:37具有至少80％同一性的序列。
  33. 根据权利要求28-31任一项所述的双特异性抗体，其特征在于，所述VHH特异性结合TNFR2，所述VHH重链可变区VH如SEQ ID NO:41所示，或为与SEQ ID NO:41具有至少80％同一性的序列。
  34. 根据权利要求8-33任一项所述的双特异性抗体，其特征在于，所述的双特异性抗体的第一多肽链选自SEQ ID NO:46-50、59-63、71-76的任一氨基酸序列，或为与SEQ ID NO:46-50、59-63、71-76的任一氨基酸序列具有至少80％同一性的氨基酸序列；所述双特异性抗体的第二多肽链选自SEQ ID NO:51-58、64-70、77-82的任一氨基酸序列，或为与SEQ ID NO:51-58、64-70、77-82的任一氨基酸序列具有至少80％同一性的氨基酸序列。
  35. 根据权利要求34所述的双特异性抗体，其特征在于，所述的双特异性抗体包含：
    (1)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:51所示的第二多肽链；或
    (2)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:56所示的第二多肽链；或
    (3)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:53所示的第二多肽链；或
    (4)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:54所示的第二多肽链；或
    (5)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:57所示的第二多肽链；或
    (6)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:55所示的第二多肽链；或
    (7)如SEQ ID NO:46所示的第一多肽链，如SEQ ID NO:58所示的第二多肽链；或
    (8)如SEQ ID NO:47所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
    (9)如SEQ ID NO:50所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
    (10)如SEQ ID NO:48所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
    (11)如SEQ ID NO:49所示的第一多肽链，如SEQ ID NO:52所示的第二多肽链；或
    (12)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:64所示的第二多肽链；或
    (13)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:69所示的第二多肽链；或
    (14)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:66所示的第二多肽链；或
    (15)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:67所示的第二多肽链；或
    (16)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:68所示的第二多肽链；或
    (17)如SEQ ID NO:59所示的第一多肽链，如SEQ ID NO:70所示的第二多肽链；或
    (18)如SEQ ID NO:60所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
    (19)如SEQ ID NO:61所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
    (20)如SEQ ID NO:62所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
    (21)如SEQ ID NO:63所示的第一多肽链，如SEQ ID NO:65所示的第二多肽链；或
    (22)如SEQ ID NO:71所示的第一多肽链，如SEQ ID NO:77所示的第二多肽链；或
    (23)如SEQ ID NO:72所示的第一多肽链，如SEQ ID NO:78所示的第二多肽链；或
    (24)如SEQ ID NO:71所示的第一多肽链，如SEQ ID NO:81所示的第二多肽链；或
    (25)如SEQ ID NO:72所示的第一多肽链，如SEQ ID NO:82所示的第二多肽链；或
    (26)如SEQ ID NO:73所示的第一多肽链，如SEQ ID NO:79所示的第二多肽链；或
    (27)如SEQ ID NO:74所示的第一多肽链，如SEQ ID NO:80所示的第二多肽链；或
    (28)如SEQ ID NO:75所示的第一多肽链，如SEQ ID NO:79所示的第二多肽链；或
    (29)如SEQ ID NO:76所示的第一多肽链，如SEQ ID NO:80所示的第二多肽链。
  36. 一种分离的核酸分子，其包含编码权利要求1-35任一项所述的双特异性抗体的核苷酸序列；优选地，所述分离的核酸分子包含编码权利要求8-35任一项所述的双特异性抗体的第一多肽链的核苷酸序列；优选地，所述分离的核酸分子包含编码权利要求8-35任一项所述的双特异性抗体的第二多肽链的核苷酸序列。
  37. 一种多功能融合蛋白，其包含权利要求1-35任一项所述的双特异性抗体。
  38. 根据权利要求37所述的多功能融合蛋白，其特征在于，还包含一个或多个与其他抗原特异性结合的第三抗体或其抗原结合部分。
  39. 根据权利要求38所述的多功能融合蛋白，其特征在于，所述结合第三抗体或其抗原结合部分的抗原选自肿瘤相关抗原(TAA)或免疫检查点。
  40. 根据权利要求39所述的多功能融合蛋白，其特征在于，所述结合第三抗体或其抗原结合部分的抗原选自GPC3、CD19、CD20(MS4A1)、CD22、CD24、CD30、CD33、CD38、CD40、CD123、CD133、CD138、CDK4、CEA、Claudin18.2、AFP、 ALK、B7H3、BAGE蛋白质、BCMA、BIRC5(存活素)、BIRC7、β-连环蛋白(β-catenin)、brc-ab1、BRCA1、BORIS、CA9、CA125、碳酸酐酶IX、半胱天冬酶-8(caspase-8)、CALR、CCR5、NA17、NKG2D、NY-BR1、NY-BR62、NY-BR85、NY-ESO1、OX40、p15、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、PRAME、PSMA(FOLH1)、RAGE蛋白质、周期素-B1、CYP1B1、EGFR、EGFRvIII、ErbB2/Her2、ErbB3、ErbB4、ETV6-AML、EpCAM、EphA2、Fra-1、FOLR1、GAGE蛋白、GD2、GD3、GloboH、GM3、gp100、Her2、HLA/B-raf、HLA/k-ras、HLA/MAGE-A3、hTERT、IL13Rα2、LMP2、κ-Light、LeY、MAGE-1、MAGE-2、MAGE-3、MAGE-4、MAGE-6、MAGE-12、MART-1、间皮素、ML-IAP、MOv-γ、Muc1、Muc2、Muc3、Muc4、Muc5、Muc16、MUM1、Ras、RGS5、Rho、ROR1、SART-1、SART-3、STEAP1、STEAP2、TAG-72、TGF-β、TMPRSS2、汤-诺氏抗原、TRP-1、TRP-2、酪氨酸酶和尿溶蛋白-3、5T4、PD-L1、CTLA4、PD-L2、PD-1、4-1BB、CD47、TIGIT、GITR、TIM3、ILT4、TREM2、LAG3、CD27或B7H4。
  41. 根据权利要求37-40任一项所述的多功能融合蛋白，其特征在于，还包含细胞因子。
  42. 根据权利要求41所述的多功能融合蛋白，其特征在于，所述细胞因子选自IL-1、IL-2、IL-2 Rα、IL-2 Rβ、IL-3、IL-3 Rα、IL-4、IL-4 Rα、IL-5、IL-5 Rα、IL-6、IL-6 Rα、IL-7、IL-7 Rα、IL-8、IL-9、IL-9 Rα、IL-10、IL-10R1、IL-10R2、IL-11、IL-11 Rα、IL-12、IL-12 Rα、IL-12 Rβ2、IL-12 Rβ1、IL-13、IL-13 Rα、IL-13 Rα2、IL-14、IL-15、IL-15Rαsushi、IL-16、IL-17、IL-18、IL-19、IL-20、IL-20R1、IL-20R2、IL-21、IL-21 Rα、IL-22、IL-23、IL-23R、IL-27 R、IL-31 R、TGF、VEGF、IFNγ、IFNα或GM-CSF。
  43. 权利要求1-35任一项所述的双特异性抗体或权利要求37-42任一项所述的多功能融合蛋白在制备治疗癌症的药物中的用途。
  44. 根据权利要求43所述的用途，其特征在于，所述癌症选自人脑星形胶质母细胞瘤、人咽头癌、肾上腺肿瘤、AIDS-相关癌症、腺泡状软组织肉瘤、星形细胞瘤、膀胱癌、骨癌、脑和脊髓癌、转移性脑瘤、乳腺癌、颈动脉体瘤、宫颈癌、软骨肉瘤、脊索瘤、肾嫌色细胞癌、透明细胞癌、结肠癌、结肠直肠癌、促结缔组织增生性小圆细胞肿瘤、室管膜细胞瘤、尤文肿瘤、骨外黏液样软骨肉瘤、骨纤维发育不全、骨纤维性发育不良、胆囊或胆管癌、胃癌、妊娠滋养细胞病、生殖细胞瘤、头颈癌、肝细胞癌、胰岛细胞瘤、卡波西肉瘤、肾癌、白血病、脂肪肉瘤/恶性脂肪瘤性肿瘤、肝癌、淋巴瘤、肺癌、成神经管细胞瘤、黑色素瘤、脑膜瘤、多发性内分泌瘤病、多发性骨髓瘤、骨髓增生异常综合征、成神经细胞瘤、神经内分泌肿瘤、卵巢癌、胰腺癌、乳头状甲状腺癌、甲状旁腺瘤、小儿癌症、外周神经鞘瘤、嗜铬细胞瘤、垂体肿瘤、前列腺癌、后葡萄膜黑色素瘤、肾转移性癌、横纹肌样瘤、横纹肌肉瘤、肉瘤、皮肤癌、软组织肉瘤、鳞状细胞癌、滑膜肉瘤、睾丸癌、胸腺癌、胸腺瘤、甲状腺转移性癌或子宫癌。
  45. 权利要求1-35任一项所述的双特异性抗体或权利要求37-42任一项所述的多功能融合蛋白在制备用于治疗自身免疫性疾病的药物中的用途。
  46. 根据权利要求45所述的用途，其特征在于，所述自身免疫性疾病选自移植物抗宿主病、类风湿性关节炎、克罗恩病、多发性硬化症、结肠炎、牛皮癣、自身免疫性葡萄膜炎、天疱疮、大疱性表皮松解症或I型糖尿病。
  47. 根据权利要求43-46任一项所述的用途，其特征在于，所述用途通过肿瘤免疫 疗法、细胞疗法或基因疗法中的一种或多种来实现。
  48. 一种药物组合物，其包含权利要求1-35任一项所述的双特异性抗体和药学上可接受的载体、稀释剂或赋形剂。
  49. 一种药物组合物，其包含权利要求37-42任一项所述的多功能融合蛋白和药学上可接受的载体、稀释剂或赋形剂。
  50. 一种抗体药物偶联物，其包含权利要求1-35任一项所述的双特异性抗体。
  51. 根据权利要求50所述的抗体药物偶联物，其特征在于，所述偶联药物选自细胞毒素、小分子化学药物或免疫毒素。
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