WO2021238932A1 - 多特异性抗体及其应用 - Google Patents

多特异性抗体及其应用 Download PDF

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WO2021238932A1
WO2021238932A1 PCT/CN2021/095852 CN2021095852W WO2021238932A1 WO 2021238932 A1 WO2021238932 A1 WO 2021238932A1 CN 2021095852 W CN2021095852 W CN 2021095852W WO 2021238932 A1 WO2021238932 A1 WO 2021238932A1
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seq
sequence shown
sequence
antigen
amino acid
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PCT/CN2021/095852
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English (en)
French (fr)
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黄俊杰
苏紫琪
梁世忠
徐振前
梁炳辉
李胜峰
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百奥泰生物制药股份有限公司
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Priority to CN202180037470.3A priority Critical patent/CN115803343A/zh
Publication of WO2021238932A1 publication Critical patent/WO2021238932A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to multispecific antibodies and their applications.
  • the current powerful tools for cancer immunotherapy include monoclonal antibodies, tumor vaccines, immune checkpoint inhibitors, CAR-T cell immunotherapy, bispecific antibodies (BsAb), and multispecific antibodies.
  • CAR-T and BsAb have received more and more attention as new strategies for anti-tumor immunotherapy.
  • Bispecific antibody is an artificially engineered antibody that can simultaneously bind to two specific epitopes or target proteins, and it can perform some special biological functions. Compared with the combination therapy of two monoclonal antibody drugs, BsAb improves antibody selectivity and functionality, and reduces the cost of treatment.
  • the preparation of BsAb mainly includes chemical coupling, two-hybridoma cell method, recombinant gene preparation and other methods.
  • the present invention provides multivalent and multispecific antibodies or antigen-binding fragments and applications thereof.
  • the invention provides bispecific antibodies or antigen-binding fragments.
  • the antibody or antigen-binding fragment provided by the present invention can bind two or more antigens, or two or more epitopes of the same antigen, or two or more copies of the same epitope.
  • the antibodies or antigen-binding fragments provided by the present invention are used to treat or improve inflammatory diseases, autoimmune diseases, cancer or spinal cord injury; the antibodies or antigen-binding fragments provided by the present invention are also used for the diagnosis and prognosis of related diseases.
  • the antibody or antigen-binding fragment binds to two different epitopes, the first epitope x and the second epitope y, and the antibody or antigen-binding fragment comprises at least two polypeptide chains; wherein , The first polypeptide chain contains VHa, CLa, VHb and CH1 sequentially from the amino terminal.
  • VHa is the variable region of the heavy chain that binds to the first epitope x
  • CLa is the constant region of the first light chain
  • VHb is the first constant region of the light chain.
  • the variable region of the heavy chain bound by the two epitope y, CH1 is the first constant region of the heavy chain.
  • first epitope x and the second epitope y are respectively different antigens, epitopes on the first antigen a and the second antigen b. In some embodiments, the first epitope x and the second epitope y are different epitopes on the same antigen a (or antigen b).
  • the antibody or antigen-binding fragment binds to two different antigens, the first antigen a and the second antigen b, and the antibody or antigen-binding fragment comprises at least two polypeptide chains; wherein, the first polypeptide The chain contains VHa, CLa, VHb and CH1 in sequence starting from the amino terminal.
  • VHa is the variable region of the heavy chain that binds to the first antigen a
  • CLa is the constant region of the first light chain
  • VHb is the heavy chain that binds to the second antigen b.
  • Variable region, CH1 is the first constant region of the heavy chain.
  • VHa and CLa are covalently linked via a linker L1, which contains 2 to 6 amino acids; and/or
  • CLa and VHb are covalently linked through a linker L2; where L2 contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine.
  • VHa and CLa are covalently linked through a linker L1
  • L1 contains 2 to 6 amino acids
  • CLa and VHb are covalently linked through a linker L2
  • L2 contains 10 to 30 amino acids, and at least 50% The amino acid is glycine.
  • L1 contains about 2, about 3, about 4, about 5, or about 6 amino acids.
  • L2 contains about 10, about 11, about 13, about 14, about 17, about 18, about 20, about 21, about 22, about 25, about 27, about 28, about 29, about 30 Amino acid, or the range between any two of these values (including the end point) or any value therein.
  • L2 contains serine.
  • L2 contains about 8, about 10, about 11, about 13, about 15, about 17, about 21, about 25 glycine, or any two of these values The range between (including the end point) or any value in it.
  • the second polypeptide chain sequentially includes VLa, CH1, VLb, and CLb starting from the amino terminus.
  • VLa is the light chain variable region that binds to the first epitope x
  • VLb is the light chain variable region that binds to the first epitope x.
  • y binds the variable region of the light chain
  • CLb is the second constant region of the light chain.
  • the first epitope x and the second epitope y are respectively different antigens, epitopes on the first antigen a and the second antigen b.
  • the first epitope x and the second epitope y are different epitopes on the same antigen a (or antigen b).
  • the second polypeptide chain includes VLa, CH1, VLb, and CLb sequentially from the amino terminal; wherein, VLa is the light chain variable region that binds to the first antigen a, and VLb is the light chain variable region that binds to the first antigen a. b binds the variable region of the light chain, CLb is the second constant region of the light chain.
  • VLa and CH1 are covalently linked through a linker L3, which contains 2 to 6 amino acids; and/or
  • CH1 and VLb are covalently linked through a linker L4; wherein, L4 contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine.
  • VLa and CH1 are covalently linked through a linker L3, and L3 contains 2 to 6 amino acids; CH1 and VLb are covalently linked through a linker L4; wherein, L4 contains 10 to 30 amino acids, and at least 50% of the amino acid is glycine.
  • L3 contains about 2, about 3, about 4, about 5, or about 6 amino acids.
  • L4 contains about 10, about 11, about 13, about 14, about 17, about 18, about 20, about 21, about 22, about 25, about 27, about 28, about 29, about 30 Amino acid, or the range between any two of these values (including the end point) or any value therein.
  • L4 contains serine.
  • L4 contains about 8, about 10, about 11, about 13, about 14, about 18, about 21, about 22 glycines, or any two of these values The range between (including the end point) or any value in it.
  • one of the two polypeptide chains further includes an Fc, which includes the hinge region, the second constant region, and the third constant region of the heavy chain.
  • the Fc is a variant Fc region.
  • the variant Fc region has one or more amino acid modifications, such as substitutions, deletions, or insertions.
  • the amino acid modification of the Fc region changes the effector function activity relative to the activity of the parent Fc region.
  • the variant Fc region may have altered (ie, increased or decreased) antibody-dependent cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding .
  • the Fc region amino acid modification can change the affinity of the variant Fc region for Fc ⁇ R (Fc ⁇ receptor).
  • the first polypeptide chain comprises Fc.
  • the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1
  • the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb.
  • the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1-Fc
  • the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb.
  • the CLa of the first polypeptide chain and the CH1 of the second polypeptide chain are connected by a disulfide bond
  • the CH1 of the first polypeptide chain and the CLb of the second polypeptide chain are connected by a disulfide bond. connect.
  • the antigens a and b are cytokines, cytokine receptors, chemokines, chemokine receptors, or cell surface proteins.
  • antibodies or antigen-binding fragments can specifically bind to cytokines.
  • cytokines include IL-1 ⁇ (interleukin IL-1 ⁇ ), IL-1 ⁇ (interleukin IL-1 ⁇ ), IL-13 (interleukin IL-13), IL-5 (interleukin IL-5), TNF - ⁇ (Tumor Necrosis Factor ⁇ ), TNF- ⁇ and (Tumor Necrosis Factor ⁇ ), etc.
  • the antibody or antigen-binding fragment can specifically bind to immune checkpoint proteins.
  • the immune checkpoint protein includes TIM-3 (T cell immunoglobin domain and mucin domain-3), LAG-3 (lymphocyte activation gene-3 molecule), CTLA-4 (cytotoxic T lymphocyte) Related antigen), TIGIT (T cell Ig and ITIM domain), CD27 (Cluster of differentiation 27), OX40 (Tumor necrosis factor receptor superfamily member 4), ICOS (inducible costimulator), BTLA (B and T lymphocyte weakening factor) , PD-1 (programmed death receptor 1) and CD137 (cluster of differentiation 137) and so on.
  • TIM-3 T cell immunoglobin domain and mucin domain-3
  • LAG-3 lymphocyte activation gene-3 molecule
  • CTLA-4 cytotoxic T lymphocyte
  • TIGIT T cell Ig and ITIM domain
  • CD27 Cluster of differentiation 27
  • OX40 Tumor necrosis factor receptor superfamily member 4
  • ICOS inducible costimulator
  • BTLA B and T lymphocyte weakening factor
  • PD-1 programmee
  • the antibody or antigen-binding fragment can specifically bind to cell surface proteins, such as tumor cell surface protein PD-L1 (programmed death ligand 1), galectin 9, CD48 (cluster of differentiation 48), CD40 (Cluster of differentiation 40), CD70 (Cluster of differentiation 70), B7H3 (CD276, Cluster of differentiation 276), HVEM (Herpesvirus Entry Mediator), etc.
  • the antibody or antigen-binding fragment can bind to chemokines or chemokine receptors, such as CCL1, CCL3, CCL5, CCL7, CCL8, etc. in the CC chemokine subgroup.
  • antigens a and b are respectively selected from the group consisting of TIGIT and CTLA-4, OX40 and CTLA-4, TIGIT and PD-1, PD-L1 and CD47 (cluster of differentiation 47), TIGIT and OX40, VEGF (vascular endothelial growth factor) and cMET (encoded by the c-met proto-oncogene), VEGF and DLL4 (delta-like ligand 4), VEGF and HGF (hepatocyte growth factor), VEGF and ANGPT2 (angiogenin) 2), TfR (transferrin receptor, CD71) and CD20 (cluster of differentiation 20), PD-L1 and 4-1BB (CD137, a member of the tumor necrosis factor receptor superfamily), PSMA (prostate specific membrane antigen) And CD28 (costimulatory molecule), PD-1 and PD-L1, HER2 (human epidermal growth factor receptor 2) and 4-1BB, PD-1 and TIM-3,
  • the antigen a is TIGIT and the antigen b is CTLA-4. In some embodiments, the antigen a is OX40 and the antigen b is CTLA-4. In some embodiments, the antigen a is OX40 and the antigen b is TIGIT.
  • the antigen a is TIGIT and the antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:
  • the VHa contains the heavy chain CDR or heavy chain variable region disclosed in US20190100591A1 or US20180169239A1;
  • the VHb contains the heavy chain CDR or heavy chain variable region disclosed in CN1404876A or US9963508; and/or
  • the VLa contains the light chain CDR or light chain variable region disclosed in US20190100591A1 or US20180169239A1; and/or
  • the VLb contains the light chain CDR or light chain variable region disclosed in CN1404876A or US9963508.
  • the antigen a is TIGIT and the antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:
  • the VHa contains amino acids at positions 30-35 (VHaCDR1, SSYGMS), and/or amino acids at positions 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG), and/or amino acids at positions 99-108 in the sequence shown in SEQ ID NO:1 (VHaCDR3, LGTGTLGFAY) amino acid; and/or
  • the VHb contains amino acids 31-35 (VHbCDR1, SYTMH), and/or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG), and/or amino acids 99-107 in the sequence shown in SEQ ID NO: 2 (VHbCDR3, TGWLGPFDY); and/or
  • the VLa includes amino acids at positions 24-34 (VLaCDR1, KASQDVKTAVS), and/or amino acids at positions 50-56 (VLaCDR2, WASTRAT), and/or amino acids at positions 50-56 (VLaCDR2, WASTRAT) in the sequence shown in SEQ ID NO: 3 -97 amino acid (VLaCDR3, QQHYSTPWT); and/or
  • the VLb contains amino acids 24-35 (VLbCDR1, RASQSVGSSYLA), and/or amino acids 51-57 (VLbCDR2, GAFSRAT), and/or amino acids 90-98 in the sequence shown in SEQ ID NO: 4 (VLbCDR3, QQYGSSPWT).
  • the antigen a is TIGIT and the antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:
  • the VHa includes the sequence shown in SEQ ID NO:1, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO:1, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO:1
  • the VHb contains the sequence shown in SEQ ID NO: 2, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 2, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 2
  • the VLa contains the sequence shown in SEQ ID NO: 3, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 3, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 3
  • the VLb contains the sequence shown in SEQ ID NO: 4, has at least 80% identity with the sequence shown in SEQ ID NO: 4, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 4 Amino acid sequence of amino acid substitution;
  • the CLa contains the sequence shown in SEQ ID NO: 5, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 5, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 5 Amino acid sequence of amino acid substitution;
  • the CLb contains the sequence shown in SEQ ID NO: 6, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 6, or has one or more conservative sequences compared to the sequence shown in SEQ ID NO: 6 Amino acid sequence of amino acid substitution;
  • the CH1 contains the sequence shown in SEQ ID NO: 7, a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 7
  • the amino acid sequence of the amino acid substitution is the amino acid sequence of the amino acid substitution.
  • the antigen a is OX40 and the antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:
  • the VHa contains the heavy chain CDR or heavy chain variable region disclosed in CN101331150A1 or US20150307617A1;
  • the VHb contains the heavy chain CDR or heavy chain variable region disclosed in CN1404876A or US9963508; and/or
  • the VLa contains the light chain CDR or light chain variable region disclosed in CN101331150A1 or US20150307617A1; and/or
  • the VLb contains the light chain CDR or light chain variable region disclosed in CN1404876A or US99635.
  • the antigen a is OX40 and the antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:
  • the VHa contains amino acids 31-35 (VHaCDR1, SYGMH), and/or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG), and/or amino acids 99-111 in the sequence shown in SEQ ID NO: 8 (VHaCDR3, DNQDTSPDVGIDY); and/or
  • the VHb contains amino acids 31-35 (VHbCDR1, SYTMH), and/or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG), and/or amino acids 99-107 in the sequence shown in SEQ ID NO: 9 (VHbCDR3, TGWLGPFDY); and/or
  • the VLa contains amino acids 24-34 (VLaCDR1, RASQNISPFLN), and/or amino acids 50-56 (VLaCDR2, AAVGLQS), and/or 89th in the sequence shown in SEQ ID NO: 10 -97 amino acid (VLaCDR3, QQYTDYPLT); and/or
  • the VLb contains amino acids 24-35 (VLbCDR1, RASQSVGSSYLA), and/or amino acids 51-57 (VLbCDR2, GAFSRAT), and/or amino acids 90-98 in the sequence shown in SEQ ID NO: 11 (VLbCDR3, QQYGSSPWT).
  • the antibody or antigen-binding fragment comprises the following:
  • the VHa contains the sequence shown in SEQ ID NO: 8, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 8, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 8
  • the VHb contains the sequence shown in SEQ ID NO: 9, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 9, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 9
  • the VLa contains the sequence shown in SEQ ID NO: 10, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 10, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 10
  • the VLb contains the sequence shown in SEQ ID NO: 11, has at least 80% identity with the sequence shown in SEQ ID NO: 11, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 11 Amino acid sequence of amino acid substitution;
  • the CLa contains the sequence shown in SEQ ID NO: 12, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 12, or has one or more conservative sequences compared to the sequence shown in SEQ ID NO: 12 Amino acid sequence of amino acid substitution;
  • the CLb contains the sequence shown in SEQ ID NO: 13, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 13, or has one or more conservative sequences compared to the sequence shown in SEQ ID NO: 13 Amino acid sequence of amino acid substitution;
  • the CH1 contains the sequence shown in SEQ ID NO: 14, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 14, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 14
  • the amino acid sequence of the amino acid substitution is the amino acid sequence of the amino acid substitution.
  • the antigen a is OX40 and the antigen b is TIGIT, and the antibody or antigen-binding fragment comprises the following:
  • the VHa contains the heavy chain CDR or heavy chain variable region disclosed in CN101331150A1 or US20150307617A1;
  • the VHb contains the heavy chain CDR or heavy chain variable region disclosed in US20190100591A1 or US20180169239A1; and/or
  • the VLa contains the light chain CDR or light chain variable region disclosed in CN101331150A1 or US20150307617A1; and/or
  • the VLb contains the light chain CDR or light chain variable region disclosed in US20190100591A1 or US20180169239A1.
  • the antigen a is OX40 and the antigen b is TIGIT, and the antibody or antigen-binding fragment comprises the following:
  • the VHa contains amino acids 31-35 (VHaCDR1, SYGMH), and/or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG), and/or amino acids 99-111 in the sequence shown in SEQ ID NO: 15 (VHaCDR3, DNQDTSPDVGIDY); and/or
  • VHb contains amino acids at positions 30-35 (VHbCDR1, SSYGMS), and/or amino acids at positions 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG), and/or amino acids at positions 99-108 in the sequence shown in SEQ ID NO: 16 (VHbCDR3, LGTGTLGFAY) amino acid; and/or
  • the VLa contains amino acids 24-34 (VLaCDR1, RASQNISPFLN), and/or amino acids 50-56 (VLaCDR2, AAVGLQS), and/or 89th in the sequence shown in SEQ ID NO: 17, -97 amino acid (VLaCDR3, QQYTDYPLT); and/or
  • VLb contains amino acids 24-34 (VLbCDR1, KASQDVKTAVS), and/or amino acids 50-56 (VLbCDR2, WASTRAT), and/or amino acids 89-97 in the sequence shown in SEQ ID NO: 18 (VLbCDR3, QQHYSTPWT).
  • the antigen a is OX40 and the antigen b is TIGIT, and the antibody or antigen-binding fragment comprises the following:
  • the VHa contains the sequence shown in SEQ ID NO: 15, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 15, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 15
  • the VHb contains the sequence shown in SEQ ID NO: 16, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 16, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 16
  • the VLa contains the sequence shown in SEQ ID NO: 17, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 17, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 17
  • the VLb contains the sequence shown in SEQ ID NO: 18, a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 18, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 18 Amino acid sequence of amino acid substitution;
  • the CLa contains the sequence shown in SEQ ID NO: 19, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 19, or has one or more conservative sequences compared to the sequence shown in SEQ ID NO: 19 Amino acid sequence of amino acid substitution;
  • the CLb contains the sequence shown in SEQ ID NO: 20, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 20, or has one or more conservative sequences compared with the sequence shown in SEQ ID NO: 20 Amino acid sequence of amino acid substitution;
  • the CH1 contains the sequence shown in SEQ ID NO: 21, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 21, or has one or more conservative sequences compared to the sequence shown in SEQ ID NO: 21
  • the amino acid sequence of the amino acid substitution is not limited to: 21.
  • a sequence that is at least 80% identical is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, About 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99 % Identity, or the range (including the end point) between any two of these values, or any value therein.
  • one or more conservative amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, About 11, about 13, about 14, about 15 conservative amino acid substitutions, or the range (including the end point) between any two of these values, or any value therein.
  • the L1 contains the sequence shown in SEQ ID NO: 22;
  • the L2 contains a sequence selected from the sequence shown in any one of SEQ ID NO: 23-27, a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO: 23-27, or a sequence with SEQ ID Compared with the sequence shown in any one of NO: 23-27, an amino acid sequence having one or more conservative amino acid substitutions; and/or
  • the L3 contains the sequence shown in SEQ ID NO: 28; and/or
  • the L4 contains a sequence selected from the sequence shown in any one of SEQ ID NO: 29-33, a sequence having at least 90% identity with the sequence shown in any one of SEQ ID NO: 29-33, or a sequence with SEQ ID Compared with the sequence shown in any one of NO: 29-33, the amino acid sequence has one or more conservative amino acid substitutions.
  • the L2 contains the sequence shown in SEQ ID NO: 23, a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 23, or is compared with the sequence shown in SEQ ID NO: 23 An amino acid sequence with one or more conservative amino acid substitutions;
  • the L4 contains the sequence shown in SEQ ID NO: 29, a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 29, or a sequence with SEQ ID NO: Compared with the sequence shown in 29, an amino acid sequence with one or more conservative amino acid substitutions.
  • the L2 contains the sequence shown in SEQ ID NO: 24, a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 24, or is compared with the sequence shown in SEQ ID NO: 24 An amino acid sequence with one or more conservative amino acid substitutions;
  • the L4 contains the sequence shown in SEQ ID NO: 30, a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 30, or a sequence with SEQ ID NO: Compared with the sequence shown in 30, the amino acid sequence has one or more conservative amino acid substitutions.
  • the L2 contains the sequence shown in SEQ ID NO: 25, a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 25, or is compared with the sequence shown in SEQ ID NO: 25 An amino acid sequence with one or more conservative amino acid substitutions;
  • the L4 contains the sequence shown in SEQ ID NO: 31, a sequence with at least 90% identity with the sequence shown in SEQ ID NO: 31, or a sequence with SEQ ID NO: Compared with the sequence shown in 31, an amino acid sequence with one or more conservative amino acid substitutions.
  • the L2 contains the sequence shown in SEQ ID NO: 26, a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 26, or is compared with the sequence shown in SEQ ID NO: 26 An amino acid sequence with one or more conservative amino acid substitutions;
  • the L4 contains the sequence shown in SEQ ID NO: 32, a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 32, or a sequence with SEQ ID NO: Compared with the sequence shown in 32, the amino acid sequence has one or more conservative amino acid substitutions.
  • the L2 contains the sequence shown in SEQ ID NO: 27, a sequence that is at least 90% identical to the sequence shown in SEQ ID NO: 27, or is compared with the sequence shown in SEQ ID NO: 27 An amino acid sequence with one or more conservative amino acid substitutions;
  • the L4 contains the sequence shown in SEQ ID NO: 33, a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 33, or a sequence with SEQ ID NO: Compared with the sequence shown in 33, the amino acid sequence has one or more conservative amino acid substitutions.
  • a sequence that is at least 90% identical is about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, About 97% identity, about 98% identity, about 99% identity, or the range between any two of these values (including the end point) or any value therein.
  • the one or more conservative amino acid substitutions are about 1, about 2, about 3, about 4, or about 5 conservative amino acid substitutions.
  • the L1 contains the sequence shown in SEQ ID NO: 22
  • the L2 contains the sequence shown in SEQ ID NO: 23
  • the L3 contains the sequence selected from SEQ ID NO: 28
  • the L4 contains the sequence shown in SEQ ID NO:29.
  • the L1 contains the sequence shown in SEQ ID NO: 22
  • the L2 contains the sequence shown in SEQ ID NO: 24
  • the L3 contains the sequence shown in SEQ ID NO: 28
  • the L4 contains the sequence shown in SEQ ID NO: 30.
  • the L1 contains the sequence shown in SEQ ID NO: 22
  • the L2 contains the sequence shown in SEQ ID NO: 25
  • the L3 contains the sequence shown in SEQ ID NO: 28
  • the L4 contains the sequence shown in SEQ ID NO: 31.
  • the L1 contains the sequence shown in SEQ ID NO: 22
  • the L2 contains the sequence shown in SEQ ID NO: 26
  • the L3 contains the sequence shown in SEQ ID NO: 28.
  • L4 contains the sequence shown in SEQ ID NO: 32.
  • the L1 contains the sequence shown in SEQ ID NO: 22
  • the L2 contains the sequence shown in SEQ ID NO: 27
  • the L3 contains the sequence shown in SEQ ID NO: 28
  • the L4 contains the sequence shown in SEQ ID NO: 33.
  • the Fc contains the sequence shown in any one of SEQ ID NO: 34-36, and has a sequence that is at least 80% identical to the sequence shown in any one of SEQ ID NO: 34-36, Or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in any one of SEQ ID NO: 34-36.
  • a sequence that is at least 80% identical is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, About 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99 % Identity, or the range (including the end point) between any two of these values, or any value therein.
  • one or more conservative amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, About 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, about 25 conservative amino acid substitutions, or between any two of these values The range (including the end point) or any value in it.
  • the Fc contains the sequence shown in SEQ ID NO:34. In some embodiments, the Fc contains the sequence shown in SEQ ID NO:35. In some embodiments, the Fc contains the sequence shown in SEQ ID NO:36.
  • the present invention also provides an antibody or antigen-binding fragment that binds two different antigens TIGIT and CTLA-4; the antibody or antigen-binding fragment comprises:
  • SEQ ID NO: 1 in the sequence shown in amino acids 30-35 (VHaCDR1, SSYGMS), and/or amino acids 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG), and/or amino acids 99-108 (VHaCDR3, LGTGTLGFAY) ) Amino acid; and
  • amino acids 31-35 amino acids 31-35 (VHbCDR1, SYTMH), and/or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG), and/or amino acids 99-107 (VHbCDR3, TGWLGPFDY) );and / or
  • Amino acids 24-34 (VLaCDR1, KASQDVKTAVS), and/or amino acids 50-56 (VLaCDR2, WASTRAT), and/or amino acids 89-97 in the sequence shown in SEQ ID NO: 3 (VLaCDR3, QQHYSTPWT); and
  • amino acids 24-35 (VLbCDR1, RASQSVGSSYLA), and/or amino acids 51-57 (VLbCDR2, GAFSRAT), and/or amino acids 90-98 (VLbCDR3, QQYGSSPWT) ).
  • the antibody or antigen-binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain;
  • the first polypeptide chain contains amino acids 30-35 (VHaCDR1, SSYGMS), and/or amino acids 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG) in the sequence shown in SEQ ID NO:1, and/or 99th -108 amino acid (VHaCDR3, LGTGTLGFAY) amino acid; and
  • amino acids 31-35 amino acids 31-35 (VHbCDR1, SYTMH), and/or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG), and/or amino acids 99-107 (VHbCDR3, TGWLGPFDY) );and / or
  • the second polypeptide chain contains amino acids at positions 24-34 (VLaCDR1, KASQDVKTAVS), and/or amino acids at positions 50-56 (VLaCDR2, WASTRAT) in the sequence shown in SEQ ID NO: 3, and / Or amino acids 89-97 (VLaCDR3, QQHYSTPWT); and
  • amino acids 24-35 (VLbCDR1, RASQSVGSSYLA), and/or amino acids 51-57 (VLbCDR2, GAFSRAT), and/or amino acids 90-98 (VLbCDR3, QQYGSSPWT) ).
  • the present invention also provides an antibody or antigen-binding fragment that binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA- 4;
  • the antibody or antigen-binding fragment contains at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 37, and SEQ ID NO:
  • the sequence shown in 37 has a sequence with at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 37;
  • the second polypeptide chain contains SEQ ID NO: 38
  • the sequence shown is a sequence with at least 80% identity with the sequence shown in SEQ ID NO: 38, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 38; or
  • the first polypeptide chain contains the sequence shown in SEQ ID NO: 39, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 39, or has one sequence compared with the sequence shown in SEQ ID NO: 39 Or an amino acid sequence of multiple conservative amino acid substitutions;
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 40, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 40, or is identical to SEQ ID An amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in NO: 40; or
  • the first polypeptide chain contains the sequence shown in SEQ ID NO: 41, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 41, or has one sequence compared with the sequence shown in SEQ ID NO: 41 Or an amino acid sequence of multiple conservative amino acid substitutions;
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 42, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 42, or is identical to SEQ ID An amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in NO: 42; or
  • the first polypeptide chain contains the sequence shown in SEQ ID NO: 43, a sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 43, or has one sequence compared with the sequence shown in SEQ ID NO: 43 Or an amino acid sequence of multiple conservative amino acid substitutions;
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 44, a sequence that has at least 80% identity with the sequence shown in SEQ ID NO: 44, or is identical to SEQ ID Compared with the sequence shown in NO:44, the amino acid sequence has one or more conservative amino acid substitutions.
  • a sequence that is at least 80% identical is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, About 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99 % Identity, or the range (including the end point) between any two of these values, or any value therein.
  • one or more conservative amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, About 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, about 25, about 27, about 29, about 31, about 33 1, about 35, about 38, about 41, about 42, about 47, about 49 conservative amino acid substitutions, or the range between any two of these values (including the end point), or any value therein .
  • the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT, the second antigen b is CTLA-4, and the antibody or antigen
  • the binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 37, and the second polypeptide chain contains SEQ ID NO: 38 shown in the sequence.
  • the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT, the second antigen b is CTLA-4, and the antibody or antigen
  • the binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 39, and the second polypeptide chain contains SEQ ID NO: 40 shown in the sequence.
  • the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT, the second antigen b is CTLA-4, and the antibody or antigen
  • the binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 41, and the second polypeptide chain contains SEQ ID NO: 42 shown in the sequence.
  • the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT, the second antigen b is CTLA-4, and the antibody or antigen
  • the binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 43, and the second polypeptide chain contains SEQ ID NO: 44 shown in the sequence.
  • the present invention also provides an antibody or antigen-binding fragment that binds two different antigens OX40 and CTLA-4; the antibody or antigen-binding fragment comprises:
  • the 31-35 amino acids (VHaCDR1, SYGMH), and/or the 50-66 amino acids (VHaCDR2, VIAEVGSNQYYADSVKG), and/or the 99-111 amino acids (VHaCDR3, DNQDTSPDVGIDY) );
  • amino acids 31-35 amino acids 31-35 (VHbCDR1, SYTMH), and/or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG), and/or amino acids 99-107 (VHbCDR3, TGWLGPFDY) );and / or
  • Amino acids at positions 24-34 (VLaCDR1, RASQNISPFLN), and/or amino acids at positions 50-56 (VLaCDR2, AAVGLQS), and/or amino acids at positions 89-97 in the sequence shown in SEQ ID NO: 10 (VLaCDR3, QQYTDYPLT); and
  • amino acids 24-35 amino acids 24-35 (VLbCDR1, RASQSVGSSYLA), and/or amino acids 51-57 (VLbCDR2, GAFSRAT), and/or amino acids 90-98 (VLbCDR3, QQYGSSPWT) ).
  • the antibody or antigen-binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain;
  • the first polypeptide chain contains amino acids 31-35 (VHaCDR1, SYGMH), and/or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) in the sequence shown in SEQ ID NO: 8, and/or 99th -111 amino acid (VHaCDR3, DNQDTSPDVGIDY); and
  • amino acids 31-35 amino acids 31-35 (VHbCDR1, SYTMH), and/or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG), and/or amino acids 99-107 (VHbCDR3, TGWLGPFDY) );and / or
  • the second polypeptide chain contains amino acids at positions 24-34 (VLaCDR1, RASQNISPFLN), and/or amino acids at positions 50-56 (VLaCDR2, AAVGLQS) in the sequence shown in SEQ ID NO: 10, and / Or amino acids 89-97 (VLaCDR3, QQYTDYPLT); and
  • amino acids 24-35 amino acids 24-35 (VLbCDR1, RASQSVGSSYLA), and/or amino acids 51-57 (VLbCDR2, GAFSRAT), and/or amino acids 90-98 (VLbCDR3, QQYGSSPWT) ).
  • the present invention also provides an antibody or antigen-binding fragment that binds two different antigens, the first antigen a and the second antigen b, wherein the first antigen a is OX40 and the second antigen b is CTLA- 4;
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 45, and SEQ ID NO:
  • the sequence shown in 45 has a sequence with at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 45;
  • the second polypeptide chain contains SEQ ID NO: 46
  • the sequence shown is a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 46, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 46.
  • a sequence that is at least 80% identical is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, About 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99 % Identity, or the range (including the end point) between any two of these values, or any value therein.
  • one or more conservative amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, About 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, about 25, about 27, about 29, about 31, about 33 1, about 35, about 38, about 41, about 42, about 47, about 49 conservative amino acid substitutions, or the range between any two of these values (including the end point), or any value therein .
  • the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40, the second antigen b is CTLA-4, and the antibody or antigen
  • the binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 45, and the second polypeptide chain contains SEQ ID NO: 46 shown in the sequence.
  • the present invention also provides an antibody or antigen-binding fragment that binds two different antigens OX40 and TIGIT; the antibody or antigen-binding fragment comprises:
  • amino acids 31-35 amino acids 31-35 (VHaCDR1, SYGMH), and/or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG), and/or amino acids 99-111 (VHaCDR3, DNQDTSPDVGIDY) );
  • amino acids 30-35 amino acids 30-35 (VHbCDR1, SSYGMS), and/or amino acids 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG), and/or amino acids 99-108 (VHbCDR3, LGTGTLGFAY) ) Amino acid; and/or
  • Amino acids 24-34 (VLaCDR1, RASQNISPFLN), and/or amino acids 50-56 (VLaCDR2, AAVGLQS), and/or amino acids 89-97 in the sequence shown in SEQ ID NO:17 (VLaCDR3, QQYTDYPLT); and
  • amino acids at positions 24-34 (VLbCDR1, KASQDVKTAVS), and/or amino acids at positions 50-56 (VLbCDR2, WASTRAT), and/or amino acids at positions 89-97 (VLbCDR3, QQHYSTPWT) ).
  • the antibody or antigen-binding fragment includes at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain;
  • the first polypeptide chain contains amino acids 31-35 (VHaCDR1, SYGMH), and/or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) in the sequence shown in SEQ ID NO: 15, and/or 99th -111 amino acid (VHaCDR3, DNQDTSPDVGIDY); and
  • amino acids 30-35 amino acids 30-35 (VHbCDR1, SSYGMS), and/or amino acids 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG), and/or amino acids 99-108 (VHbCDR3, LGTGTLGFAY) ) Amino acid; and/or
  • the second polypeptide chain contains amino acids 24-34 (VLaCDR1, RASQNISPFLN), and/or amino acids 50-56 (VLaCDR2, AAVGLQS) in the sequence shown in SEQ ID NO: 17, and / Or amino acids 89-97 (VLaCDR3, QQYTDYPLT); and
  • amino acids at positions 24-34 (VLbCDR1, KASQDVKTAVS), and/or amino acids at positions 50-56 (VLbCDR2, WASTRAT), and/or amino acids at positions 89-97 (VLbCDR3, QQHYSTPWT) ).
  • the present invention also provides an antibody or antigen-binding fragment that binds two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is TIGIT;
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 47 or 49, and SEQ ID NO:
  • the sequence shown in 47 or 49 has a sequence with at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 47 or 49;
  • the second polypeptide chain contains SEQ The sequence shown in ID NO: 48 or 50, a sequence that has at least 80% identity with the sequence shown in SEQ ID NO: 48 or 50, or has one or more sequences compared with the sequence shown in SEQ ID NO: 48 or 50 Amino acid sequence of conservative amino acid substitution
  • a sequence that is at least 80% identical is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, About 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99 % Identity, or the range (including the end point) between any two of these values, or any value therein.
  • one or more conservative amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, About 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, about 25, about 27, about 29, about 31, about 33 1, about 35, about 38, about 41, about 42, about 47, about 49 conservative amino acid substitutions, or the range between any two of these values (including the end point), or any value therein .
  • the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is TIGIT, the antibody or antigen-binding fragment It contains at least two polypeptide chains: the first polypeptide chain and the second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 47, and the second polypeptide chain contains the sequence shown in SEQ ID NO: 48. Sequence shown.
  • the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is TIGIT, the antibody or antigen-binding fragment It includes at least two polypeptide chains: the first polypeptide chain and the second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 49, and the second polypeptide chain contains the sequence shown in SEQ ID NO: 50. Sequence shown.
  • the antibody or antigen-binding fragment contains a first polypeptide chain and a third polypeptide chain with the same sequence, and a second polypeptide and a fourth polypeptide chain with the same sequence. In some embodiments, the antibody or antigen-binding fragment contains a first polypeptide chain and a third polypeptide chain with the same sequence, and a second polypeptide and a fourth polypeptide chain with the same sequence. The first polypeptide chain Pairs with the Fc region of the third polypeptide chain to form a disulfide bond.
  • the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.
  • the present invention also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments.
  • the nucleic acid molecule is an isolated nucleic acid molecule.
  • the present invention also provides a vector containing the nucleic acid molecule.
  • the carrier is an isolated carrier.
  • the present invention also provides a host cell containing the nucleic acid molecule or vector.
  • the host cell is an isolated host cell.
  • the host cells are CHO cells, 293 cells, Cos1 cells, Cos7 cells, CV1 cells, and murine L cells.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned antibody or antigen-binding fragment, and pharmaceutically acceptable excipients.
  • the invention also provides treatment methods and uses.
  • a method for treating or ameliorating various diseases such as inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancer, or spinal cord injury
  • the method comprising administering to a patient an effective dose of The antibody or antigen-binding fragment.
  • there is provided the application of the antibody or antigen-binding fragment in the treatment or amelioration of various diseases such as inflammatory diseases, autoimmune diseases, cancer, or spinal cord injury.
  • the application of the antibody or antigen-binding fragment in the preparation of drugs for the treatment or amelioration of various diseases is provided.
  • the autoimmune disease or inflammatory disease is selected from the group consisting of Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid joints) Inflammation, psoriatic arthritis, osteoarthritis or juvenile idiopathic arthritis), multiple sclerosis, ankylosing spondylitis, spondylosing arthropathy, systemic lupus erythematosus, uveitis, sepsis, neurodegeneration Diseases, neuronal regeneration, spinal cord injury, primary and metastatic cancers, respiratory disorders, asthma, allergic and non-allergic asthma, asthma caused by infection, asthma caused by infection with respiratory syncytial virus (RSV), Chronic obstructive pulmonary disease (COPD), conditions involving airway inflammation, eosinophilia, fibrosis and excessive mucus production, cystic fibrosis, pulmonary fibrosis, atopic disorders, atopic dermatitis
  • RSV
  • the cancer is selected from the group consisting of hepatocellular carcinoma, glioblastoma, lymphoma, or Hodgkin's lymphoma.
  • the cancer is selected from the group consisting of: melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate Adenocarcinoma), pancreatic cancer, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, neuroglia Plasma, leukemia, lymphoma and other neoplastic malignant diseases.
  • melanoma e.g., metastatic malignant melanoma
  • kidney cancer e.g., clear cell carcinoma
  • prostate cancer e.g., hormone refractory prostate Aden
  • the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], precursor B-cell lymphoblastic leukemia/lymphoma Tumor, mature B-cell neoplasm, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell young lymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, skin follicular center Lymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt’s lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorders, metastatic disease Waldenstrom's macroglobulinemia and anaplastic large cell lymphoma.
  • NHL non-Hodgkin's lymphoma
  • NHL non-Hodgkin
  • the invention also provides diagnostic methods and uses.
  • a method for detecting the expression of antigen a and/or antigen b in a sample is provided, and the sample is contacted with the antibody or antigen-binding fragment, so that the antibody or antigen-binding fragment binds to antigen a and/or Antigen b, and detect its binding, that is, the content of antigen a and/or antigen b in the sample.
  • the antigens a and b are cytokines, cytokine receptors, chemokines, chemokine receptors, or cell surface proteins.
  • antigens a and b are selected from the group consisting of TIGIT and CTLA-4, OX40 and CTLA-4, TIGIT and PD-1, PD-L1 and CD47, TIGIT and OX40, VEGF and cMET, respectively.
  • VEGF and DLL4 VEGF and HGF, VEGF and ANGPT2, TfR and CD20, PD-L1 and 4-1BB, PSMA and CD28, PD-1 and PD-L1, HER2 and 4-1BB, PD-1 and TIM-3 , PD-1 and CD47, GITR and CTLA-4, CD40 and 4-1BB, OX40 and 4-1BB, LAG-3 and TIM-3, EGFR and CTLA-4, CD19 and CD22, CD16 and CD30, CD3 and CD123 , BCMA and CD47, MSLN and CD47, EGFR and cMET, CD73 and TGF ⁇ , EGFR and TGF ⁇ , CCR2 and CSF1R, CD20 and CD3, CD19 and CD47, CDH17 and TRAILR2, APLP2 and Her2, IL-1 ⁇ and IL-1 ⁇ , IL -17 and IL-13, IL-4 and IL-13, BAFF and IL-17A, CD3 and PD-1, IL-4
  • the application of the antibody or antigen-binding fragment in the preparation of a kit for diagnosing inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancer or spinal cord injury is provided.
  • a diagnostic kit comprising the antibody or antigen-binding fragment is provided.
  • the present invention provides multivalent and multispecific antibodies or antigen-binding fragments and applications thereof.
  • the multispecific antibodies or antigen-binding fragments of the present invention can bind two or more antigens, or two or more of the same antigen gauge.
  • the antibody or antigen-binding fragment of the present invention is used to treat or ameliorate various diseases, such as inflammatory diseases, autoimmune diseases, cancer, or spinal cord injury, as well as the diagnosis and prognosis of related diseases.
  • Figure 1 is a schematic diagram of the structure of the bispecific antibody of the present invention in some embodiments.
  • Figure 2 is an SDS-PAGE chart of antibody 1 in the example of the present invention; wherein, lane 1 indicates that antibody 1 is in a non-reduced state, lane M indicates maker, and lane 2 indicates that antibody 1 is in a reduced state.
  • Figure 3 is the SDS-PAGE chart of antibody 2 and antibody 3 in the example of the present invention; in the figure, CT-20BiAb represents antibody 2, CT-25BiAb represents antibody 3, lane 1 represents antibody 3 in a non-reduced state, and lane 2 represents antibody 2 In a non-reduced state, lane 3 indicates that antibody 3 is in a reduced state, lane 4 indicates that antibody 2 is in a reduced state, and M represents maker.
  • Figure 4 shows the binding activity of antibody 1, antibody 2, and antibody 3 to TIGIT-Fc; wherein CT BiAb represents antibody 1, CT-20BiAb represents antibody 2, and CT-25BiAb represents antibody 3.
  • Figure 5 shows the binding activity of antibody 1, antibody 2, and antibody 3 to CTLA-4-Fc; wherein CT BiAb represents antibody 1, CT-20BiAb represents antibody 2, and CT-25BiAb represents antibody 3.
  • Figure 6 shows the use of Promega's anti-CTLA-4 reporter gene detection system to detect the biological activity of antibodies; where CTLA-4/TIGIT BiAb represents antibody 1.
  • FIG. 7 shows the use of Promega's anti-CTLA-4 reporter gene detection system to detect the biological activity of antibodies; among them, CT-20BiAb represents antibody 2.
  • Figure 8 shows the use of anti-TIGIT reporter gene detection system to detect the biological activity of antibodies; where CT BiAb represents antibody 1.
  • Figure 9 shows the use of anti-TIGIT reporter gene detection system to detect the biological activity of antibodies; where CT-20BiAb represents antibody 2.
  • Figure 10 shows the use of anti-TIGIT reporter gene detection system to detect the biological activity of antibodies; where CT-25BiAb represents antibody 3.
  • Fig. 11 is an SDS-PAGE chart of antibody 5 (CTLA-4-OX40 bispecific antibody) in an embodiment of the present invention; where lane represents a lane, lane 1, lane 2, and lane 3 are arranged in order from left to right.
  • CTLA-4-OX40 bispecific antibody CTL-4-OX40 bispecific antibody
  • Figure 12 is the binding curve of antibody 5 and OX40 + cells in the example of the present invention
  • OX40 + cells are cells expressing OX40
  • the abscissa represents the logarithmic value of the antibody concentration
  • the ordinate represents the 2 channels read on a flow cytometer Average fluorescence value; among them, O4 represents antibody 5.
  • FIG. 13 is a fitting curve of the affinity kinetics of antibody 5 binding to OX40 antigen and CTLA-4 antigen in an embodiment of the present invention, where O4 represents antibody 5.
  • Figure 14 is the SDS-PAGE chart of antibodies 6 and 7 in the example of the present invention; wherein, lane M indicates maker, lane 1 indicates antibody 6 is in a non-reduced state, lane 2 indicates antibody 6 is in a reduced state, and lane 3 indicates antibody 7 is in a reduced state. In a non-reduced state, lane 4 indicates that antibody 7 is in a reduced state; OT-4D-13aa antibody indicates antibody 6, and OT-4D-30aa antibody indicates antibody 7.
  • Fig. 15 is a fitting curve of affinity kinetics of binding of antibody 7 to OX40 antigen and TIGIT antigen in an embodiment of the present invention.
  • Figure 16 is the binding curve of antibody 7 to Jurkat-OX40 cells in the example of the present invention; wherein, anti-OX40Ab represents OX40 monoclonal antibody (ie anti-OX40), and both OT-4D-30a and 4D-30a represent antibody 7 ;
  • the unit of EC50 in the figure is nM.
  • Fig. 17 is a binding curve of antibody 7 and Jurkat-TIGIT cells in an embodiment of the present invention; wherein, anti-Tigit Ab represents TIGIT monoclonal antibody (ie anti-TIGIT), and OT-4D-30a represents antibody 7.
  • anti-Tigit Ab represents TIGIT monoclonal antibody (ie anti-TIGIT)
  • OT-4D-30a represents antibody 7.
  • an entity refers to one or more of the entity, for example, "an antibody” should be understood as one or more antibodies, therefore, the term “an” (or “an” ), “one or more” and “at least one” can be used interchangeably herein.
  • compositions, method, etc. include the listed elements, such as components or steps, but do not exclude others.
  • Consisting essentially of means that the composition and method exclude other elements that have a fundamental influence on the characteristics of the combination, but does not exclude elements that do not substantially affect the composition or method.
  • Consisting of means excluding elements not specifically listed.
  • polypeptide is intended to encompass both singular “polypeptide” and plural “polypeptide”, and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also called peptide bonds).
  • polypeptide refers to any single chain or multiple chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, the definition of “polypeptide” includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", “amino acid chains” or any other term used to refer to two or more amino acid chains, and the term “polypeptide” can Used in place of any of the above terms, or used interchangeably with any of the above terms.
  • polypeptide is also meant to refer to the product of a modified polypeptide after expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-natural The amino acid modification that occurred.
  • the polypeptide can be derived from natural biological sources or produced by recombinant technology, but it does not need to be translated from a specified nucleic acid sequence, and it may be produced in any manner including chemical synthesis.
  • amino acid refers to an organic compound containing both amino and carboxyl groups, such as ⁇ -amino acids, which can be directly or in the form of precursors encoded by nucleic acids.
  • a single amino acid is encoded by a nucleic acid composed of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The same amino acid encoded by different codons is called “the degeneracy of the genetic code”.
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine Acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
  • Constant amino acid substitution refers to the substitution of an amino acid residue by another amino acid residue containing a side chain (R group) that is chemically similar (eg, charge or hydrophobicity). Generally speaking, conservative amino acid substitutions are unlikely to substantially change the functional properties of the protein.
  • amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threon Amino acid; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.
  • isolated used in the present invention with regard to cells, nucleic acids, polypeptides, antibodies, etc., for example, "isolated" DNA, RNA, polypeptides, and antibodies refer to other components in the cell's natural environment, such as DNA or RNA. Of one or more separated molecules.
  • isolated as used in the present invention also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA technology, or chemical precursors or other chemicals during chemical synthesis.
  • isolated nucleic acid is intended to include nucleic acid fragments that do not exist in a natural state, and do not exist in a natural state.
  • isolated is also used in the present invention to refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptides are meant to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are usually prepared through at least one purification step. In some embodiments, the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or some of these values. The range (including the end point) between any two values of, or any value in it.
  • recombinant refers to a polypeptide or polynucleotide, and means a form of a polypeptide or polynucleotide that does not exist in nature.
  • An unrestricted embodiment can be combined to produce polynucleotides or polynucleotides that do not normally exist. Peptides.
  • “Homology” or “identity” or “similarity” refers to the sequence similarity between two peptides or between two nucleic acid molecules.
  • the homology can be determined by comparing the positions that can be aligned in each sequence. When a position in the sequence being compared is occupied by the same base or amino acid, then the molecules are homologous at that position.
  • the degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
  • a polynucleotide or polynucleotide sequence has a certain percentage (for example, 90%, 95%, 98%, or 99%) of "identity or sequence identity" with another sequence It means that when the sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared is the same.
  • Visual inspection or software programs known in the art can be used to determine the alignment and percent identity or sequence identity, such as the software program described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. It is preferable to use the default parameters for comparison.
  • a biologically equivalent polynucleotide is a polynucleotide that has the aforementioned specified percentage of identity and encodes a polypeptide having the same or similar biological activity.
  • Polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or as a polynucleotide When it is RNA, thymine is replaced with uracil (U).
  • the "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter indicates that it can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymerized form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • genes or gene fragments such as probes, primers, EST or SAGE tags
  • exons introns
  • messenger RNA mRNA
  • transfer RNA ribose Body RNA
  • ribozyme cDNA
  • dsRNA siRNA
  • miRNA miRNA
  • recombinant polynucleotide branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe And primers.
  • Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • the structural modification of the nucleotide can be performed before or after the polynucleotide is assembled.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide can be further modified, for example, by conjugation with a labeling component.
  • This term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, embodiments of any polynucleotide of the present disclosure include a double-stranded form and each of two complementary single-stranded forms that are known or predicted to constitute a double-stranded form.
  • encoding refers to the polynucleotides referred to as “encoding” polypeptides, which have been transcribed and/ Or translation can produce the polypeptide and/or fragments thereof.
  • Antibody and antigen-binding fragment refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • the antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Therefore, the term “antibody” includes any protein or peptide whose molecule contains at least a part of an immunoglobulin molecule that has the biological activity of binding to an antigen.
  • Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), and heavy chain constant region of a heavy chain or light chain or its ligand binding portion (CH), light chain constant region (CL), framework region (FR) or any part thereof, or at least part of a binding protein.
  • the CDR region includes the CDR region of the light chain (VL CDR1-3) and the CDR region of the heavy chain (VH CDR1-3).
  • the antibody or antigen-binding fragment described in the embodiment of the present invention is a bispecific antibody, which is a fusion of fragments that specifically bind antigen a and antigen b: the first polypeptide chain includes VHa, CLa, VHb, CH1, and the first polypeptide The chain is similar to the heavy chain or heavy chain fragment of an immunoglobulin; the second polypeptide chain contains the structures VLa, CH1, VLb, and CLb, and the second polypeptide chain is similar to the light chain of an immunoglobulin.
  • antibody fragment refers to a part of an antibody.
  • the composition of the antibody fragment of the present invention can be similar to F(ab') 2 , F(ab) 2 , Fab', Fab in monospecific antibody fragments. , Fv, scFv, etc. Regardless of its structure, antibody fragments bind to the same antigen recognized by the intact antibody.
  • antibody fragment includes aptamers, spiegelmers and diabodies.
  • antigen-binding fragment also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.
  • Single chain variable fragment refers to a fusion protein of the variable regions of an immunoglobulin heavy chain (VH) and light chain (VL). In some aspects, these regions are linked to short linker peptides of 10 to about 25 amino acids.
  • the linker can be rich in glycine to increase flexibility, and rich in serine or threonine to increase solubility, and can connect the N-terminus of VH and the C-terminus of VL, and vice versa.
  • the protein has the constant region removed and the linker introduced, it retains the specificity of the original immunoglobulin.
  • ScFv molecules are generally known in the art, such as described in U.S. Patent No. 5,892,019.
  • antibody includes a wide variety of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that the types of heavy chains include gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ ), and there are some subclasses (for example, ⁇ 1- ⁇ 4). The nature of this chain determines the "class" of the antibody is IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and the functional specificity conferred is also known.
  • the immunoglobulin molecule is of the IgG class.
  • IgG usually contains two identical light chain polypeptides with a molecular weight of about 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000-70,000. These four chains are connected in a "Y" configuration by disulfide bonds, where the light chain starts at the "Y" port and continues to surround the heavy chain through the variable region.
  • the antibodies, antigen-binding fragments, or derivatives disclosed in the present invention include but are not limited to polyclonal, monoclonal, multispecific, fully humanized, humanized, primatized, chimeric antibodies, single-chain antibodies, epitopes Binding fragments such as Fab-like, Fab'-like and F(ab') 2 , and single-chain Fvs (scFv)-like.
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can be combined with a kappa or lambda light chain.
  • ⁇ kappa
  • ⁇ lambda
  • the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of the immunoglobulin ⁇ light chain is V ⁇ ; the variable region of the immunoglobulin ⁇ light chain is V ⁇ .
  • variable regions of the light chain (VL) and heavy chain (VH) chain parts determine antigen recognition and specificity.
  • the constant regions of the light and heavy chains confer important biological properties, such as secretion, transplacental movement, Fc receptor binding, complement fixation, and so on. By convention, the numbering of constant regions increases as they become further away from the antigen binding site or amino terminus of the antibody.
  • the N-terminal part is the variable region, and the C-terminal part is the constant region; the CH3 and CL domains actually contain the carboxy terminus of the heavy chain and light chain, respectively.
  • the six “complementarity determining regions" or “CDRs” present in each antigen-binding domain are short, A non-contiguous amino acid sequence that specifically binds to an antigen.
  • the remaining other amino acids in the antigen-binding domain called the "framework” region show less intermolecular variability.
  • Most of the framework regions adopt a ⁇ -sheet conformation, and CDRs form a loop structure connected to it, or in some cases form part of a ⁇ -sheet structure. Therefore, the framework region forms a scaffold to position the CDR in the correct position through non-covalent interactions between chains.
  • the antigen-binding domain of the CDR with a specific position forms a surface complementary to the epitope on the antigen, and the complementary surface promotes the non-covalent binding of the antibody and its epitope.
  • those of ordinary skill in the art can identify amino acids containing CDR and framework regions by known methods (see Kabat, E., et al., USDepartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
  • CDR complementarity determining region
  • the CDRs defined by Kabat and Chothia include overlaps or subsets of amino acid residues when compared with each other. Nevertheless, it is within the scope of the present invention to apply any definition to refer to the CDRs of antibodies or variants thereof.
  • the exact residue number that contains a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues the CDR contains based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those of ordinary skill in the art can apply the "Kabat numbering" system to any variable region sequence without relying on other experimental data other than the sequence itself. "Kabat number” refers to the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest” (1983). Antibodies can also use the EU numbering system.
  • the antibodies disclosed in the present invention can be derived from any animal, including birds and mammals.
  • the antibody is a human, murine, donkey, rabbit, goat, camel, llama, horse, or chicken antibody.
  • the variable region may be of condricthoid origin (e.g., from sharks).
  • the heavy chain constant region includes at least one of a CH1 domain, a hinge (for example, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment.
  • the heavy chain constant region of an antibody can be derived from different immunoglobulin molecules.
  • heavy chain constant region polypeptide may comprise a CH1 domain derived from IgG 1 molecule and a hinge region derived from IgG 3 molecule.
  • the heavy chain constant region may include a hinge region derived partly from an IgG 1 molecule and partly from an IgG 3 molecule.
  • part of the heavy chain may include a chimeric hinge region derived partly from an IgG 1 molecule and partly from an IgG 4 molecule.
  • the "light chain constant region” includes a portion of the amino acid sequence derived from the light chain of an antibody.
  • the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.
  • the "light chain-heavy chain pair” refers to a collection of light and heavy chains that can form a dimer through the disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • the "VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain
  • the "CH1 domain” includes the first (mostly amino terminal) constant region of an immunoglobulin heavy chain.
  • the CH2 domain is not closely paired with other domains, but two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete natural IgG molecule. It is also documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.
  • the "hinge region” includes a part of the heavy chain region connecting the CH1 domain and the CH2 domain. The hinge region contains about 25 residues and is flexible, so that the two N-terminal antigen binding regions can move independently. The hinge region can be subdivided into three different domains: upper, middle and lower hinge domains (Rouxetal., J. Immunol 161:4083 (1998)).
  • Disulfide bond refers to a covalent bond formed between two sulfur atoms.
  • the thiol group of cysteine can form a disulfide bond or bridge with the second thiol group.
  • the CH1 and CL regions are connected by disulfide bonds.
  • Chimeric antibody refers to any antibody whose variable region is obtained or derived from a first species, and whose constant region (which can be complete, partial or modified) is derived from a second species.
  • the variable region is derived from a non-human source (e.g., mouse or primate), and the constant region is derived from a human source.
  • Specific binding or “specific to” generally means that an antibody or antigen-binding fragment binds to a specific antigen through the complementary binding of its antigen-binding domain to an epitope to form a relatively stable complex.
  • Specificity can be expressed by the relative affinity of an antibody or antigen-binding fragment that binds to a specific antigen or epitope. For example, if antibody “A” has a greater relative affinity for the same antigen than antibody “B”, it can be considered that antibody “A” has a higher specificity for that antigen than antibody "B”.
  • Specific binding can be described by equilibrium dissociation constant (KD), a smaller KD means tighter binding.
  • Antibodies that "specifically bind" to antigen a include antibodies with an equilibrium dissociation constant KD of antigen a less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, or less than or equal to about 1 nM.
  • Treatment refers to therapeutic treatment and preventive or preventive measures, the purpose of which is to prevent, slow down, ameliorate or stop undesirable physiological changes or disorders, such as disease progression, including but not limited to the following whether detectable or undetectable As a result, the symptoms are alleviated, the degree of the disease is reduced, the disease state is stabilized (that is, it does not worsen), the disease progression is delayed or slowed, the disease state is improved, alleviated, alleviated or disappeared (whether partial or full), prolonged and Expected survival time when not receiving treatment, etc.
  • Patients in need of treatment include patients who have already suffered from a disease or disorder, patients who are prone to suffer from a disease or disorder, or patients who need to prevent the disease or disorder, can or are expected to be used for detection from the administration of the antibody or pharmaceutical composition disclosed in the present invention , Patients benefiting from the diagnosis process and/or treatment.
  • Patient refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, etc.
  • EC 50 is the concentration for 50% of maximal effect (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximal effect.
  • the present invention provides, for example, the novel bispecific antibody or antigen-binding fragment shown in Figure 1, which can be adjusted by adjusting the position of the variable region that binds to the first antigen and the variable region that binds to the second antigen and the linker between them.
  • the present invention provides bispecific antibodies or antigen binding fragments with high affinity to TIGIT and CTLA-4 proteins.
  • the tested antibody exhibits effective binding activity, biological activity, and can be used for therapeutic and diagnostic purposes.
  • these antibodies or antigen-binding fragments effectively block immune checkpoints, activate lymphocytes to release cytokines, and are used to treat various types of cancer, tumors or infections and other related diseases.
  • an embodiment disclosed in the present invention provides an antibody or antigen-binding fragment that targets TIGIT and CTLA-4, and the antibody or antigen-binding fragment specifically binds to TIGIT and CTLA-4.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains the sequence shown in SEQ ID NO: 37, so The second polypeptide chain contains the sequence shown in SEQ ID NO:38.
  • the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains SEQ ID NO: 37 except for the Fc region sequence.
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 38.
  • the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains the sequence shown in SEQ ID NO: 39, so The second polypeptide chain contains the sequence shown in SEQ ID NO:40.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains SEQ ID NO: 39 in addition to the Fc region sequence.
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 40.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains the sequence shown in SEQ ID NO: 41, so The second polypeptide chain contains the sequence shown in SEQ ID NO:42.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains SEQ ID NO: 41 except for the Fc region sequence.
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 42.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains the sequence shown in SEQ ID NO: 43, so The second polypeptide chain contains the sequence shown in SEQ ID NO:44.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains SEQ ID NO: 43 except for the Fc region sequence.
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 44.
  • the present invention provides bispecific antibodies or antigen binding fragments with high affinity to OX40 and CTLA-4 proteins.
  • the tested antibody exhibits effective binding activity, biological activity, and can be used for therapeutic and diagnostic purposes.
  • these antibodies or antigen-binding fragments effectively activate T cells, activate lymphocytes to release cytokines, and are used to treat various types of cancer, tumors or infections and other related diseases.
  • an embodiment disclosed in the present invention provides an antibody or antigen-binding fragment that targets OX40 and CTLA-4, and the antibody or antigen-binding fragment specifically binds to OX40 and CTLA-4.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains the sequence shown in SEQ ID NO: 45, so The second polypeptide chain contains the sequence shown in SEQ ID NO: 46.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains SEQ ID NO: 45 except for the Fc region sequence. And the Fc region shown in SEQ ID NO: 36, and the second polypeptide chain contains the sequence shown in SEQ ID NO: 46.
  • the present invention provides bispecific antibodies or antigen binding fragments with high affinity to OX40 and TIGIT proteins.
  • the tested antibody exhibits effective binding activity, biological activity, and can be used for therapeutic and diagnostic purposes.
  • these antibodies or antigen-binding fragments effectively block immune checkpoints, activate T cells, activate lymphocytes to release cytokines, and are used to treat various types of cancer, tumors, infections and other related diseases.
  • an embodiment disclosed in the present invention provides an antibody or antigen-binding fragment that targets OX40 and TIGIT, and the antibody or antigen-binding fragment specifically binds to OX40 and TIGIT.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains the sequence shown in SEQ ID NO: 47, so The second polypeptide chain contains the sequence shown in SEQ ID NO: 48.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains SEQ ID NO: 47 except for the Fc region sequence.
  • the second polypeptide chain contains the sequence shown in SEQ ID NO: 48.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains the sequence shown in SEQ ID NO: 49, so The second polypeptide chain contains the sequence shown in SEQ ID NO:50.
  • the antibody or antigen-binding fragment includes at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain contains SEQ ID NO: 49 except for the Fc region sequence.
  • the second polypeptide chain contains the sequence shown in SEQ ID NO:50.
  • the sequence of the antibody or antigen-binding fragment disclosed in the present invention can be replaced, and the amino acid sequence after the replacement is different from the naturally-occurring amino acid sequence of the antibody.
  • the substituted amino acid sequence may be similar to the starting sequence, such as having a certain proportion of identity with the starting sequence, for example, it may be about 80%, about 85%, or about 90% identical to the starting sequence. , About 95%, about 98%, or about 99%, or the range (including the end point) between any two of these values, or any value therein.
  • the amino acid-containing sequence contained in the antibody has one or more modification groups.
  • the bispecific antibodies disclosed in the present invention may include A flexible linker sequence, or can be modified to add functional groups (e.g., PEG, drugs, toxins, or tags).
  • the antibodies and antigen-binding fragments disclosed in the present invention include modified derivatives, that is, modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope.
  • antibodies can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or Other proteins, etc. Any one of numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • the antibody may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • the antibody may be conjugated or fused to a therapeutic agent, which may include cells that are detectable labels (such as radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photosensitizers, diagnostic agents, drugs, or toxins Toxic agents, ultrasound enhancers, non-radioactive markers and combinations thereof, and other such agents known in the art.
  • a therapeutic agent may include cells that are detectable labels (such as radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photosensitizers, diagnostic agents, drugs, or toxins Toxic agents, ultrasound enhancers, non-radioactive markers and combinations thereof, and other such agents known in the art.
  • the antibody can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of chemiluminescent labeled antibodies is then determined by detecting the luminescence that occurs during the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.
  • the present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the present invention.
  • the polynucleotide disclosed in the present invention can encode the first polypeptide (similar to the heavy chain of immunoglobulin or fragments thereof), the second polypeptide (similar to the light chain of immunoglobulin), and the variable region of the heavy chain , Light chain variable region, Fc region, part of heavy chain variable region or part of light chain variable region.
  • Methods of preparing antibodies are well known in the art and are described in the present invention.
  • the variable regions and constant regions included in the antibodies and antigen-binding fragments disclosed in the present invention are all of fully human origin.
  • Fully human antibodies and antigen-binding fragments can be prepared using techniques disclosed in the art and the techniques described in the present invention.
  • fully human antibodies directed against specific antigens can be prepared by administering the antigen to transgenic animals that have been modified to respond to antigen challenge to produce fully human antibodies.
  • Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patent Nos. 6,458,592; 6,420,140, the entire contents of which are incorporated herein by reference.
  • the bispecific antibody in the present invention is a fusion of fragments that specifically bind to antigen a and antigen b.
  • fragments of the bispecific antibody refer to the above-mentioned preparation method of antibodies that bind to a single antigen.
  • the prepared antibody does not cause a harmful immune response in the animal (e.g., human) to be treated.
  • the antibodies, antigen-binding fragments, or derivatives disclosed in the present invention are modified using art-recognized techniques to reduce their immunogenicity.
  • antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, which retain or basically retain the antigen-binding properties of the parent antibody but are less immunogenic in humans.
  • framework substitutions can be identified by methods known in the art, for example, by simulating the interaction of CDR and framework residues to identify framework residues that play an important role in antigen binding and by sequence alignment to identify abnormal framework residues at specific positions. (Refer to US Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); the entire contents of which are incorporated herein by reference). Various techniques known in the art can be used to humanize antibodies, such as CDR grafting (EP 239,400; WO 91/09967; U.S. Patent Nos.
  • Deimmunization can also be used to reduce the immunogenicity of antibodies.
  • the term "deimmunization” includes altering antibodies to modify T cell epitopes (see, for example, WO/9852976A1 and WO/0034317A2). For example, analyze the heavy chain variable region sequence and light chain variable region sequence from the starting antibody, and generate a "map" of human T cell epitopes from each variable region, showing the epitopes relative to the complementarity determining regions (CDRs) And the positions of other key residues within the sequence. The individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a lower risk of altering antibody activity.
  • CDRs complementarity determining regions
  • a series of optional heavy chain variable region sequences and light chain variable region sequences containing combinations of amino acid substitutions are designed, and then these sequences are incorporated into a series of binding polypeptides. Then the genes containing the complete heavy and light chains of the modified variable regions and human constant regions are cloned into expression vectors, and then the plasmids are transferred into cell lines to produce complete antibodies. Then use appropriate biochemical and biological experiments to compare antibodies to identify the best antibody.
  • the binding specificity of the bispecific antibody or antigen-binding fragment disclosed in the present invention can be tested by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro experiments such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • the scFv in the bispecific antibody of the present invention can be found in the technology of producing single chain units (US Patent 4,694,778; Bird, Science 242:423-442 (1988), Huston et al., Proc. Natl. Acad. Sci. USA55: 5879-5883 (1988) and Ward et al., Nature 334:544-554 (1989) and Nie et al., Antibody Therapeutics 3(1): 18-62 (2020)).
  • the heavy chain and light chain fragments of the Fv region are bridged by amino acids to form a single-chain unit, resulting in a single-chain fusion peptide.
  • the technique of assembling functional Fv fragments in E. coli can also be used.
  • scFv single-chain Fv
  • antibodies include, for example, U.S. Patent Nos. 4,946,778 and 5,258,498, and Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90: 1995-1999 (1993) and Skerra et al., Science 240: 1038-1040 (1988).
  • chimeric antibodies, humanized antibodies, or fully human antibodies can be used.
  • a chimeric antibody is a class of molecules in which different parts of the antibody are derived from different animal species, such as antibodies with the variable regions of murine monoclonal antibodies and the constant regions of human immunoglobulins.
  • Methods of producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191 -202 (1989); Neuberger et al., Nature372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patent Nos. 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated by reference Into this article.
  • Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. You can also refer to U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each The entire content of the patent is incorporated herein by reference.
  • Fully human antibodies that recognize selective epitopes can be produced using a technique called "guided selection".
  • selected non-human monoclonal antibodies eg, mouse antibodies
  • guided selection is used to guide the screening of fully human antibodies that recognize the same epitope.
  • the DNA encoding the desired monoclonal antibody can be isolated and processed. Sequencing. Isolated and subcloned hybridoma cells can be used as a source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma that does not produce other immunoglobulins In the cell.
  • prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma that does not produce other immunoglobulins In the cell.
  • Isolated DNA (which may be synthetic as described herein) can also be used to prepare the sequences of the constant and variable regions of antibodies, as described in U.S. Patent No. 5,658,570, the entire contents of which are incorporated herein by reference. This method extracts RNA from selected cells and converts it into cDNA, and then uses Ig-specific primers to amplify by PCR technology. Suitable probes for this purpose are also mentioned in U.S. Patent No. 5,658,570.
  • one or more CDRs of the antibody of the present invention can be inserted into the framework region, for example, inserted into the human framework region to construct a humanized non-fully human antibody.
  • the framework region may be a naturally occurring or shared framework region, preferably a human framework region (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human framework regions).
  • Some polynucleotides can encode antibodies that specifically bind to at least one epitope of the target antigen produced by a combination of framework regions and CDRs.
  • One or more amino acid substitutions can be made in the framework region, and amino acid substitutions that can improve the binding of the antibody to its antigen can be selected.
  • this method can be used to perform substitution or deletion of cysteine residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby producing antibody molecules lacking one or more interchain disulfide bonds.
  • Other changes made to polynucleotides within the technical scope of the art are also encompassed in the present invention.
  • the antibody-producing cell line can be selected, constructed and cultured by using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, the technology suitable for use in the present invention described below refers to Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), and its entire contents include The supplementary content is incorporated in full by reference.
  • the antibody expression vector includes at least one promoter element, antibody coding sequence, transcription termination signal, and polyA tail.
  • Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the inserted sequence. Efficient transcription can be obtained through the early and late promoters of SV40, the long terminal repeats from retroviruses such as RSV, HTLV1, HIVI and the early promoters of cytomegalovirus, and the promoters of other cells such as muscle Kinesin promoter.
  • Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2 etc.
  • Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.
  • the inserted gene fragment needs to contain selection markers.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes to facilitate transfection Screening and isolation of successful cells.
  • the constructed plasmid is transfected into host cells without the above-mentioned genes, and then cultured in a selective medium, the successfully transfected cells grow in large numbers to produce the desired target protein.
  • variants encode less than 50 amino acid substitutions or less than the original heavy chain variable region VH CDR1, VH CDR2, VH CDR3 and light chain variable region VL CDR1, VL CDR2 or VL CDR3.
  • amino acid substitutions less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, and less than 5 amino acid substitutions Amino acid substitutions, substitutions of less than 4 amino acids, substitutions of less than 3 amino acids, or substitutions of less than 2 amino acids.
  • mutations can be introduced randomly along all or part of the coding sequence, for example, through saturation mutations, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
  • substitutions described herein are conservative amino acid substitutions.
  • the invention also provides treatment methods and uses.
  • a method for treating or ameliorating various types of cancers, tumors or infections and other related diseases comprising administering to the patient an effective dose of the bispecific antibody: targeting TIGIT and CTLA -4 antibodies, antibodies targeting OX40 and CTLA-4, or antibodies targeting OX40 and TIGIT.
  • the application of the bispecific antibody in the treatment or improvement of related diseases such as cancer, tumor or infection is provided.
  • the application of the bispecific antibody in the preparation of drugs for the treatment or improvement of related diseases such as cancer, tumor or infection is provided.
  • the specific dosage and treatment plan for any particular patient will depend on various factors, including the specific antibody or derivative used, the patient’s age and weight, general health, gender and diet, as well as the time of administration, frequency of excretion, and drugs Combination, and the severity of the specific disease being treated. These factors are judged by medical and nursing staff included within the scope of those of ordinary skill in the art.
  • the dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect.
  • the dosage used can be determined by the principles of pharmacology and pharmacokinetics well known in the art.
  • Methods of administration of antibodies or derivatives include, but are not limited to, intradermal, intramuscular, abdominal, intravenous, subcutaneous, nasal, epidural, and oral injections.
  • the pharmaceutical composition can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelial or skin mucosa (e.g., oral mucosa, rectum and intestinal mucosa, etc.), and can be co-administered with other biologically active agents.
  • the pharmaceutical composition containing the antibody of the present invention can be administered orally, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, or topical (such as by powder, ointment, drops or Transdermal patch), oral administration, or oral or nasal spray administration.
  • parenteral refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the mode of administration may be systemic administration or local administration.
  • intracerebroventricular injection can be connected to, for example, a reservoir (which can be an Ommaya reservoir) through an intraventricular catheter.
  • Auxiliary injection It can also be administered via the lungs, for example by using an inhaler or nebulizer, and using aerosolized formulations.
  • the antibody of the present invention can be locally applied to the area to be treated; it can be through but not limited to the following methods: local infusion during surgery, such as local application combined with post-operative wound dressing, by injection, through a catheter, by suppository or by implant
  • the implant is a porous, non-porous or gel-like material, including membranes (such as silicone rubber membranes) or fibers.
  • membranes such as silicone rubber membranes
  • care must be taken to use materials that do not absorb the protein.
  • the composition of the present invention comprises a nucleic acid or polynucleotide encoding an antibody, which can be administered in vivo by constructing it as part of a suitable nucleic acid expression vector to promote the expression of the protein encoded thereby, The part of the vector described above is then applied to make it an intracellular part, for example, by using a retroviral vector (see U.S.
  • Patent 4,980,286) or by direct injection, or by using particle bombardment (for example, gene gun; Biolistic, Dupont) , Or coated with lipids or cell surface receptors or transfection reagents, or administered by linking with homeobox peptides known to enter the nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci .USA 88:1864-1868) and so on.
  • the nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.
  • the antibody of the present invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight, or 0.1 mg/kg to 20 mg/kg of the patient's body weight.
  • a second or multiple doses of the antibody or antigen-binding fragment may be subsequently administered, the dose of which is approximately the same or less than the initial dose, wherein the subsequent doses may be separated by at least 1 day to 3 days; or at least one week.
  • Modifications such as lipidation can be used to enhance antibody uptake and tissue penetration (for example, into the brain), thereby reducing the dosage and frequency of administration of the antibody of the present invention.
  • Various known delivery systems can be used to administer the antibody or derivative of the present invention or the polynucleotide encoding it, such as encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated Endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.
  • the antibodies of the invention can be combined with other treatment or prevention regimens, including administration of one or more antibodies of the invention and one or more other therapeutic agents or methods together or in combination.
  • the antibody and the other therapeutic agent can be administered simultaneously or separately.
  • the antibody of the present invention may be administered before or after the administration of another other therapeutic agent.
  • the bispecific antibody of the invention is administered in combination with a chemotherapeutic agent.
  • the bispecific antibody of the present invention is an antibody that targets TIGIT and CTLA-4, an antibody that targets OX40 and CTLA-4, or an antibody that targets OX40 and TIGIT.
  • the antibodies of the invention are administered in combination with cytokines.
  • Cytokines that can be administered with the antibody of the present invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, And IL-15 etc.
  • the bispecific antibody of the invention is administered in combination with a chemotherapeutic agent.
  • chemotherapeutic agents include immunotherapeutics, including but not limited to therapeutic antibodies suitable for treating patients.
  • Some examples of therapeutic antibodies include secretuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab (alemtuzumab), altumomab, amatuximab, anatumomab, arcitumomab, bavituximab ), Bectumomab, Bevacizumab, Bivatuzumab, Blinatumomab, Brentuximab, Cantor Cantuzumab, catumaxomab, cetuzumab, cetuzumab, cixutumumab, krivartuzumab Anti-clivatuzumab, conatumumab, daratumum
  • the antibodies of the invention can be used with immune checkpoint inhibitors. In some embodiments, the antibodies of the invention are administered in combination with other treatment or prevention regimens, such as radiotherapy.
  • the invention also provides a pharmaceutical composition.
  • a pharmaceutical composition includes an effective dose of the antibody or antigen-binding fragment and an acceptable carrier.
  • the pharmaceutical composition further includes an anticancer agent (e.g., immune checkpoint inhibitor).
  • the term “pharmaceutically acceptable” refers to a substance approved by a government regulatory agency or listed in a recognized pharmacopoeia for use in animals, especially humans.
  • pharmaceutically acceptable excipients generally refer to any type of non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids.
  • adjuvant refers to a diluent, adjuvant, excipient or carrier that can be administered to a patient together with the active ingredient.
  • Such pharmaceutical excipients can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • water is the preferred carrier. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, especially for injection solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, Propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition may also contain small amounts of wetting or emulsifying agents, or pH buffering agents such as acetate, citrate or phosphate.
  • Antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity agents such as sodium chloride or dextrose are also foreseen.
  • These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations and the like.
  • the composition can be formulated as a suppository with traditional binders and carriers such as triglycerides.
  • Oral preparations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in a purified form, together with an appropriate number of carriers, to provide a dosage form suitable for the patient.
  • the formulation should be suitable for the mode of administration.
  • the parent preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
  • the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures.
  • Compositions for intravenous administration are usually solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.
  • the active ingredients are supplied separately or mixed together in a unit dosage form, such as in the form of a dry freeze-dried powder or an anhydrous concentrate in a sealed container (such as an ampoule or a sachet) that can indicate the amount of the active agent.
  • the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be used so that the active ingredients can be mixed before administration.
  • the compounds of the present invention can be formulated in neutral or salt form.
  • Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., as well as salts derived from such as sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2- Ethylaminoethanol, histidine, procaine and other salts formed with cations.
  • TIGIT-Fc antigen CTLA-4-Fc antigen
  • TIGIT-his antigen can be purchased from Shanghai Nearshore Technology Co., Ltd. or AcroBiosystems
  • the anti-TIGIT reporter gene detection system can be from Promega (product number is JA1400), or can be prepared according to known methods.
  • Anti-TIGIT antibody (reference antibody) was expressed and purified in CHO cells.
  • the heavy chain of anti-TIGIT antibody is:
  • the light chain of anti-TIGIT antibody is:
  • Anti-CTLA-4 antibody (reference antibody) was expressed and purified in CHO cells.
  • the heavy chain of anti-CTLA-4 antibody is:
  • the light chain of anti-CTLA-4 antibody is:
  • the anti-OX40 antibody (the heavy chain sequence of the reference antibody is SEQ ID NO: 75, and the light chain sequence is SEQ ID NO: 74) was expressed and purified in CHO cells.
  • the heavy chain of anti-OX40 is:
  • the light chain of anti-OX40 is:
  • the structure of the bispecific antibody is shown in Figure 1.
  • the structure is similar to immunoglobulin.
  • the antibody contains a first polypeptide chain and a third polypeptide chain with the same sequence, and a second polypeptide and a fourth polypeptide chain with the same sequence.
  • antigen a is TIGIT
  • Antigen b is CTLA-4.
  • the amino acid sequences of the first polypeptide and the second polypeptide of the antibody are sequence-optimized according to the codon preference characteristics of the host cell CHO (Chinese Hamster Ovary Cell), and the DNA sequences of the first polypeptide and the second polypeptide are obtained.
  • CHO Choinese Hamster Ovary Cell
  • a signal peptide and Kozak sequence were added to the sequences of the first polypeptide and the second polypeptide respectively (Kozak sequence is a nucleic acid sequence located behind the 5'cap structure of eukaryotic mRNA), A stop codon was added to the end of the sequence.
  • the optimized and synthesized sequence clones were cloned into the pCDNA3.1 TM (+) vector, and then a large number of plasmids were extracted separately.
  • the first polypeptide and the second polypeptide were based on the plasmid molar ratio of 1:1 using ExpiCHO TM expression system (Gibco , The item number is A29133) for instantaneous expression.
  • the protein was harvested according to the instructions and purified with protein A.
  • the related amino acid sequence of the bispecific antibody targeting TIGIT and CTLA-4 is shown in Table 1, and the related nucleic acid sequence of the bispecific antibody targeting TIGIT and CTLA-4 is shown in Table 2.
  • the linker in Table 1 is shown in bold italics and underlined; the first polypeptide of antibody 1 is shown in SEQ ID NO: 37, the second polypeptide of antibody 1 is shown in SEQ ID NO: 38; the first polypeptide of antibody 2 The first polypeptide is shown in SEQ ID NO: 39, and the second polypeptide of Antibody 2 is shown in SEQ ID NO: 40; the first polypeptide of Antibody 3 is shown in SEQ ID NO: 41, and the second polypeptide of Antibody 3 is shown in SEQ ID NO: 41.
  • the first polypeptide of antibody 4 is shown in SEQ ID NO: 43
  • the second polypeptide of antibody 4 is shown in SEQ ID NO: 44.
  • the 5'end italicized and underlined small letters are Hind III restriction site, Kozak sequence and leader peptide sequence in sequence
  • the 3'end italicized and underlined small letters are the stop codon and the EcoR I restriction site in turn; since antibodies 1, 2, 3, and 4 only have different linker sequences, only the first polypeptide and the second polypeptide of antibody 1 are listed in Table 2.
  • the nucleic acid sequence of the polypeptide, and the nucleic acid sequence of the linker are listed in Table 2.
  • Perform gel electrophoresis detection on purified antibodies detect the purity of the purified antibodies. As shown in Figure 2, the purity of antibody 1 is relatively high; as shown in Figure 3, the purity of antibody 2 and antibody 3 is relatively high.
  • the binding ability of antibody 1 and antibody 2 to human TIGIT-Fc is close to the result of anti-TIGIT, while the binding ability of antibody 3 to human TIGIT-Fc is relatively low.
  • the specific test process is as follows: The hTIGIT-his antigen is diluted with HBS-EP+buffer, a total of 5 gradients, the initial concentration is 50nM, and then half-diluted; the test protein anti-TIGIT, antibody 1, antibody 2 and antibody 3 Dilute with HBS-EP+buffer to 2.5 ⁇ g/ml; use the instrument BiacoreT200, Protein A chip (GE healthcare, item number 29127556) for detection, the instrument settings are as follows: antibody capture binding time (contact time) 25s, flow rate 5 ⁇ l/ min; sample contact time 120s, dissociation time 300s, flow rate 30 ⁇ l/min; regeneration conditions: Glycine-Hcl (pH value 1.5), regeneration time 60s, flow rate 30 ⁇ l/min ; The detection channel (flow path) is 2-1 and 4-3.
  • the Biacore T200 instrument was used to detect the affinity constant of anti-CTLA-4, Antibody 1, Antibody 2, and Antibody 3 and CTLA-4-his protein (Shanghai Nearshore Technology Co., Ltd., CP33).
  • the specific test process is as follows: The hCTLA-4-his antigen is diluted with HBS-EP+buffer in 6 gradients, the initial concentration is 100nM, and then half-diluted; the test protein anti-CTLA-4, antibody 1, antibody 2 and antibody 3 are respectively diluted with HBS-EP+buffer to 5 ⁇ g/ml; the instrument is Biacore T200, Protein A chip (GE healthcare, item number: 29127556) for detection, the instrument settings are as follows: antibody capture binding time (contact time) 15s, Flow rate 10 ⁇ l/min; sample binding time (contact time) 180s, dissociation time (dissociation time) 300s, flow rate 30 ⁇ l/min; regeneration conditions: Glycine-Hcl (pH value 1.5), regeneration time 60s, flow rate 30 ⁇ l/
  • the anti-CTLA-4 reporter gene detection system (Promega, item number is JA1400) is used to detect the biological activity of the antibody. Refer to the product manual for the detection process.
  • control antibody anti-CTLA-4 has good biological activity and can activate the IL-2 promoter in the reporter system to activate the expression of downstream luciferase; the activation ability of antibody 1 is relatively weak and biologically active relatively low.
  • antibody 2 compared with anti-CTLA-4, antibody 2 has a relatively weak activation ability and relatively low biological activity.
  • the anti-TIGIT reporter gene detection system is used to determine the biological activity of the antibody.
  • the detection process is similar to the above-mentioned Promega anti-CTLA-4 reporter gene detection system.
  • the increase in the length of the linker L2 and L4 has no significant impact on the function of the TIGIT target, but it has an impact on the function of the CTLA-4 target: the longer the linker is, the longer the linker is, the longer it is for CTLA- 4
  • the biological activity of the target is lower; the biological activity of the antibody 1 of the present invention is better.
  • the structure of the bispecific antibody is shown in Figure 1.
  • the structure is similar to immunoglobulin.
  • the antibody contains a first polypeptide chain and a third polypeptide chain with the same sequence, and a second polypeptide and a fourth polypeptide chain with the same sequence.
  • antigen a is OX40
  • Antigen b is CTLA-4.
  • sequence of the second polypeptide of the synthetic antibody contains the HindIII restriction site and the leader peptide, the 3'end contains the stop codon and the EcoRI restriction site; the sequence of the first polypeptide of the synthetic antibody: 5'end Contains HindIII restriction site and leader peptide, 3'end contains stop codon and EcoRI restriction site.
  • the plasmids carrying the nucleic acid sequences of the first polypeptide and the second polypeptide were transiently transfected into 293F cells at the same time.
  • 293F cells were cultured with CD 293 TGE Medium (BPM Cell Culture, Item No.: CM-1156). When the cell viability was above 95%, the cells were subcultured to 800,000-1,000,000/mL with fresh medium. After 24 hours, the cell density was about When 1.5-2 million/mL, transfection can be carried out.
  • plasmid First put the plasmid in a water bath at 65°C for 30 minutes, use 0.5 ⁇ g of plasmid per 1 million cells, and 3 ⁇ L of PEI (Polysciences, catalog number 24765-2) for every 1 ⁇ g of total plasmid. Dilute the plasmid and PEI with culture medium (the volume of the two solutions add up to 5% of the total volume), and let stand at room temperature for 5 minutes. Then add the PEI solution to the plasmid solution, mix well, and let stand at room temperature for 20 minutes.
  • PEI Polysciences, catalog number 24765-2
  • the related amino acid sequence of the bispecific antibody 5 targeting OX40 and CTLA-4 is shown in Table 5, and the related nucleic acid sequence of the bispecific antibody 5 targeting TIGIT and CTLA-4 is shown in Table 6.
  • linkers are indicated by bold italics and underline.
  • Antibody 5 was subjected to reduction and non-reduction treatments respectively, and then used for SDS-PAGE electrophoresis. As shown in Figure 11, the reduced and non-reduced bands are clear and free of contaminants, indicating high sample purity: the size of the non-reduced antibody 5 on the electropherogram is greater than 250kDa; the size of the first polypeptide of the reduced antibody 5 on the electropherogram It is about 90kDa, and the size of the second polypeptide of reduced antibody 5 is about 50kDa on the electropherogram; the size is in line with expectations. It can be seen that bispecific antibody 5 can be expressed normally in cells; antibody 5 expressed in 293F cells can be used in the next step after purification.
  • CTLA-4-Fc antigen and OX40-Fc antigen were respectively coated on the ELISA plate at 4°C overnight. The next day, it was blocked with 5% BSA blocking solution at 37°C for 2 hours. After washing the plate, add antibody 5, CTLA-4 monoclonal antibody (i.e. anti-CTLA-4) and OX40 monoclonal antibody (i.e. anti-OX40) in 2-fold dilutions starting from 2 ⁇ g/mL, and dilute 12 concentrations in total (The last one is blank 0 concentration). Make 2 replicate wells for each concentration and incubate at 37°C for 2 hours.
  • CTLA-4 monoclonal antibody i.e. anti-CTLA-4
  • OX40 monoclonal antibody i.e. anti-OX40
  • the preparation method of OX40-Fc antigen is: from the protein database Uniprot, find the amino acid sequence of human OX40 (P43489), wherein the amino acid sequence of the extracellular region of human OX40 is residues 1 to 216; from the protein database Uniprot, find The amino acid sequence of IgG1-Fc (P01857), residues 104 to 330; then artificially synthesized (General Company) to obtain the nucleotide sequence corresponding to the extracellular region of OX40 and Fc, which was ligated by restriction enzymes and inserted into pCDNA3 .0 vector (Invitrogen) to obtain the recombinant plasmid; then the above plasmid was transiently transformed into HEK293 cells by PEI, the supernatant was collected after 7 days of culture, and finally purified to obtain OX40-Fc (i.e. hOX40-Fc).
  • OX40-Fc i.e. hOX40-F
  • Antibody 5 can retain the binding ability to OX40 and CTLA-4; Compared with monoclonal antibodies, Antibody 5 has better binding ability to OX40, and Antibody 5 has better binding ability to CTLA-4. Difference.
  • the OX40 monoclonal antibody, CTLA-4 monoclonal antibody, and antibody 5 to be tested were coupled to biotin respectively. Pre-wet the SA sensor in PBS for 10 minutes. Dilute the biotin-labeled antibody with PBST (hereinafter referred to as dilution buffer) containing 0.5% BSA with a pH of 6.8 to 10 ⁇ g/mL. The 3 biotinylated antibodies solidified to a signal of about 4nm. Dilute the CTLA-4-Fc antigen and hOX40-Fc antigen to be tested with dilution buffer to 400nM, and use this as the starting concentration to make a 2-fold gradient dilution.
  • PBST biotin-labeled antibody
  • dilution buffer containing 0.5% BSA with a pH of 6.8 to 10 ⁇ g/mL.
  • the 3 biotinylated antibodies solidified to a signal of about 4nm. Dilute the CTLA-4-Fc antigen and hO
  • a total of 6 concentration points 400, 200, 100, 50, 25, 0nM.
  • 1Baseline in the dilution buffer Baseline detection in solution for 60 seconds
  • 2Association Combine in antibody gradient dilution and sample blank (dilution buffer) for 300 seconds
  • 3Dissociation Dissociate in dilution buffer for 360 seconds
  • 4Regeneration Regener
  • ForteBio Data Analysis 8.2 is used to process and analyze the data.
  • the sample data is subtracted from the reference (sample blank) signal and then fitted to obtain the affinity constant K D.
  • the affinity results of antibody 5 and CTLA-4-Fc antigen are shown in Table 8, and the affinity results of antibody 5 and OX40-Fc antigen are shown in Table 9.
  • antibody 5 As shown in Table 8, compared with monoclonal antibodies, antibody 5 has a higher affinity for CTLA-4 antigen; as shown in Table 9, compared with monoclonal antibodies, antibody 5 has a similar affinity for OX40 antigen.
  • OX40 monoclonal antibody and antibody 5 were respectively diluted 3 times from the antibody concentration of 100 nM, and a total of 10 concentrations were diluted. Resuspend the corresponding cell pellets (OX40 + cells) with these antibody solutions of different concentrations. Each concentration requires about 2 million cells. After incubating for 2 hours at 4°C, the supernatant was removed by centrifugation and washed once with PBS.
  • Anti-Fc-PE invitrogen, article number 12-4998-82
  • diluted 1:1000 was added to each sample, and incubated for half an hour at 4°C in the dark. Centrifuge to remove the supernatant and wash once with PBS. Each sample was resuspended in PBS, and then loaded and tested on a C6 flow cytometer, and channel 2 was selected as the light source.
  • the binding curve is shown in Figure 12, and the statistics of the positive rate and fluorescence value are shown in Table 10-11.
  • Example 11 Antibody simultaneously binds OX40 antigen and CTLA-4 antigen
  • the OX40-Fc antigen is coupled to biotin, and the SA sensor is placed in PBS to pre-wet for 10 minutes.
  • Use the biotin-labeled OX40-Fc antigen in PBST with pH 6.8 containing 0.5% BSA (hereinafter referred to as dilution buffer) Dilute to 5 ⁇ g/Ml, and solidify the SA sensor in the labeled biotinylated OX40-Fc antigen solution to a signal of about 0.8nM.
  • the antibody 5 to be tested was diluted to 50 nM with dilution buffer; CTLA-4-Fc antigen was diluted to 200 nM with dilution buffer.
  • the senor binds to antibody 5 after curing the OX40-Fc antigen, and has obvious binding; at this time, it binds to the CTLA-4-Fc antigen, and there is still a certain binding signal value; and it is not seen during the dissociation step. To a significant signal drop; indicating that antibody 5 can bind to OX40 antigen and CTLA-4 antigen at the same time.
  • the structure of the bispecific antibody is shown in Figure 1.
  • the structure is similar to immunoglobulin.
  • the antibody contains a first polypeptide chain and a third polypeptide chain with the same sequence, and a second polypeptide and a fourth polypeptide chain with the same sequence.
  • antigen a is OX40
  • Antigen b is TIGIT.
  • the nucleic acid sequences corresponding to the first polypeptide and the second polypeptide of the bispecific antibody are artificially synthesized, and the nucleic acid sequences are respectively connected to the pCDNA3.0 vector (purchased from Invitrogen) by restriction enzyme digestion and connection; 2 kinds of recombinant plasmids.
  • ForteBio affinity determination can refer to the existing conventional methods (Estep Patricia, et al. High throughput solution-based measurement of antibody-antigen affinity and epitope binning.MAbs, 2013, 5(2): 270-8), and the specific test process is as follows: : The sensor is equilibrated in the analytical buffer (such as PBS) for 20 minutes, and then tested on the computer for 60 seconds to establish a signal baseline, and loaded the purified antibodies 6 and 7 to the corresponding sensor (ForteBio) on the computer, and finally perform the ForteBio affinity measurement; Use protein sensor to adsorb antibodies 6 and 7, and then separately detect the binding and dissociation with OX40-his (AcroBiosystems) and Tigit-his (AcroBiosystems), each for about 5 minutes; finally, the 1:1 binding model is used for kinetic analysis analyze.
  • the analytical buffer such as PBS
  • antibody 6 can obviously bind to OX40 antigen, but its affinity is weaker than anti-OX40; antibody 7 can obviously bind to OX40 antigen, and the affinity is equivalent to anti-OX40; antibody 7 can also obviously bind to TIGIT antigen, its affinity Basically close to anti-TIGIT.
  • Example 15 Antibody binds to OX40 antigen and TIGIT antigen at the same time
  • the bispecific antibody can simultaneously bind to OX40-his antigen (AcroBiosystems) and TIGIT-his antigen (AcroBiosystems).
  • the protein sensor is used to adsorb the antibody 7 in the above example, and the binding and dissociation with the OX40 antigen is detected first, and then the binding and dissociation with the TIGIT antigen is detected.
  • antibody 7 can obviously bind OX40 and TIGIT antigens at the same time without steric hindrance.
  • Example 16 Binding activity of antibody to Jurkat cells overexpressed by OX40 or TIGIT
  • Jurkat-OX40 cells overexpressing human OX40 were produced by transfection of pCMV vector (Invirogen) with human OX40cDNA, and Jurkat-hOX40 cells (0.5 ⁇ 10 6 cells) were combined with different concentrations of Jurkat-OX40 cells.
  • the antibody was incubated in PBS on ice for 40 minutes.
  • the cells were then washed twice and incubated with the secondary antibody in PBS (containing 0.1% BSA) on ice for 25 minutes.
  • the cells were washed twice and analyzed by flow cytometry on the Accuri C6 system (BD Biosciences).
  • antibody 7 (OT-4D-30a) can obviously bind to Jurkat-OX40 cells, and its binding force is close to anti-OX40.
  • antibody 7 can obviously bind to Jurkat-Tigit cells, and its binding force is slightly weaker than anti-TIGIT.

Abstract

提供了多特异性抗体或其抗原结合片段，该多特异性抗体或其抗原结合片段可以结合两个或多个抗原、或同一抗原的两个或多个表位；还提供了该抗体或抗原结合片段用于炎性疾病、自身免疫性疾病或脊髓损伤的治疗、诊断和预后中的用途。

Description

多特异性抗体及其应用 技术领域
本发明属于生物医药领域，尤其涉及多特异性抗体及其应用。
背景技术
目前癌症免疫治疗的有力工具包含单克隆抗体、肿瘤疫苗、免疫检查点抑制剂、CAR-T细胞免疫疗法、双特异性抗体(BsAb)以及多特异性抗体。CAR-T和BsAb作为抗肿瘤免疫疗法的新策略受到越来越多的关注。
双特异性抗体是一种可以同时结合两种特异性表位或目的蛋白的人工工程化抗体，其可以起到一些特殊的生物学功能。与两种单克隆抗体药物联合用药治疗相比，BsAb提高了抗体选择性和功能性，降低了治疗成本。BsAb的制备主要有化学偶联、双杂交瘤细胞法、重组基因制备等方法。
然而，双特异性抗体制备存在着难点，如抗体制备中会产生大量的副产物、生物活性受到抑制。
发明内容
本发明提供了多价和多特异性抗体或抗原结合片段及其应用。在一些实施方案中，本发明提供了双特异性抗体或抗原结合片段。本发明提供的抗体或抗原结合片段可以结合两个或更多个抗原，或同一抗原的两个或更多个表位，或同一表位的两个或更多个拷贝。本发明提供的抗体或抗原结合片段用于治疗或改善炎性疾病、自体免疫性疾病、癌症或脊髓损伤；本发明提供的抗体或抗原结合片段也用于相关疾病的诊断和预后。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原表位第一抗原表位x和第二抗原表位y，并且所述抗体或抗原结合片段至少包含2条多肽链；其中，第一条多肽链从氨基末端开始顺序包含VHa、CLa、VHb和CH1，VHa是与第一抗原表位x结合的重链可变区，CLa是第一轻链恒定区，VHb是与第二抗原表位y结合的重链可变区，CH1是重链的第一恒定区。在一些实施方案中，所述第一抗原表位x和第二抗原表位y分别为不同抗原，第一抗原a和第二抗原b上的表位。在一些实施方案中，所述第一抗原表位x和第二抗原表位y为相同抗原a(或抗原b)上的不同表位。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原即第一抗原a和 第二抗原b，并且所述抗体或抗原结合片段至少包含2条多肽链；其中，第一条多肽链从氨基末端开始顺序包含VHa、CLa、VHb和CH1，VHa是与第一抗原a结合的重链可变区，CLa是第一轻链恒定区，VHb是与第二抗原b结合的重链可变区，CH1是重链的第一恒定区。
在一些实施方案中，VHa与CLa通过连接子L1进行共价连接，L1含2到6个氨基酸；和/或
CLa与VHb通过连接子L2进行共价连接；其中，L2含10到30个氨基酸，并且至少50％的氨基酸为甘氨酸。
在一些实施方案中，VHa与CLa通过连接子L1进行共价连接，L1含2到6个氨基酸；CLa与VHb通过连接子L2进行共价连接，L2含10到30个氨基酸，并且至少50％的氨基酸为甘氨酸。在一些实施方案中，L1含约2、约3、约4、约5或约6个氨基酸。在一些实施方案中，L2含约10、约11、约13、约14、约17、约18、约20、约21、约22、约25、约27、约28、约29、约30个氨基酸，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，L2含有丝氨酸。在一些实施方案中，L2含约8个、约10个、约11个、约13个、约15个、约17个、约21个、约25个甘氨酸，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述第二条多肽链从氨基末端开始顺序包含VLa、CH1、VLb和CLb，VLa是与第一表位x结合的轻链可变区，VLb是与第二表位y结合的轻链可变区，CLb是第二轻链恒定区。在一些实施方案中，所述第一抗原表位x和第二抗原表位y分别为不同抗原，第一抗原a和第二抗原b上的表位。在一些实施方案中，所述第一抗原表位x和第二抗原表位y为相同抗原a(或抗原b)上的不同表位。
在一些实施方案中，所述第二条多肽链从氨基末端开始顺序包含VLa、CH1、VLb和CLb；其中，VLa是与第一抗原a结合的轻链可变区，VLb是与第二抗原b结合的轻链可变区，CLb是第二轻链恒定区。
在一些实施方案中，VLa与CH1通过连接子L3进行共价连接，L3含2到6个氨基酸；和/或
CH1与VLb通过连接子L4进行共价连接；其中，L4含10到30个氨基酸，并且至少50％的氨基酸为甘氨酸。
在一些实施方案中，VLa与CH1通过连接子L3进行共价连接，L3含2到6个氨基酸；CH1与VLb通过连接子L4进行共价连接；其中，L4含10到30个氨基酸，并且至少50％的氨基酸为甘氨酸。在一些实施方案中，L3含约2、约3、约4、约5 或约6个氨基酸。在一些实施方案中，L4含约10、约11、约13、约14、约17、约18、约20、约21、约22、约25、约27、约28、约29、约30个氨基酸，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，L4含有丝氨酸。在一些实施方案中，L4含约8个、约10个、约11个、约13个、约14个、约18个、约21个、约22个甘氨酸，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述2条多肽链中的一条还包含Fc，Fc包含重链的铰链区、第二恒定区和第三恒定区。在一些实施方案中，Fc是变体Fc区。在一些实施方案中，相对于亲本Fc区，变体Fc区具有一个或多个氨基酸修饰，如取代、缺失或插入。在一些实施方案中，相对于亲本Fc区活性，Fc区的氨基酸修饰改变了效应功能活性。在一些实施方案中，变体Fc区可以具有改变的(即，增加的或降低的)抗体依赖性细胞毒性(ADCC)、补体介导的细胞毒性(CDC)、吞噬作用、调理作用或细胞结合。在一些实施方案中，相对于亲本Fc区，Fc区氨基酸修饰可以改变变体Fc区对FcγR(Fcγ受体)的亲和力。
在一些实施方案中，所述第一条多肽链包含Fc。
在一些实施方案中，所述第一条多肽链包含结构VHa-L1-CLa-L2-VHb-CH1，所述第二条多肽链包含结构VLa-L3-CH1-L4-VLb-CLb。在一些实施方案中，所述第一条多肽链包含结构VHa-L1-CLa-L2-VHb-CH1-Fc，所述第二条多肽链包含结构VLa-L3-CH1-L4-VLb-CLb。
在一些实施方案中，所述第一条多肽链的CLa与第二条多肽链的CH1通过二硫键连接，所述第一条多肽链的CH1与第二条多肽链的CLb通过二硫键连接。
在一些实施方案中，所述抗原a、b为细胞因子、细胞因子受体、趋化因子、趋化因子受体或细胞表面蛋白。在一些实施方案中，抗体或抗原结合片段能特异性结合细胞因子。在一些实施方案中，细胞因子包括IL-1α(白介素IL-1α)、IL-1β(白介素IL-1β)、IL-13(白介素IL-13)、IL-5(白介素IL-5)、TNF-α(肿瘤坏死因子α)、TNF-β和(肿瘤坏死因子β)等。在一些实施方案中，抗体或抗原结合片段能特异性结合免疫检查点蛋白。在一些实施方案中，所述免疫检查点蛋白包括TIM-3(T cell immunoglobin domain and mucin domain-3)、LAG-3(淋巴细胞活化基因-3分子)、CTLA-4(细胞毒性T淋巴细胞相关抗原)、TIGIT(T cell Ig and ITIM domain)、CD27(分化簇27)、OX40(肿瘤坏死因子受体超家族成员4)、ICOS(inducible costimulator)、BTLA(B和T淋巴细胞弱化因子)、PD-1(程序性死亡受体1)和CD137(分化簇137)等。在一些实施方案中，抗体或抗原结合片段能特异性结合细胞表面蛋白，如肿瘤细胞表面蛋白PD-L1(程序性死亡配体1)、半乳糖凝集素9、CD48(分化簇48)、CD40 (分化簇40)、CD70(分化簇70)、B7H3(CD276，分化簇276)和HVEM(Herpesvirus Entry Mediator)等。在一些实施方案中，所述抗体或抗原结合片段能结合趋化因子或趋化因子受体，如CC趋化因子亚组中的CCL1、CCL3、CCL5、CCL7、CCL8等。
在一些实施方案中，抗原a、b分别选自于以下组成的组：TIGIT和CTLA-4，OX40和CTLA-4，TIGIT和PD-1，PD-L1和CD47(分化簇47)，TIGIT和OX40，VEGF(血管内皮生长因子)和cMET(由c-met原癌基因编码)，VEGF和DLL4(delta-like ligand 4)，VEGF和HGF(肝细胞生长因子)，VEGF和ANGPT2(血管生成素2)，TfR(转铁蛋白受体，CD71)和CD20(分化簇20)，PD-L1和4-1BB(CD137，属于肿瘤坏死因子受体超家族成员)，PSMA(前列腺特异性膜抗原)和CD28(共刺激分子)，PD-1和PD-L1，HER2(人表皮生长因子受体2)和4-1BB，PD-1和TIM-3，PD-1和CD47(分化簇47)，GITR(糖皮质激素诱导的肿瘤坏死因子受体)和CTLA-4，CD40(分化簇40，肿瘤坏死因子受体超家族成员5)和4-1BB，OX40和4-1BB，LAG-3和TIM-3，EGFR(表皮生长因子受体1)和CTLA-4，CD19(分化簇19)和CD22(分化簇22)，CD16(分化簇16)和CD30(分化簇30)，CD3(分化簇3)和CD123(分化簇123)，BCMA(B细胞成熟抗原)和CD47，MSLN(间皮素)和CD47，EGFR和cMET，CD73和TGFβ(转化生长因子β)，EGFR和TGFβ，CCR2(CC趋化因子受体2)和CSF1R(集落刺激因子1受体)，CD20和CD3，CD19和CD47，CDH17(肝肠钙黏着蛋白)和TRAILR2(TRAIL受体2，TRAIL为肿瘤坏死相关凋亡诱导配体)，APLP2(淀粉样肽前体样蛋白2)和HER2，IL-1α和IL-1β，IL-17和IL-13，IL-4和IL-13，BAFF(B细胞活化因子)和IL-17A(白介素17A)，CD3和PD-1，IL-4Ra(白介素4受体亚基α)和IL-5，VEGF和IL-6(白介素IL-6)，FGFR1(成纤维细胞生长因子受体1)和KLB(klotho beta蛋白)。在一些实施方案中，抗体或抗原结合片段能同时特异性结合抗原a和抗原b。
在一些实施方案中，所述抗原a为TIGIT，抗原b为CTLA-4。在一些实施方案中，所述抗原a为OX40，抗原b为CTLA-4。在一些实施方案中，所述抗原a为OX40，抗原b为TIGIT。
在一些实施方案中，所述抗原a为TIGIT且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
所述VHa含US20190100591A1或US20180169239A1中公开的重链CDR或重链可变区；
所述VHb含CN1404876A或US9963508中公开的重链CDR或重链可变区；和/或
所述VLa含US20190100591A1或US20180169239A1中公开的轻链CDR或轻链可变区；和/或
所述VLb含CN1404876A或US9963508中公开的轻链CDR或轻链可变区。
US20190100591A1、US20180169239A1、CN1404876A、US9963508的全部内容通过引用并入本文。
在一些实施方案中，所述抗原a为TIGIT且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
所述VHa含SEQ ID NO:1所示的序列中第30-35位氨基酸(VHaCDR1，SSYGMS)，和/或第50-66位氨基酸(VHaCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHaCDR3，LGTGTLGFAY)氨基酸；和/或
所述VHb含SEQ ID NO:2所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
所述VLa含SEQ ID NO:3所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLaCDR2，WASTRAT)，和/或第89-97位氨基酸(VLaCDR3，QQHYSTPWT)；和/或
所述VLb含SEQ ID NO:4所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
在一些实施方案中，所述抗原a为TIGIT且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
所述VHa含SEQ ID NO:1所示的序列，与SEQ ID NO:1所示序列具有至少80％同一性的序列，或与SEQ ID NO:1所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VHb含SEQ ID NO:2所示的序列，与SEQ ID NO:2所示序列具有至少80％同一性的序列，或与SEQ ID NO:2所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VLa含SEQ ID NO:3所示的序列，与SEQ ID NO:3所示序列具有至少80％同一性的序列，或与SEQ ID NO:3所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VLb含SEQ ID NO:4所示的序列，与SEQ ID NO:4所示序列具有至少80％同一性的序列，或与SEQ ID NO:4所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CLa含SEQ ID NO:5所示的序列，与SEQ ID NO:5所示序列具有至少80％同一性的序列，或与SEQ ID NO:5所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CLb含SEQ ID NO:6所示的序列，与SEQ ID NO:6所示序列具有至少80％同一性的序列，或与SEQ ID NO:6所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CH1含SEQ ID NO:7所示的序列，与SEQ ID NO:7所示序列具有至少80％同一性的序列，或与SEQ ID NO:7所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗原a为OX40且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
所述VHa含CN101331150A1或US20150307617A1中公开的重链CDR或重链可变区；
所述VHb含CN1404876A或US9963508中公开的重链CDR或重链可变区；和/或
所述VLa含CN101331150A1或US20150307617A1中公开的轻链CDR或轻链可变区；和/或
所述VLb含CN1404876A或US99635中公开的轻链CDR或轻链可变区。
CN101331150A1、US20150307617A1、CN1404876A、US9963508的全部内容通过引用并入本文。
在一些实施方案中，所述抗原a为OX40且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
所述VHa含SEQ ID NO:8所示的序列中第31-35位氨基酸(VHaCDR1，SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和/或
所述VHb含SEQ ID NO:9所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
所述VLa含SEQ ID NO:10所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和/或
所述VLb含SEQ ID NO:11所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
在一些实施方案中，当所述抗原a为OX40且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
所述VHa含SEQ ID NO:8所示的序列，与SEQ ID NO:8所示序列具有至少80％同一性的序列，或与SEQ ID NO:8所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VHb含SEQ ID NO:9所示的序列，与SEQ ID NO:9所示序列具有至少80％同一性的序列，或与SEQ ID NO:9所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VLa含SEQ ID NO:10所示的序列，与SEQ ID NO:10所示序列具有至少80％同一性的序列，或与SEQ ID NO:10所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VLb含SEQ ID NO:11所示的序列，与SEQ ID NO:11所示序列具有至少80％同一性的序列，或与SEQ ID NO:11所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CLa含SEQ ID NO:12所示的序列，与SEQ ID NO:12所示序列具有至少80％同一性的序列，或与SEQ ID NO:12所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CLb含SEQ ID NO:13所示的序列，与SEQ ID NO:13所示序列具有至少80％同一性的序列，或与SEQ ID NO:13所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CH1含SEQ ID NO:14所示的序列，与SEQ ID NO:14所示序列具有至少80％同一性的序列，或与SEQ ID NO:14所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗原a为OX40且抗原b为TIGIT，所述抗体或抗原结合片段包含以下：
所述VHa含CN101331150A1或US20150307617A1中公开的重链CDR或重链可变区；
所述VHb含US20190100591A1或US20180169239A1中公开的重链CDR或重链可变区；和/或
所述VLa含CN101331150A1或US20150307617A1中公开的轻链CDR或轻链可变区；和/或
所述VLb含US20190100591A1或US20180169239A1中公开的轻链CDR或轻链可变区。
在一些实施方案中，所述抗原a为OX40且抗原b为TIGIT，所述抗体或抗原结合片段包含以下：
所述VHa含SEQ ID NO:15所示的序列中第31-35位氨基酸(VHaCDR1， SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和/或
所述VHb含SEQ ID NO:16所示的序列中第30-35位氨基酸(VHbCDR1，SSYGMS)，和/或第50-66位氨基酸(VHbCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHbCDR3，LGTGTLGFAY)氨基酸；和/或
所述VLa含SEQ ID NO:17所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和/或
所述VLb含SEQ ID NO:18所示的序列中第24-34位氨基酸(VLbCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLbCDR2，WASTRAT)，和/或第89-97位氨基酸(VLbCDR3，QQHYSTPWT)。
在一些实施方案中，所述抗原a为OX40且抗原b为TIGIT，所述抗体或抗原结合片段包含以下：
所述VHa含SEQ ID NO:15所示的序列，与SEQ ID NO:15所示序列具有至少80％同一性的序列，或与SEQ ID NO:15所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VHb含SEQ ID NO:16所示的序列，与SEQ ID NO:16所示序列具有至少80％同一性的序列，或与SEQ ID NO:16所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VLa含SEQ ID NO:17所示的序列，与SEQ ID NO:17所示序列具有至少80％同一性的序列，或与SEQ ID NO:17所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述VLb含SEQ ID NO:18所示的序列，与SEQ ID NO:18所示序列具有至少80％同一性的序列，或与SEQ ID NO:18所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CLa含SEQ ID NO:19所示的序列，与SEQ ID NO:19所示序列具有至少80％同一性的序列，或与SEQ ID NO:19所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CLb含SEQ ID NO:20所示的序列，与SEQ ID NO:20所示序列具有至少80％同一性的序列，或与SEQ ID NO:20所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
所述CH1含SEQ ID NO:21所示的序列，与SEQ ID NO:21所示序列具有至少80％同一性的序列，或与SEQ ID NO:21所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，至少80％同一性的序列为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，一个或多个保守氨基酸取代为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述L1含SEQ ID NO:22所示的序列；
所述L2含选自SEQ ID NO:23-27中任一项所示的序列，与SEQ ID NO:23-27中任一项所示序列具有至少90％同一性的序列，或与SEQ ID NO:23-27中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
所述L3含SEQ ID NO:28所示的序列；和/或
所述L4含选自SEQ ID NO:29-33中任一项所示的序列，与SEQ ID NO:29-33中任一项所示序列具有至少90％同一性的序列，或与SEQ ID NO:29-33中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述L2含SEQ ID NO:23所示的序列，与SEQ ID NO:23所示序列具有至少90％同一性的序列，或与SEQ ID NO:23所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述L4含SEQ ID NO:29所示的序列，与SEQ ID NO:29所示序列具有至少90％同一性的序列，或与SEQ ID NO:29所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述L2含SEQ ID NO:24所示的序列，与SEQ ID NO:24所示序列具有至少90％同一性的序列，或与SEQ ID NO:24所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述L4含SEQ ID NO:30所示的序列，与SEQ ID NO:30所示序列具有至少90％同一性的序列，或与SEQ ID NO:30所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述L2含SEQ ID NO:25所示的序列，与SEQ ID NO:25所示序列具有至少90％同一性的序列，或与SEQ ID NO:25所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述L4含SEQ ID NO:31所示的序列，与SEQ ID NO:31所示序列具有至少90％同一性的序列，或与SEQ ID NO:31所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述L2含SEQ ID NO:26所示的序列，与SEQ ID NO:26所示序列具有至少90％同一性的序列，或与SEQ ID NO:26所示序列相比具有一或多个 保守氨基酸取代的氨基酸序列；所述L4含SEQ ID NO:32所示的序列，与SEQ ID NO:32所示序列具有至少90％同一性的序列，或与SEQ ID NO:32所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述L2含SEQ ID NO:27所示的序列，与SEQ ID NO:27所示序列具有至少90％同一性的序列，或与SEQ ID NO:27所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述L4含SEQ ID NO:33所示的序列，与SEQ ID NO:33所示序列具有至少90％同一性的序列，或与SEQ ID NO:33所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，至少90％同一性的序列为约90％同一性、约91％同一性、约92％同一性、约93％同一性、约95％同一性、约96％同一性、约97％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，一个或多个保守氨基酸取代为约1个、约2个、约3个、约4个或约5个保守氨基酸取代。
在一些实施方案中，所述L1含SEQ ID NO:22所示的序列，所述L2含SEQ ID NO:23所示的序列，所述L3含选自SEQ ID NO:28所示的序列，所述L4含SEQ ID NO:29所示的序列。
在一些实施方案中，所述L1含SEQ ID NO:22所示的序列，所述L2含SEQ ID NO:24所示的序列，所述L3含SEQ ID NO:28所示的序列，所述L4含SEQ ID NO:30所示的序列。
在一些实施方案中，所述L1含SEQ ID NO:22所示的序列，所述L2含SEQ ID NO:25所示的序列，所述L3含SEQ ID NO:28所示的序列，所述L4含SEQ ID NO:31所示的序列。
在一些实施方案中，所述L1含SEQ ID NO:22所示的序列，所述L2含SEQ ID NO:26所示的序列，所述L3含SEQ ID NO:28所示的序列，所述L4含SEQ ID NO:32所示的序列。
在一些实施方案中，所述L1含SEQ ID NO:22所示的序列，所述L2含SEQ ID NO:27所示的序列，所述L3含SEQ ID NO:28所示的序列，所述L4含SEQ ID NO:33所示的序列。
在一些实施方案中，所述Fc含SEQ ID NO:34-36中任一项所示的序列，与SEQ ID NO:34-36中任一项所示序列具有至少80％同一性的序列，或与SEQ ID NO:34-36中任一项所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，至少80％同一性的序列为约80％同一性、约81％同一性、约82％同一性、约83％同一 性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，一个或多个保守氨基酸取代为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约17个、约19个、约21个、约22个、约25个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述Fc含SEQ ID NO:34所示的序列。在一些实施方案中，所述Fc含SEQ ID NO:35所示的序列。在一些实施方案中，所述Fc含SEQ ID NO:36所示的序列。
本发明还提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段结合两个不同抗原TIGIT和CTLA-4；所述抗体或抗原结合片段包含：
SEQ ID NO:1所示的序列中第30-35位氨基酸(VHaCDR1，SSYGMS)，和/或第50-66位氨基酸(VHaCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHaCDR3，LGTGTLGFAY)氨基酸；和
SEQ ID NO:2所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
SEQ ID NO:3所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLaCDR2，WASTRAT)，和/或第89-97位氨基酸(VLaCDR3，QQHYSTPWT)；和
SEQ ID NO:4所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；
所述第一条多肽链含SEQ ID NO:1所示的序列中第30-35位氨基酸(VHaCDR1，SSYGMS)，和/或第50-66位氨基酸(VHaCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHaCDR3，LGTGTLGFAY)氨基酸；和
SEQ ID NO:2所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
所述第二条多肽链含SEQ ID NO:3所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLaCDR2，WASTRAT)， 和/或第89-97位氨基酸(VLaCDR3，QQHYSTPWT)；和
SEQ ID NO:4所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
本发明还提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为TIGIT，第二抗原b为CTLA-4；所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:37所示的序列，与SEQ ID NO:37所示序列具有至少80％同一性的序列，或与SEQ ID NO:37所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:38所示的序列，与SEQ ID NO:38所示序列具有至少80％同一性的序列，或与SEQ ID NO:38所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；或
所述第一条多肽链含SEQ ID NO:39所示的序列，与SEQ ID NO:39所示序列具有至少80％同一性的序列，或与SEQ ID NO:39所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:40所示的序列，与SEQ ID NO:40所示序列具有至少80％同一性的序列，或与SEQ ID NO:40所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；或
所述第一条多肽链含SEQ ID NO:41所示的序列，与SEQ ID NO:41所示序列具有至少80％同一性的序列，或与SEQ ID NO:41所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:42所示的序列，与SEQ ID NO:42所示序列具有至少80％同一性的序列，或与SEQ ID NO:42所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；或
所述第一条多肽链含SEQ ID NO:43所示的序列，与SEQ ID NO:43所示序列具有至少80％同一性的序列，或与SEQ ID NO:43所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:44所示的序列，与SEQ ID NO:44所示序列具有至少80％同一性的序列，或与SEQ ID NO:44所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，至少80％同一性的序列为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，一个或多个保守氨基酸取代为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约17个、约19个、约21个、约22个、约25个、约27个、 约29个、约31个、约33个、约35个、约38个、约41个、约42个、约47个、约49个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为TIGIT，第二抗原b为CTLA-4，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:37所示的序列，所述第二条多肽链含SEQ ID NO:38所示的序列。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为TIGIT，第二抗原b为CTLA-4，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:39所示的序列，所述第二条多肽链含SEQ ID NO:40所示的序列。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为TIGIT，第二抗原b为CTLA-4，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:41所示的序列，所述第二条多肽链含SEQ ID NO:42所示的序列。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为TIGIT，第二抗原b为CTLA-4，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:43所示的序列，所述第二条多肽链含SEQ ID NO:44所示的序列。
本发明还提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段结合两个不同抗原OX40和CTLA-4；所述抗体或抗原结合片段包含：
SEQ ID NO:8所示的序列中第31-35位氨基酸(VHaCDR1，SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和
SEQ ID NO:9所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
SEQ ID NO:10所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和
SEQ ID NO:11所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；
所述第一条多肽链含SEQ ID NO:8所示的序列中第31-35位氨基酸(VHaCDR1，SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和
SEQ ID NO:9所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
所述第二条多肽链含SEQ ID NO:10所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和
SEQ ID NO:11所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
本发明还提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为OX40，第二抗原b为CTLA-4；所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:45所示的序列，与SEQ ID NO:45所示序列具有至少80％同一性的序列，或与SEQ ID NO:45所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:46所示的序列，与SEQ ID NO:46所示序列具有至少80％同一性的序列，或与SEQ ID NO:46所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，至少80％同一性的序列为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，一个或多个保守氨基酸取代为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约17个、约19个、约21个、约22个、约25个、约27个、约29个、约31个、约33个、约35个、约38个、约41个、约42个、约47个、约49个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为OX40，第二抗原b为CTLA-4，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID  NO:45所示的序列，所述第二条多肽链含SEQ ID NO:46所示的序列。
本发明还提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段结合两个不同抗原OX40和TIGIT；所述抗体或抗原结合片段包含:
SEQ ID NO:15所示的序列中第31-35位氨基酸(VHaCDR1，SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和
SEQ ID NO:16所示的序列中第30-35位氨基酸(VHbCDR1，SSYGMS)，和/或第50-66位氨基酸(VHbCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHbCDR3，LGTGTLGFAY)氨基酸；和/或
SEQ ID NO:17所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和
SEQ ID NO:18所示的序列中第24-34位氨基酸(VLbCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLbCDR2，WASTRAT)，和/或第89-97位氨基酸(VLbCDR3，QQHYSTPWT)。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；
所述第一条多肽链含SEQ ID NO:15所示的序列中第31-35位氨基酸(VHaCDR1，SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和
SEQ ID NO:16所示的序列中第30-35位氨基酸(VHbCDR1，SSYGMS)，和/或第50-66位氨基酸(VHbCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHbCDR3，LGTGTLGFAY)氨基酸；和/或
所述第二条多肽链含SEQ ID NO:17所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和
SEQ ID NO:18所示的序列中第24-34位氨基酸(VLbCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLbCDR2，WASTRAT)，和/或第89-97位氨基酸(VLbCDR3，QQHYSTPWT)。
本发明还提供了一种抗体或抗原结合片段，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为OX40，第二抗原b为TIGIT；所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:47或49所示的序列，与SEQ ID NO:47或49所示序列具有至少80％同一性的序列，或与SEQ ID NO:47或49所示序列相比具有一个或多个保守氨 基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:48或50所示的序列，与SEQ ID NO:48或50所示序列具有至少80％同一性的序列，或与SEQ ID NO:48或50所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，至少80％同一性的序列为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。在一些实施方案中，一个或多个保守氨基酸取代为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约17个、约19个、约21个、约22个、约25个、约27个、约29个、约31个、约33个、约35个、约38个、约41个、约42个、约47个、约49个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为OX40，第二抗原b为TIGIT，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:47所示的序列，所述第二条多肽链含SEQ ID NO:48所示的序列。
在一些实施方案中，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为OX40，第二抗原b为TIGIT，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:49所示的序列，所述第二条多肽链含SEQ ID NO:50所示的序列。
在一些实施方案中，所述抗体或抗原结合片段含序列相同的第一条多肽链和第三条多肽链以及序列相同的第二条多肽和第四条多肽链。在一些实施方案中，所述抗体或抗原结合片段含序列相同的第一条多肽链和第三条多肽链以及序列相同的第二条多肽和第四条多肽链，所述第一条多肽链和第三条多肽链的Fc区配对形成二硫键。
在一些实施方案中，所述抗体或抗原结合片段为分离的抗体或抗原结合片段。
本发明还提供了编码所述的抗体或抗原结合片段的核酸分子。在一些实施方案中，所述核酸分子为分离的核酸分子。
本发明还提供了包含所述的核酸分子的载体。在一些实施方案中，所述载体为分离的载体。
本发明还提供了包含所述核酸分子或载体的宿主细胞。在一些实施方案中，所述宿主细胞为分离的宿主细胞。在一些实施方案中，所述宿主细胞为CHO细胞、293细胞、Cos1细胞、Cos7细胞、CV1细胞和鼠L细胞。
本发明还提供了药物组合物，所述药物组合物包含上述的抗体或抗原结合片段，以及药学上可接受的辅料。
本发明还提供了治疗方法和用途。在一些实施方案中，提供了用于治疗或改善各种疾病(如炎性疾病、自体免疫性疾病、神经退行性疾病、癌症或脊髓损伤)的方法，所述方法包括向患者施用有效剂量的所述的抗体或抗原结合片段。在一些实施方案中，提供了所述的抗体或抗原结合片段在治疗或改善各种疾病(如炎性疾病、自体免疫性疾病、癌症或脊髓损伤)的药物中的应用。在一些实施方案中，提供了所述的抗体或抗原结合片段在制备用于治疗或改善各种疾病(如炎性疾病、自体免疫性疾病、癌症或脊髓损伤)的药物中的应用。
在一些实施方案中，所述自体免疫性疾病或炎性疾病选自由以下组成的组：克罗恩氏病(Crohn's disease)、牛皮癣(包括斑块状牛皮癣)、关节炎(包括类风湿性关节炎、牛皮癣性关节炎、骨关节炎或青少年特发性关节炎)、多发性硬化、强直性脊椎炎、强直性关节病变(spondylosing arthropathy)、全身性红斑狼疮、葡萄膜炎、败血症、神经退行性疾病、神经元再生、脊髓损伤、原发性和转移性癌症、呼吸道病症、哮喘、过敏性和非过敏性哮喘、由感染引起的哮喘、由感染呼吸道合胞病毒(RSV)引起的哮喘、慢性阻塞性肺病(COPD)、涉及气道炎症的病状、嗜酸性粒细胞增多症、纤维化和过量粘液产生、囊性纤维化、肺纤维化、异位性病症、异位性皮炎、荨麻疹、湿疹、过敏性鼻炎、过敏性肠胃炎、炎性和/或自体免疫性皮肤病状、炎性和/或自体免疫性胃肠器官病状、炎性肠病(IBD)、溃疡性结肠炎、炎性和/或自体免疫性肝病、肝硬化、肝纤维化、由乙型和/或丙型肝炎病毒引起的肝纤维化、硬皮病。在一些实施方案中，所述癌症选自以下组成的组：肝细胞癌、成胶质细胞瘤、淋巴瘤、或霍奇金氏淋巴瘤。在一些实施方案中，所述癌症选自由以下组成的组：黑素瘤(例如，转移性恶性黑素瘤)、肾癌(例如，透明细胞癌)、前列腺癌(例如，激素难治性前列腺腺癌)、胰腺癌、乳癌、结肠癌、肺癌(例如，非小细胞肺癌)、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病。在一些实施方案中，所述癌症为选自由以下组成的组：霍奇金氏淋巴瘤(Hodgkin's lymphoma)、非霍奇金氏淋巴瘤[NHL]、前体B细胞淋巴母细胞性白血病/淋巴瘤、成熟B细胞赘瘤、B细胞慢性淋巴细胞白血病/小淋巴细胞性淋巴瘤、B细胞幼淋巴细胞白血病、淋巴浆细胞淋巴瘤、套细胞淋巴瘤、滤泡性淋巴瘤、皮肤滤泡中心淋巴瘤、边缘区B细胞淋巴瘤、毛细胞白血病、弥漫性大B细胞淋巴瘤、伯基特氏淋巴瘤(Burkitt's lymphoma)、浆细胞瘤、浆细胞性骨髓瘤、移植后淋巴增生病症、移瓦尔登斯特伦氏巨球蛋白血症(Waldenstrom's macroglobulinemia)及间变性大细胞淋巴瘤。
本发明还提供了诊断方法和用途。在一些实施方案中，提供了检测样品中抗原a和/或抗原b表达的方法，使样品与所述的抗体或抗原结合片段进行接触，使得所述抗 体或抗原结合片段结合抗原a和/或抗原b，并检测其结合，即样品中抗原a和/或抗原b的含量。在一些实施方案中，所述抗原a、b为细胞因子、细胞因子受体、趋化因子、趋化因子受体或细胞表面蛋白。在一些实施方案中，抗原a、b分别选自于以下组成的组：TIGIT和CTLA-4，OX40和CTLA-4，TIGIT和PD-1，PD-L1和CD47，TIGIT和OX40，VEGF和cMET，VEGF和DLL4，VEGF和HGF，VEGF和ANGPT2，TfR和CD20，PD-L1和4-1BB，PSMA和CD28，PD-1和PD-L1，HER2和4-1BB，PD-1和TIM-3，PD-1和CD47，GITR和CTLA-4，CD40和4-1BB，OX40和4-1BB，LAG-3和TIM-3，EGFR和CTLA-4，CD19和CD22，CD16和CD30，CD3和CD123，BCMA和CD47，MSLN和CD47，EGFR和cMET，CD73和TGFβ，EGFR和TGFβ，CCR2和CSF1R，CD20和CD3，CD19和CD47，CDH17和TRAILR2，APLP2和Her2，IL-1α和IL-1β，IL-17和IL-13，IL-4和IL-13，BAFF和IL-17A，CD3和PD-1，IL-4Ra和IL-5，VEGF和IL-6，FGFR1和KLB。
在一些实施方案中，提供了所述的抗体或抗原结合片段在制备用于诊断炎性疾病、自体免疫性疾病、神经退行性疾病、癌症或脊髓损伤的试剂盒中的应用。在一些实施方案中，提供了包含所述抗体或抗原结合片段的诊断试剂盒。
本发明提供了多价和多特异性抗体或抗原结合片段及其应用，本发明的多特异性抗体或抗原结合片段可以结合两个或更多个抗原，或同一抗原的两个或更多个表位。本发明抗体或抗原结合片段用于治疗或改善各种疾病，比如炎性疾病、自体免疫性疾病癌症或脊髓损伤，也用于相关疾病的诊断和预后。
附图说明
图1为一些实施方案中本发明双特性抗体的结构示意图。
图2为本发明实施例中抗体1的SDS-PAGE图谱；其中，泳道1表示抗体1处于非还原状态，泳道M表示maker，泳道2表示抗体1处于还原状态。
图3为本发明实施例中抗体2和抗体3的SDS-PAGE图谱；图中CT-20BiAb表示抗体2，CT-25BiAb表示抗体3，泳道1表示抗体3处于非还原状态，泳道2表示抗体2处于非还原状态，泳道3表示抗体3处于还原状态，泳道4表示抗体2处于还原状态，M表示maker。
图4显示抗体1、抗体2、抗体3与TIGIT-Fc的结合活性；其中，CT BiAb表示抗体1，CT-20BiAb表示抗体2，CT-25BiAb表示抗体3。
图5显示抗体1、抗体2、抗体3与CTLA-4-Fc的结合活性；其中，CT BiAb表示抗体1，CT-20BiAb表示抗体2，CT-25BiAb表示抗体3。
图6为采用Promega的anti-CTLA-4报告基因检测系统检测抗体的生物学活性； 其中，CTLA-4/TIGIT BiAb表示抗体1。
图7为采用Promega的anti-CTLA-4报告基因检测系统检测抗体的生物学活性；其中，CT-20BiAb表示抗体2。
图8为采用anti-TIGIT报告基因检测系统检测抗体的生物学活性；其中，CT BiAb表示抗体1。
图9为采用anti-TIGIT报告基因检测系统检测抗体的生物学活性；其中，CT-20BiAb表示抗体2。
图10为采用anti-TIGIT报告基因检测系统检测抗体的生物学活性；其中，CT-25BiAb表示抗体3。
图11为本发明实施例中抗体5(CTLA-4-OX40双特抗体)的SDS-PAGE图谱；其中，lane表示泳道，lane 1、lane 2和lane 3依次从左往右排列。
图12为本发明实施例中抗体5与OX40 +细胞的结合曲线；OX40 +细胞为表达OX40的细胞；横坐标表示抗体浓度的对数值，纵坐标表示在流式细胞仪上读出的2通道平均荧光值；其中，O4表示抗体5。
图13为本发明实施例中抗体5与OX40抗原和CTLA-4抗原结合的亲和力动力学拟合曲线，其中O4表示抗体5。
图14为本发明实施例中抗体6和7的SDS-PAGE图谱；其中，泳道M表示maker，泳道1表示抗体6处于非还原状态，泳道2表示抗体6处于还原状态，泳道3表示抗体7处于非还原状态，泳道4表示抗体7处于还原状态；OT-4D-13aa抗体表示抗体6，OT-4D-30aa抗体表示抗体7。
图15为本发明实施例中抗体7与OX40抗原和TIGIT抗原结合的亲和力动力学拟合曲线。
图16为本发明实施例中抗体7与Jurkat-OX40细胞的结合曲线；其中，anti-OX40Ab表示为OX40单克隆抗体(即anti-OX40)，OT-4D-30a和4D-30a均表示抗体7；图中EC50的单位为nM。
图17为本发明实施例中抗体7与Jurkat-TIGIT细胞的结合曲线；其中，anti-Tigit Ab表示TIGIT单克隆抗体(即anti-TIGIT)，OT-4D-30a表示抗体7。
术语
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。
定义
应当注意的是，术语“一种”实体是指一种或多种该实体，例如“一种抗体”应当被理解为一种或多种抗体，因此，术语“一种”(或“一个”)、“一种或多种”和“至少一种”可以在本文中互换使用。
本文所用的术语“包含”或“包括”意味着组合物和方法等包括所列举的元素，例如组份或步骤，但不排除其它。“基本上由……组成”意味着组合物和方法排除对组合的特征有根本影响的其它元素，但不排除对组合物或方法无本质上影响的元素。“由……组成”意味着排除未特别列举的元素。
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体组成的分子。术语“多肽”是指两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：ala，一字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨酸(leu，L)、赖氨酸(lys，K)、甲硫氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp，W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。
本发明中关于细胞、核酸、多肽、抗体等所使用的术语“分离的”，例如“分离 的”DNA、RNA、多肽、抗体是指分别于细胞天然环境中的其它组分如DNA或RNA中的一种或多种所分离的分子。本发明使用的术语“分离的”还指当通过重组DNA技术产生时基本上不含细胞材料、病毒材料或细胞培养基的核酸或肽，或化学合成时的化学前体或其他化学品。此外，“分离的核酸”意在包括不以天然状态存在的核酸片段，并且不会以天然状态存在。术语“分离的”在本发明中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。分离的多肽、抗体等通常通过至少一个纯化步骤制备。在一些实施方案中，分离的核酸、多肽、抗体等的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式，不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。
多聚核苷酸或多聚核苷酸序列(或多肽或抗体序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性或序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对和同一性百分比或序列同一性，比如Ausubel et al.eds.(2007)在Current Protocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。
多聚核苷酸是由四个核苷酸碱基的特定序列组成：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)，或当多聚核苷酸是RNA时胸腺嘧啶换为尿嘧啶(U)。“多聚核苷酸序列”可以以多聚核苷酸分子的字母表示。该字母表示可以被输入到具有中央处理单元的计算机中的数据库中，并用于生物信息学应用，例如用于功能基因组学和同源性搜索。
术语“多聚核苷酸”和“寡核苷酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多聚核苷酸可以具有任何三维结 构并且可以执行已知或未知的任何功能。以下是不受限制的多聚核苷酸的实施例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、dsRNA、siRNA、miRNA、重组多聚核苷酸、分支的多聚核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。多聚核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在该修饰，则对核苷酸的结构修饰可以在组装多聚核苷酸之前或之后进行。核苷酸的序列可以被非核苷酸组分中断。聚合后可以进一步修饰多聚核苷酸，例如通过与标记组分缀合。这个术语也指双链和单链分子。除另有说明或要求外，本公开的任何多聚核苷酸的实施例包括双链形式和已知或预测构成双链形式的两种可互补单链形式中的每一种。
术语“编码”应用于多聚核苷酸时，是指被称为“编码”多肽的多聚核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。
“抗体”、“抗原结合片段”是指特异性识别和结合抗原的多肽或多肽复合物。抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的任何蛋白质或肽。抗体和抗原结合片段包括但不局限重链或轻链或其配体结合部分的互补决定区(CDR)、重链可变区(VH)、轻链可变区(VL)、重链恒定区(CH)、轻链恒定区(CL)、框架区(FR)或其任何部分，或结合蛋白的至少一部分。CDR区包括轻链的CDR区(VL CDR1-3)和重链的CDR区(VH CDR1-3)。本发明实施例所述抗体或抗原结合片段为双特异性抗体，是将特异性结合抗原a、抗原b的片段进行融合：第一条多肽链包含VHa、CLa、VHb、CH1，第一条多肽链类似于免疫球蛋白的重链或重链片段；第二条多肽链包含结构VLa、CH1、VLb和CLb，第二条多肽链类似于免疫球蛋白的轻链。
术语“抗体片段”或“抗原结合片段”指抗体的一部分，本发明抗体片段的组成形式可类似于单特异性抗体片段中的F(ab’) 2、F(ab) 2、Fab'、Fab、Fv、scFv等。不管其结构如何，抗体片段与被完整抗体识别的同一抗原结合。术语“抗体片段”包括适体、镜像异构体和双价抗体。术语“抗原结合片段”还包括通过与特定抗原结合形成复合物起抗体作用的任何合成或基因工程蛋白质。
“单链可变片段”或“scFv”是指免疫球蛋白的重链(VH)和轻链(VL)的可变区的融合蛋白。在一些方面，这些区域与10个至约25个氨基酸的短接头肽连接。接头可以富含甘氨酸以增加柔韧性，以及富含丝氨酸或苏氨酸以增加溶解性，并且可以连接VH的N端和VL的C端，反之亦然。尽管该蛋白质被除去了恒定区和引入了接头，但其保留了原始免疫球蛋白的特异性。ScFv分子通常是本领域中已知的，例如在美国 专利5,892,019中有相关描述。
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。本领域技术人员将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgG5等已被充分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一些实施方案中，免疫球蛋白分子为IgG种类。IgG通常包含分子量约23,000道尔顿的两条相同的轻链多肽和分子量约为53,000-70,000的两条相同的重链多肽。这四条链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。
本发明公开的抗体、抗原结合片段、或衍生物包括但不限于多克隆、单克隆、多特异性，全人源、人源化、灵长类化、嵌合抗体、单链抗体、表位结合片段例如类Fab、类Fab'和类F(ab') 2、类单链Fvs(scFv)。
轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤，B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为V λ。
轻链和重链都分成结构和功能同源性的区域。术语“恒定的”和“可变的”根据功能被使用。轻链(VL)和重链(VH)链部分的可变区决定了抗原识别和特异性。轻链和重链的恒定区赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可变区，C端部分是恒定区；CH3和CL结构域实际上分别包含重链和轻链的羧基端。
在天然存在的抗体中，假设抗体在含水环境中呈现其三维构型时，存在于每个抗原结合域中的六个“互补决定区”或“CDR”是形成抗原结合结构域的短的、非连续的与抗原特异性结合的氨基酸序列。抗原结合结构域中被称为“构架”区域的剩余其它氨基酸显示出较小的分子间可变性。构架区大部分采用β-折叠构象，CDR形成与之连接的环状结构，或在某些情况下形成β折叠结构的一部分。因此，框架区通过形成支架从而通过链间非共价相互作用使CDR定位在正确的方位上。具有特定位置的CDR的抗原结合域形成了与抗原上的表位互补的表面，该互补表面促进抗体和其抗原表位的非共价结合。对于给定的重链或轻链可变区，本领域普通技术人员都可以通过已知方法鉴定出包含CDR和框架区的氨基酸(参见Kabat,E.,et al.,U.S.Department of Health  and Human Services,Sequences of Proteins of Immunological Interest,(1983)和Chothia and Lesk,J.Mol.Biol.,196:901-917(1987))。
在本领域中使用和/或接受的术语有两个或多个定义的情况下，除非明确地对立指出，否则本文使用的术语的定义包括所有这些含义。一个具体的例子是使用“互补决定区”(“CDR”)一词来描述在重链和轻链多肽的可变区内发现的非连续的抗原结合位点。这一特定区域在Kabat et al.,U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)和Chothia等在J.Mol.Biol.196:901-917(1987)有相关描述，其通过引用全部并入本文。
根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。包含特定CDR的确切残基编号将根据CDR的序列和大小而变化。本领域技术人员通常可以根据抗体的可变区氨基酸序列确定出CDR包含哪些特定的残基。
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.,U.S.Dept.of Health and Human Services在“Sequence of Proteinsof Immunological Interest”(1983)提出的编号系统。抗体还可以用EU编号系统。
本发明公开的抗体可以来源于任何动物，包括鸟类和哺乳动物。在一些实施方案中，抗体是人源、鼠源、驴源、兔源、山羊源、骆驼源、美洲驼源、马源或鸡源抗体。在另一实施方案中，可变区可以是软骨鱼纲(condricthoid)来源(例如来自鲨鱼)。
重链恒定区包括CH1结构域、铰链(例如上、中和/或下铰链区)结构域、CH2结构域、CH3结构域，或变体或片段中的至少一种。抗体的重链恒定区可以来源于不同的免疫球蛋白分子。例如，多肽的重链恒定区可以包括源自IgG 1分子的CH1结构域和源自IgG 3分子的铰链区。在另一实施方案中，重链恒定区可以包括部分源自IgG 1分子和部分源自IgG 3分子的铰链区。在另一实施方案中，部分重链可以包括部分源自IgG 1分子和部分源自IgG 4分子的嵌合铰链区。
“轻链恒定区”包括来自抗体轻链的一部分氨基酸序列。较佳地，轻链恒定区包含恒定κ结构域或恒定λ结构域中的至少一个。“轻链-重链对”是指可通过轻链的CL结构域和重链的CH1结构域之间的二硫键形成二聚体的轻链和重链的集合。
“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，“CH1结构域”包括免疫球蛋白重链的第一个(大部分氨基末端)恒定区。CH2结构域不与其它结构域紧密配对，而是在完整的天然IgG分子的两个CH2结构域之间插入两个N-连接的分支碳水 化合物链。还有文献记载，CH3结构域从CH2结构域开始延伸到IgG分子的C-末端，大约包含108个残基。“铰链区”包括连接CH1结构域和CH2结构域的部分重链区域。所述铰链区包含约25个残基并且是有韧性的，从而使得两个N端抗原结合区能够独立移动。铰链区可以被细分为三个不同的结构域：上、中和下铰链结构域(Rouxetal.,J.Immunol 161:4083(1998))。
“二硫键”指两个硫原子之间形成的共价键。半胱氨酸的硫醇基团可以与第二个硫醇基团形成二硫键或桥接。在大多数天然存在的IgG分子中，CH1和CL区通过二硫键连接。
“嵌合抗体”指其可变区从第一个物种中获得或衍生，而其恒定区(可以是完整的、部分的或修饰过的)来源于第二个物种的任何抗体。某些实施方案中，可变区来自非人源(例如小鼠或灵长类动物)，而恒定区来自人源。
“特异性结合”或“对……具有特异性”通常是指抗体或抗原结合片段与特定抗原通过其抗原结合结构域与表位互补性结合形成相对稳定的复合物。“特异性”可以用抗体或抗原结合片段与特定抗原或表位结合的相对亲和力表达。例如，如果抗体“A”比抗体“B”与同一抗原的相对亲和力大，可以认为抗体“A”比抗体“B”对该抗原具有更高的特异性。特异性结合可以用平衡解离常数(KD)来描述，较小的KD意味着较紧密的结合。确定两个分子是否特异性结合的方法是本领域内众所周知的，并包括例如平衡透析、表面等离子共振、生物膜层光学干涉测量法等。“特异性结合”抗原a的抗体包括与抗原a平衡解离常数KD小于或等于约100nM、小于或等于约10nM、小于或等于约5nM、小于或等于约1nM的抗体。
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善或停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善、缓和、减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的抗体或药物组合物用于检测、诊断过程和/或治疗中受益的患者。
“患者”指需要诊断、预后或治疗的任何哺乳动物，包括人类、狗、猫、兔子、鼠、马、牛等。
“约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。
“EC 50”即半最大效应浓度(concentration for 50％of maximal effect,EC 50)是指能 引起50％最大效应的浓度。
双特异性抗体
本发明提供了例如图1所示的新型双特异抗体或抗原其结合片段，可以通过调节与第一抗原结合的可变区和与第二抗原结合的可变区的位置及之间的连接子来调节双特异抗体或抗原其结合片段对其结合的两个抗原的结合活性，以增强抗体治疗效果并减少副作用。
1)靶向TIGIT和CTLA-4的抗体
本发明提供了对TIGIT和CTLA-4蛋白具有高亲和力的双特异抗体或抗原其结合片段。受测抗体表现出有效的结合活性、生物学活性，并可用于治疗和诊断用途。比如，这些抗体或抗原结合片段有效阻断免疫检查点，激活淋巴细胞释放细胞因子，用于治疗各种类型的癌症、肿瘤或感染等相关疾病。
因此，本发明公开的一个实施方案提供了靶向TIGIT和CTLA-4的抗体或抗原结合片段，该抗体或抗原结合片段特异性结合TIGIT和CTLA-4。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:37所示的序列，所述第二条多肽链含SEQ ID NO:38所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:37中除Fc区序列以外的序列，所述第二条多肽链含SEQ ID NO:38所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:39所示的序列，所述第二条多肽链含SEQ ID NO:40所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:39中除Fc区序列以外的序列，所述第二条多肽链含SEQ ID NO:40所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:41所示的序列，所述第二条多肽链含SEQ ID NO:42所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:41中除Fc区序列以外的序列，所述第二条多肽链含SEQ ID NO:42所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:43所示的序列，所述第二条多肽链含SEQ ID NO:44所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:43中除Fc区序列以外的序列，所述第二条多肽链含SEQ ID NO:44所示的序列。
2)靶向OX40和CTLA-4的抗体
本发明提供了对OX40和CTLA-4蛋白具有高亲和力的双特异抗体或抗原其结合片段。受测抗体表现出有效的结合活性、生物学活性，并可用于治疗和诊断用途。比如，这些抗体或抗原结合片段有效活化T细胞，激活淋巴细胞释放细胞因子，用于治疗各种类型的癌症、肿瘤或感染等相关疾病。
因此，本发明公开的一个实施方案提供了靶向OX40和CTLA-4的抗体或抗原结合片段，该抗体或抗原结合片段特异性结合OX40和CTLA-4。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:45所示的序列，所述第二条多肽链含SEQ ID NO:46所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:45中除Fc区序列以外的序列以及SEQ ID NO:36所示的Fc区，所述第二条多肽链含SEQ ID NO:46所示的序列。
3)靶向OX40和TIGIT的抗体
本发明提供了对OX40和TIGIT蛋白具有高亲和力的双特异抗体或抗原其结合片段。受测抗体表现出有效的结合活性、生物学活性，并可用于治疗和诊断用途。比如，这些抗体或抗原结合片段有效效阻断免疫检查点，活化T细胞，激活淋巴细胞释放细胞因子，用于治疗各种类型的癌症、肿瘤或感染等相关疾病。
因此，本发明公开的一个实施方案提供了靶向OX40和TIGIT的抗体或抗原结合片段，该抗体或抗原结合片段特异性结合OX40和TIGIT。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:47所示的序列，所述第二条多肽链含SEQ ID NO:48所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:47中除Fc区序列以外的序列，所 述第二条多肽链含SEQ ID NO:48所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:49所示的序列，所述第二条多肽链含SEQ ID NO:50所示的序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链，所述第一条多肽链含SEQ ID NO:49中除Fc区序列以外的序列，所述第二条多肽链含SEQ ID NO:50所示的序列。
本领域普通技术人员还应当理解，本发明所公开抗体或抗原结合片段序列是可以被替换的，替换后其氨基酸序列不同于该抗体的天然存在的氨基酸序列。例如，替换后的氨基酸序列可以是与起始序列相似的，比如与起始序列具有一定比例的同一性，比如它可以与起始序列的同一性是约80％、约85％、约90％、约95％、约98％或约99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
在某些实施方案中，抗体包的含氨基酸序列具有一个或多个修饰基团。例如，本发明公开的双特异性抗体(靶向TIGIT和CTLA-4的双特异性抗体、靶向OX40和CTLA-4的双特异性抗体以及靶向OX40和TIGIT的双特异性抗体)可以包含有韧性的接头序列，或者可以被修饰以添加功能性基团(例如PEG、药物、毒素或标签)。
本发明公开的抗体、抗原结合片段包括被修饰的衍生物，即通过任何类型的分子与抗体的共价连接进行修饰，其中共价连接不会阻止抗体与表位结合。包括但不限制以下实例，抗体可以被糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割、连接至细胞配体或其他蛋白质等。众多化学修饰中的任一种修饰可以通过现有技术进行，包括但不限于特异性化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等。
在一些实施方案中，抗体可以与治疗剂、药物前体、肽、蛋白质、酶、病毒、脂类、生物反应调节剂、药剂或PEG缀合。
抗体可以与治疗剂缀合或融合，所述治疗剂可包括可检测标记(如放射性标记)、免疫调节剂、激素、酶、寡核苷酸、光敏治疗剂、诊断剂、药物或毒素的细胞毒性剂、超声增强剂、非放射性标记物及其组合物，和本领域已知的其它此类试剂。
抗体可通过将其偶联至化学发光化合物来被可检测地标记。然后通过检测在化学反应过程中出现的发光从而确定化学发光标记的抗体的存在。化学发光标记化合物的实例包括鲁米诺、异鲁米诺、芳香吖啶酯、咪唑、吖啶盐和草酸酯。
抗体和编码抗体的多聚核苷酸的制备方法
本发明还公开了编码本发明所述抗体、抗原结合片段、及其衍生物的多聚核苷酸或核酸分子。本发明公开的多聚核苷酸可以编码第一条多肽(类似于免疫球蛋白的重链或其片段)、第二条多肽(类似于免疫球蛋白的的轻链)、重链可变区、轻链可变区、Fc区、部分重链可变区或部分轻链可变区。制备抗体的方法是本领域公知的并且在本发明中有所描述。在某些实施方案中，本发明公开的抗体、抗原结合片段包括的可变区和恒定区都是全人源的。全人源抗体和抗原结合片段可以使用本领域中公开的技术和本发明所述的技术制备。例如，针对特定抗原的全人源抗体可以通过将抗原施用于转基因动物中来制备，所述转基因动物已经被改良过以响应抗原攻击而产生全人源抗体。可用于制备这类抗体的示例性技术参见美国专利6,458,592；6,420,140，其全部内容通过引用并入本文。本发明中所述双特异性抗体是将特异性结合抗原a、抗原b的片段进行融合，双特异性抗体中部分片段可参见上述结合单个抗原的抗体的制备方法。
在某些实施方案中，制备的抗体不会在待治疗的动物(例如人类)中引起有害的免疫应答。在一实施方案中，本发明公开的抗体、抗原结合片段、或衍生物使用本领域公认的技术修饰以降低其免疫原性。例如，抗体可以被人源化、灵长类化、去免疫化或者可以制备嵌合抗体。这些类型的抗体来源于非人抗体，通常是鼠类或灵长类抗体，其保留或基本保留亲本抗体的抗原结合特性但在人体中免疫原性较低。其可以通过多种方法来实现，包括(a)将整个非人源的可变区移植到人源的恒定区以产生嵌合抗体；(b)将一个或多个非人类互补决定区(CDR)的至少一部分移植到人源的框架和恒定区中，保留或不保留关键的框架残基；或(c)移植整个非人源的可变区，但通过用类人源的部分置换表面残基从而“隐藏”它们。通常人框架区中的框架残基将被来自CDR供体抗体的相应残基取代，比如能够改善抗原结合的残基。这些框架替换可以通过本领域公知的方法鉴定，例如通过模拟CDR和框架残基的相互作用以鉴定对抗原结合起重要作用的框架残基和通过序列对比以鉴定特定位置上异常的框架残基。(参考美国专利5,585,089；Riechmann et al.,Nature 332:323(1988)；其全部内容通过引用并入本文)。可以使用本领域公知的多种技术使抗体人源化，例如CDR移植(EP 239,400；WO 91/09967；美国专利5,225,539,5,530,101和5,585,089)，修复或者表面重排(EP592,106；EP519,596；Padlan,et al.,Molecular Immunology 28(4/5):489-498(1991)；Studnicka et al.,Protein Engineering 7(6):805-814(1994)；Roguska,et al.,Proc.Natl.Sci.USA 91:969-973(1994))，以及链的重排(美国专利5,565,332)，其全部内容通过引用并入本文。
去免疫化也可用于降低抗体的免疫原性。在本发明中，术语“去免疫化”包括改变抗体以修饰T细胞表位(参见例如WO/9852976A1和WO/0034317A2)。例如，分析来自起始抗体的重链可变区序列和轻链可变区序列，并产生来自每个可变区的人T细胞表位“图谱”，显示表位相对于互补决定区(CDRs)和序列内其它关键残基的位置。分 析来自T细胞表位图的单个T细胞表位，以鉴定具有较低改变抗体活性风险的可选择的氨基酸取代。设计包含氨基酸取代组合的一系列可选的重链可变区序列和轻链可变区序列，随后将这些序列掺入到一系列结合多肽中。然后将包含修饰过的可变区和人类恒定区的完整重链和轻链的基因克隆到表达载体中，随后将质粒转入细胞系以产生完整的抗体。然后利用合适的生物化学和生物学实验中比较抗体，鉴定出最佳的抗体。
本发明公开的双特异性抗体或抗原结合片段的结合特异性可以通过体外实验，例如免疫共沉淀、放射免疫实验(RIA)或酶联免疫吸附实验(ELISA)来检测。
或者，本发明双特异性抗体中scFv可参见生产单链单元的技术(美国专利4,694,778；Bird,Science 242:423-442(1988)、Huston et al.,Proc.Natl.Acad.Sci.USA55:5879-5883(1988)和Ward et al.,Nature 334:544-554(1989)和Nie et al.,Antibody Therapeutics 3(1):18-62(2020))。通过氨基酸桥接Fv区的重链和轻链片段形成单链单元，产生单链融合肽。也可以使用在大肠杆菌中组装功能性Fv片段的技术(Skerra et al.,Science 242:1038-1041(1988))。
可用于生产单链Fv(scFv)和抗体的技术的实例包括如美国专利4,946,778和5,258,498，以及Huston et al.,Methods in Enzymology 203:46-88(1991)、Shu et al.,Proc.Natl.Sci.USA 90:1995-1999(1993)和Skerra et al.,Science 240:1038-1040(1988)中所述。对于包括在人体内使用抗体和体外检测实验的某些用途，可以使用嵌合抗体、人源化抗体或全人源抗体。嵌合抗体是抗体的不同部分源自不同动物物种的一类分子，例如具有鼠源单克隆抗体的可变区和人源免疫球蛋白恒定区的抗体。生产嵌合抗体的方法是本领域已知的，参见Morrison,Science 229:1202(1985)；Oi et al.,BioTechniques 4:214(1986)；Gillies et al.,J.Immunol.Methods 125:191-202(1989)；Neuberger et al.,Nature372:604-608(1984)；Takeda et al.,Nature 314:452-454(1985)；和美国专利5,807,715、4,816,567和4,816,397，其全部内容通过引用并入本文。
此外，在Newman,Biotechnology 10:1455-1460(1992)中公开了另一种生产重组抗体的高效方法，特别地，该技术能产生含有猴可变区和人恒定区序列的灵长类抗体，该参考文献的全部内容通过引用并入本文。此外，该技术也在共同转让的美国专利5,658,570、5,693,780和5,756,096中有所提及，每个专利的全部内容通过引用并入本文。
抗体可以通过本领域已知的多种方法制备，包括使用来自免疫球蛋白序列的抗体文库进行的噬菌体展示方法。也可参考美国专利4,444,887和4,716,111，以及PCT公布文本WO 98/46645、WO 98/50433、WO 98/24893、WO 98/16654、WO 96/34096、WO 96/33735和WO 91/10741，每个专利的全部内容通过引用并入本文。
识别选择性表位的全人源抗体可以使用被称为“引导选择”的技术来生产。在该方法中，使用选择的非人单克隆抗体(例如小鼠抗体)来引导识别相同表位的全人源抗体的筛选(参见美国专利5,565,332，其全部内容通过引用并入本文)。
在另一实施方案中，使用常规方法(例如使用能够特异性结合编码鼠抗体重链和轻链的基因的寡核苷酸探针)，可以分离编码所需单克隆抗体的DNA并对其进行测序。分离的和亚克隆的杂交瘤细胞可以作为此类DNA的来源。一旦分离出来，DNA可以被置于表达载体中，然后被转染到原核或真核宿主细胞如大肠杆菌细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞或不产生其他免疫球蛋白的骨髓瘤细胞中。分离的DNA(如本文所述可以是合成的)也可用于制备抗体的恒定区和可变区的序列，如美国专利5,658,570中所述，其全部内容通过引用并入本文。该方法从所选细胞中提取RNA并转化成cDNA，然后使用Ig特异性引物通过PCR技术进行扩增。适于此目的的合适的探针在美国专利5,658,570中也有所提及。
此外，使用常规重组DNA技术，可将本发明的抗体的一个或多个CDR插入框架区，例如插入到人类框架区以构建人源化非全人源抗体。框架区可以是天然存在的或共有的框架区，优选人类框架区(参见Chothia et al.,J.Mol.Biol.278:457-479(1998)，其列出一系列人类框架区)。一些多核苷酸可以编码框架区和CDR组合产生的与目标抗原的至少一个表位特异性结合的抗体。在框架区内可以进行一个或多个氨基酸取代，可以选择能够改善抗体与其抗原结合的氨基酸取代。另外，可用此法进行参与链间二硫键形成的一个或多个可变区中半胱氨酸残基的取代或缺失，从而产生缺少一个或多个链间二硫键的抗体分子。本领域技术范围内的对多核苷酸进行的其他改变也涵盖于本发明中。
通过使用本领域技术人员公知的技术可以选择、构建和培养生产抗体的细胞系。这些技术在各种实验室手册和主要出版物中均有描述。在这方面，下文描述的适合本发明使用的技术参考Current Protocols in Immunology,Coligan et al.,Eds.,Green Publishing Associates and Wiley-Interscience,John Wiley and Sons,New York(1991)，其全部内容包括补充内容通过引用并入全文。
在一些实施方案中，表达抗体载体包括至少一个启动子元件，抗体编码序列，转录终止信号和polyA尾。其他元件包括增强子，Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，或者Plncx，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI和pCS2等。 常使用的哺乳动物细胞包括293细胞，Cos1细胞，Cos7细胞，CV1细胞，鼠L细胞和CHO细胞等。
在一些实施方案中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶，谷氨酰胺合成酶，新霉素抗性，潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。
此外，可以使用本领域技术人员已知的标准技术在编码本发明所述抗体的核苷酸序列中引入突变，包括但不限于导致氨基酸取代的定点突变和PCR介导的突变。变体(包括衍生物)编码相对于原重链可变区VH CDR1、VH CDR2、VH CDR3和轻链可变区VL CDR1、VL CDR2或VL CDR3来说少于50个氨基酸的取代、少于40个氨基酸的替换、少于30个氨基酸的取代、少于25个氨基酸的取代、少于20个氨基酸的取代、少于15个氨基酸的取代、少于10个氨基酸的取代、少于5个氨基酸的取代、少于4个氨基酸的取代、少于3个氨基酸的取代或少于2个氨基酸的取代。或者可以沿着全部或部分编码序列时随机引入突变，例如通过饱和突变，以及可以筛选所得突变体的生物活性以鉴定保留活性的突变体。
在一些实施方案中，本文所述取代为保守氨基酸取代。
治疗方法
本发明还提供了治疗方法和用途。在一些实施方案中，提供了用于治疗或改善各种类型的癌症、肿瘤或感染等相关疾病的方法，所述方法包括向患者施用有效剂量的所述的双特异抗体：靶向TIGIT和CTLA-4的抗体，靶向OX40和CTLA-4的抗体，或靶向OX40和TIGIT的抗体。在一些实施方案中，提供了所述的双特异性抗体在治疗或改善癌症、肿瘤或感染等相关疾病中的应用。在一些实施方案中，提供了所述的双特异抗体在制备用于治疗或改善癌症、肿瘤或感染等相关疾病的药物中的应用。
对于任何特定患者的具体剂量和治疗方案将取决于各种因素，包括所使用的特定抗体或衍生物、患者的年龄和体重、一般健康状况、性别和饮食，以及给药时间、排泄频率、药物组合，以及所治疗的特定疾病的严重程度。由包括在本领域普通技术人员范围内的医疗护理人员对这些因素进行判断。所述剂量还将取决于待治疗的个体患者、给药途径、制剂类型、所用化合物的特性、疾病的严重程度以及所需的效果。所用剂量可以通过本领域熟知的药理学和药代动力学原理确定。
抗体或衍生物的施用方法包括但不限于真皮内、肌肉、腹腔、静脉、皮下、鼻腔、硬脊膜外和口服注射。药物组合物可以通过任何方便的途径施用，例如通过输注或推注，通过上皮或皮肤粘膜(例如口腔粘膜、直肠和肠粘膜等)吸收，并且可以与其他 生物活性剂共同施用。因此，含有本发明抗体的药物组合物可以口服给药、直肠给药、肠胃外给药、脑池内给药、阴道内给药、腹腔内给药、外敷(如通过粉末，软膏，滴剂或透皮贴剂)、口腔给药或通过口服或鼻腔喷雾给药。
本发明使用的术语“肠胃外”是指包括静脉内、肌肉内、腹腔内、胸骨内、皮下和关节内注射和输注的施用方式。
施用方式可以是全身施用或局部施用。此外，可能需要通过任何合适的途径将本发明的抗体引入中枢神经系统，包括脑室内和鞘内注射；脑室内注射可以通过脑室内导管连接到如贮液囊(可以是Ommaya贮液囊)来辅助注射。也可以通过肺部给药，例如通过使用吸入器或喷雾器，以及使用雾化的制剂。
本发明抗体可以局部施用于需要治疗的区域；可以通过但不限于以下方式：手术期间局部输注，例如与手术后伤口敷料联合的局部应用，通过注射，通过导管，借助栓剂或借助植入物来实现，所述植入物是多孔的、无孔的或凝胶状的材料，包括膜(例如硅橡胶膜)或纤维。优选地，当施用本发明的蛋白质(包括抗体)时，必须注意使用不吸收蛋白质的材料。
在一些实施方案中，本发明组合物包含编码抗体的核酸或多聚核苷酸，可以通过将其构建为合适的核酸表达载体的一部分来体内施用所述核酸以促进其编码的蛋白质的表达，然后通过下述方式施用上述部分载体使其变为胞内部分，例如通过使用逆转录病毒载体(参见美国专利4,980,286)，或通过直接注射，或通过使用微粒轰击(例如基因枪；Biolistic，Dupont)，或用脂质或细胞表面受体或转染试剂包被，或者通过与已知进入细胞核的同源异型盒类肽连接施用(参见例如Joliot et al.,1991,Proc.Natl.Acad.Sci.USA 88:1864-1868)等等。可选地，核酸可以通过同源重组在引入细胞内并整合至宿主细胞DNA中用于表达。
在一些实施方案中，本发明抗体施用于患者的剂量为0.01mg/kg至100mg/kg患者体重，或0.1mg/kg至20mg/kg患者的体重。在初始剂量之后可随后给予第二剂或多剂该抗体或抗原结合片段，其剂量与初始剂量大致相同或较少，其中该随后的剂量可相隔至少1天至3天；或至少一星期。可以通过例如脂质化等修饰来增强抗体的摄取和组织穿透能力(例如进入脑内)，从而减少本发明抗体的施用的剂量和频率。
各种已知输送系统可用于施用本发明抗体或衍生物或编码其的多核苷酸，例如包封于脂质体、微粒、微胶囊、能够表达所述化合物的重组细胞、受体介导的内吞作用(参见例如Wu and Wu,1987,J.Biol.Chem.262:4429-4432)、作为逆转录病毒或其它载体的一部分的核酸的构建等。
联合疗法
在一些实施方案中，本发明抗体可以结合其它治疗或预防方案，包括施用一种或多种本发明抗体以及一种或多种其它治疗剂或方法一起使用或组合使用。对于组合治疗，抗体可以与其它治疗剂可同时或分开施用。当分开施用时，可以在施用另一种其它治疗剂之前或之后施用本发明抗体。
在一些实施方案中，本发明双特异抗体与化疗剂组合施用。在一些实施方案中，本发明双特异性抗体为靶向TIGIT和CTLA-4的抗体，靶向OX40和CTLA-4的抗体，或靶向OX40和TIGIT的抗体。在一些实施方案中，可与本发明抗体一起施用的化疗剂包括但不限于抗生素衍生物(例如阿霉素、博来霉素、柔红霉素和放线菌素D)、抗雌激素药(如他莫昔芬)、抗代谢物(如氟尿嘧啶、5-FU、甲氨蝶呤、氟尿苷、干扰素α-2b、谷氨酸、光神霉素，巯基嘌呤和6-硫基鸟嘌呤)、细胞毒性剂(如卡莫司汀、BCNU、洛莫司汀、CCNU、阿糖胞苷、环磷酰胺、雌莫司汀、羟基脲、甲基苄肼、丝裂霉素、白消安、顺铂和硫酸长春新碱)、激素(如甲羟孕酮、雌莫司汀磷酸钠、炔雌醇、雌二醇、醋酸甲地孕酮、甲睾酮、己烯雌酚二磷酸、氯烯雌醚和睾内酯)、氮芥衍生物(例如美法仑、苯丁酸氮芥、二氯甲基二乙铵(氮芥)和噻替哌)、类固醇及其组合(如倍他米松磷酸钠)，以及其它化合物(如氮烯唑胺、天冬酰胺酶、米托坦、硫酸长春新碱、硫酸长春碱和依托泊苷)。
在一些实施方案中，本发明抗体与细胞因子联合施用。可以与本发明抗体一起施用的细胞因子包括但不限于IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-10、IL-12、IL-13、和IL-15等。
在一些实施方案中，本发明双特异性抗体与化疗剂联合施用。化疗剂的实例包括免疫治疗剂，包括但不限于适用于治疗患者的治疗性抗体。治疗性抗体的一些实例包括辛妥珠单抗(simtuzumab)、阿巴伏单抗(abagovomab)、阿德卡妥姆单抗(adecatumumab)、阿富妥珠单抗(afutuzumab)、阿仑单抗(alemtuzumab)、阿妥莫单抗(altumomab)、阿马妥昔单抗(amatuximab)、阿纳妥姆单抗(anatumomab)、阿西莫单抗(arcitumomab)、巴维妥昔单抗(bavituximab)、贝妥莫单抗(bectumomab)、贝伐单抗(bevacizumab)、比伐单抗(bivatuzumab)、布莱纳妥姆单抗(blinatumomab)、布伦妥昔单抗(brentuximab)、坎妥珠单抗(cantuzumab)、卡妥马索单抗(catumaxomab)、西妥昔单抗(cetuximab)、西妥珠单抗(citatuzumab)、西妥木单抗(cixutumumab)、克里瓦妥珠单抗(clivatuzumab)、克那妥姆单抗(conatumumab)、达拉妥姆单抗(daratumumab)、乔奇妥单抗(drozitumab)、杜利戈妥单抗(duligotumab)、杜西吉妥单抗(dusigitumab)、地莫单抗(detumomab)、达塞妥珠单抗(dacetuzumab)、达洛妥珠单抗(dalotuzumab)、埃克洛莫昔单抗(ecromeximab)、埃洛妥珠单抗(elotuzumab)、恩西妥昔单抗(ensituximab)、厄妥马索单抗(ertumaxomab)、伊他拉昔珠单抗(etaracizumab)、法勒妥珠单抗(farletuzumab)、菲可拉妥珠单抗(ficlatuzumab)、菲 吉妥姆单抗(figitumumab)、弗兰沃妥单抗(flanvotumab)、富妥昔单抗(futuximab)、加尼妥单抗(ganitumab)、吉妥珠单抗(gemtuzumab)、吉伦妥昔单抗(girentuximab)、格兰巴妥姆单抗(glembatumumab)、替伊莫单抗(ibritumomab)、伊戈伏单抗(igovomab)、伊姆加妥珠单抗(imgatuzumab)、英达妥昔单抗(indatuximab)、伊诺妥珠单抗(inotuzumab)、因替妥姆单抗(intetumumab)、伊匹单抗(ipilimumab)、伊拉妥姆单抗(iratumumab)、拉贝妥珠单抗(labetuzumab)、来沙木单抗(lexatumumab)、林妥珠单抗(lintuzumab)、洛沃妥珠单抗(lorvotuzumab)、卢卡妥姆单抗(lucatumumab)、玛帕妥姆单抗(mapatumumab)、玛妥珠单抗(matuzumab)、米拉妥珠单抗(milatuzumab)、明瑞莫单抗(minretumomab)、米妥姆单抗(mitumomab)、莫斜妥姆单抗(moxetumomab)、纳那妥单抗(narnatumab)、纳妥姆单抗(naptumomab)、内吉妥姆单抗(necitumumab)、尼莫妥珠单抗(nimotuzumab)、诺费妥单抗(nofetumomab)、奥卡妥珠单抗(ocaratuzumab)、奥法木单抗(ofatumumab)、奥拉妥珠单抗(olaratumab)、奥那妥珠单抗(onartuzumab)、奥泼妥珠单抗(oportuzumab)、奥戈伏单抗(oregovomab)、帕尼单抗(panitumumab)、帕萨妥珠单抗(parsatuzumab)、帕崔妥单抗(patritumab)、彭妥姆单抗(pemtumomab)、帕妥珠单抗(pertuzumab)、平妥姆单抗(pintumomab)、普拖木单抗(pritumumab)、拉蔻妥姆单抗(racotumomab)、拉吉妥姆单抗(radretumab)、里洛妥姆单抗(rilotumumab)、利妥昔单抗(rituximab)、洛巴妥姆单抗(robatumumab)、沙妥莫单抗(satumomab)、思布妥珠单抗(sibrotuzumab)、思妥昔单抗(siltuximab)、索力图单抗(solitomab)、塔卡妥珠单抗(tacatuzumab)、塔普利妥珠单抗(taplitumomab)、特纳妥姆单抗(tenatumomab)、特普洛妥姆单抗(teprotumumab)、提咖妥珠单抗(tigatuzumab)、拖西莫单抗(tositumomab)、曲妥珠单抗(trastuzumab)、图库图珠单抗(tucotuzumab)、尤不理妥昔单抗(ublituximab)、维尔妥珠单抗(veltuzumab)、沃思妥珠单抗(vorsetuzumab)、伏妥莫单抗(votumumab)和扎鲁妥姆单抗(zalutumumab)等。
在一些实施例中，本发明抗体可以与免疫检查点抑制剂一起使用。在一些实施方案中，本发明抗体与其它治疗或预防方案联合施用，例如放射性疗法。
药物组合物
本发明还提供了药物组合物。这样的组合物包含有效剂量的抗体或抗原结合片段和可接受的载体。在一些实施方案中，药物组合物还包含抗癌剂(例如免疫检查点抑制剂)。
在一些实施方案中，术语“药学上可接受的”是指由政府的监管机构批准的或公认药典中列出的用于动物，特别是用于人类的物质。此外，“药学上可接受的辅料”通常指是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂等。
术语“辅料”是指施用于可以与活性成分一起施用于患者的稀释剂、佐剂、赋形剂或载体。这此类药物辅料可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等。当药物组合物静脉内给药时，水是优选的载体。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如有需要，组合物还可以含有少量的润湿剂或乳化剂，或pH缓冲剂如乙酸盐、柠檬酸盐或磷酸盐。抗菌剂如苯甲醇或对羟基苯甲酸甲酯、抗氧化剂如抗坏血酸或亚硫酸氢钠、螯合剂如乙二胺四乙酸，以及调节张力的试剂如氯化钠或右旋葡萄糖也是可以预见的。这些组合物可以采取溶液、悬液、乳剂、片剂、丸剂、胶囊、散剂、缓释制剂等形式。该组合物可以用传统的粘合剂和载体如甘油三酯配制成栓剂。口服制剂可以包括标准载体，例如药物等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例在E.W.Martin的Remington's Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的抗体或抗原结合片段，优选以纯化后的形式，连同合适数量的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。亲本制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。
在一些实施方案中，根据常规步骤将组合物配制成适合静脉内注射于人体的药物组合物。用于静脉内给药的组合物通常是在无菌等渗水性缓冲液中的溶液。组合物还可包含增溶剂和局部麻醉剂如利多卡因，从而缓解注射部位的疼痛。一般而言，有效成分以单位剂量形式单独供给或混在一起供给，如以干燥的冻干粉末或无水浓缩物的形式装在可指示活性剂份量的密封容器(如安瓿瓶或小袋)中。在通过输注施用组合物的情况下，可以用含有无菌药用级水或盐水的输液瓶来分装组合物。在通过注射施用组合物的情况下，可以使用注射用的无菌水或盐水的安瓿瓶，使得可以在施用之前混合有效成分。
本发明的化合物可以配制成中性的或盐的形式。药学上可接受的盐包括衍生自如盐酸、磷酸、乙酸、草酸、酒石酸等的与阴离子形成的盐，以及衍生自如钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙氨基乙醇、组氨酸、普鲁卡因等的与阳离子形成的盐。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到；比如，TIGIT-Fc抗原、CTLA-4-Fc抗原、TIGIT-his抗原可购自于上海近岸科技有限公司或AcroBiosystems，anti-TIGIT报告基因检测系统可够自Promega(货号为JA1400)，或者可根据已知方法制备。
参照抗体
anti-TIGIT抗体(参照抗体)在CHO细胞中表达并纯化后所得。anti-TIGIT抗体的重链为：
Figure PCTCN2021095852-appb-000001
anti-TIGIT抗体的轻链为：
Figure PCTCN2021095852-appb-000002
anti-CTLA-4抗体(参照抗体)在CHO细胞中表达并纯化后所得。anti-CTLA-4抗体的重链为：
Figure PCTCN2021095852-appb-000003
anti-CTLA-4抗体的轻链为：
Figure PCTCN2021095852-appb-000004
Figure PCTCN2021095852-appb-000005
anti-OX40抗体(参照抗体的重链序列为SEQ ID NO:75，轻链序列为SEQ ID NO:74)在CHO细胞中表达并纯化后所得。anti-OX40的重链为：
Figure PCTCN2021095852-appb-000006
或
Figure PCTCN2021095852-appb-000007
anti-OX40的轻链为：
Figure PCTCN2021095852-appb-000008
靶向TIGIT和CTLA-4的双特异性抗体
实施例1抗体制备方法
本双特异抗体的结构如图1所示，该结构类似于免疫球蛋白，所述抗体含序列相同的第一条多肽链和第三条多肽链，以及序列相同的第二条多肽和第四条多肽链；其中，第一条多肽链包含结构VHa-L1-CLa-L2-VHb-CH1-Fc，第二条多肽链包含结构 VLa-L3-CH1-L4-VLb-CLb；抗原a为TIGIT，抗原b为CTLA-4。
将抗体的第一条多肽和第二条多肽的氨基酸序列依照宿主细胞CHO(中国仓鼠卵巢细胞)密码子偏好性特点进行序列优化，得到第一条多肽和第二条多肽的DNA序列。为了便于在CHO细胞中表达，分别在第一条多肽和第二条多肽的序列中添加了信号肽和Kozak序列(Kozak序列是位于真核生物mRNA 5’端帽子结构后面的一段核酸序列)，序列的末端添加了终止密码子。同时为了方便克隆至哺乳动物表达载体pCDNA3.1 TM(+)(Invitrogen，货号为V79020)，序列的两端也同时添加了酶切位点，5’端为Hind III和3’端为EcoR I。
将优化并合成的序列克隆分别克隆至pCDNA3.1 TM(+)载体中，然后分别抽提大量质粒，第一条多肽和第二条多肽按质粒摩尔比1:1采用ExpiCHO TM表达系统(Gibco，货号为A29133)进行瞬时表达。按照说明书要求收获蛋白，并用protein A进行纯化。
靶向TIGIT和CTLA-4的双特异性抗体相关的氨基酸序列见表1，靶向TIGIT和CTLA-4的双特异性抗体相关的核酸序列见表2。表1中连接子用加粗斜体和下划线表示；抗体1的第一条多肽如SEQ ID NO:37所示，抗体1的第二条多肽如SEQ ID NO:38所示；抗体2的第一条多肽如SEQ ID NO:39所示，抗体2的第二条多肽如SEQ ID NO:40所示；抗体3的第一条多肽如SEQ ID NO:41所示，抗体3的第二条多肽如SEQ ID NO:42所示；抗体4的第一条多肽如SEQ ID NO:43所示，抗体4的第二条多肽如SEQ ID NO:44所示。表2中，5’端斜体加下划线的小字母(5’端到3’端)依次是Hind III酶切位点、Kozak序列和前导肽序列，3’端斜体加下划线的小字母(5’端到3’端)依次是终止密码子和EcoR I酶切位点；由于抗体1、2、3和4仅连接子序列不同，表2中仅列出抗体1的第一条多肽和第二条多肽的核酸序列，以及连接子的核酸序列。
表1靶向TIGIT和CTLA-4的双特异性抗体的氨基酸序列
Figure PCTCN2021095852-appb-000009
Figure PCTCN2021095852-appb-000010
Figure PCTCN2021095852-appb-000011
Figure PCTCN2021095852-appb-000012
Figure PCTCN2021095852-appb-000013
表2编码靶向TIGIT和CTLA-4的双特异性抗体的核酸序列
Figure PCTCN2021095852-appb-000014
Figure PCTCN2021095852-appb-000015
Figure PCTCN2021095852-appb-000016
Figure PCTCN2021095852-appb-000017
实施例2抗体纯度检测
对纯化后抗体进行凝胶电泳检测：检测纯化后抗体的纯度。如图2所示，抗体1的纯度较高；如图3所示，抗体2和抗体3的纯度较高。
实施例3抗体结合活性的检测
1)对纯化后双特异抗体进行结合活性的检测，检测抗体是否能正常结合TIGIT或CTLA-4。抗体与TIGIT结合试验的过程如下：用PBS包被抗原TIGIT-Fc抗原1μg/ml，每孔100μl，然后放置2-8℃冰箱中过夜包被；第二天加入250μl含3％BSA的PBS在37℃恒温箱中封闭2小时；之后用PBST洗涤2次，然后分别加入梯度稀释的anti-TIGIT、抗体1、抗体2和抗体3，起始浓度3μg/ml，1/3倍稀释，共10个稀释梯度；在对应孔中每孔加入100μl稀释好的抗体，在37℃恒温箱中孵育1小时；用PBST洗涤3次，加入1:5000倍稀释的酶标二抗(Sigma山羊抗人IgG kappa，IgG轻链偶联HRP，货号为A7164-1ML)每孔100μl，在37℃恒温箱中孵育1小时；用PBST洗涤5次，加入TMB显色液显色约20min后加入1M H 2SO 4进行终止，30min之内读OD450值。
如附图4所示，抗体1、抗体2与人TIGIT-Fc的结合能力与anti-TIGIT结果接近，抗体3与人TIGIT-Fc结合能力比较低。
2)本发明抗体与CTLA-4-Fc的结合试验的步骤与上述试验类似。如图5所示，抗体1、抗体2和抗体3均可以与CTLA-4-Fc结合，抗体1、抗体2的结合能力较强。实施例4抗体亲和力的检测
1)为了判断连接子长度(L2和L4)对双特异抗体结合hTIGIT-his蛋白(上海近岸科技有限公司，CJ58)能力的影响，采用Biacore T200仪器检测anti-TIGIT、抗体1、抗体2和抗体3与hTIGIT-his蛋白的亲和力常数测。具体试验过程如下：将hTIGIT-his抗原用HBS-EP+buffer梯度稀释，共5个梯度，初始浓度为50nM，然后对半稀释；将待测蛋白anti-TIGIT、抗体1、抗体2和抗体3用HBS-EP+buffer分别稀释为2.5μg/ml；使用仪器为BiacoreT200，Protein A芯片(GE healthcare，货号为29127556)进行检测，仪器设置如下：抗体捕获结合时间(contact time)25s，流速5μl/min；样品结合时间(contact time)120s，解离时间(dissociation time)300s，流速30μl/min；再生(regeneration)条件为：Glycine-Hcl(pH值为1.5)，再生时间60s，流速30μl/min；检测通道(flow path)为2-1和4-3。
如表3所示，与anti-TIGIT相比，抗体1与hTIGIT-his的亲和力未降低，甚至有 一定程度的升高；与anti-TIGIT相比，抗体2、抗体3与hTIGIT-his的亲和力基本接近。
2)采用Biacore T200仪器检测anti-CTLA-4、抗体1、抗体2和抗体3与h CTLA-4-his蛋白(上海近岸科技有限公司，CP33)的亲和力常数测。具体试验过程如下：将hCTLA-4-his抗原用HBS-EP+buffer梯度稀释，共6个梯度，初始浓度为100nM，然后对半稀释；将待测蛋白anti-CTLA-4、抗体1、抗体2和抗体3用HBS-EP+buffer分别稀释为5μg/ml；使用仪器为BiacoreT200，Protein A芯片(GE healthcare，货号：29127556)进行检测，仪器设置如下：抗体捕获结合时间(contact time)15s，流速10μl/min；样品结合时间(contact time)180s，解离时间(dissociation time)300s，流速30μl/min；再生(regeneration)条件为：Glycine-Hcl(pH值为1.5)，再生时间60s，流速30μl/min；检测通道(flow path)为2-1和4-3。
如表4所示，与anti-CTLA-4相比，抗体1、抗体2与hCTLA-4-his的亲和力基本接近；与anti-CTLA-4相比，抗体3与hCTLA-4-his的亲和力较低。
表3抗体与hTIGIT-his的亲和力
Figure PCTCN2021095852-appb-000018
表4抗体与hCTLA-4-his的亲和力
Figure PCTCN2021095852-appb-000019
实施例5抗体生物学活性的检测
1)为了进一步鉴定抗体的生物学活性，采用anti-CTLA-4报告基因检测系统(Promega，货号为JA1400)对抗体的生物学活性进行检测，该检测过程参考产品说明书。
如图6所示，对照抗体anti-CTLA-4具有良好的生物学活性，可以激活报告系统中IL-2启动子从而激活下游荧光素酶的表达；抗体1的激活能力相对较弱，生物活性相 对较低。如图7所示，与anti-CTLA-4相比，抗体2的激活能力相对较弱，生物活性相对较低。
2)采用anti-TIGIT报告基因检测系统对抗体的生物学活性进行了测定，检测过程类似于上述Promega的anti-CTLA-4报告基因检测系统。
如图8-10所示，对照抗体anti-TIGIT-4相比，抗体1、抗体2和抗体3并没有表现出更差的生物学活性，甚至抗体1和抗体2激活能力相对较好。
综合上述抗体生物学活性的检测，连接子L2和L4长度的增加对TIGIT靶点的功能未产生明显影响，但对CTLA-4靶点的功能产生了影响：连接子长度越长，针对CTLA-4靶点的生物学活性越低；本发明抗体1的生物活性较优。
从当前TIGIT靶点的临床试验(Genentech MTIG-7129临床试验(NCT03563716)或OncoMed公司OMP-313M32临床试验(NCT03119428))可以看出，该靶点药物一般需要较高的剂量，2018年癌症免疫治疗学会摘要(SITC 2018)公开TIGIT靶点抗体OMP-313M32没有严重的不良事件发生；但是对于抗CTLA-4抗体而言，临床上发现高达60％接受抗CTLA-4抗体Yervoy治疗的患者会发生免疫相关不良事件：10％-30％属于严重(3-4级)免疫相关不良事件，且接受Yervoy治疗的患者发生免疫相关不良事件的风险是剂量依赖性的(Martins F,Sofiya L,Sykiotis G P,et al.Adverse effects of immune-checkpoint inhibitors:epidemiology,management and surveillance[J].Nature Reviews Clinical Oncology,2019,16(9):563-580.)；本发明抗体1针对CTLA-4的生物学活性相对于anti-CTLA-4有一定程度降低，预计既可以保证对TIGIT及CTLA-4靶点的阻断，也可以减轻或避免抗CTLA-4抗体所带来的副作用。
靶向OX40和CTLA-4的双特异性抗体
实施例6抗体制备方法
本双特异抗体的结构如图1所示，该结构类似于免疫球蛋白，所述抗体含序列相同的第一条多肽链和第三条多肽链，以及序列相同的第二条多肽和第四条多肽链；其中，第一条多肽链包含结构VHa-L1-CLa-L2-VHb-CH1-Fc，第二条多肽链包含结构VLa-L3-CH1-L4-VLb-CLb；抗原a为OX40，抗原b为CTLA-4。
合成抗体的第二条多肽的序列：5’端含有HindIII酶切位点及前导肽，3’端含有终止密码子及EcoRI酶切位点；合成抗体的第一条多肽的序列：5’端含有HindIII酶切位点及前导肽，3’端含有终止密码子及EcoRI酶切位点。
将分别载有第一条多肽和第二条多肽的核酸序列的质粒同时瞬时转染到293F细胞中。293F细胞用CD 293 TGE Medium(BPM Cell Culture，货号：CM-1156)培养，待细胞活力在95％以上时，用新鲜培养基将细胞传代到80-100万/mL，24h后细胞密 度大概在150-200万/mL时，可进行转染。先将质粒在65℃水浴30分钟，每100万细胞用0.5μg质粒，每1μg质粒总量用3μL的PEI(Polysciences，货号为24765-2)。分别将质粒和PEI用培养基稀释(两个溶液的体积加起来是总体积的5％)，室温静置5分钟。然后把PEI溶液加到质粒溶液中，混匀，室温静置20分钟。将复合物溶液加入到细胞中，37℃摇床培养24小时后，加入总体积比为10％的CD Feed X Supplement(BPM Cell Culture，货号为CF-1116)到细胞中。5天后收获细胞上清，上清用Protein A进行纯化，得到双特异抗体5。
靶向OX40和CTLA-4的双特异性抗体5相关的氨基酸序列见表5，靶向TIGIT和CTLA-4的双特异性抗体5相关的核酸序列见表6。表5中连接子用加粗斜体和下划线表示。
表5靶向OX40和CTLA-4的双特异性抗体的氨基酸序列
Figure PCTCN2021095852-appb-000020
Figure PCTCN2021095852-appb-000021
表6编码靶向OX40和CTLA-4的双特异性抗体的核酸酸序列
Figure PCTCN2021095852-appb-000022
Figure PCTCN2021095852-appb-000023
Figure PCTCN2021095852-appb-000024
实施例7抗体纯度检测
对抗体5分别进行还原和非还原处理，然后用于SDS-PAGE电泳。如图11所示，还原和非还原的条带清晰无杂带，显示样品纯度较高：非还原抗体5在电泳图上大小为大于250kDa；还原抗体5的第一条多肽在电泳图上大小约为90kDa，还原抗体5的第二条多肽在电泳图上大小约为50kDa；大小符合预期。可见，双特异性抗体5能在细胞中正常表达；在293F细胞中表达的抗体5纯化后可用于下一步试验。
实施例8抗体结合活性的检测
CTLA-4-Fc抗原、OX40-Fc抗原分别包被ELISA板，4℃过夜。第二天用5％BSA封闭液于37℃封闭2小时。洗板后分别加上从2μg/mL开始2倍梯度稀释的抗体5、CTLA-4单克隆抗体(即anti-CTLA-4)、OX40单克隆抗体(即anti-OX40)，一共稀释12个浓度(最后一个为空白0浓度)，每个浓度做2个复孔，37℃孵育2小时。洗板后加入1:10000倍稀释的抗人Kappa轻链二抗，37℃孵育0.5小时。洗板后加入TMB单组分显色液避光显色10-15分钟，用0.1M硫酸终止反应，在450nm波长下读数。由ELISA结合曲线计算EC 50值，结果见表7。
其中，OX40-Fc抗原的制备方法为：从蛋白数据库Uniprot，找到人OX40的氨基酸序列(P43489)，其中人OX40的胞外区的氨基酸序列是1到216位残基；从蛋白数据库Uniprot，找到IgG1-Fc的氨基酸序列(P01857)第104到330位残基；然后通过人工合成(通用公司)，得到OX40胞外区和Fc对应的核苷酸序列，通过酶切连接，把其插入到pCDNA3.0载体(Invitrogen)得到重组质粒；再把上述质粒通过PEI瞬转HEK293细胞，在培养7天后收集上清液，最后通过纯化得到OX40-Fc(即hOX40-Fc)。
如表7所示，抗体5能保留对OX40和CTLA-4两个抗原的结合能力；与单克隆抗 体相比，抗体5与OX40的结合能力较好，抗体5与CTLA-4的结合能力较差。
表7抗体与抗原结合的EC 50值
Figure PCTCN2021095852-appb-000025
实施例9抗体亲和力的检测
将待测OX40单克隆抗体、CTLA-4单克隆抗体、抗体5分别耦联生物素。SA传感器置于PBS中预湿10分钟备用，将生物素标记的抗体用pH值为6.8的含有0.5％BSA的PBST(以下简称稀释缓冲液)稀释至10μg/mL，将SA传感器在分别在上述3个生物素化抗体中固化至信号约为4nm。将待测CTLA-4-Fc抗原、hOX40-Fc抗原用稀释缓冲液稀释至400nM，并以此为起始浓度作2倍梯度稀释，一共准备6个浓度点(400、200、100、50、25、0nM)。将稀释缓冲液、梯度浓度的药物稀释液、再生缓冲液(pH为8.4的1M MgCl 2)、中和缓冲液(PBST)依次加入96孔板相应列中，运行如下步骤：①Baseline：在稀释缓冲液中检测基线60秒；②Association：在抗体梯度稀释液及样品空白(稀释缓冲液)中结合300秒；③Dissociation：在稀释缓冲液中解离360秒；④Regeneration：在再生缓冲液中再生5秒；⑤Neutralization：在中和缓冲液中中和5秒；⑥Regeneration和Neutralization循环进行3次。
采用ForteBio Data Analysis 8.2对数据进行处理分析，样品数据经参比(样品空白)信号扣减后进行拟合，得到亲和常数K D。抗体5与CTLA-4-Fc抗原的亲和力结果见表8，抗体5与OX40-Fc抗原的亲和力结果见表9。
表8抗体与CTLA-4抗原亲和结合的动力学参数
Figure PCTCN2021095852-appb-000026
表9抗体与OX40抗原亲和结合的动力学参数
Figure PCTCN2021095852-appb-000027
Figure PCTCN2021095852-appb-000028
如表8所示，与单克隆抗体相比，抗体5对CTLA-4抗原的亲和力较高；如表9所示，与单克隆抗体相比，抗体5对OX40抗原的亲和力基本接近。
实施例10抗体与OX40 +细胞的结合活性
通过转染带有的人OX40cDNA的pCMV载体(Invitrogen)产生过表达人OX40的Jurkat细胞(OX40 +细胞)，分别取OX40 +细胞、离心去掉培养基，用PBS洗一次，保留细胞沉淀。OX40单克隆抗体、抗体5分别从100nM的抗体浓度开始3倍稀释，一共稀释10个浓度。把这些不同浓度的抗体溶液分别重悬相对应的细胞沉淀(OX40 +细胞)，每个浓度需要约200万细胞。4℃孵育2小时后，离心去上清，用PBS洗一次。每个样品加入1:1000稀释的抗Fc-PE(invitrogen，货号为12-4998-82)，4℃避光孵育半小时。离心去上清，用PBS洗一次。每个样品再用PBS重悬，然后在C6流式细胞仪进行上样检测，光源选择通道2。结合曲线如图12，阳性率及荧光值统计见表10-11。
如图12和表10-11，与单克隆抗体相比，抗体5与OX40 +细胞的结合活性相当。
表10 OX40单克隆抗体与OX40 +细胞的结合阳性率及荧光值
Figure PCTCN2021095852-appb-000029
表11抗体5与OX40 +细胞的结合阳性率及荧光值
Figure PCTCN2021095852-appb-000030
实施例11抗体同时结合OX40抗原和CTLA-4抗原
将OX40-Fc抗原耦联生物素，SA传感器置于PBS中预湿10分钟备用，将生物素标记的OX40-Fc抗原用pH值为6.8的含有0.5％BSA的PBST(以下简称稀释缓冲液)稀释至5μg/Ml，将SA传感器在上述标记好的生物素化OX40-Fc抗原溶液中固化至信号约为0.8nM。将待测抗体5用稀释缓冲液稀释至50nM；CTLA-4-Fc抗原用稀释缓冲液稀释至200nM。将稀释缓冲液、抗体5稀释液、CTLA-4-Fc抗原稀释液、再生缓冲液(pH为8.4的1M MgCl 2)、中和缓冲液(PBST)依次加入96孔板相应列中，运 行如下步骤：①Baseline：在稀释缓冲液中检测基线60秒；②Association：在抗体梯度稀释液及样品空白(稀释缓冲液)中结合300秒；③Baseline：在稀释缓冲液中检测基线60秒；④Association：在抗体梯度稀释液及样品空白(稀释缓冲液)中结合300秒；⑤Dissociation：在稀释缓冲液中解离300秒；⑥Regeneration：在再生缓冲液中再生5秒；⑦Neutralization：在中和缓冲液中中和5秒；⑧Regeneration和Neutralization循环进行3次。
如图13所示，传感器在固化了OX40-Fc抗原后结合抗体5，有明显的结合；此时再结合CTLA-4-Fc抗原，仍有一定的结合信号值；并且解离步骤时没有看到明显的信号下降；说明抗体5能同时结合OX40抗原和CTLA-4抗原。
靶向OX40和TIGIT的双特异性抗体
实施例12抗体的制备方法
本双特异抗体的结构如图1所示，该结构类似于免疫球蛋白，所述抗体含序列相同的第一条多肽链和第三条多肽链，以及序列相同的第二条多肽和第四条多肽链；其中，第一条多肽链包含结构VHa-L1-CLa-L2-VHb-CH1-Fc，第二条多肽链包含结构VLa-L3-CH1-L4-VLb-CLb；抗原a为OX40，抗原b为TIGIT。
人工合成得到双特异性抗体第一条多肽和第二条多肽对应的核酸序列，再通过酶切连接分别把上述核酸序列连接到pCDNA3.0载体(购自Invitrogen公司)；得到用于表达全抗的2种重组质粒。根据制造商的说明书使用Freedom CHO-S试剂盒(购自Invitrogen)，再把上述质粒通过PEI瞬转HEK293细胞，在培养7天后收集上清液，最后通过纯化得到双特异抗体蛋白样品。
靶向OX40和TIGIT的双特异性抗体6和7相关的氨基酸序列见表12，靶向TIGIT和TIGIT的双特异性抗体6和7相关的核酸序列见表13。表12中连接子用加粗斜体和下划线表示。
表12靶向OX40和TIGIT的双特异性抗体的氨基酸序列
Figure PCTCN2021095852-appb-000031
Figure PCTCN2021095852-appb-000032
Figure PCTCN2021095852-appb-000033
表13编码靶向OX40和TIGIT的双特异性抗体的核酸酸序列
Figure PCTCN2021095852-appb-000034
Figure PCTCN2021095852-appb-000035
Figure PCTCN2021095852-appb-000036
Figure PCTCN2021095852-appb-000037
实施例13抗体纯度检测
通过SDS-PAGE，在还原条件和非还原条件下分析双特异抗体的组成和纯度。如图14所示，在非还原条件下，抗体6和抗体7以单一带迁移；在还原条件下，抗体6和抗体7各自产生两条带；抗体6和抗体7为单一物质，并且这2条多肽链有效配对形成类IgG蛋白。
实施例14抗体亲和力的检测
ForteBio亲和力测定可参照现有的常规方法(Estep Patricia,et al.High throughput solution-based measurement of antibody-antigen affinity and epitope binning.MAbs,2013,5(2):270-8)，具体试验过程为：传感器在分析缓冲液(如PBS)中线下平衡20分钟，然后上机检测60秒建立信号基线，上机加载纯化后的抗体6和7至相应传感器(ForteBio)上，最后进行ForteBio亲和力测量；采用protein传感器吸附抗体6和7，再分别单独检测与OX40-his(AcroBiosystems公司)和Tigit-his(AcroBiosystems公司)的结合与解离，各约5min；最后采用1:1结合模型进行动力学的分析。
表14抗体与OX40-his和TIGIT-his的亲和力
Figure PCTCN2021095852-appb-000038
如表14所示，抗体6能明显结合OX40抗原，但其亲和力弱于anti-OX40；抗体7能明显结合OX40抗原，且亲和力和anti-OX40相当；抗体7还能明显结合TIGIT抗原，其亲和力与anti-TIGIT基本接近。
实施例15抗体同时结合OX40抗原和TIGIT抗原
与上述Fortebio测定亲和力方法类似，检测双特异抗体能否同时结合OX40-his抗 原(AcroBiosystems公司)和TIGIT-his抗原(AcroBiosystems公司)。采用protein传感器吸附上述实施例中的抗体7，先检测与OX40抗原的结合和解离，再检测与TIGIT抗原的结合和解离。
如图15所示，抗体7明显能同时结合OX40和TIGIT抗原，而不存在空间位阻。实施例16抗体与OX40或TIGIT过量表达的Jurkat细胞的结合活性
1)通过转染带有人OX40cDNA的pCMV载体(Invirogen公司)产生过表达人OX40的Jurkat-OX40细胞(即上述OX40 +细胞)，将Jurkat-hOX40细胞(0.5×10 6个细胞)与不同浓度的抗体在PBS中冰上孵育40分钟。然后将细胞洗涤两次，并与二抗在PBS(含0.1％BSA)中冰上孵育25分钟。将细胞洗涤两次，在Accuri C6系统(BD Biosciences)上进行流式细胞术分析。
如图16所示，抗体7(OT-4D-30a)能明显结合Jurkat-OX40细胞，其结合力接近于anti-OX40。
2)通过转染带有人TIGIT cDNA的pCMV载体产生过表达人TIGIT的Jurkat细胞Jurkat-Tigit细胞，将Jurkat-Tigit细胞(0.5×10 6个细胞)与不同浓度的抗体在PBS含0.1％BSA中在冰上孵育40分钟。然后将细胞洗涤两次，并与二抗在PBS(含0.1％BSA)中冰上孵育25分钟。将细胞洗涤两次，在Accuri C6系统(BD Biosciences)上进行流式细胞术分析。
如图17所示，抗体7能明显结合Jurkat-Tigit细胞，其结合力稍弱于anti-TIGIT。

Claims (28)

  1. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，并且所述抗体或抗原结合片段至少包含2条多肽链；其中，第一条多肽链从氨基末端开始顺序包含VHa、CLa、VHb和CH1，VHa是与第一抗原a结合的重链可变区，CLa是第一轻链恒定区，VHb是与第二抗原b结合的重链可变区，CH1是重链的第一恒定区。
  2. 如权利要求1所述的抗体或抗原结合片段，其特征在于，VHa与CLa通过连接子L1进行共价连接，L1含2到6个氨基酸；和/或
    CLa与VHb通过连接子L2进行共价连接；其中，L2含10到30个氨基酸，并且至少50％的氨基酸为甘氨酸。
  3. 如权利要求1或2所述的抗体或抗原结合片段，其特征在于，所述第二条多肽链从氨基末端开始顺序包含VLa、CH1、VLb和CLb；其中，VLa是与第一抗原a结合的轻链可变区，VLb是与第二抗原b结合的轻链可变区，CLb是第二轻链恒定区。
  4. 如权利要求3所述的抗体或抗原结合片段，其特征在于，VLa与CH1通过连接子L3进行共价连接，L3含2到6个氨基酸；和/或
    CH1与VLb通过连接子L4进行共价连接；其中，L4含10到30个氨基酸，并且至少50％的氨基酸为甘氨酸。
  5. 如权利要求4所述的抗体或抗原结合片段，其特征在于，所述L1含SEQ ID NO:22所示的序列；和/或
    所述L2含选自SEQ ID NO:23-27中任一项所示的序列，与SEQ ID NO:23-27中任一项所示序列具有至少90％同一性的序列，或与SEQ ID NO:23-27中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述L3含SEQ ID NO:28所示的序列；和/或
    所述L4含选自SEQ ID NO:29-33中任一项所示的序列，与SEQ ID NO:29-33中任一项所示序列具有至少90％同一性的序列，或与SEQ ID NO:29-33中任一项所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  6. 如权利要求1-5任一项所述的抗体或抗原结合片段，其特征在于，所述2条 多肽链中的一条还包含Fc，Fc包含重链的铰链区、第二恒定区和第三恒定区。
  7. 如权利要求6所述的抗体或抗原结合片段，其特征在于，第一条多肽链包含结构VHa-L1-CLa-L2-VHb-CH1，第二条多肽链包含结构VLa-L3-CH1-L4-VLb-CLb；或
    第一条多肽链包含结构VHa-L1-CLa-L2-VHb-CH1-Fc，第二条多肽链包含结构VLa-L3-CH1-L4-VLb-CLb。
  8. 如权利要求1-7任一项所述的抗体或抗原结合片段，其特征在于，抗原a、b分别选自于以下组成的组：TIGIT和CTLA-4，OX40和CTLA-4，TIGIT和PD-1，PD-L1和CD47，TIGIT和OX40，VEGF和cMET，VEGF和DLL4，VEGF和HGF，VEGF和ANGPT2，TfR和CD20，PD-L1和4-1BB，PSMA和CD28，PD-1和PD-L1，HER2和4-1BB，PD-1和TIM-3，PD-1和CD47，GITR和CTLA-4，CD40和4-1BB，OX40和4-1BB，LAG-3和TIM-3，EGFR和CTLA-4，CD19和CD22，CD16和CD30，CD3和CD123，BCMA和CD47，MSLN和CD47，EGFR和cMET，CD73和TGFβ，EGFR和TGFβ，CCR2和CSF1R，CD20和CD3，CD19和CD47，CDH17和TRAILR2，APLP2和Her2，IL-1α和IL-1β，IL-17和IL-13，IL-4和IL-13，BAFF和IL-17A，CD3和PD-1，IL-4Ra和IL-5，VEGF和IL-6，FGFR1和KLB。
  9. 如权利要求8所述的抗体或抗原结合片段，其特征在于，所述抗原a为TIGIT且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
    所述VHa含SEQ ID NO:1所示的序列中第30-35位氨基酸(VHaCDR1，SSYGMS)，和/或第50-66位氨基酸(VHaCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHaCDR3，LGTGTLGFAY)氨基酸；和/或
    所述VHb含SEQ ID NO:2所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
    所述VLa含SEQ ID NO:3所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLaCDR2，WASTRAT)，和/或第89-97位氨基酸(VLaCDR3，QQHYSTPWT)；和/或
    所述VLb含SEQ ID NO:4所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
  10. 如权利要求9所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含以下：
    所述VHa含SEQ ID NO:1所示的序列，与SEQ ID NO:1所示序列具有至少80％同一性的序列，或与SEQ ID NO:1所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VHb含SEQ ID NO:2所示的序列，与SEQ ID NO:2所示序列具有至少80％同一性的序列，或与SEQ ID NO:2所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VLa含SEQ ID NO:3所示的序列，与SEQ ID NO:3所示序列具有至少80％同一性的序列，或与SEQ ID NO:3所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VLb含SEQ ID NO:4所示的序列，与SEQ ID NO:4所示序列具有至少80％同一性的序列，或与SEQ ID NO:4所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CLa含SEQ ID NO:5所示的序列，与SEQ ID NO:5所示序列具有至少80％同一性的序列，或与SEQ ID NO:5所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CLb含SEQ ID NO:6所示的序列，与SEQ ID NO:6所示序列具有至少80％同一性的序列，或与SEQ ID NO:6所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CH1含SEQ ID NO:7所示的序列，与SEQ ID NO:7所示序列具有至少80％同一性的序列，或与SEQ ID NO:7所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  11. 如权利要求8所述的抗体或抗原结合片段，其特征在于，所述抗原a为OX40且抗原b为CTLA-4，所述抗体或抗原结合片段包含以下：
    所述VHa含SEQ ID NO:8所示的序列中第31-35位氨基酸(VHaCDR1，SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和/或
    所述VHb含SEQ ID NO:9所示的序列中第31-35位氨基酸(VHbCDR1，SYTMH)，和/或第50-66位氨基酸(VHbCDR2，FISYDGNNKYYADSVKG)，和/或第99-107位氨基酸(VHbCDR3，TGWLGPFDY)；和/或
    所述VLa含SEQ ID NO:10所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和/或
    所述VLb含SEQ ID NO:11所示的序列中第24-35位氨基酸(VLbCDR1，RASQSVGSSYLA)，和/或第51-57位氨基酸(VLbCDR2，GAFSRAT)，和/或第90-98位氨基酸(VLbCDR3，QQYGSSPWT)。
  12. 如权利要求11所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含以下：
    所述VHa含SEQ ID NO:8所示的序列，与SEQ ID NO:8所示序列具有至少80％同一性的序列，或与SEQ ID NO:8所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VHb含SEQ ID NO:9所示的序列，与SEQ ID NO:9所示序列具有至少80％同一性的序列，或与SEQ ID NO:9所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VLa含SEQ ID NO:10所示的序列，与SEQ ID NO:10所示序列具有至少80％同一性的序列，或与SEQ ID NO:10所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VLb含SEQ ID NO:11所示的序列，与SEQ ID NO:11所示序列具有至少80％同一性的序列，或与SEQ ID NO:11所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CLa含SEQ ID NO:12所示的序列，与SEQ ID NO:12所示序列具有至少80％同一性的序列，或与SEQ ID NO:12所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CLb含SEQ ID NO:13所示的序列，与SEQ ID NO:13所示序列具有至少80％同一性的序列，或与SEQ ID NO:13所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CH1含SEQ ID NO:14所示的序列，与SEQ ID NO:14所示序列具有至少80％同一性的序列，或与SEQ ID NO:14所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  13. 如权利要求8所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含以下：
    所述VHa含SEQ ID NO:15所示的序列中第31-35位氨基酸(VHaCDR1，SYGMH)，和/或第50-66位氨基酸(VHaCDR2，VIAEVGSNQYYADSVKG)，和/或第99-111位氨基酸(VHaCDR3，DNQDTSPDVGIDY)；和/或
    所述VHb含SEQ ID NO:16所示的序列中第30-35位氨基酸(VHbCDR1， SSYGMS)，和/或第50-66位氨基酸(VHbCDR2，TINSNGGSTYYPDSVKG)，和/或第99-108位氨基酸(VHbCDR3，LGTGTLGFAY)氨基酸；和/或
    所述VLa含SEQ ID NO:17所示的序列所示的序列中第24-34位氨基酸(VLaCDR1，RASQNISPFLN)，和/或第50-56位氨基酸(VLaCDR2，AAVGLQS)，和/或第89-97位氨基酸(VLaCDR3，QQYTDYPLT)；和/或
    所述VLb含SEQ ID NO:18所示的序列中第24-34位氨基酸(VLbCDR1，KASQDVKTAVS)，和/或第50-56位氨基酸(VLbCDR2，WASTRAT)，和/或第89-97位氨基酸(VLbCDR3，QQHYSTPWT)。
  14. 如权利要求13所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含以下：
    所述VHa含SEQ ID NO:15所示的序列，与SEQ ID NO:15所示序列具有至少80％同一性的序列，或与SEQ ID NO:15所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VHb含SEQ ID NO:16所示的序列，与SEQ ID NO:16所示序列具有至少80％同一性的序列，或与SEQ ID NO:16所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VLa含SEQ ID NO:17所示的序列，与SEQ ID NO:17所示序列具有至少80％同一性的序列，或与SEQ ID NO:17所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；和/或
    所述VLb含SEQ ID NO:18所示的序列，与SEQ ID NO:18所示序列具有至少80％同一性的序列，或与SEQ ID NO:18所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CLa含SEQ ID NO:19所示的序列，与SEQ ID NO:19所示序列具有至少80％同一性的序列，或与SEQ ID NO:19所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CLb含SEQ ID NO:20所示的序列，与SEQ ID NO:20所示序列具有至少80％同一性的序列，或与SEQ ID NO:20所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；
    所述CH1含SEQ ID NO:21所示的序列，与SEQ ID NO:21所示序列具有至少80％同一性的序列，或与SEQ ID NO:21所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  15. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段结合两个 不同抗原第一抗原a和第二抗原b，其中第一抗原a为TIGIT，第二抗原b为CTLA-4；所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:37所示的序列，与SEQ ID NO:37所示序列具有至少80％同一性的序列，或与SEQ ID NO:37所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:38所示的序列，与SEQ ID NO:38所示序列具有至少80％同一性的序列，或与SEQ ID NO:38所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；或
    所述第一条多肽链含SEQ ID NO:39所示的序列，与SEQ ID NO:39所示序列具有至少80％同一性的序列，或与SEQ ID NO:39所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:40所示的序列，与SEQ ID NO:40所示序列具有至少80％同一性的序列，或与SEQ ID NO:40所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；或
    所述第一条多肽链含SEQ ID NO:41所示的序列，与SEQ ID NO:41所示序列具有至少80％同一性的序列，或与SEQ ID NO:41所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:42所示的序列，与SEQ ID NO:42所示序列具有至少80％同一性的序列，或与SEQ ID NO:42所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；或
    所述第一条多肽链含SEQ ID NO:43所示的序列，与SEQ ID NO:43所示序列具有至少80％同一性的序列，或与SEQ ID NO:43所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:44所示的序列，与SEQ ID NO:44所示序列具有至少80％同一性的序列，或与SEQ ID NO:44所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  16. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；
    所述第一条多肽链含SEQ ID NO:37所示的序列，所述第二条多肽链含SEQ ID NO:38所示的序列；或
    所述第一条多肽链含SEQ ID NO:39所示的序列，所述第二条多肽链含SEQ ID NO:40所示的序列；或
    所述第一条多肽链含SEQ ID NO:41所示的序列，所述第二条多肽链含SEQ ID NO:42所示的序列；或
    所述第一条多肽链含SEQ ID NO:43所示的序列，所述第二条多肽链含SEQ ID  NO:44所示的序列。
  17. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为OX40，第二抗原b为CTLA-4；所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:45所示的序列，与SEQ ID NO:45所示序列具有至少80％同一性的序列，或与SEQ ID NO:45所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:46所示的序列，与SEQ ID NO:46所示序列具有至少80％同一性的序列，或与SEQ ID NO:46所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  18. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:45所示的序列，所述第二条多肽链含SEQ ID NO:46所示的序列。
  19. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段结合两个不同抗原第一抗原a和第二抗原b，其中第一抗原a为OX40，第二抗原b为TIGIT；所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；所述第一条多肽链含SEQ ID NO:47所示的序列，与SEQ ID NO:47所示序列具有至少80％同一性的序列，或与SEQ ID NO:47所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:48所示的序列，与SEQ ID NO:48所示序列具有至少80％同一性的序列，或与SEQ ID NO:48所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；或
    所述第一条多肽链含SEQ ID NO:49所示的序列，与SEQ ID NO:49所示序列具有至少80％同一性的序列，或与SEQ ID NO:49所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列；所述第二条多肽链含SEQ ID NO:50所示的序列，与SEQ ID NO:50所示序列具有至少80％同一性的序列，或与SEQ ID NO:50所示序列相比具有一个或多个保守氨基酸取代的氨基酸序列。
  20. 一种抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段至少包含第一条多肽链和第二条多肽链2条多肽链；
    所述第一条多肽链含SEQ ID NO:47所示的序列，所述第二条多肽链含SEQ ID  NO:48所示的序列；或
    所述第一条多肽链含SEQ ID NO:49所示的序列，所述第二条多肽链含SEQ ID NO:50所示的序列。
  21. 一种编码如权利要求1-20任一项所述的抗体或抗原结合片段的核酸分子。
  22. 一种包含如权利要求21所述的核酸分子的载体。
  23. 一种包含如权利要求21所述核酸分子或如权利要求22所述的载体的宿主细胞。
  24. 一种包含如权利要求1-20任一项所述的抗体或抗原结合片段的药物组合物。
  25. 一种治疗或改善各种疾病的方法，其包括向患者施用有效剂量的如权利要求1-20任一项所述的抗体或抗原结合片段。
  26. 如权利要求1-20任一项所述的抗体或抗原结合片段在制备用于治疗或改善各种疾病的药物中的应用。
  27. 如权利要求26所述的应用，所述疾病选自于以下组成的组：炎性疾病、自体免疫性疾病、癌症和脊髓损伤。
  28. 一种于诊断的试剂盒，其包含如权利要求1-20任一项所述的抗体或抗原结合片段。
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WO2024032662A1 (zh) * 2022-08-09 2024-02-15 上海济煜医药科技有限公司 一种靶向pd-1和vegf的抗体及其应用
WO2024046389A1 (zh) * 2022-08-31 2024-03-07 百奥泰生物制药股份有限公司 抗tigit抗体和抗ctla-4抗体联合用于治疗肿瘤中的应用

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