WO2022184155A1 - 抗ctla-4抗体及其应用 - Google Patents

抗ctla-4抗体及其应用 Download PDF

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WO2022184155A1
WO2022184155A1 PCT/CN2022/079170 CN2022079170W WO2022184155A1 WO 2022184155 A1 WO2022184155 A1 WO 2022184155A1 CN 2022079170 W CN2022079170 W CN 2022079170W WO 2022184155 A1 WO2022184155 A1 WO 2022184155A1
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seq
amino acid
acid sequence
antibody
ctla
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French (fr)
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肖扬
李雪
周新然
赵立文
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南京圣和药业股份有限公司
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/28—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the technical field of antibody drugs, in particular to an anti-CTLA-4 antibody or an antigen-binding fragment thereof, a double antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, and their application.
  • T lymphocytes play a key role in the adaptive immune response to antigens.
  • Naive T cells require two signals for full activation.
  • the first signal is antigen-specific, provided by the interaction of T cell receptors (TCRs) with MHC/peptide complexes on antigen presenting cells (APCs).
  • the second signal is a costimulatory signal, provided by the interaction between the receptor on the T cell and its ligand on the APC.
  • CTLA-4 cytotoxic T lymphocyte-associated protein 4, also known as CD152
  • CTLA-4 is structurally homologous to CD28, but binds more tightly to B7-1 and B7-2 ligands.
  • CTLA-4 suppresses immune responses in two major ways, it competes with CD28 for B7-1 and B7-2 ligands to block co-stimulation, and it also signals negatively to inhibit T cell activation.
  • Blockade of CTLA-4 has been reported to increase T cell responses in vitro and in vivo, enhance antitumor immunity, and enhance induced autoimmune disease.
  • Antibodies to CTLA-4 have been described as modulators of immune stimulation in a number of disease conditions, such as in the treatment or prevention of viral and bacterial infections and in the treatment of cancer.
  • Ipilimumab is a human anti-human CTLA-4 antibody that blocks the binding of CTLA-4 to B7-1 and B7-2 expressed on APCs, thereby blocking the negative downregulation of immune responses elicited by the interaction of these molecules.
  • PD-1 is also a key immune checkpoint receptor, which is expressed by activated T and B cells and mediates immune suppression.
  • PD-1 regulates T cell activation by binding to its ligands programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2).
  • PD-1 ligands are expressed on antigen-presenting cells and in a variety of human cancers, and have been shown to downregulate T-cell activation and cytokine secretion when PD-1 ligands bind to PD-1.
  • the two checkpoints CTLA-4 and PD-1 may be complementary immune checkpoints with different pathways. Therefore, the research and development of double-antibody drugs that can act on these two checkpoints at the same time also has significant clinical significance and broad market space.
  • One aspect of the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises the complementarity of the heavy chain variable region Determining region 1 (H1CDR1), heavy chain variable region complementarity determining region 2 (H1CDR2) and/or heavy chain variable region complementarity determining region 3 (H1CDR3) comprising the light chain variable region
  • H1CDR1 heavy chain variable region complementarity determining region 2
  • H1CDR3 heavy chain variable region complementarity determining region 3
  • the invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3 selected from the group consisting of:
  • the light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the group consisting of:
  • the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein H1CDR1, H1CDR2 of the heavy chain variable region and H1CDR3 are SEQ ID NOs: 1, 2 and 3, respectively, and L1CDR1, L1CDR2 and L1CDR3 of the light chain variable region are SEQ ID NOs: 5, 6 and 7, respectively.
  • the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein H1CDR1, H1CDR2 of the heavy chain variable region and H1CDR3 are SEQ ID NOs: 1, 2 and 4, respectively, and L1CDR1, L1CDR2 and L1CDR3 of the light chain variable region are SEQ ID NOs: 5, 6 and 7, respectively.
  • the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 26, and SEQ ID NO: 26 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 26 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 26, and the amino acids of the light chain variable region The sequence is SEQ ID NO:27, the amino acid sequence of SEQ ID NO:27 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:27 or having at least 85% sequence with SEQ ID NO:27 identical amino acid sequences.
  • the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 28, and SEQ ID NO: 28 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 28 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 28, and the amino acids of the light chain variable region The sequence is SEQ ID NO:27, the amino acid sequence of SEQ ID NO:27 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:27 or having at least 85% sequence with SEQ ID NO:27 identical amino acid sequences.
  • the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 29, and SEQ ID NO: 29 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 29 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 29, and the amino acids of the light chain variable region
  • the sequence is SEQ ID NO:32, the amino acid sequence of SEQ ID NO:32 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:32 or having at least 85% sequence with SEQ ID NO:32 identical amino acid sequences.
  • the present invention provides an anti-CTLA-4 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 30, SEQ ID NO: 30 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 30 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 30, and the amino acids of the light chain variable region
  • the sequence is SEQ ID NO:32, the amino acid sequence of SEQ ID NO:32 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:32 or having at least 85% sequence with SEQ ID NO:32 identical amino acid sequences.
  • the anti-CTLA-4 antibody according to the present invention is a murine antibody which further contains the heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and a murine heavy chain constant region.
  • the anti-CTLA-4 murine antibody according to the present invention further contains the heavy chain constant regions of murine IgG1, IgG2a, IgG2b, IgG2c or variants thereof, and murine kappa chains or variants thereof The light chain constant region of the body.
  • the antibody heavy chain of the anti-CTLA-4 chimeric antibody or its antigen-binding fragment further comprises a heavy chain of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutant sequence thereof
  • the constant region preferably comprises a human IgG1 or IgG2 heavy chain constant region, or an IgG4 constant region that significantly reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.
  • the present invention provides an anti-CTLA-4 humanized antibody or antigen-binding fragment thereof, wherein the heavy chain comprises the heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4 or variant thereof , the light chain comprises the light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1 or IgG2 or a variant thereof, and a human kappa chain or a variant thereof light chain constant region.
  • the present invention provides an anti-CTLA-4 antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, or F(ab) 2 .
  • an anti-PD-1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region
  • the heavy chain variable region comprises a Complementarity determining region 1 (H2CDR1), heavy chain variable region complementarity determining region 2 (H2CDR2) and/or heavy chain variable region complementarity determining region 3 (H2CDR3) comprising light chain variable regions
  • H2CDR1 Complementarity determining region 1
  • H2CDR2CDR2 heavy chain variable region complementarity determining region 2
  • H2CDR3 heavy chain variable region complementarity determining region 3
  • the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the group consisting of:
  • the light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the group consisting of:
  • the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof, which has the H2CDR1, H2CDR2 and H2CDR3 respectively as SEQ ID NO: 8, 9 and 10 or with SEQ ID NO:
  • the amino acid sequences shown in 8, 9 and 10 have the heavy chain variable region of the amino acid sequence of at least 85% sequence identity
  • the L2CDR1, L2CDR2 and L2CDR3 are respectively SEQ ID NOs: 11, 12 and 13 or with SEQ ID
  • the amino acid sequences shown in NO: 11, 12 and 13 have light chain variable regions of amino acid sequences of at least 85% sequence identity.
  • the anti-PD-1 antibody or antigen-binding fragment thereof according to the invention is a monoclonal antibody or antigen-binding fragment thereof.
  • the anti-PD-1 antibody or antigen-binding fragment thereof according to the invention is a murine antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof .
  • the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 43, SEQ ID NO: 43 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 43 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 43, and the amino acids of the light chain variable region
  • the sequence is SEQ ID NO:44, the amino acid sequence of SEQ ID NO:44 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:44 or having at least 85% sequence with SEQ ID NO:44 identical amino acid sequences.
  • the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 46, SEQ ID NO: 46 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 46, and the amino acids of the light chain variable region
  • the sequence is SEQ ID NO:51, the amino acid sequence of SEQ ID NO:51 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:51 or having at least 85% sequence with SEQ ID NO:51 identical amino acid sequences.
  • the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 46, SEQ ID NO: 46 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 46, and the amino acids of the light chain variable region
  • the sequence is SEQ ID NO:52, the amino acid sequence of SEQ ID NO:52 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:52 or having at least 85% sequence with SEQ ID NO:52 identical amino acid sequences.
  • the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 46, SEQ ID NO: 46 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 46, and the amino acids of the light chain variable region
  • the sequence is SEQ ID NO:53, the amino acid sequence of SEQ ID NO:53 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:53 or having at least 85% sequence with SEQ ID NO:53 identical amino acid sequences.
  • the present invention provides an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 46, SEQ ID NO: 46 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO:46 or having at least 85% sequence identity with SEQ ID NO:46 and said H2CDR1, H2CDR2 and H2CDR3 as SEQ ID NO:8
  • the amino acid sequences shown in , 9 and 10 and the amino acid sequence of the light chain variable region is SEQ ID NO: 52, SEQ ID NO: 52 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID NO: 52
  • the amino acid sequence of NO:52 is functionally identical or has at least 85% sequence identity with SEQ ID NO:52 and the L2CDR1, L2CDR2 and L2CDR3 amino acid sequences shown in SEQ ID NO:11, 12 and 13.
  • the anti-PD-1 antibody according to the present invention is a murine antibody further comprising the heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and a murine heavy chain constant region The light chain constant region of the kappa chain or its variants.
  • the anti-PD-1 murine antibody according to the present invention further contains the heavy chain constant regions of murine IgG1, IgG2a, IgG2b, IgG2c or variants thereof, and murine kappa chains or variants thereof light chain constant region.
  • the antibody light chain of the anti-PD-1 chimeric antibody or its antigen-binding fragment further comprises a light chain constant region of a murine ⁇ , ⁇ chain or a mutant sequence thereof.
  • the antibody heavy chain of the anti-PD-1 chimeric antibody or its antigen-binding fragment further comprises the heavy G chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutated sequence thereof, preferably human IgG1, IgG2a , IgG2b, IgG2c heavy chain constant regions, or IgG4 constant regions with amino acid mutations that significantly reduce ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity.
  • the anti-PD-1 humanized antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region of human IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, or a variant thereof, and Light chain constant regions of human kappa, lambda chains or variants thereof.
  • the anti-PD-1 humanized antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region of human IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, or a variant thereof, and The light chain constant region of a human kappa chain or a variant thereof.
  • the present invention provides an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, or F(ab)2.
  • Another aspect of the present invention provides an isolated nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof according to the present invention.
  • the isolated nucleic acid according to the present invention comprises an amino acid sequence encoding a heavy chain variable region such as SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 , the nucleotide sequence of SEQ ID NO:48, SEQ ID NO:49 or SEQ ID NO:50; and the amino acid sequence encoding the light chain variable region such as SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO: 52.
  • the nucleotide sequence of SEQ ID NO:53 is an amino acid sequence encoding a heavy chain variable region such as SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 , the nucleotide sequence of SEQ ID NO:48, SEQ ID NO:49 or SEQ ID NO:50; and the amino acid sequence encoding the light chain variable region such as SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO: 52.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO:43; and a nucleotide sequence encoding a light chain variable region of SEQ ID NO:44 acid sequence.
  • the isolated nucleic acid according to the present invention comprises a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO:46; and a nucleotide sequence encoding a light chain variable region of SEQ ID NO:52 acid sequence.
  • Another aspect of the present invention provides an expression vector expressing the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention.
  • the expression vector according to the present invention comprises the isolated nucleic acid molecule of the present invention.
  • Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
  • the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells.
  • the host cell is a bacterium, preferably E. coli.
  • the host cell is a mammalian cell.
  • Another aspect of the present invention provides a method of preparing an anti-PD-1 antibody or antigen-binding fragment thereof of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the anti-PD-1 humanized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising the anti-PD-1 humanized antibody or antigen-binding fragment thereof of the present invention, and other active components, such as other antibodies, targeted drugs, and the like.
  • the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • the pharmaceutically acceptable carrier is in the form of a liposome.
  • the anti-PD-1 humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration.
  • Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, intranasal routes.
  • the formulations may be administered by any route, such as by infusion or bolus injection, by route of absorption through the epithelium or mucocutaneous (eg, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the formulations can be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the art of pharmaceutical formulations.
  • Another aspect of the present invention provides a method of inhibiting PD-1 activity, the method comprising administering an anti-PD-1 antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition of the present invention to an individual in need thereof.
  • Another aspect of the present invention provides a method for detecting or measuring human PD-1, the method comprising the step of using the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention.
  • Another aspect of the present invention provides a reagent for detecting or measuring human PD-1, the reagent comprising the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention.
  • Another aspect of the present invention provides a method of treating a disease associated with PD-1, the method comprising administering to a subject a pharmaceutically effective amount of the PD-1 antibody or antigen-binding fragment thereof of the present invention, or comprising the above-mentioned drug A composition, or an isolated nucleic acid molecule as described above.
  • Another aspect of the present invention provides use of the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for PD-1-related diseases.
  • the medicament for a disease associated with PD-1 is used to treat a T cell dysfunctional disorder, such as a tumor, an immune disease, or an infectious disorder.
  • the tumor is non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma tumor, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, adrenocortical carcinoma, Liver hepatocellular carcinoma, pancreatic duct adenocarcinoma, pheochromocytoma, ganglion cell tumor, endometrial carcinoma and ovarian serous cystadenocarcinoma, etc.
  • the immune disease is arthritis, inflammatory bowel disease, psoriasis.
  • the infectious disease is a chronic viral infection.
  • an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-CTLA- 4
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises the complementarity determining region 1 (H1CDR1) of the heavy chain variable region, the heavy chain variable region The complementarity determining region 2 (H1CDR2) of the light chain variable region and/or the complementarity determining region 3 (H1CDR3) of the heavy chain variable region, the light chain variable region comprises the complementarity determining region 1 (L1CDR1) of the light chain variable region, the light chain variable region The complementarity determining region 2 (L1CDR2) of the variable region and/or the complementarity determining region 3 (L1CDR3) of the light chain variable region; and the anti-PD-1 antibody or antigen-bind
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or antigen-binding fragment thereof and an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof
  • the CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3 selected from the group consisting of:
  • the light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the group consisting of:
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof has:
  • the H1CDR1, H1CDR2 and H1CDR3 are the heavy chain variable regions of SEQ ID NOs: 1, 2 and 3 or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively , and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 5, 6 and 7 or the light chain of the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, 6 and 7 can be variable area; or
  • the H1CDR1, H1CDR2 and H1CDR3 are respectively SEQ ID NOs: 1, 2 and 4 or heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NOs: 1, 2 and 4
  • the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 5, 6 and 7 or the light chain of the amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, 6 and 7 can be variable area.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 26, the amino acid sequence of SEQ ID NO: 26 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 26 or the same amino acid sequence as SEQ ID NO: 26.
  • ID NO: 26 has an amino acid sequence of at least 85% sequence identity and the H1CDR1, H1CDR2 and H1CDR3 amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, and the amino acid sequence of the light chain variable region is SEQ ID NO:27, the amino acid sequence of SEQ ID NO:27 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:27 or having at least 85% sequence identity with SEQ ID NO:27
  • the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 28, the amino acid sequence of SEQ ID NO: 28 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 28 or the same amino acid sequence as SEQ ID NO: 28.
  • H1CDR1, H1CDR2 and H1CDR3 are as shown in SEQ ID NOs: 1, 2 and 4, and the amino acid sequence of said light chain variable region is SEQ ID NO:27, the amino acid sequence of SEQ ID NO:27 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:27 or having at least 85% sequence identity with SEQ ID NO:27 and the amino acid sequences of L1CDR1, L1CDR2 and L1CDR3 are shown in SEQ ID NOs: 5, 6 and 7.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable region and the light chain variable region, where:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable region and light chain variable regions, where
  • the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 29, the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in SEQ ID NO: 29 and having the same function as SEQ ID NO: 29 or the same amino acid sequence as SEQ ID NO: 29.
  • ID NO: 29 has an amino acid sequence of at least 85% sequence identity
  • the amino acid sequence of the light chain variable region is SEQ ID NO: 32 obtained by substitution, deletion or addition of one or more amino acids and an amino acid sequence that is functionally identical to SEQ ID NO:32 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:32; or
  • the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 30, the amino acid sequence of SEQ ID NO: 30 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 30 or the same amino acid sequence as SEQ ID NO: 30.
  • ID NO:30 has an amino acid sequence of at least 85% sequence identity
  • the amino acid sequence of the light chain variable region is SEQ ID NO:32 obtained by substitution, deletion or addition of one or more amino acids and an amino acid sequence that is functionally identical to SEQ ID NO:32 or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:32.
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the The anti-CTLA-4 antibody or antigen-binding fragment thereof is as defined in the above embodiments; and the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region, wherein the The heavy chain variable region comprises the complementarity determining region 1 (H2CDR1) of the heavy chain variable region, the complementarity determining region 2 (H2CDR2) of the heavy chain variable region and/or the complementarity determining region 3 (H2CDR3) of the heavy chain variable region,
  • the light chain variable region comprises complementarity determining region 1 (L2CDR1) of the light chain variable region, complementarity determining region 2 (L2CDR2) of the light chain variable region and/or complementarity determining region 3 (L2CDR3) of the light chain
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof is as defined in the above embodiments, and the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the group consisting of:
  • (A2) an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (A1);
  • the light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the group consisting of:
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein:
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3 selected from the group consisting of:
  • the light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the group consisting of:
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the group consisting of:
  • (A2) an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (A1);
  • the light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the group consisting of:
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the Said anti-CTLA-4 antibody or antigen-binding fragment thereof comprises said H1CDR1, H1CDR2 and H1CDR3 are respectively SEQ ID NO: 1, 2 and 3 or have at least 85% with the amino acid sequence shown in SEQ ID NO: 1, 2 and 3
  • the heavy chain variable region of the amino acid sequence of sequence identity, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 5, 6 and 7 or have at least the amino acid sequence shown in SEQ ID NO: 5, 6 and 7
  • a light chain variable region of an amino acid sequence of 85% sequence identity; and said anti-PD-1 antibody or antigen-binding fragment thereof comprising said H2CDR1, H2CDR2 and H2CDR3, respectively, are SEQ ID NOs: 8, 9 and 10 or the same
  • amino acid sequences shown in ID NOs: 8, 9 and 10 have the heavy chain variable regions of amino acid sequences of at least 85% sequence identity, and the L2CDR1, L2CDR2 and L2CDR3 are respectively SEQ ID NOs: 11, 12 and 13 or A light chain variable region of an amino acid sequence having at least 85% sequence identity with the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13.
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the Said anti-CTLA-4 antibody or antigen-binding fragment thereof comprises said H1CDR1, H1CDR2 and H1CDR3 are respectively SEQ ID NO: 1, 2 and 4 or have at least 85% with the amino acid sequence shown in SEQ ID NO: 1, 2 and 4
  • the heavy chain variable region of the amino acid sequence of sequence identity, and the L1CDR1, L1CDR2 and L1CDR3 are respectively SEQ ID NO: 5, 6 and 7 or have at least the amino acid sequence shown in SEQ ID NO: 5, 6 and 7
  • a light chain variable region of an amino acid sequence of 85% sequence identity; and said anti-PD-1 antibody or antigen-binding fragment thereof comprising said H2CDR1, H2CDR2 and H2CDR3, respectively, are SEQ ID NOs: 8, 9 and 10 or the same
  • amino acid sequences shown in ID NOs: 8, 9 and 10 have the heavy chain variable regions of amino acid sequences of at least 85% sequence identity, and the L2CDR1, L2CDR2 and L2CDR3 are respectively SEQ ID NOs: 11, 12 and 13 or A light chain variable region of an amino acid sequence having at least 85% sequence identity with the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13.
  • the anti-CTLA-4 antibody or antigen-binding fragment thereof and the anti-PD-1 antibody or antigen-binding fragment thereof are each independently Murine antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, in:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the The amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or antigen-binding fragment thereof is SEQ ID NO: 30, which is obtained by substitution, deletion or addition of one or more amino acids and is the same as that of SEQ ID NO: 30.
  • amino acid sequence of SEQ ID NO: 32 is SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 32 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 32 or an amino acid with at least 85% sequence identity to SEQ ID NO: 32 sequence.
  • the present invention provides an anti-CTLA-4/anti-PD-1 antibody comprising an anti-CTLA-4 antibody or an antigen-binding fragment thereof and an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the The amino acid sequence of the heavy chain variable region of the anti-CTLA-4 antibody or antigen-binding fragment thereof is SEQ ID NO: 30, which is obtained by substitution, deletion or addition of one or more amino acids and is the same as that of SEQ ID NO: 30.
  • amino acid sequence of the variable region is SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 32 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 32 or the amino acid sequence of SEQ ID NO: 32 have at least 85% sequence identity and the L1CDR1, L1CDR2 and L1CDR3 amino acid sequences shown in SEQ ID NOs: 5, 6 and 7.
  • the anti-CTLA-4 antibody or anti-PD-1 antibody may be a murine antibody, which further contains murine IgG1 , the heavy chain constant region of IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and the light chain constant region of murine kappa chain or variants thereof.
  • the anti-CTLA-4/anti-PD-1 antibody according to the present invention wherein the anti-CTLA-4 murine antibody further contains the heavy chain constant region of murine IgG1 or IgG2 or a variant thereof , and the light chain constant region of a murine kappa chain or a variant thereof.
  • the invention provides an anti-CTLA-4/anti-PD-1 antibody, wherein the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • (C2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (C1) and having the same or similar function to the amino acid sequence shown in (C1);
  • (C3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (C1);
  • amino acid sequence of the light chain variable region is selected from:
  • (C5) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in (C4) and having the same or similar function to the amino acid sequence shown in (C4);
  • the present invention provides anti-CTLA-4/anti-PD-1 antibodies comprising anti-CTLA-4 antibodies or antigen-binding fragments thereof and anti-PD-1 antibodies or antigen-binding fragments thereof, wherein anti-CTLA- 4
  • the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is SEQ ID NO: 30, which is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 30
  • the amino acid sequence of SEQ ID NO:30 has at least 85% sequence identity and the H1CDR1, H1CDR2 and H1CDR3 are as shown in SEQ ID NO:1, 2 and 4, and the anti-CTLA-4 antibody or
  • the amino acid sequence of the light chain variable region of its antigen-binding fragment is SEQ ID NO: 32, and SEQ ID NO: 32 is the amino acid sequence obtained by replacing, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 32 or have at least 85% sequence identity with SEQ ID NO:32 and
  • Amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 and said H2CDR1, H2CDR2 and H2CDR3 are as shown in SEQ ID NO:8, 9 and 10, and said anti-PD-1 antibody
  • the amino acid sequence of the light chain variable region of the antigen-binding fragment thereof is SEQ ID NO: 52, and SEQ ID NO: 52 is an amino acid obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 52
  • Sequence or amino acid sequence with at least 85% sequence identity to SEQ ID NO:52 and said L2CDR1, L2CDR2 and L2CDR3 are as shown in SEQ ID NO:11, 12 and 13.
  • the present invention provides anti-CTLA-4/anti-PD-1 antibodies comprising anti-CTLA-4 antibodies or antigen-binding fragments thereof and anti-PD-1 antibodies or antigen-binding fragments thereof, wherein anti-CTLA- 4
  • the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is SEQ ID NO: 28, which is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 28
  • the amino acid sequence of SEQ ID NO:28 has at least 85% sequence identity and the H1CDR1, H1CDR2 and H1CDR3 amino acid sequences shown in SEQ ID NO:1, 2 and 4, and the anti-CTLA-4 antibody or
  • the amino acid sequence of the light chain variable region of its antigen-binding fragment is SEQ ID NO: 27, the amino acid sequence obtained by substitution, deletion or addition of one or more amino acids in SEQ ID NO: 27 and having the same function as SEQ ID NO: 27 or have at least 85% sequence identity with SEQ ID NO: 27 and
  • amino acid sequence of the light chain variable region of the antigen-binding fragment thereof is SEQ ID NO: 52
  • SEQ ID NO: 52 is an amino acid obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO: 52
  • Sequence or amino acid sequence with at least 85% sequence identity to SEQ ID NO:52 and said L2CDR1, L2CDR2 and L2CDR3 are as shown in SEQ ID NO:11, 12 and 13.
  • the present invention provides an anti-CTLA-4/anti-PD-1 humanized antibody, wherein the heavy chain comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof , the light chain comprises the light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the murine anti-CTLA-4/anti-PD-1 antibody may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, and/or further comprise Heavy chain constant regions of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof.
  • the antibody light chain of the anti-CTLA-4 antibody or its antigen-binding fragment further comprises a murine ⁇ , ⁇ chain or The light chain constant region of its mutated sequence.
  • the antibody heavy chain of the anti-CTLA-4 antibody or its antigen-binding fragment further comprises the heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutant sequence thereof, preferably human IgG1, IgG2, IgG4 heavy chain. chain constant region.
  • the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2a, IgG2b, IgG2c, Heavy chain constant regions of IgG3 or variants thereof, and light chain constant regions of human kappa, lambda chains or variants thereof.
  • the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG4 or a variant thereof, and a human kappa chain or its The light chain constant region of the variant.
  • the antibody heavy chain of the anti-PD-1 antibody or its antigen-binding fragment further comprises murine IgG1, IgG2a, IgG2b , IgG2c, IgG3 or the heavy chain constant region of mutated sequence thereof, preferably comprising the heavy chain constant region of human IgG or its mutated sequence;
  • the antibody light chain of the anti-PD-1 antibody or its antigen-binding fragment further comprises murine kappa , the light chain constant region of the lambda chain or a mutated sequence thereof.
  • the anti-PD-1 humanized antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2, IgG3 or IgG4 or Heavy chain constant regions of variants thereof, and light chain constant regions of human kappa, lambda chains or variants thereof.
  • the anti-CTLA-4 humanized antibody or antigen-binding fragment thereof of the present invention further comprises the heavy chain constant region of human IgG4 or a variant thereof, and the light weight of a human kappa chain or a variant thereof chain constant region.
  • the present invention provides anti-CTLA-4/anti-PD-1 antibody, wherein the anti-CTLA-4 antibody or its antigen-binding fragment and anti-PD-1 antibody or its antigen-binding fragment are Fab, Fv, respectively , sFv or F(ab) 2 .
  • the anti-CTLA-4/anti-PD-1 antibody provided by the present invention is scF(ab) 2 .
  • the anti-CTLA-4/anti-PD-1 antibody in the above embodiment of the invention is an anti-CTLA-4/anti-PD-1 bispecific antibody.
  • the bispecific antibody is a human antibody or a humanized antibody.
  • one of the binding specificities is for CTLA-4 and the other binding specificity is for any other antigen.
  • one of the binding specificities is for CTLA-4 and the other binding specificity is for PD-1.
  • the bispecific antibody binds two different epitopes of CTLA-4.
  • the bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CTLA-4.
  • bispecific antibodies of the invention possess a CTLA-4 binding arm and a cytotoxic agent such as saporin, anti-interferon-alpha, vinca alkaloids, ricin A chain, methotrexate or radioisotopes hapten.
  • cytotoxic agent such as saporin, anti-interferon-alpha, vinca alkaloids, ricin A chain, methotrexate or radioisotopes hapten.
  • Bispecific antibodies of the invention can be prepared as full-length antibodies or antibody fragments (eg, F(ab') 2 bispecific antibodies).
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of this correct molecule is usually carried out by an affinity chromatography step, which is rather cumbersome and yields low product. Similar methods are disclosed in WO93/08829 and Traunecker et al., EMBOJ. 10:3655 (1991).
  • antibody variable regions with the desired binding specificities are fused to immunoglobulin constant region sequences.
  • the fusion is to an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2 and CH3 regions.
  • the heavy chain constant region (CH1) comprising the site necessary for binding to the light chain is present in at least a portion of the fusion.
  • the DNA encoding the immunoglobulin heavy chain fusion fragment and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism.
  • the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (provides a second binding specificity) composition. Since the presence of the immunoglobulin light chain in only one half of the bispecific molecule provides a convenient separation route, this asymmetric structure was found to facilitate separation of the desired bispecific material from the undesired immunoglobulin chain composition . This method is disclosed in WO 94/04690. For further information on the production of bispecific antibodies see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture.
  • the interface comprises at least a portion of the CH3 domain of the antibody constant region.
  • one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan).
  • larger side chains eg, tyrosine or tryptophan.
  • a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other undesired end products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one heteroconjugated antibody can be conjugated to avidin and the other heteroconjugated antibody can be conjugated to biotin.
  • Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of crosslinking techniques.
  • Bispecific antibodies of the present invention can be produced from antibody fragments.
  • bispecific antibodies can be prepared using chemical ligation techniques.
  • Brennetal., Science 229:81 (1985) describes a method for proteolytic cleavage of intact antibodies to generate F(ab') 2 fragments. These fragments are decomposed in the presence of the dithiol complexing agent sodium arsenite (to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds).
  • the resulting Fab' fragments were then converted into thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives was then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • Fab'-SH fragments can be recovered directly from E. coli and these fragments can be chemically coupled to form bispecific antibodies.
  • Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the generation of fully humanized bispecific antibody F(ab') 2 molecules.
  • Each Fab' fragment is secreted separately from E. coli and subjected to targeted chemical coupling in vitro to form bispecific antibodies.
  • bispecific antibody fragments of the invention can be produced and isolated directly from recombinant cell culture.
  • bispecific antibodies can be generated using leucine zippers (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)).
  • Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by genetic fusion.
  • Antibody homodimers break down at the hinge region to form monomers, which are then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers.
  • Diabody technology provides additional mechanisms for making bispecific antibody fragments.
  • the bispecific antibody fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL) joined by a linker that is too short to allow pairing between the two domains on the same chain .
  • VH and VL domains on one fragment are forced to pair with the complementary VL and VH domains on the other fragment, thereby forming two antigen binding sites.
  • bispecific antibody fragments can be constructed by using single-chain Fv (sFv) dimers.
  • the present invention encompasses multivalent antibodies with more than two valences, eg, trispecific antibodies can be prepared.
  • Multivalent antibodies can be internalized (and/or dissimilated) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds.
  • Antibodies of the invention may be multivalent antibodies having three or more antigen binding sites (eg, tetravalent antibodies) that can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains.
  • Multivalent antibodies may contain a dimerization domain and three or more antigen binding sites. In some embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region.
  • the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region.
  • the multivalent antibody comprises (or consists of) three to about eight antigen binding sites.
  • the multivalent antibody comprises four antigen binding sites.
  • Multivalent antibodies comprise at least one polypeptide chain (eg, two polypeptide chains), wherein the polypeptide chains comprise two or more variable regions.
  • the multivalent antibodies of the present invention may further comprise at least two (eg, four) light chain variable region polypeptides.
  • the multivalent antibodies of the invention may comprise, for example, from about two to about eight light chain variable region polypeptides.
  • the light chain variable region polypeptides of the invention comprise a light chain variable region, and optionally further comprise a CL domain.
  • one of the CTLA-4 targeting moiety and the PD-1 targeting moiety may be a full-length antibody, and the other may be a heavy Antigen-binding fragments (eg, scFvs) of chain CDRs, light chain CDRs, or combinations thereof.
  • a full-length antibody targeting one of the CTLA-4 and PD-1 proteins and an antigen-binding fragment targeting the other protein can be linked (e.g., covalently) directly or chemically via a linking peptide.
  • Antigen-binding fragments can be directly or by linking peptides to the N-terminus of a full-length antibody (eg, the N-terminus of a light or heavy chain of a full-length antibody), the C-terminus of a full-length antibody (eg, of a full-length antibody the C-terminus of the heavy chain (or Fc or CH3 domains) or both.
  • the bispecific antibodies of the invention comprise full-length anti-CTLA-4 antibodies, antigen-binding fragments of anti-PD-1 antibodies (eg, scFab, scFv), and linking peptides therebetween.
  • the bispecific antibodies of the invention comprise a full-length anti-PD-1 antibody, an antigen-binding fragment of an anti-CTLA-4 antibody (eg, scFab, scFv), and linking peptides therebetween.
  • the scFv comprised in the bispecific antibodies of the invention may comprise a heavy chain variable region and a light chain variable region in any order.
  • the scFv contained in a bispecific antibody may comprise a heavy chain variable region and a light chain variable region and optionally a linking peptide between them in the N-terminal to C-terminal direction, or alternatively
  • the scFv comprised in the bispecific antibody of the invention may comprise a light chain variable region and a heavy chain variable region and optionally a linking peptide between them in the direction from the N-terminus to the C-terminus.
  • the linker peptide may include, for example, Gly, Asn, and/or Ser residues, and may also include neutral amino acids, such as Thr and/or Ala.
  • Amino acid sequences suitable for linking peptides may be those known in the relevant art.
  • the length of the linker peptide can be determined differently within such limits that the function of the fusion protein is not affected.
  • the linker peptide can be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala .
  • the linker peptide can be represented as (GmSl)n (m, 1, and n are independently integers from about 1 to about 10, particularly from about 2 to about 5).
  • the PD-1 targeting moiety and the CTLA-4 targeting moiety can both be full-length antibodies or antigen-binding fragments comprising heavy chain CDRs, light chain CDRs, or a combination thereof.
  • the bispecific antibody may be in the form of a heterodimer comprising a first arm and a second arm comprising a pair of heavy chains targeting one of CTLA-4 and PD-1 and light chain, the second arm includes a pair of heavy and light chains targeting the other.
  • full-length antibodies may be in the form of full-length immunoglobulins (eg, IgG, IgM, IgA, IgE, or IgD, eg, human IgG, human IgM, human IgA, human IgE, or human IgD), and antigen-binding fragments It can be selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, scFv, scFab, single chain antibody, sdFv, and the like.
  • immunoglobulins eg, IgG, IgM, IgA, IgE, or IgD
  • antigen-binding fragments It can be selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, scFv, scFab, single chain antibody, sdFv, and the like.
  • the full-length antibody can be in the form of a full-length human IgG (human IgGl, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment can be an scFv.
  • the antibodies described herein can contain flexible linker sequences, or can be modified to add functional moieties (eg, PEG, drugs, toxins, or labels).
  • the structure of the anti-CTLA-4 antibody or antigen-binding fragment thereof is (VH)-linking peptide-(VL )
  • the structure of the anti-PD-1 antibody or its antigen-binding fragment is (VL-CL)-linking peptide-(VH).
  • the linker peptide is in the form of (GGGGS)n, wherein n is 1-12, preferably 3-10, more preferably 3-8, such as 3, 4, 5, 6, 7, 8 GGGGS repeats.
  • the anti-CTLA-4/anti-PD-1 bispecific antibody according to the invention consists of two peptide chains, wherein:
  • the anti-PD-1 antibody VH is the heavy chain variable region of the anti-PD-1 antibody
  • Connecting peptide 1 and connecting peptide 2 are each a flexible connecting peptide of a repeat sequence in the form of (GGGGS) n, wherein n is 1-10;
  • the anti-CTLA-4 antibody VH is the heavy chain variable region of the anti-CTLA-4 antibody
  • the anti-CTLA-4 antibody VL is the light chain variable region of the anti-CTLA-4 antibody
  • the anti-PD-1 antibody VL-CL is the light chain of the anti-PD-1 antibody.
  • the anti-CTLA-4/anti-PD-1 bispecific antibody according to the present invention consists of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 55, and the peptide chain The amino acid sequence of 2 is SEQ ID NO:54.
  • the anti-CTLA-4/anti-PD-1 bispecific antibody according to the present invention consists of two peptide chains, wherein the amino acid sequence of peptide chain 1 is SEQ ID NO: 56, the peptide The amino acid sequence of chain 2 is SEQ ID NO:54.
  • Another aspect of the invention provides isolated nucleic acids.
  • the isolated nucleic acid according to the present invention encodes an anti-CTLA-4/anti-PD-1 antibody of the present invention. In some embodiments, an isolated nucleic acid according to the present invention encodes an anti-CTLA-4 antibody or antigen-binding fragment thereof of the present invention. In other embodiments, the isolated nucleic acid according to the present invention encodes an anti-PD-1 antibody or antigen-binding fragment thereof of the present invention.
  • the isolated nucleic acid according to the present invention comprises a heavy chain variable region encoding an anti-CTLA-4 antibody or antigen-binding fragment thereof such as SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID
  • the nucleotide sequence of NO:29, SEQ ID NO:30 and the light chain variable region encoding the anti-CTLA-4 antibody or antigen-binding fragment thereof such as SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:32 , the nucleotide sequence of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35.
  • the isolated nucleic acid according to the present invention comprises a heavy chain variable region encoding an anti-PD-1 antibody or antigen-binding fragment thereof such as SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:45, Nucleotide sequences of ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and variable light chains encoding anti-PD-1 antibodies or antigen-binding fragments thereof Regions are the nucleotide sequences of SEQ ID NO:44, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53.
  • the expression vectors of the present invention express the anti-CTLA-4/anti-PD-1 bispecific antibodies of the present invention.
  • the expression vector of the present invention expresses an anti-CTLA-4 antibody or antigen-binding fragment thereof of the present invention.
  • the expression vector of the present invention expresses an anti-PD-1 antibody or antigen-binding fragment thereof of the present invention.
  • the vector expressing the anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention and the vector expressing the anti-PD-1 antibody or antigen-binding fragment thereof of the invention are homologous expression vector.
  • the expression vector according to the present invention comprises the isolated nucleic acid molecule of the present invention.
  • a chimeric antigen receptor (CAR) fusion protein comprising the anti-CTLA-4 antibody or antigen-binding fragment thereof and/or anti-PD-1 antibody or antigen-binding fragment thereof of the present invention.
  • the chimeric antigen receptor fusion protein comprises an anti-CTLA-4 antibody or antigen-binding fragment thereof of the invention, which is a single-chain variable fragment (scFv) directed against the VH and VL of the CTLA-4 antigen .
  • the chimeric antigen receptor fusion protein comprises an anti-PD-1 antibody or antigen-binding fragment thereof of the present invention, which is a single-chain variable fragment (scFv) directed against the VH and VL of the PD-1 antigen. ).
  • the chimeric antigen receptor fusion protein comprises a first single-chain variable fragment (scFv) against the VH and VL of the CTLA-4 antigen and a second single-chain variable fragment (scFv) against the VH and VL of the PD-1 antigen Single-chain variable fragments (scFv).
  • the first scFv against the VH and VL of the CTLA-4 antigen has H1CDR1, H1CDR2 and H1CDR3 of the heavy chain variable region and L1CDR1, L1CDR2 and L1CDR3 of the light chain variable region as described in the above embodiments.
  • the second scFv against the VH and VL of the PD-1 antigen has H2CDR1, H2CDR2 and H2CDR3 of the heavy chain variable region and L2CDR1, L2CDR2 and L2CDR3 of the light chain variable region as described in the above embodiments.
  • the present invention provides an anti-CTLA-4/anti-PD-1 bispecific antibody or an antigen-binding fragment thereof that is a bispecific antibody or an antigen-binding fragment thereof in an scFv structure, comprising a C-terminal
  • the CTLA-4 binding part and the N-terminus are the PD-1 binding part, wherein
  • the amino acid sequence of the C-terminal CTLA-4 binding part is selected from:
  • the amino acid sequence of the N-terminal PD-1 binding portion is selected from:
  • the present invention provides an anti-CTLA-4 and PD-1 bispecific antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the N-terminal PD-1 binding moiety is SEQ ID NO:54 , the amino acid sequence of SEQ ID NO:54 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:54 or an amino acid sequence with at least 85% sequence identity to SEQ ID NO:54, and
  • the amino acid sequence of the C-terminal CTLA-4 binding part is SEQ ID NO: 55, and SEQ ID NO: 55 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 55.
  • SEQ ID NO:55 has an amino acid sequence of at least 85% sequence identity.
  • the present invention provides an anti-CTLA-4 and PD-1 bispecific antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the N-terminal PD-1 binding moiety is SEQ ID NO:54 , the amino acid sequence of SEQ ID NO:54 obtained by substitution, deletion or addition of one or more amino acids and having the same function as SEQ ID NO:54 or an amino acid sequence with at least 85% sequence identity to SEQ ID NO:54, and
  • the amino acid sequence of the C-terminal CTLA-4 binding part is SEQ ID NO: 56, and SEQ ID NO: 56 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 56.
  • SEQ ID NO:56 has an amino acid sequence of at least 85% sequence identity.
  • the anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, and light chain constant regions of human kappa, lambda chains or variants thereof.
  • the anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the invention further comprises the heavy chain constant region of human IgG1 or IgG2 or a variant thereof, and a human kappa The light chain constant region of the chain or a variant thereof.
  • the present invention provides an anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv, or F(ab)2.
  • Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
  • the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells.
  • the host cell is a bacterium, preferably E. coli.
  • the host cell is a mammalian cell.
  • Another aspect of the present invention provides a method for preparing an anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the present invention, comprising expressing the antibody in the host cell and isolating the antibody from the host cell A step of.
  • compositions comprising the anti-CTLA-4/anti-PD-1 bispecific humanized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides pharmaceutical compositions comprising an anti-CTLA-4/anti-PD-1 bispecific humanized antibody or antigen-binding fragment thereof of the present invention, and other active components, such as other antibodies , targeted drugs, etc.
  • the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • the pharmaceutically acceptable carrier is in the form of a liposome.
  • the anti-CTLA-4/anti-PD-1 bispecific humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for Oral or parenteral administration.
  • Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, intranasal routes.
  • the formulations may be administered by any route, such as by infusion or bolus injection, by route of absorption through the epithelium or mucocutaneous (eg, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the formulations can be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the art of pharmaceutical formulations.
  • Another aspect of the invention provides a method of inhibiting CTLA-4 and/or PD-1 activity, the method comprising administering to an individual in need thereof an anti-CTLA-4/anti-PD-1 bispecific antibody of the invention or its Antigen-binding fragments or pharmaceutical compositions of the present invention.
  • Another aspect of the present invention provides the anti-CTLA-4/anti-PD-1 bispecific antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in preparation for inhibiting CTLA-4 and/or PD-1 activity application in medicines.
  • the drug that inhibits the activity of CTLA-4 and/or PD-1 is used to treat leukemia, lymphoma, breast, lung, stomach, bowel, esophagus, ovary, cervix, kidney cancer , bladder cancer, pancreatic cancer, glioma and/or melanoma.
  • the present invention provides the use of the above-mentioned anti-B7-H3 antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of an anti-tumor drug, preferably, the tumor is selected from leukemia, lymphoma, Breast, Lung, Stomach, Bowel, Esophagus, Ovarian, Cervical, Kidney, Bladder, Pancreatic, Glioma, and Melanoma.
  • the anti-CTLA-4/anti-PD-1 bispecific antibody or the antigen-binding fragment thereof provided by the present invention has higher affinity and stability, significant anti-tumor effect and low toxicity, and can be used in the preparation of anti-CTLA-4/anti-PD-1 bispecific antibodies for treating various tumor diseases.
  • the application in medicine has broad market prospects.
  • the term "at least 80% sequence identity” means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the term “at least 85% sequence identity” means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% %. Sequence comparison and percent identity determination between two sequences can be performed by the BLASTN/BLASTP algorithm on the National Center For Biotechnology Institute website.
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementarity determining regions" or "CDRs.”
  • CDRs complementarity determining regions
  • the “antibody” of the present invention refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments or single chains thereof.
  • An “antibody” of the present invention includes any protein or peptide containing at least a portion of an Ig molecule that has the biological activity of binding an antigen.
  • Examples of “antibodies” of the invention include, but are not limited to, heavy or light chain CDRs or ligand binding portions thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework regions, or any portion thereof.
  • the "antigen-binding fragments" of the present invention include Fab fragments, Fab' fragments, F(ab')2 fragments with antigen-binding activity, Fv fragments and scFv fragments that bind to human CTLA-4 or PD-1.
  • Fv fragments contain antibody heavy and light chain variable regions, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Different linkers can also be used to link the two antibody variable regions into a single polypeptide chain, called a single-chain antibody or single-chain Fv (scFv).
  • the anti-CTLA-4 or anti-PD-1 antibody of the present invention may be a single-chain variable fragment (scFv), which is derived from the single-chain polypeptide of the antibody and retains the ability to bind antigen.
  • scFvs include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence.
  • H chain immunoglobulin heavy chain
  • L chain light chain
  • Antibodies in the context of the present invention refer to immunoglobulin molecules or immunologically active portions thereof, ie molecules comprising an antigen-binding site that specifically binds to (immunoreacts with) an antigen.
  • "Specifically binds” means that an antibody reacts with one or more epitopes of an antigen but does not react with other polypeptides or binds other polypeptides with very low affinity (Kd>10<" 6 >).
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAbs (domain antibodies), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv, and Fab expression libraries.
  • Monoclonal antibodies are antibodies obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant techniques such as hybridoma technology, recombinant technology, phage display technology, and synthetic technology such as CDR grafting or other existing techniques.
  • the "murine antibody” in the present invention is a monoclonal antibody to human CTLA-4 prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with CTLA-4 antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the "chimeric antibody” of the present invention is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the variable region of the mouse.
  • the gene is linked with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the "humanized antibody” of the present invention is also referred to as a CDR-grafted antibody, and is an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework (FR).
  • FR human antibody variable region framework
  • Such variable region framework sequences can be obtained from public DNA databases or published references, eg from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from J. Immunoglobulins, 2001 ISBN012441351.
  • Bispecific antibodies as used in the present invention refer to monoclonal antibodies that have binding specificities for at least two different antigens.
  • the “peptide linkers” according to the present invention may be those comprising any amino acid from 1 to 10, especially 2 to 50, and may comprise any kind of amino acid without any limitation.
  • Figure 1 is a schematic diagram of the structure of the peptide chain 1 of the anti-CTLA-4/anti-PD-1 bispecific antibody.
  • Figure 2 is a schematic diagram of the structure of the peptide chain 2 of the anti-CTLA-4/anti-PD-1 bispecific antibody.
  • Figure 3 is the experimental result (FACS) of anti-CTLA-4 humanized antibody blocking the activity of CTLA-4 binding to CD80, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • Figure 4 is the experimental result (FACS) of anti-CTLA-4 humanized antibody blocking the activity of CTLA-4 binding to CD86, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • FIG. 5 is a schematic diagram of the bispecific anti-CTLA-4/PD-1 antibody structure of the ScFv structure of the present invention.
  • Fig. 6 is the result of the binding experiment (ELISA) of the bispecific antibody to CTLA-4, wherein the abscissa is the antibody concentration (nM), and the ordinate is the absorbance value at OD450.
  • Fig. 7 is the result of the binding experiment of bispecific antibody to PD-1 (ELISA), wherein the abscissa is the antibody concentration (nM), and the ordinate is the absorbance value at OD450.
  • FIG. 8 is the result of the binding experiment (FACS) of the bispecific antibody to the cell surface CTLA-4, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • FIG. 9 is the result of the binding experiment (FACS) between the bispecific antibody and the cell surface PD-1, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • Figure 10 is the experimental result (FACS) of bispecific antibody blocking CTLA-4 binding to CD80 activity, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • Figure 11 is the experimental result (FACS) of the bispecific antibody blocking the activity of CTLA-4 binding to CD86, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • Figure 12 is the experimental result (FACS) of the bispecific antibody blocking the activity of PD-1 binding to PD-L1, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • Figure 13 shows the results of the CD80 blocking activity experiment (FACS) of bispecific antibodies on bistable transfected cell lines, where the abscissa is the antibody concentration (nM) and the ordinate is the fluorescence intensity.
  • Figure 14 shows the results of the CD86 blocking activity experiment (FACS) of bispecific antibodies on bi-stable transfected cell lines, where the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • Figure 15 is the experimental result of PD-1 blocking activity (FACS) of bispecific antibodies on bi-stable transfected cell lines, wherein the abscissa is the antibody concentration (nM), and the ordinate is the fluorescence intensity.
  • FACS PD-1 blocking activity
  • a gene fragment encoding the full-length extracellular region of human CTLA-4 protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 14. Its nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pTargetT-hCTLA-4.
  • a gene fragment encoding the full-length extracellular region of the monkey-derived CTLA-4 protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 15. Its nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pTargetT-cynoCTLA-4.
  • the amino acid sequences of the fused human CTLA-4 protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO: 16 and SEQ ID NO: 17, and the above amino acid sequences are codon-optimized to synthesize a tagged
  • the nucleotide sequences of CTLA-4-hFc and CTLA-4-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-CTLA-4-hFc and pHR-CTLA-4-His.
  • the amino acid sequence of the fused human CTLA-4 protein extracellular region and the mIgG1-Fc tag is shown in SEQ ID NO: 18. After codon optimization of the extracellular region sequence of human CTLA-4 protein, the nucleotide sequence of the tagged CTLA-4-mFc was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain the expression plasmid pHR- CTLA-4-mFc.
  • the amino acid sequence of the fused monkey CTLA-4 protein extracellular region and hIgG1-Fc tag is shown in SEQ ID NO: 19, and the nucleosides of the tagged cynoCTLA-4-hFc are synthesized after codon optimization of the above amino acid sequence
  • the acid sequence was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-cynoCTLA-4-hIgG1-Fc.
  • a gene fragment encoding the full-length extracellular region of human CD80 protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 20. Its nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pTargetT-CD80.
  • a gene fragment encoding the full-length extracellular region of human CD86 protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 21. Its nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain its expression plasmid pTargetT-CD86.
  • the amino acid sequence of the fused human CD80 protein extracellular region and the mIgG1-Fc tag is shown in SEQ ID NO: 22, and the nucleotide sequence of the tagged CD80-mFc is synthesized after the above amino acid sequence is codon-optimized. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CD80-mFc.
  • the amino acid sequence of the fused human CD86 protein extracellular region and the mIgG1-Fc tag is shown in SEQ ID NO: 23, and the nucleotide sequence of the tagged CD86-mFc is synthesized after the above amino acid sequence is codon-optimized. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CD86-mFc.
  • the amino acid sequence of the fused human CD80 protein extracellular region and hIgG1-Fc tag is as SEQ ID NO: 24. After codon optimization of the above amino acid sequence, a tagged CD80-hFc nucleotide sequence is synthesized and cloned On the eukaryotic expression plasmid pHR, the expression plasmid pHR-CD80-hFc was obtained.
  • the amino acid sequence of the fused human CD86 protein extracellular region and hIgG1-Fc tag is as SEQ ID NO: 25. After codon optimization of the above amino acid sequence, a tagged CD86-hFc nucleotide sequence is synthesized and cloned On the eukaryotic expression plasmid pHR, the expression plasmid pHR-CD86-hFc was obtained.
  • the eukaryotic expression plasmid pTargeT-hCTLA-4 was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation under the square pulse of 160V and 15msec, and placed at 37°C, 5% CO2 concentration in an incubator. After 24 h, pressurized culture was performed with medium containing 1000 ⁇ g/ml G418 (Gibco, #10131-027).
  • the positive rate of the transfection pool was detected by flow cytometry, and the cells in the pool with a higher positive rate were plated (according to the cell density of 1 ⁇ 10 6 cells/ml, 100 ⁇ l/well, plated in a 96-well plate), using Ipilimumab antibody (trade name Yervoy, purchased from Bristol-Myers Squibb Company) and Goat pAb to Hu IgG(PE) (Abcam, ab98596) antibody were incubated with cells, and the mean at 585 nm wavelength was detected by flow cytometry (ACEABIO, Novocyte 2060R). Values were generated using GraphPad for data analysis.
  • the positive cell line was subcloned, and the cloned CHO-K1 cell line was selected, which expressed CTLA-4 molecule at a high level and named it CHO-K1-CTLA-4.
  • the gene fragments encoding human CTLA-4 protein (SEQ ID NO: 14) and the full-length extracellular region encoding human PD1 protein (SEQ ID NO: 41) were synthesized respectively. Then it was cloned into the eukaryotic plasmid pTargetT to obtain its expression plasmid pTargetT-hPD-1-hCTLA-4.
  • the pTargeT-hPD1-hCTLA-4 was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation under the square pulse of 160V and 15msec, and placed at 37°C in a 5% CO2 concentration. Cultivated in an incubator.
  • pressurized culture was carried out in a medium containing 1000 ⁇ g/ml G418 (Gibco, #10131-027) and 500 ⁇ g/ml hygromycin B.
  • the positive rate of the transfection pool was detected by flow cytometry, and the cells in the pool with a higher positive rate were plated (according to the cell density of 1 ⁇ 10 6 cells/ml, 100 ⁇ l/well, plated in a 96-well plate), using Ipilimumab antibody and Nivolumab antibody (purchased from Bristol-Myers Squibb Company) and Goat pAb to Hu IgG(PE) (Abcam, ab98596) antibody were incubated with cells, and the mean value at 585 nm wavelength was detected by flow cytometry (ACEABIO, Novocyte 2060R).
  • the positive cell line was subcloned, and the cloned CHO-K1 cell line was selected.
  • the cell line expressed PD-1 and CTLA-4 molecules at a high level and named it CHO-K1-PD1-CTLA-4.
  • Inoculate 293E cells (derived from ATCC) at a density of 0.5 x 10 6 cells/ml in a 1L cell culture flask, add fresh pre-warmed FreeStyle 293 expression medium to make the total volume after seeding reach 250mL, set at 37°C, Incubate overnight in a humidified CO 2 incubator with 8% CO 2 .
  • the mixed solution of PEI and FreeStyle293 expression medium was added to the plasmid, mixed evenly, then added to the cell culture, and cultured in a humidified CO 2 incubator at 37° C., 8% CO 2 .
  • Cells were fed on days 1 and 3 post-transfection with 2.5 ml of glutamine (200 mM stock concentration) and 5 ml of glucose (180 g/L stock concentration) per flask. When the cell viability dropped to 65%-75%, the cell supernatant was collected. The cell culture was centrifuged at 1500 rpm for 5 min to collect the supernatant, and then centrifuged at 8000 rpm for 20 min to collect the supernatant.
  • hCTLA-4 antigen proteins and adjuvants were used to co-immunize experimental animals, including Balb/c strain mice and SD rats.
  • the immunized animals were immunized with 50 ⁇ g of antigen for the first immunization, and one animal was immunized with 25 ⁇ g of antigen in the later period.
  • the immune adjuvant can be Freund's adjuvant (Sigma) or Quick Antibody-Mouse5W (Beijing Boaolong Immunotechnology Co., Ltd.). Freund's adjuvant was used to emulsify the antigen, and the hCTLA-4 antigen protein samples with different labels were added dropwise to the adjuvant solution, and vortexed to mix thoroughly while the adjuvant was added.
  • mice were immunized after mixing to form a water-in-oil milk.
  • Quick Antibody-Mouse5W as an adjuvant
  • the hCTLA-4 antigen protein samples with different tags and Quick Antibody-Mouse5W were mixed at a volume ratio of 1:1, and after mixing, the SD rats were immunized by intramuscular injection.
  • the immunization schedule is shown in Table 1.
  • splenocytes The boosted mice/rats were sacrificed and immersed in 75% alcohol. The spleen was dissected out, ground with a grinding rod, and filtered through a cell mesh to prepare a single-cell suspension. The spleen cell suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. Add 2 mL of erythrocyte lysis solution, lyse the erythrocytes for 2 min at room temperature, add PBS to 20 mL, centrifuge at 1500 rpm for 7 min, discard the supernatant, resuspend and count the viable cells.
  • the Sp2/0 cells in the culture flask were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the viable cells were counted after resuspension.
  • Cells were resuspended in 20 mL of electroporation buffer and centrifuged at 1500 rpm for 7 min. Discard the supernatant and repeat once. Resuspend the cells with an appropriate amount of electroporation buffer to ensure the cell concentration is about 2 ⁇ 10 7 cells/mL.
  • the cell suspension was added to a 9mL electroporation fusion tank for fusion. After fusion, the cell suspension was transferred to 15 mL of RPMI 1640 complete medium containing 20% FBS, and placed at room temperature for 20 min. Confluent cells were resuspended in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, 20% FBS. Add 100 ⁇ l/well of the cell suspension to several 96-well cell culture plates to ensure that the amount of cells in each well is about 4 ⁇ 10 4 cells/well, and culture in a 37°C cell incubator. After 5 days, 100 ⁇ L/well of RPMI 1640 complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3) was supplemented.
  • the cell culture supernatant of the hybridoma parent clone was taken, and the hybridoma parent clone that bound human hCTLA-4 protein and cynomolgus monkey CTLA-4 protein was screened by ELISA, and the hybridoma parent clone that could bind to CHO-K1 was further screened by flow cytometry.
  • - Parent clone of CTLA-4 stably transfected cell line.
  • the positive parent clones were subcloned by limiting dilution method, and the binding activity of the subclone supernatants to hCTLA-4 molecules was detected by ELISA after one week of culture, and the monoclonal cell lines secreting anti-hCTLA-4 antibodies were screened. A number of better monoclonal cell lines were obtained.
  • One of the anti-hCTLA-4 monoclonal antibodies was labeled SHS010-C92.
  • the monoclonal antibody parent clone was determined and expanded.
  • the culture conditions are 1640 medium containing 10% fetal bovine serum, 1x NAEE, 1x sodium pyruvate, and 1% penicillin-streptomycin double antibody.
  • the cell confluence is greater than 80%, the cells are subcultured and expanded, and the cells are cultured to about The supernatant was collected at 50 ml and the antibody was purified. The obtained antibody was confirmed to be of good purity by SDS-PAGE gel electrophoresis.
  • the subcloned positive hybridoma cells were expanded and cultured, and an appropriate amount of cells were taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134), and reverse transcribed using Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A).
  • the kit synthesizes the first strand of cDNA.
  • Design specific primers according to the variable region of the mouse antibody subtype (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector), and use the cDNA as the template to carry out PCR amplification of the variable region gene of the antibody , to obtain the gene fragments of mouse antibody light chain and heavy chain variable regions respectively; design primers (reference: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201: 35–55; 2.
  • Antibody Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence SHS010-C92 SEQ ID NO: 26 SEQ ID NO: 27
  • the CDR sequence of the VH of the antibody SHS010-C92 the sequences of CDR1, CDR2, and CDR3 are respectively SEQ ID NOs: 1, 2, and 3
  • the CDR sequences of the VL are respectively SEQ ID NOs: 5, 6 , 7.
  • the purified mouse antibody light chain and heavy chain variable region gene fragments were co-transformed into E. coli DH5 ⁇ with the linearized eukaryotic expression plasmids containing human antibody light chain or heavy chain constant regions, respectively.
  • competent cells the mixture was evenly spread on the surface of the agar plate containing the corresponding antibiotics, and after overnight incubation in a constant temperature incubator at 37°C, several single colonies were picked for DNA sequencing; the correctly sequenced chimeric antibody was labeled as SHS010-C92 -english.
  • the chimeric antibody heavy and light chain plasmids were co-transfected into HEK293E cells, and the antibody was obtained by expression and purification, and then the purity detection, activity analysis and affinity detection were carried out.
  • the chimeric antibody SHS010-C92-CHI was genetically mutated to screen for better antibodies.
  • SHS010-C92-CHI heavy chain CDR position 24 G was mutated to E, the stability of the antibody was improved, and it was marked as SHS010-C92-G24E-CHI.
  • the sequencing results of the chimeric antibodies are shown in Table 3.
  • the CDR sequences of the VH of the antibody SHS010-C92-CHI are respectively SEQ ID NO: 1, 2, and 3, and the CDR sequences of the VL: the sequences of CDR1, CDR2, and CDR3 are respectively SEQ ID NO: 5 , 6, 7.
  • multiple active chimeric antibodies were selected for humanized antibody transformation.
  • the humanization of the antibody was firstly compared with the mouse antibody sequence in the immune gene database (IMGT) to confirm the murine germline of the variable region of the SHS010-C92-CHI antibody. After homology alignment, SHS010 - The FR sequence of the heavy chain variable region of the C92-CHI antibody is most similar to the mouse antibody germline gene IGHV3-48*01; the FR sequence of the antibody light chain variable region is the most similar to the mouse antibody IGKV3-11*01 resemblance.
  • IMGT immune gene database
  • SHS010-C92-CHI antibody framework region sequence FR1-FR3 as a template, a fully human framework with similar 3D structure but lower immunogenicity was found in the human framework region library to replace the FR1-FR3 sequence of SHS010-C92-CHI, heavy chain 3D modeling of the full-length sequence of the light chain/light chain and structural alignment analysis with the heavy chain/light chain sequence of the original antibody, comprehensive consideration of antigenicity and 3D structural similarity, and finally two humanized recombinants of SHS010-C92-CHI were selected.
  • the chain variable regions see SEQ ID NOs: 29, 30
  • the five humanized light chain variable regions see SEQ ID NOs: 31, 32, 33, 34, 35
  • the non-CDR region sequence of the humanized antibody of SHS010-C92-CHI is more than 95% humanized.
  • the above-designed humanized antibody light chain and heavy chain variable region amino acid sequences are reverse transcribed into corresponding nucleotide sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments are generated.
  • the oligonucleotide fragments are annealed and connected by PCR, and then the complete light chain and heavy chain are amplified by specific primers (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector).
  • Variable region nucleotide fragment; the purified light chain variable region nucleotide fragment and the linearized eukaryotic expression plasmid (pHR-hK) containing the constant region of human kappa light chain were co-transformed into E.
  • the purified heavy chain variable region nucleotide fragments and the eukaryotic expression plasmid (pHR-IgG4) of the human IgG4 heavy chain constant region were co-transformed into E. coli DH5 ⁇ competent cells, and the competent cells of the transformed plasmid were uniformly coated. On the surface of the agar plate containing the corresponding antibiotics, after overnight incubation in a constant temperature incubator at 37°C, several single colonies were picked for DNA sequencing.
  • the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid medium containing corresponding antibiotics, shaken at 37°C for more than 12 hours, and then collected the cells for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids , use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the plasmid was transfected into HEK293E cells, expressed and purified to obtain a large number of antibodies, and the purity detection, activity analysis and affinity detection were carried out.
  • the CDR sequences of VH of antibody HuC92-11 are respectively SEQ ID NOs: 1, 2, and 4, and the CDR sequences of VL: the sequences of CDR1, CDR2, and CDR3 are respectively SEQ ID NOs: 5, 6 , 7.
  • Human CTLA-4-His protein (1 ⁇ g/well, prepared in Examples 1 and 2) was coated on a 96-well microtiter plate and incubated at 4°C overnight. After washing three times with 1xPBST, the cells were blocked with 5% skim milk at 37°C for 2h.
  • the anti-CTLA-4 antibody of the present invention was used as the primary antibody from 10 ⁇ g/mL, 5-fold gradient dilution was added to the ELISA plate, a total of 8 concentrations, the concentrations were 10000ng/mL, 2000ng/mL, 400ng /mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, incubated at 37°C for 2h, the control antibody was Ipilimumab (purchased from Bristol-Myers Squibb Company); washed 5 times with 1xPBST Afterwards, the secondary antibody was incubated with Anti-Human IgG HRP (Jackson, 109-035-003, 1:5000) at 37°C for 1 h. After washing 5 times with 1 ⁇ PBST, the color developing solution TMB was added, and the OD450 value was read by a microplate reader (thermo, Multiskan FC) after termination.
  • a microplate reader thermo,
  • the experimental results show that the humanized anti-CTLA-4 antibody HuC92-11 of the present invention has an EC 50 of 0.01 nM, which is equivalent to that of the control antibody Ipilimumab (EC 50 : 0.01 nM), and has better binding to human CTLA-4. ability.
  • the binding activity of the antibodies was analyzed by ELISA. Cynomolgus monkey CTLA-4-His (1 ⁇ g/well, prepared in Examples 1 and 2) was coated on a 96-well ELISA plate.
  • the anti-CTLA-4 antibody of the present invention is used as the primary antibody from 10 ⁇ g/mL, 5-fold gradient dilution is added to the ELISA plate, a total of 8 concentrations, the concentrations are 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, incubated at 37°C for 2h, and the control antibody was Ipilimumab.
  • Anti-Human IgG HRP Jackson, 109-035-003, 1:10000 was used as the secondary antibody, and TMB (3,3',5,5'-tetramethylbenzidine) was added to the chromogenic solution. After termination, enzyme labeling was used. The OD450 value was read by a thermometer (thermo, Multiskan FC). EC50s were generated using GraphPad and the results are shown in Table 5 .
  • Example 7 Determination of blocking activity of anti-CTLA-4 antibodies against CTLA-4 and its ligands (ELISA)
  • the blocking activity of the antibodies against CTLA-4 and its ligands was analyzed by ELISA.
  • Human CTLA-4-His protein (1 ⁇ g/well, prepared in Examples 1 and 2) was coated on a 96-well microtiter plate and incubated at 4°C overnight. After washing three times with 1xPBST, the cells were blocked with 5% skim milk at 37°C for 2h. After washing 3 times with 1xPBST, prepare CD80-mFc and 5 ⁇ g/mL CD86-mFc ligand solutions with a concentration of 1 ⁇ g/mL.
  • the anti-CTLA-4 antibody of the present invention is used as the primary antibody from Starting from 25 ⁇ g/mL, 5-fold gradient dilution was added to the microtiter plate, a total of 8 concentrations, the concentrations were 25000ng/mL, 5000ng/mL, 1000ng/mL, 200ng/mL, 40ng/mL, 8ng/mL, 1.6ng/mL , 0.32ng/mL, incubated at 37°C for 2h, and the control antibody was Ipilimumab; after 5 washes with 1xPBST, the secondary antibody was Anti-mouse IgG HRP (Jackson, 109-035-003, 1:5000), incubated at 37°C for 1h.
  • the experimental results show that the humanized anti-CTLA-4 antibody HuC92-11 of the present invention can block the binding of CTLA-4 to its ligand, and the blocking activity is stronger than that of the control antibody Ipilimumab.
  • the binding activity of the antibody to CTLA-4 on the surface of CHO-K1-CTLA-4 cells was analyzed by FACS. After CHO-K1-CTLA-4 cells were digested, they were resuspended in 2% FBS-PBS and counted. The above cells were plated in a cell plate with 1 ⁇ 10 5 cells per well.
  • the anti-CTLA-4 antibody of the present invention was used as a primary antibody from 20 ⁇ g/ml, and was added to the cell plate by gradient dilution, with a total of 8 concentrations, the concentrations were 20000ng/mL.
  • control antibody was Ipilimumab
  • secondary antibody was PE-Anti- Human IgG (Biolegend, Cat. No. 409303, 1.25 ⁇ l/well)
  • fluorescence intensity generated by the binding of the antibody to the cell surface was detected by flow cytometry. The results are shown in Table 8.
  • the experimental results show that the humanized anti-CTLA-4 antibody HuC92-11 of the present invention has better binding ability to cell surface CTLA-4.
  • the ability of the antibodies to block the binding of CTLA-4 on the surface of CHO-K1-CTLA-4 cells was analyzed by FACS. After CHO-K1-CTLA-4 cells were digested, they were resuspended in 2% FBS-PBS and counted. The above cells were plated in a cell plate with 1 ⁇ 10 5 cells per well.
  • the anti-CTLA-4 antibody of the present invention was used as a primary antibody from 20 ⁇ g/ml, and was added to the cell plate by gradient dilution, with a total of 8 concentrations, the concentrations were 20000ng/mL.
  • the experimental results show that the humanized anti-CTLA-4 antibody HuC92-11 of the present invention can block the binding of CTLA-4 to its ligand, and the blocking activity is stronger than that of the control antibody Ipilimumab.
  • the amino acid sequence design is shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38 respectively. Show. After codon optimization of the above amino acid sequence, the tagged PD-1 protein extracellular region gene fragments PD-1-hFc, PD-1-mFc and PD-1-His were synthesized and cloned into eukaryotic expression. In the plasmid pHR, the expression plasmids pHR-PD-1-hFc, pHR-PD-1-mFc and pHR-PD-1-His were obtained.
  • the amino acid sequence of the extracellular region of the human PD-L1 protein and the amino acid sequence of the hIgG1-Fc or mIgG1-Fc tag were designed, and the amino acid sequence designs are shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.
  • the tagged PD-L1 protein extracellular region gene fragments PD-L1-hFc, PD-L1-mFc and PD-L1-His were synthesized and cloned into eukaryotic expression.
  • the expression plasmids pHR-PD-L1-hFc and pHR-PD-L1-mFc were obtained.
  • the full-length gene fragment encoding the PD-1 protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 41, and then cloned into the eukaryotic expression plasmid pTargeT to obtain the expression plasmid pTargeT-PD-1.
  • the eukaryotic expression plasmid pTargeT-PD-1 was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation at a voltage of 160V and a square pulse of 15msec. Cultivated in an incubator. After 24 h, the cells were cultured under pressure in DME/F12 complete medium containing 1000 ⁇ g/ml G418. After 16 days, the positive rate of the pool was detected by FACS, and the cell viability was calculated. The cells were incubated with PE anti-human PD-1 antibody (Biolegend, Inc., 621607), the mean value and parent% value were read by flow cytometer (ACEA, Novocyte), and data analysis was performed using GraphPad generation.
  • PE anti-human PD-1 antibody Biolegend, Inc., 621607
  • the mean value and parent% value were read by flow cytometer (ACEA, Novocyte), and data analysis was performed using GraphPad generation.
  • the positive pool cells with cell viability above 90% were subcloned and plated (cell density of 0.5 cells/well, 200 ⁇ l/well), and cultured in a 37° C., 5% CO 2 incubator. After the subclone was grown for 12 days, the cloned CHO-K1 cell line was selected, and the subclone with high PD-1 expression was screened by FACS and named hPD-1-CHO-K1.
  • a gene fragment encoding the full-length PD-L1 protein was synthesized, and the amino acid sequence design was shown in SEQ ID NO: 42, and then cloned into the eukaryotic expression plasmid pTargeT to obtain its expression plasmid pTargeT-PD-L1.
  • the eukaryotic expression plasmid pTargeT-PD-L1 was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation under the square pulse of 160V and 15msec, and placed at 37°C, 5% CO2 cultured in an incubator. After 24 h, the cells were cultured under pressure in DME/F12 complete medium containing 1000 ⁇ g/ml G418. After 16 days, the positive rate of the pool was detected by FACS, and the cell viability was calculated.
  • the cells were incubated with PE anti-human PD-L1 antibody (Sino Biological Inc., 10084-R312-P), the mean and parent% values were read by flow cytometry (ACEA, Novocyte), and data analysis was performed using GraphPad generation .
  • the positive pool cells with cell viability above 90% were subcloned and plated (cell density of 0.5 cells/well, 200 ⁇ l/well), and cultured in a 37° C., 5% CO 2 incubator. After the subclone was grown for 12 days, the cloned CHO-K1 cell line was selected, and the subclone with high PD-L1 expression was screened by FACS and named hPD-L1-CHO-K1.
  • FreeStyle 293 expression medium Take 8.5ml of FreeStyle 293 expression medium, add 500 ⁇ l of 1mg/ml PEI solution, mix well, take 250 ⁇ g of plasmid to be transfected and add it to 8.5ml of FreeStyle 293 expression medium, mix well, among which the tag antigen protein particle pHR-PD-1 -hFc, pHR-PD-1-mFc, pHR-PD-1-His were transfected respectively; tag ligand protein particles pHR-PD-L1-hFc, pHR-PD-L1-mFc were respectively transfected.
  • the mixed solution of PEI and FreeStyle 293 expression medium was added to the plasmid, mixed well, then added to the cell culture, and cultured in a humidified CO 2 incubator at 37° C., 8% CO 2 .
  • Cells were fed on days 1 and 3 post-transfection with 2.5 ml of glutamine (200 mM stock concentration) and 5 ml of glucose (180 g/L stock concentration) per flask. When the cell viability dropped to 65%-75%, the cell supernatant was collected. The cell culture was centrifuged at 1500 rpm for 5 min to collect the supernatant, and then centrifuged at 8000 rpm for 20 min to collect the supernatant.
  • Anti-PD-1 antigen protein with different tags (PD-1-His, purchased from Sino Biological Inc., 10377-H08H), PD-1-hFc, PD-1-mFc) and adjuvant were used to immunize C57
  • the experimental mice of SJL strain were immunized with 50 ⁇ g antigen for the first immunization, and then 25 ⁇ g antigen was used for immunization; the experimental rats of SD strain were immunized by co-immunization with anti-PD-1 antigen proteins of different labels and adjuvant, and 100 ⁇ g antigen was used for the first antigen. , 50 ⁇ g antigen was used for immunization in the later stage.
  • the immune adjuvant can be Quick Antibody-Mouse5W (Beijing Boaolong Immune Technology Co., Ltd.) or Titer Max (Sigma) and CpG (GenScript Biotechnology Co., Ltd.)/Alum (thermo) adjuvant spacer.
  • the PD-1 antigen protein samples with different labels were added dropwise to the adjuvant solution, vortexed while adding dropwise to mix thoroughly, and the dosage of the adjuvant was carried out with reference to the instructions. Mice and rats were immunized after mixing to form a water-in-oil emulsion.
  • Cell lines expressing high levels of PD-1 molecules such as hPD-1-CHO-K1 were also used to immunize rats to produce antibodies. Treat the hPD-1-CHO-K1 positive single cells obtained in Example 1 under culture with trypsinization, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cell pellet with PBS, take samples and count with a cell counter, and 1000 rpm for the remaining samples Centrifuge for 5 min, discard the supernatant, resuspend the cell pellet with PBS, and add an appropriate amount of PBS to obtain a cell suspension of 1 ⁇ 10 8 cells/ml. Each mouse in the experimental group was immunized with 1 ⁇ 10 7 cells.
  • the immunization program is shown in Table 9 and Table 10:
  • spleen cells The boosted mice/rat were sacrificed and immersed in 75% alcohol. The spleen was dissected out, ground with a grinding rod, and filtered through a cell mesh to prepare a single-cell suspension. The spleen cell suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. Add 2 mL of erythrocyte lysis solution, lyse the erythrocytes for 2 min at room temperature, add PBS to 20 mL, centrifuge at 1500 rpm for 7 min, discard the supernatant, resuspend and count the viable cells.
  • the Sp2/0 cells in the culture flask were collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the viable cells were counted after resuspension.
  • Cells were resuspended in 20 mL of electroporation buffer and centrifuged at 1500 rpm for 7 min. Discard the supernatant and repeat once. Resuspend the cells with an appropriate amount of electroporation buffer to ensure the cell concentration is about 2 ⁇ 10 7 cells/mL.
  • the cell suspension was added to a 9mL electroporation fusion tank for fusion. After fusion, the cell suspension was transferred to 15 mL of H-SFM complete medium containing 20% FBS, and placed at room temperature for 20 min. Confluent cells were resuspended in H-SFM medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC-3, 2% FBS. Add 100 ⁇ l/well of the cell suspension to several 96-well cell culture plates to ensure that the amount of cells in each well is about 4 ⁇ 10 4 cells/well, and culture in a 37°C cell incubator. After 5 days, 100 ⁇ L/well of H-SFM complete medium (containing 2% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3) was supplemented.
  • the specific method is as follows: coat PD-L1-mFc on an ELISA plate, add recombinant human protein PD-1-hFc and a mixture of hybridoma supernatant for 2 h, add HRP-labeled anti-human IgG Fc specific antibody ( Jackson Immuno Research) was incubated for 1 h, and the absorbance at 450 nm was detected by a microplate reader.
  • the hybridoma parent clones with binding ability and blocking ability obtained by screening were expanded and cultured, and the retest of ELISA binding activity was carried out; the hybridomas that could bind to PD-1 on the surface of hPD-1-CHO-K1 cells were screened by FACS. The supernatant of the hybridoma was screened by ELISA, and the supernatant of the hybridoma that could bind to the cyno-PD-1-His protein was screened by ELISA;
  • the positive cell lines were subcloned by the limiting dilution method, and after one week of culture, the binding activity of the subclone supernatant to PD-1 molecules and the activity of blocking the interaction between PD-1 and PD-L1 were detected by ELISA, and the double-positive cell lines were screened. .
  • One of the anti-PD-1 monoclonal antibodies was labeled SHS006-P01.
  • the monoclonal antibody parent clone was expanded.
  • the culture conditions are 1640 medium containing 10% fetal bovine serum, 1 ⁇ NAEE, 1 ⁇ sodium pyruvate, and 1% penicillin-streptomycin double antibody.
  • the cell confluence is greater than 80%, the cells are subcultured and expanded.
  • the culture reaches about 50ml, collect the supernatant and purify the antibody.
  • the obtained antibody was confirmed to be of good purity by SDS-PAGE gel electrophoresis.
  • the subcloned positive hybridoma cells were expanded and cultured, and an appropriate amount of cells were taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134), and reverse transcribed using Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A).
  • the kit synthesizes the first strand of cDNA.
  • Design specific primers according to the variable region of the rat antibody subtype (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector), and use cDNA as the template to carry out PCR amplification of the variable region gene of the antibody , so as to obtain the gene fragments of the variable region of the light chain and heavy chain of the rat antibody respectively; design primers (reference: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester etal. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201: 35–55; 2.
  • the CDR sequences of VH of antibody SHS006-P01 the sequences of CDR1, CDR2, and CDR3 are respectively SEQ ID NOs: 8, 9, and 10
  • the CDR sequences of VL the sequences of CDR1, CDR2, and CDR3 are respectively SEQ ID NOs: 11, 12 , 13.
  • the purified mouse anti-light chain and heavy chain variable region gene fragments were co-transformed with the linearized eukaryotic expression plasmids containing the human antibody light chain or heavy chain constant region respectively.
  • the mixed solution was evenly coated on the surface of the agar plate containing the corresponding antibiotics, and after overnight incubation in a constant temperature incubator at 37 °C, several single colonies were picked for DNA sequencing; the correctly sequenced chimeric antibody was labeled as SHS006-P01CHI.
  • the heavy chain variable region and light chain variable region of the chimeric antibody SHS006-P01CHI are the same as those of the murine antibody SHS006-P01.
  • the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid medium containing the corresponding antibiotics, shaken and cultured at 37°C for more than 12 hours, and then the cells were collected for plasmid extraction to obtain chimeric antibody light chain and heavy chain expression plasmids. Use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the chimeric antibody was transfected into HEK293E cells, expressed and purified to obtain a large number of antibodies, and the purity detection, activity analysis and affinity detection were carried out.
  • the humanization of the antibody was firstly compared with the rat antibody sequence in the immune gene database (IMGT) to confirm the murine germline of the variable region of the SHS006-P01CHI antibody. After homology alignment, SHS006-P01CHI The FR region of the variable region of the heavy chain of the antibody is most similar to the rat antibody germline gene IGHV2-26*01; the FR sequence of the variable region of the antibody light chain is the most similar to that of the rat antibody IGKV2-28*01.
  • SHS006-P01CHI antibody framework region sequence FR1-FR3 as a template, search for a fully human framework with similar 3D structure but lower immunogenicity in the human framework region library to replace the FR1-FR3 sequence of SHS006-P01CHI, heavy chain / light chain full 3D modeling of long sequences and structural alignment analysis with the original antibody heavy chain/light chain sequence, comprehensive consideration of antigenicity and 3D structural similarity, and the amino acids that play a key role in the stability of the antibody structure will be displayed in the structural simulation. Site backmutation to murine amino acid residues.
  • SHS006-P01CHI humanized heavy chain variable regions (see SEQ ID NO: 45, 46, 47, 48, 49, 50) and 3 humanized light chain variable regions (see SEQ ID NO: 45, 46, 49, 50) of SHS006-P01CHI were finally selected :51, 52, 53) for further optimization.
  • the non-CDR region sequence of SHS006-P01CHI humanized antibody is more than 95% humanized.
  • the above-designed humanized antibody light chain and heavy chain variable region amino acid sequences are reverse transcribed into corresponding nucleotide sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments are generated.
  • the oligonucleotide fragments are annealed and connected by PCR, and then the complete light chain and heavy chain are amplified by specific primers (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector).
  • Variable region nucleotide fragment; the purified light chain variable region nucleotide fragment and the linearized eukaryotic expression plasmid containing the IgG4 light chain constant region were co-transformed into E.
  • variable region nucleotide fragments and the eukaryotic expression plasmid containing the S228P/L235E mutated IgG4 heavy chain constant region were co-transformed into E. coli DH5 ⁇ competent cells, and the competent cells of the transformed plasmid were uniformly spread on agar containing the corresponding antibiotics. The surface of the plate was cultured overnight in a constant temperature incubator at 37°C, and several single colonies were picked for DNA sequencing.
  • the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid medium containing corresponding antibiotics, shaken at 37°C for more than 12 hours, and then collected the cells for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids , use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the plasmid was transfected into HEK293E cells, expressed and purified to obtain a large number of antibodies, and the purity detection, activity analysis and affinity detection were carried out.
  • HuP01-22 A humanized antibody with better purity, activity and affinity was selected, one of which was labeled SHS006-HuP01-22 (herein referred to as HuP01-22), and the sequence is shown in Table 12.
  • the CDR sequences of VH of antibody SHS006-HuP01-22 are respectively SEQ ID NOs: 8, 9, and 10
  • the CDR sequences of VL are respectively SEQ ID NO: 11 , 12, 13.
  • Antibody binding activity was analyzed by Protein based ELISA. Cynomolgus monkey PD-1-His (0.1 ⁇ g/well, SB, Cat. No. 90311-C08H) was coated on a 96-well ELISA plate and incubated at 4°C overnight.
  • the anti-PD-1 antibody of the present invention is used as the primary antibody, starting from 10 ⁇ g/mL, 5-fold gradient dilution is added to the ELISA plate, a total of 8 concentrations, the concentrations are 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.13ng/mL, and incubated at 37°C for 1.5h.
  • Anti-Human IgG HRP Jackson, 109-035-003, 1:10000 was used as the secondary antibody, and TMB (3,3',5,5'-tetramethylbenzidine) was added to the chromogenic solution. After termination, enzyme labeling was used. The OD450 value was read by a thermometer (thermo, Multiskan FC). EC50s were generated using GraphPad .
  • the humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention has an EC 50 of 0.088 nM, and has a good binding ability to cynomolgus monkey PD-1.
  • Example 15 Anti-PD-1 antibody and human PD-1 binding activity assay (ELISA)
  • the binding activity of the antibodies was analyzed by ELISA.
  • Human PD-1-His protein (2 ⁇ g/ml, self-produced) was coated on a 96-well microtiter plate and incubated at 4°C overnight. After washing three times with 1 ⁇ PBST, the cells were blocked with 5% skim milk overnight at 4°C. After washing three times with 1 ⁇ PBST, the anti-PD-1 antibody of the present invention was used as the primary antibody, starting from 1 ⁇ g/mL, 5-fold gradient dilution was added to the ELISA plate, a total of 8 concentrations, the concentrations were 1000ng/mL and 200ng/mL.
  • control antibody was Nivolumab
  • secondary antibody was used Anti-Human IgG HRP (Jackson, 109-035-003, 1:10000), incubated at 37°C for 40 min.
  • the color developing solution TMB was added, and the OD450 value was read by a microplate reader (thermo, Multiskan FC) after termination.
  • EC50s were generated using GraphPad .
  • the humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention has an EC 50 of 0.18 nM for binding to human PD-1, and has a good binding ability to human PD-1.
  • Example 16 Anti-PD-1 antibody and cell surface PD-1 binding activity assay (FACS)
  • Antibody binding activity was analyzed by FACS. hPD-1-CHO-K1 cells were plated into a 96-well round-bottom cell culture plate at the rate of 1 ⁇ 10 5 cells per well.
  • the anti-PD-1 antibody of the present invention was used as the primary antibody starting from 3.3 ⁇ g/mL, and was added in gradient dilution.
  • the concentrations are 3333ng/mL, 1111ng/mL, 370ng/mL, 123ng/mL, 41ng/mL, 13.7ng/mL, 4.57ng/mL, 1.52ng/mL
  • the control antibody is Nivolumab , incubate at 37°C for 1 h; wash 3 times with Cell stanining buffer; use PE anti-human IgG Fc (Biolegend, 409304, 0.8 ⁇ l/well) as the secondary antibody, incubate at 4°C for 30 min in the dark; wash with Cell stanining buffer 3 times and then use flow A cytometer (ACEABIO, Novocyte) was used to measure the mean value at a wavelength of 585 nm. EC50s were generated using GraphPad .
  • the humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention has an EC 50 of 0.11 nM for binding to PD-1 on the cell surface, and has better binding ability to PD-1 on the cell surface.
  • Example 17 Affinity determination of anti-PD-1 antibody and human PD-1 protein
  • the binding affinity of the humanized anti-PD-1 antibody prepared in the above example to the antigen PD-1-His was determined using Fortebio Octet.
  • the humanized anti-PD-1 antibody was diluted with SD buffer (PBS + 0.02% Tween20 + 0.1% BSA) to a concentration of 10 ⁇ g/ml, and the antigen PD-1-His was diluted with SD buffer 5-fold concentration gradient to make The concentrations were 5 ⁇ g/ml, 1 ⁇ g/ml, 0.2 ⁇ g/ml, and 0 ⁇ g/ml.
  • the AHC sensor was used to immobilize the antibody, and the affinity was determined according to the operating procedures of Fortebio Octet RED96. The specific parameters and experimental results are shown in Table 13.
  • the experimental results show that the humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention has better binding affinity to human PD-1 protein.
  • FACS was used to detect the ability of the anti-PD-1 antibody of the present invention to block the binding of PD-1 to PD-L1 on the cell surface.
  • the hPD-L1-CHO-K1 positive cell line was used as a PD-L1 provider to observe the binding ability of PD-1-hFc to PD-L1-CHO-K1 in the presence of serially diluted anti-PD-1 antibody.
  • the secondary antibody used PE Goat anti-human IgG (Biolegend, 405307, 0.8 ⁇ l/well) to monitor changes in PD-1-hFc.
  • the mean value at a wavelength of 585 nm was read by a flow cytometer (ACEABIO, Novocyte), and IC 50 was generated using GraphPad.
  • the experimental results show that the humanized anti-PD-1 antibody SHS006-HuP01-22 of the present invention has an EC 50 of 0.77 nM for blocking the binding of human PD-1 to PD-L1, and has good blocking ability.
  • Resuscitate PBMC according to the required cell amount, add it to 8-9ml of IMDM complete medium, centrifuge at 1200rpm for 10min, discard the supernatant; resuspend with an appropriate amount of medium, count with a hemocytometer, add it to a 6-well plate, and add it at the same time.
  • SEB solution with a final concentration of 100ng/ml, incubated for 48h; after 48h, centrifuge at 1200rpm for 10min, discard the supernatant, wash 1-2 times with IMDM complete medium, resuspend with an appropriate amount of medium, count with a hemocytometer, and resuspend To 1M/mL, 100 ⁇ L/well was added to 96-well plate; according to 4 times concentration (ie 40 ⁇ g/mL), 50 ⁇ L/well, prepare Nivolumab and Isotype with IMDM complete medium, make a label, vortex; add antibody solution to In the corresponding wells, 50 ⁇ L/well of culture medium was added to the control group, the 96-well plate was placed in a 37°C incubator, and the cells and antibodies were incubated for 1 h; The SEB solution was prepared in IMDM complete medium and added to the corresponding wells; the 96-well plate was placed in a 37°C, 5% CO2 incubator for 72 hours, and the
  • the present invention also adopts the luciferase reporter gene method (NFAT) to detect the release of the in vitro cytokine IL-2 of the chimeric antibody, and the results are shown in Table 14.
  • NFAT luciferase reporter gene method
  • Example 20 Construction of anti-CTLA-4/anti-PD-1 bispecific antibody expression vector and protein expression purification
  • the anti-PD-1 antibody used in the present invention is a mouse monoclonal antibody SHS006-P01 (heavy chain variable region: SEQ ID NO: 43, light chain variable region : SEQ ID NO: 44), after humanization, the humanized monoclonal antibody SHS006-HuP01-22 (heavy chain variable region: SEQ ID NO: 46, light chain variable region: SEQ ID NO: SEQ ID NO: 52).
  • the anti-CTLA-4 antibody used in the present invention is a mouse monoclonal antibody obtained by immunizing mice with human CTLA-4-his protein, and then obtaining a chimeric antibody SHS010-C92-CHI-G24E (the heavy chain variable region sequence is SEQ ID NO: 28, the light chain variable region sequence is SEQ ID NO: 27), after its humanization, the humanized monoclonal antibody HuC92-11 is obtained by screening (the heavy chain variable region sequence is SEQ ID NO: 30). , the light chain variable region sequence is SEQ ID NO: 32).
  • the present invention utilizes the variable region sequences of the above-mentioned anti-PD-1 and anti-CTLA-4 humanized antibodies to construct an anti-CTLA-4/anti-PD-1 bispecific antibody.
  • This bispecific antibody is a bispecific antibody comprising a symmetrical structure of scFv, as shown in FIG. 5 .
  • the scFv of the anti-CTLA-4 antibody is linked to the C-terminus of the complete heavy chain of the anti-PD-1 antibody through a flexible linking peptide, and the linking peptide comprises the GGGGS repeat sequence.
  • Anti-PD-1 antibody VH humanized anti-PD-1 monoclonal antibody SHS006-HuP01-22 heavy chain variable region;
  • Connecting peptide 1 a flexible connecting peptide that is a repeat of 3 GGGGS;
  • Anti-CTLA-4 antibody VH humanized anti-CTLA-4 monoclonal antibody HuC92-11 heavy chain variable region
  • Connecting peptide 2 a flexible connecting peptide that is a repeating sequence of 4 GGGGS;
  • Anti-CTLA-4 antibody VL humanized anti-CTLA-4 monoclonal antibody HuC92-11 light chain variable region
  • Anti-PD-1 antibody VL-CL humanized anti-PD-1 monoclonal antibody SHS006-HuP01-22 light chain;
  • the bispecific antibody ScFv-[P01-22-C92-11] was constructed using the variable region sequences of anti-PD-1 antibody SHS006-HuP01-22 and anti-CTLA-4 antibody SHS010-HuC92-11 , the amino acid sequence of peptide chain 1 is shown in SEQ ID NO:55, and the amino acid sequence of peptide chain 2 is shown in SEQ ID NO:54.
  • the bispecific antibody ScFv-[P01-22-C92-12] was also constructed, the amino acid sequence of peptide chain 1 is shown in SEQ ID NO:56, and the amino acid sequence of peptide chain 2 is shown in SEQ ID NO:54 Show.
  • the sequences of H2CDR2 and H2CDR3 are SEQ ID NOs: 1, 2, 4, 5, 6, 7, 8, 9, and 10, respectively, and the CDR sequences included in peptide chain 2: L2CDR1, L2CDR2, and L2CDR3 sequences are respectively SEQ ID NO: 11, 12, 13.
  • the plasmid was transfected into HEK293E cells, expressed and purified to obtain a large number of antibodies, and the purity detection, activity analysis and affinity detection were carried out.
  • the bispecific antibody with better purity, activity and affinity was selected and labeled as BsAB0192-1 (ie antibody ScFv-[P01-22-C92-11]).
  • the bispecific antibody provided by the present invention was used as the primary antibody from 10 ⁇ g/mL, 5-fold gradient dilution was added to the microtiter plate, a total of 8 concentrations, the concentrations were 10000ng/mL, 2000ng/mL, 400ng /mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, incubated at 37°C for 2h, the control antibodies were Ipilimumab and Nivolumab, respectively; after 5 washes with 1xPBST, the secondary antibody was Anti -Human IgG HRP (Jackson, 109-035-003, 1:5000), incubated at 37°C for 1 h.
  • the experimental results show that the bispecific antibody BsAB0192-1 of the present invention has better binding ability to human CTLA-4 and human PD-1.
  • Example 22 Determination of antibody binding to cell surface hCTLA-4 and hPD-1 (FACS)
  • FACS was used to analyze the binding ability of the antibodies to CTLA-4 on the surface of CHO-K1-CTLA-4 and PD-1 on the surface of CHO-K1-PD-1.
  • CHO-K1-CTLA-4 cells and CHO-K1-PD-1 cells were digested, they were resuspended in 2% FBS-PBS and counted.
  • the above cells are plated in a cell plate with 1 ⁇ 10 5 cells per well.
  • the bispecific antibody provided by the present invention is used as the primary antibody from 20 ⁇ g/ml, and is added to the cell plate by gradient dilution, with a total of 8 concentrations, and the concentration is 20000ng/mL.
  • control antibodies were Ipilimumab and Nivolumab; the secondary antibody was PE -Anti-Human IgG (Biolegend, Cat. No. 409303, 1.25 ⁇ l/well), after washing, use flow cytometer to detect the fluorescence intensity generated by the binding of the antibody to the cell surface, and use GraphPad to generate EC 50 , the results are shown in Figures 8 and 9 shown.
  • the experimental results show that the bispecific antibody BsAB0192-1 of the present invention has better binding ability to cell surface CTLA-4 and PD-1.
  • the ability of the antibodies to block the binding of CTLA-4 on the surface of CHO-K1-CTLA-4 was analyzed by FACS. After CHO-K1-CTLA-4 cells were digested, they were resuspended in 2% FBS-PBS and counted. The above cells are plated in a cell plate with 1 ⁇ 10 5 cells per well.
  • the bispecific antibody provided by the present invention is used as the primary antibody from 20 ⁇ g/ml, and is added to the cell plate by gradient dilution, with a total of 8 concentrations, and the concentration is 20000ng/mL.
  • the experimental results show that the bispecific antibody BsAB0192-1 of the present invention can well block the binding of CTLA-4 on the cell surface to its ligands CD80 and CD86.
  • Example 24 Antibody blocking activity assay for PD-1 and its ligands (FACS)
  • CHO-K1-PD-1 The ability of the antibodies to block PD-1 binding ligands on the surface of CHO-K1-PD-1 was analyzed by FACS. After CHO-K1-PD-1 cells were digested, they were resuspended in 2% FBS-PBS and counted. The above-mentioned cells are plated with 1 ⁇ 10 5 cells per well.
  • the bispecific antibody provided by the present invention is used as the primary antibody, starting from 10 ⁇ g/ml, and is added to the cell plate by gradient dilution, with a total of 8 concentrations, each with a concentration of 10000ng/mL , 3333.33ng/mL, 1111.11ng/mL, 370.37ng/mL, 123.45ng/mL, 41.15ng/mL, 13.71ng/mL, 4.57ng/mL, the diluted antibody was mixed with 10 ⁇ g/mL PD-L1-mFc After mixing at 1:1, incubated at 4°C for 1 h, the control antibody was Nivolumab; the secondary antibody was PE-Anti-mouse IgG (Biolegend, Cat. No. 409303, 1.25 ⁇ l/well), which was detected by flow cytometry after washing. The fluorescence intensity generated by ligand binding to the cell surface is shown in Figure 12.
  • the experimental results show that the bispecific antibody BsAB0192-1 of the present invention can better block the binding of PD-1 on the cell surface and its ligand PD-L1.
  • Example 25 Antibody blocking activity on cells co-expressing CTLA-4 and PD-1 (FACS)
  • bispecific antibodies to block the binding of CTLA-4 to its ligand was determined using CHO-K1 cells co-expressing CTLA-4 and PD-1. After CHO-K1-PD-1-CTLA-4 cells were digested, they were resuspended in 2% FBS-PBS and counted. The above cells are plated in a cell plate with 1 ⁇ 10 5 cells per well.
  • the bispecific antibody provided by the present invention is used as the primary antibody from 20 ⁇ g/ml, and is added to the cell plate by gradient dilution, with a total of 8 concentrations, and the concentration is 20000ng/mL.
  • bispecific antibodies to block the binding of PD-1 to its ligand was determined using CHO-K1 cells co-expressing CTLA-4 and PD-1. After CHO-K1-PD-1-CTLA-4 cells were digested, the above cells were plated with 1 ⁇ 10 5 cells per well.
  • the bispecific antibody of the present invention was used as the primary antibody at 10 ⁇ g/ml, and was added in gradient dilution.
  • the concentrations are 10000ng/mL, 3333.33ng/mL, 1111.11ng/mL, 370.37ng/mL, 123.45ng/mL, 41.15ng/mL, 13.71ng/mL, 4.57ng/mL,
  • the diluted antibody was mixed 1:1 with 10 ⁇ g/mL PD-L1-mFc and incubated at 4°C for 1 h.
  • the control antibody was Nivolumab; the secondary antibody was PE-Anti-mouse IgG (Biolegend, Cat. No. 409303, 1.25 ⁇ l/well), after washing, the fluorescence intensity generated by the binding of the ligand to the cell surface was detected by flow cytometry. The results are shown in Figure 15.
  • the experimental results show that the bispecific antibody BsAB0192-1 of the present invention can better block the binding ability of CTLA-4 and PD-1 and its ligands on the bistable transfected cell line, and the blocking activity is better than the corresponding
  • the monoclonal antibodies HuC92-11 and HuP01-22 were superior to the control antibodies Ipilimumab and Nivolumab.
  • Example 26 Bispecific antibody affinity determination with human CTLA-4 and human PD-1
  • the binding affinity of the obtained bispecific antibody to the antigens CTLA-4-His and PD-1-His was determined by Fortebio Octet.
  • the three different concentrations of 1.5 ⁇ g/ml, 0.3 ⁇ g/ml, and 0.06 ⁇ g/ml were used as control antibodies.
  • Ipilimumab and Nivolumab were selected as control antibodies, and the affinity was determined according to the operating procedures of fortebio Octet RED96. The specific parameters and experimental results are shown in Table 15.
  • the experimental results show that, compared with the control antibody, the bispecific antibody of the present invention has a higher affinity for binding to human CTLA-4 and human PD-1 proteins.
  • Example 27 Anti-tumor experiment of MC38 xenograft model
  • MC38 cells were purchased from the American Type Culture Collection (ATCC);
  • B6-hPD1/hCTLA-4 mice female, 6-8 weeks old, weighing 18-20 g, were purchased from Nanjing Jicui Yaokang Biotechnology;
  • the reference substance Ipilimumab was purchased from Beijing Yiqiao Shenzhou Biotechnology (Item No. 68052-H001) and used as a positive control; the reference substance Nivolumab was purchased from Bristol-Myers Squibb Company and used as a positive control; before the test, the PD-1 antibody of the present invention was used as a positive control.
  • SHS006-P01-22 was formulated at 1 mg/mL in PBS.
  • the human tumor cell MC38 cells were inoculated on the right rear of the mice, and the number of inoculated cells was 5 ⁇ 10 6 /mice.
  • the tumor grows to an average volume of 140mm 3 , the grouping starts, the tail vein is administered twice a week, the diameter of the tumor is measured with a vernier caliper twice a week, and the tumor volume is calculated.
  • T/C tumor proliferation rate
  • the experimental results show that the anti-PD-1 antibody of the present invention can better inhibit tumor growth.
  • Example 28 Anti-tumor test of human lymphoma Raji-PDL1 xenograft model
  • Raji human lymphoma cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.
  • the Raji-PDL1 cell line was constructed by conventional methods.
  • NOD-Scid mice female, 5-8 weeks old, weighing 18-20 grams, were purchased from Biositu (Jiangsu) Gene Biotechnology Co., Ltd.;
  • Nivolumab antibody was purchased from Bristol-Myers Squibb Company, and ipilimumab was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • the anti-CTLA-4/PD-1 double antibody BsAB0192-1 of the present invention was prepared with PBS at 2.66 mg/mL, and the Nivolumab and ipilimumab of the present invention were prepared with PBS at 2 mg/mL, and stored at 4°C.
  • Human lymphoma cell Raji-PDL1 was cultured in RMPI1640 medium containing 10% fetal bovine serum, 100 U/mL of penicillin and 100 ⁇ g/mL of streptomycin in a 37°C, 5% CO2 incubator. When the cell saturation was 80%-90%, cells were harvested, counted, and seeded. 50 ⁇ l of PBS containing 5 ⁇ 10 6 cells was mixed with 50 ⁇ L of Matrigel (final volume 100 ⁇ L) and inoculated on the right rear of the mice, and the number of inoculated cells was 5 ⁇ 10 6 per mouse.
  • T/C Relative tumor proliferation rate
  • RTV V21/V0, where V0 is the tumor volume measured at the time of group administration (ie d0), and V25 is the tumor volume measured at 25 days of administration.
  • the tumor volume on the last day (Day 25) of the administration group and the vehicle group was analyzed by T-test and performed by GraphPad Prism. The results are shown in Table 17.
  • Nivolumab 20mg/kg group had the same molar concentration.
  • the experimental results show that, at the same molar concentration, the double antibody BsAB0192-1 of the present invention has a better effect of inhibiting tumor growth, and the effect is better than that of Nivolumab, Ipilimumab and the combined administration of Nivolumab and Ipilimumab.

Abstract

本发明涉及抗体药物技术领域，尤其涉及抗CTLA-4抗体或其抗原结合片段，抗CTLA-4/抗PD-1抗体或其抗原结合片段，包含抗CTLA-4抗体或其抗原结合片段或者抗CTLA-4/抗PD-1抗体或其抗原结合片段的药物组合物以及它们的应用。本发明的抗CTLA-4抗体及抗CTLA-4/抗PD-1抗体具有显著的抗肿瘤活性，可在制备抗肿瘤的药物中应用。

Description

抗CTLA-4抗体及其应用 技术领域
本发明涉及抗体药物技术领域，尤其涉及一种抗CTLA-4抗体或其抗原结合片段，包含抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段的双抗，以及它们的应用。
背景技术
T淋巴细胞在对抗原的适应性免疫应答中起关键作用。初始T细胞需要两种信号进行完全活化。第一种信号是抗原特异性的，通过T细胞受体(TCR)与抗原呈递细胞(APC)上的MHC/肽复合物相互作用来提供。第二种信号是共刺激信号，通过T细胞上的受体与在APC上的其配体之间的相互作用来提供。
T细胞上的CD28和APC上的B7-1(CD80)和B7-2(CD86)之间的共刺激通路是参与T细胞调控的最关键通路。T细胞活化之后，T细胞上的负调控受体因子CTLA-4(细胞毒性T淋巴细胞相关蛋白4，也称为CD152)被上调。CTLA-4在结构上与CD28同源，但更紧密地与B7-1和B7-2配体结合。CTLA-4以两种主要方式抑制免疫应答，它与CD28竞争B7-1和B7-2配体从而阻断共刺激，且它还以负性方式传导信号以抑制T细胞活化。
已报告，CTLA-4的阻断增大了体外和体内T细胞应答，加强了抗肿瘤免疫，并且增强了诱导的自身免疫疾病。在许多疾病病症中，例如治疗或预防病毒和细菌感染以及用于治疗癌症中，针对CTLA-4的抗体被描述作免疫刺激调节剂。Ipilimumab是人抗人CTLA-4抗体，其阻断CTLA-4与APC上表达的B7-1和B7-2的结合，从而阻断由这些分子相互作用引发的免疫应答的负性下调。
尽管Ipilimumab已经上市用于一些癌症治疗，但是仍需要替代性抗CTLA-4抗体。
此外，PD-1也是关键的免疫检查点受体，其由活化的T和B细胞表达，介导免疫抑制。PD-1通过结合其配体程序性死亡配体1(PD-L1)和程序性死亡配 体2(PD-L2)来调节T细胞的活化。PD-1配体在抗原呈递细胞上以及多种人类癌症中表达，已经表明当PD-1配体与PD-1结合时下调T细胞活化和细胞因子分泌。
CTLA-4和PD-1两个检查点可能是互相补充的具有不同作用通路的免疫检查点。因此，研究开发能够同时作用于这两个检查点的双抗药物亦具有显著的临床意义和广阔的市场空间。
发明内容
本发明一方面提供一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(H1CDR1)、重链可变区的互补决定区2(H1CDR2)和/或重链可变区的互补决定区3(H1CDR3)，所述轻链可变区包含轻链可变区的互补决定区1(L1CDR1)、轻链可变区的互补决定区2(L1CDR2)和/或轻链可变区的互补决定区3(L1CDR3)。
在一些实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
(a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(a2)如SEQ ID NO:1、2和4所示的氨基酸序列；和
(a3)与SEQ ID NO:1、2和3或SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
(a4)如SEQ ID NO:5、6和7所示的氨基酸序列；和
(a5)与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一个具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和3，和所述轻链可变区的L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7。
在一个具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和4，和所述轻链可变区的L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7。
在一些具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列 或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:29、SEQ ID NO:30所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可变区 的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗CTLA-4抗体为鼠源抗体，其还含有鼠源的IgG1、IgG2a、、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和鼠源的κ、λ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗CTLA-4鼠源抗体还含有鼠源的IgG1、IgG2a、、IgG2b、IgG2c或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，所述的抗CTLA-4嵌合抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、、IgG2b、IgG2c、IgG3或其突变序列的重链恒定区，优选包含人源IgG1或IgG2重链恒定区，或者使用氨基酸突变后显著降低ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的IgG4恒定区。
在一些实施方案中，本发明提供一种抗CTLA-4人源化抗体或其抗原结合片段，其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，所述轻链包含人源的κ、λ链或其变体的轻链恒定区。
在一些优选的实施方案中，本发明的抗CTLA-4人源化抗体或其抗原结合片段还包含人源IgG1或IgG2或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗CTLA-4抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv或F(ab) 2。
本发明另一方面提供一种抗PD-1抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(H2CDR1)、重链可变区的互补决定区2(H2CDR2)和/或重链可变区的互补决定区3(H2CDR3)，所述轻链可变区包含轻链可变区的互补决定区1(L2CDR1)、轻链可变区的互补决定区2(L2CDR2)和/或轻链可变区的互补决定区3(L2CDR3)。
在一些实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其包 含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
(a1)如SEQ ID NO:8、9和10所示的氨基酸序列；和
(a2)与(a1)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
(a3)如SEQ ID NO:11、12和13所示的氨基酸序列；和
(a4)与(a3)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一个具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其具有所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:8、9和10或与SEQ ID NO:8、9和10所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:11、12和13或与SEQ ID NO:11、12和13所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
在一些具体的实施方案中，根据本发明的抗PD-1抗体或其抗原结合片段是单克隆抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的抗PD-1抗体或其抗原结合片段是鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段或人源化抗体或其抗原结合片段。
在一些具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:44、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:43，SEQ ID NO:43经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:43功能相同的氨基酸序列或与SEQ ID NO:43具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:44，SEQ ID NO:44经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:44功能相同的氨基酸序列或与SEQ ID NO:44具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或 与SEQ ID NO:46具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:51，SEQ ID NO:51经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:51功能相同的氨基酸序列或与SEQ ID NO:51具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:52，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:53，SEQ ID NO:53经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:53功能相同的氨基酸序列或与SEQ ID NO:53具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗PD-1抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:8、9和10所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:52，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:11、12和13所示的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗PD-1抗体为鼠源抗体，其还含有鼠源的IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和鼠源 的κ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗PD-1鼠源抗体还含有鼠源的IgG1、IgG2a、IgG2b、IgG2c或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，所述的抗PD-1嵌合抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。所述的抗PD-1嵌合抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其突变序列的重G链恒定区，优选包含人源IgG1、IgG2a、IgG2b、IgG2c重链恒定区，或者使用氨基酸突变后显著降低ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的IgG4恒定区。
在一些具体的实施方案中，本发明的抗PD-1人源化抗体或其抗原结合片段还包含人源IgG1、IgG2a、IgG2b、IgG2c、IgG3、IgG4或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗PD-1人源化抗体或其抗原结合片段还包含人源IgG1、IgG2a、IgG2b、IgG2c、IgG3、IgG4或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗PD-1抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv或F(ab)2。
本发明的另一方面提供一种分离的核酸，其编码根据本发明的抗PD-1抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区氨基酸序列如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49或SEQ ID NO:50的核苷酸序列；和编码轻链可变区氨基酸序列如SEQ ID NO:44、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53的核苷酸序列。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区SEQ ID NO:43的核苷酸序列；和编码轻链可变区SEQ ID NO:44的核苷酸序列。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码重链可变区SEQ ID NO:46的核苷酸序列；和编码轻链可变区SEQ ID NO:52的核苷酸序 列。
本发明的另一方面提供一种表达载体，其表达本发明的抗PD-1抗体或其抗原结合片段。根据本发明的表达载体其包含本发明的分离的核酸分子。
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。
本发明的另一方面提供制备本发明的抗PD-1抗体或其抗原结合片段的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。
本发明的另一方面提供一种药物组合物，其包含本发明的抗PD-1人源化抗体或其抗原结合片段和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗PD-1人源化抗体或其抗原结合片段，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。
可以将本发明的抗PD-1人源化抗体或其抗原结合片段与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
本发明的另一方面提供抑制PD-1活性的方法，所述方法包括向有此需要的个体施用本发明的抗PD-1抗体或其抗原结合片段或本发明的药物组合物。
本发明的另一方面提供用于检测或测定人PD-1的方法，所述方法包括使用本发明的抗PD-1抗体或其抗原结合片段的步骤。
本发明的另一方面提供用于检测或测定人PD-1的试剂，所述试剂包本发明的抗PD-1抗体或其抗原结合片段。
本发明的另一方面提供一种治疗与PD-1相关的疾病的方法，所述方法包括向受试者施用药物有效量的本发明的PD-1抗体或其抗原结合片段，或包含上述药物组合物，或上述分离的核酸分子。本发明的另一方面提供本发明的抗PD-1抗体或其抗原结合片段或本发明的药物组合物在制备与PD-1相关的疾病的药物中的应用。在一些实施方案中，所述与PD-1相关的疾病的药物用于治疗T细胞功能障碍性病症，例如肿瘤、免疫性疾病或感染性病症。在一些实施方案中，所述肿瘤为非小细胞肺癌、小细胞肺癌、肾细胞癌、结直肠癌、卵巢癌、乳腺癌、胰腺癌、胃癌、膀胱癌、食道癌、间皮瘤、黑素瘤、头和颈癌、甲状腺癌、肉瘤、***癌、成胶质细胞瘤、***、胸腺癌、白血病、淋巴瘤、骨髓瘤、蕈样肉芽肿、梅克尔细胞癌、肾上腺皮质癌、肝脏肝细胞癌、胰管腺癌、嗜铬细胞瘤、神经节细胞瘤、子宫内膜癌和卵巢浆液性囊腺癌等。在一些实施方案中，所述免疫性疾病为关节炎、炎性肠病、银屑病。在一些实施方案中，所述感染性疾病为慢性病毒感染。
本发明的另一方面提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(H1CDR1)、重链可变区的互补决定区2(H1CDR2)和/或重链可变区的互补决定区3(H1CDR3)，所述轻链可变区包含轻链可变区的互补决定区1(L1CDR1)、轻链可变区的互补决定区2(L1CDR2)和/或轻链可变区的互补决定区3(L1CDR3)；和所述抗PD-1抗体或其抗原结合片段为特异性结合PD-1的抗体或其抗原结合片段。
在一些实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
(a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(a2)如SEQ ID NO:1、2和4所示的氨基酸序列；和
(a3)与SEQ ID NO:1、2和3或SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
(a4)如SEQ ID NO:5、6和7所示的氨基酸序列；和
(a5)与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4抗体或其抗原结合片段具有：
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；或
所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和4或与SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
在一些实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、 且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
所述重链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性的氨基酸序列且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:5、6和7所示的氨基酸序列；或
所述重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和4所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:5、6和7所示的氨基酸序列。
在一些实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4抗体或其抗原结合片段为人源化抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:29或SEQ ID NO:30所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4抗体或其抗原结合片段为人源化抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
所述重链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列；或
所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列。
在一些优选的实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段如以上实施方案中所限定；和所述抗PD-1抗体或其抗原结合片段包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(H2CDR1)、重链可变区的互补决定区2(H2CDR2)和/或重链可变区的互补决定区3(H2CDR3)，所述轻链可变区包含轻链可变区的互补决定区1(L2CDR1)、轻链可变区的互补决定区2(L2CDR2)和/或轻链可变区的互补决定区3(L2CDR3)区。
进一步优选地，在一些实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段如以上实施方案中所限定，和所述抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
(A1)如SEQ ID NO:8、9和10所示的氨基酸序列；
(A2)与(A1)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
(A3)如SEQ ID NO:11、12和13所示的氨基酸序列；
(A4)与(A3)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中：
所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
(a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(a2)如SEQ ID NO:1、2和4所示的氨基酸序列；和
(a3)与SEQ ID NO:1、2和3或SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
(a4)如SEQ ID NO:5、6和7所示的氨基酸序列；和
(a5)与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
所述抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
(A1)如SEQ ID NO:8、9和10所示的氨基酸序列；
(A2)与(A1)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
(2)所述轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
(A3)如SEQ ID NO:11、12和13所示的氨基酸序列；
(A4)与(A3)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
在一个具体的实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；和所述抗PD-1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:8、9和10或与SEQ ID NO:8、9和10所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:11、12和13或与SEQ ID NO:11、12和13所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和4或与SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；和所述抗PD-1抗体或其抗原结合片段包含所述H2CDR1、H2CDR2和H2CDR3分别为SEQ ID NO:8、9和10或与SEQ ID NO:8、9和10所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L2CDR1、L2CDR2和L2CDR3分别为SEQ ID NO:11、12和13或与SEQ ID NO:11、12和13所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
在一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4抗体或其抗原结合片段以及抗PD-1抗体或其抗原结合片段各自独立地为鼠源抗体、嵌合抗体、人源化抗体或完全人抗体。
在一些具体的实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其中所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
所述抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区的氨基酸序列选自：
(B1)如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列；
(B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B3)与(B1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(B4)如SEQ ID NO:44、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列；
(B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(B6)与(B4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述抗CTLA-4抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中所述抗CTLA-4抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和4所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:5、6和7所示的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，其中所述抗CTLA-4抗体或抗PD-1抗体可以为鼠源抗体，其还含有鼠源的IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和鼠源的κ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，其中所述抗CTLA-4鼠源抗体还含有鼠源的IgG1或IgG2或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在一些具体的实施方案中，本发明提供抗CTLA-4/抗PD-1抗体，其中所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:30所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:32所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
所述抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(1)所述重链可变区的氨基酸序列选自：
(C1)如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列；
(C2)(C1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(C1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(C3)与(C1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(C4)如SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列；
(C5)(C4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(C4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(C6)与(C4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中抗CTLA-4抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30 功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和4所示的氨基酸序列，且所述抗CTLA-4抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:5、6和7所示的氨基酸序列；和所述抗PD-1抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:46所示的氨基酸序列，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:8、9和10所示的氨基酸序列，且所述所述抗PD-1抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:52，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:11、12和13所示的氨基酸序列。
在一些具体的实施方案中，本发明提供抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中抗CTLA-4抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和4所示的氨基酸序列，且所述抗CTLA-4抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:5、6和7所示的氨基酸序列；和所述抗PD-1抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:46所示的氨基酸序列，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:8、9 和10所示的氨基酸序列，且所述所述抗PD-1抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:52，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:11、12和13所示的氨基酸序列。
在一些实施方案中，本发明提供一种抗CTLA-4/抗PD-1人源化抗体，其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，所述轻链包含人源的κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，所述的鼠源抗CTLA-4/抗PD-1抗体，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，和/或进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区。
在本发明一个优选的实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。所述的抗CTLA-4抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其突变序列的重链恒定区，优选包含人源IgG1、IgG2、IgG4重链恒定区。
在一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗CTLA-4人源化抗体或其抗原结合片段还包含人源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗CTLA-4人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG4或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗PD-1抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、IgG2b、IgG2c、IgG3或其突变序列的重链恒定区，优选包含人源IgG或其突变序列的重链恒定区；所述抗PD-1抗体或其抗原结合片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。
在一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1抗体，所述抗PD-1人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施 方案中，本发明的抗CTLA-4人源化抗体或其抗原结合片段还包含人源IgG4或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗CTLA-4/抗PD-1抗体，其中所述的抗CTLA-4抗体或其抗原结合片段以及抗PD-1抗体或其抗原结合片段分别为Fab、Fv、sFv或F(ab) 2。在一个具体的实施方案中，本发明提供的抗CTLA-4/抗PD-1抗体为scF(ab) 2。
优选地，本发明以上实施方案中的抗CTLA-4/抗PD-1抗体是抗CTLA-4/抗PD-1双特异性抗体。在一些实施方案中，所述双特异性抗体是人抗体或人源化抗体。在一些实施方案中，所述结合特异性之一是针对CTLA-4，而另一个结合特异性是针对任何其它抗原。在一些实施方案中，所述结合特异性之一是针对CTLA-4，而另一个结合特异性是针对PD-1。在一些实施方案中，所述双特异性抗体可结合CTLA-4的两种不同表位。所述双特异性抗体还可用于将细胞毒剂定位于表达CTLA-4的细胞。这些抗体拥有CTLA-4结合臂和细胞毒剂结合臂，所述细胞毒剂例如肥皂草毒蛋白、抗干扰素-α、长春花生物碱类、蓖麻毒蛋白A链、甲氨蝶呤或放射性同位素半抗原。可将本发明的双特异性抗体制备成全长抗体或抗体片段(例如F(ab') 2双特异性抗体)。
制备双特异性抗体的方法是本领域已知的。传统上，双特异性抗体的重组制备基于两个免疫球蛋白重链-轻链对的共表达，其中两个重链具有不同的特异性(Millstein and Cuello,Nature 305:537(1983))。由于免疫球蛋白重链和轻链的随机分配，这些杂交瘤(quadroma))可能产生10种不同抗体分子的混合物，其中只有一种分子具有正确的双特异性结构。该正确分子的纯化通常通过亲和层析步骤进行，相当麻烦且产物产量低。类似的方法在WO93/08829及Trauneckeretal.,EMBOJ.10:3655(1991)中有公开。
根据一种不同的方法，具有期望结合特异性(抗体-抗原结合位点)的抗体可变区与免疫球蛋白恒定区序列融合。在一些实施方案中，与包含至少部分铰链、CH2和CH3区的免疫球蛋白重链恒定区进行融合。在一些实施方案中，包含与轻链结合所必需位点的重链恒定区(CH1)存在于该融合的至少一部分中。将编码免疫球蛋白重链融合片段以及免疫球蛋白轻链(如果需要)的DNA***不同的表达载体中并共转染入合适的宿主生物体。在用于构建的三种多肽链比例不等时 提供最佳产量的实施方案中，这为调整三种多肽片段的相互比例提供极大的灵活性。不过，在至少两种多肽链以相同比例表达产生高产量时或比例没有特别意义时，有可能将两种或所有三种多肽链的编码序列***一个表达载体中。
在该方法的一个实施方案中，所述双特异性抗体由一个臂中具有第一结合特异性的杂合免疫球蛋白重链和另一个臂中的杂合免疫球蛋白重链-轻链对(提供第二结合特异性)组成。由于免疫球蛋白轻链仅在该双特异性分子的一半中存在提供了便利的分离途径，因此发现该不对称性结构便于将期望的双特异性物质与不想要的免疫球蛋白链组合物分开。该方法在WO 94/04690中公开。关于产生双特异性抗体的进一步信息参见例如Sureshetal.,Methods in Enzymology 121:210(1986)。
根据另一种方法，可改造一对抗体分子间的界面以使从重组细胞培养物回收的异二聚体的百分比最大化。该界面包含抗体恒定区CH3结构域的至少一部分。在该方法中，将第一抗体分子界面的一个或多个小氨基酸侧链用较大侧链(例如酪氨酸或色氨酸)替换。通过将大氨基酸侧链用较小氨基酸侧链(例如丙氨酸或苏氨酸)替换，在第二抗体分子的界面上产生与大侧链相同或相似大小的补偿性“空腔”。这提供了提高异二聚体相比于其它不想要的终产物诸如同二聚体的产量的机制。
双特异性抗体包括交联或“异源缀合”抗体。例如，一种异源缀合抗体可以与亲合素偶联，另一种异源缀合抗体可以与生物素偶联。可使用任何便利的交联方法来制备异源缀合抗体。合适的交联剂是本领域众所周知的，连同许多交联技术一起在美国专利No.4,676,980中公开。
本发明的双特异性抗体可以由抗体片段生成。例如，可使用化学连接技术来制备双特异性抗体。Brennanetal.,Science 229:81(1985)描述了通过蛋白水解切割完整抗体以生成F(ab') 2片段的方法。将这些片段在存在二硫醇络合剂***的情况下(用以稳定邻近的二硫醇和防止分子间二硫键的形成)分解。然后将产生的Fab'片段转变为硫代硝基苯甲酸酯(TNB)衍生物。然后将Fab'-TNB衍生物之一通过巯基乙胺的还原重新恢复成Fab'-硫醇，并与等摩尔量的另一种Fab'-TNB衍生物混合，以形成双特异性抗体。
可以从大肠杆菌直接回收Fab'-SH片段，这些片段可化学偶联以形成双特异 性抗体。Shalaby et al.,J.Exp.Med.175:217-225(1992)描述了完全人源化的双特异性抗体F(ab') 2分子的生成。每个Fab'片段由大肠杆菌单独分泌，并在体外进行定向化学偶联以形成双特异性抗体。
在一些实施方案中，本发明的双特异性抗体片段可以直接从重组细胞培养物生成和分离。例如，可以使用亮氨酸拉链生成双特异性抗体(Kostelnyetal.,J.Immunol.148(5):1547-1553(1992))。将来自Fos和Jun蛋白的亮氨酸拉链肽通过基因融合与两种不同抗体的Fab'部分连接。抗体同二聚体在铰链区分解以形成单体，然后重新氧化以形成抗体异二聚体。该方法也可用于生成抗体同二聚体。双抗体技术提供了制备双特异性抗体片段的其他机制。所述双特异性抗体片段包含通过接头相连的重链可变区(VH)和轻链可变区(VL)，所述接头太短以使得同一条链上的两个结构域之间不能配对。因此，迫使一个片段上的VH和VL结构域与另一个片段上的互补VL和VH结构域配对，由此形成两个抗原结合位点。在另一实施方案中，可以通过使用单链Fv(sFv)二聚体构建双特异性抗体片段。
本发明涵盖具有超过两价的多价抗体，例如，可制备三特异性抗体。多价抗体可以比二价抗体更快的受到表达该抗体结合的抗原的细胞的内在化(和/或异化)。本发明的抗体可以是可容易地通过编码抗体多肽链的核酸的重组表达而生成的、具有三个或更多抗原结合位点(例如四价抗体)的多价抗体。多价抗体可包含二聚化结构域和三个或更多抗原结合位点。在一些实施方案中，二聚化结构域包含(或由其组成)Fc区或铰链区。在这种情况中，抗体会包含Fc区及Fc区氨基末端的三个或更多抗原结合位点。在一些实施方案中，多价抗体包含(或由其组成)三个至大约八个抗原结合位点。在一些实施方案中，多价抗体包含四个抗原结合位点。多价抗体包含至少一条多肽链(例如两条多肽链)，其中所述多肽链包含两个或更多可变区。本发明的多价抗体可进一步包含至少两条(例如四条)轻链可变区多肽。本发明的多价抗体可包含例如约两条至约八条轻链可变区多肽。本发明的轻链可变区多肽包含轻链可变区，且任选进一步包含CL结构域。
在包含CTLA-4靶向部分和PD-1靶向部分的双特异性抗体中，CTLA-4靶向部分和PD-1靶向部分之一可以是全长抗体，并且另一个可以是包含重链CDR、轻链CDR或其组合的抗原结合片段(例如scFv)。靶向CTLA-4和PD-1蛋白之一的全长抗体和靶向另一蛋白的抗原结合片段可以直接或通过连接肽以化学方式 连接(例如共价连接)。抗原结合片段(例如scFv)可以直接或通过连接肽与全长抗体的N-末端(例如全长抗体的轻链或重链的N-末端)、全长抗体的C-末端(例如全长抗体的重链(或Fc或CH3结构域)的C-末端)或两者连接。
在一些实施方案中，本发明的双特异性抗体包含全长抗CTLA-4抗体、抗PD-1抗体的抗原结合片段(例如scFab、scFv)以及它们之间的连接肽。在另一些实施方案中，本发明的双特异性抗体包含全长抗PD-1抗体、抗CTLA-4抗体的抗原结合片段(例如scFab、scFv)以及它们之间的连接肽。
在一些实施方案中，本发明的双特异性抗体中包含的scFv可以按任何顺序包含重链可变区和轻链可变区。例如，双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包含重链可变区和轻链可变区以及任选地在它们之间的连接肽，或者可替代地，本发明的双特异性抗体中包含的scFv可以在从N-末端到C-末端的方向上包含轻链可变区和重链可变区以及任选地在它们之间的连接肽。
在一些实施方案中，所述连接肽可包括例如Gly、Asn和/或Ser残基，并且还可以包括中性氨基酸，例如Thr和/或Ala。适用于连接肽的氨基酸序列可以是相关领域中已知的那些。同时，可以在使得融合蛋白功能不受影响的这样的限度内不同地确定连接肽的长度。例如，连接肽可以通过包括总共约1至约100、约2至约50、或约5至约25个选自由Gly、Asn、Ser、Thr、和Ala组成的组的一种或多种来形成。在一个实施方案中，连接肽可以表示为(GmSl)n(m、l和n独立地是约1至约10的整数，特别是约2至约5的整数)。
在另一个实施方案中，PD-1靶向部分和CTLA-4靶向部分可以均是全长抗体或包含重链CDR、轻链CDR或其组合的抗原结合片段。
在另一个实施方案中，双特异性抗体可以是异二聚体形式，其包含第一臂和第二臂，该第一臂包括靶向CTLA-4和PD-1之一的一对重链和轻链，该第二臂包括靶向另一者的一对重链和轻链。
在一个实施方案中，全长抗体可以是全长免疫球蛋白形式(例如IgG、IgM、IgA、IgE或IgD，例如人IgG、人IgM、人IgA、人IgE或人IgD)，并且抗原结合片段可以选自由Fab、Fab’、F(ab’) 2、Fd、Fv、scFv、scFab、单链抗体、sdFv等组成的组。例如，全长抗体可以是全长人IgG(人IgG1、人IgG2、人IgG3或 人IgG4)形式，并且抗原结合片段可以是scFv。
例如，本文描述的抗体可以包含柔性接头序列，或者可以被修饰以添加功能部分(例如PEG、药物、毒素或标记)。在一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1双特异性抗体，所述抗CTLA-4抗体或其抗原结合片段的结构为(VH)-连接肽-(VL)，所述抗PD-1抗体或其抗原结合片段的结构为(VL-CL)-连接肽-(VH)。在一些具体的实施方案中，所述连接肽为(GGGGS)n形式，其中n为1-12，优选为3-10，更优选为3-8，例如3、4、5、6、7、8个GGGGS重复序列。在一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1双特异性抗体，其由两条肽链组成，其中：
肽链1的结构见图1所示；
肽链2的结构见图2所示；
其中：
抗PD-1抗体VH为抗PD-1抗体的重链可变区；
连接肽1、连接肽2各自为(GGGGS)n形式的重复序列的柔性连接肽，其中n为1-10；
抗CTLA-4抗体VH为抗CTLA-4抗体的重链可变区；
抗CTLA-4抗体VL为抗CTLA-4抗体的轻链可变区；
抗PD-1抗体VL-CL为抗PD-1抗体的轻链。
在一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:55，肽链2的氨基酸序列为SEQ ID NO:54。在另一些具体的实施方案中，根据本发明的抗CTLA-4/抗PD-1双特异性抗体，其由两条肽链组成，其中肽链1的氨基酸序列为SEQ ID NO:56，肽链2的氨基酸序列为SEQ ID NO:54。本发明的另一方面提供分离的核酸。在一些实施方案中，根据本发明的分离的核酸编码本发明的抗CTLA-4/抗PD-1抗体。在一些实施方案中，根据本发明的分离的核酸编码本发明的抗CTLA-4抗体或其抗原结合片段。在另一些实施方案中，根据本发明的分离的核酸编码本发明的抗PD-1抗体或其抗原结合片段。
在一个具体的实施方案中，根据本发明的分离的核酸，其包含编码抗CTLA-4抗体或其抗原结合片段的重链可变区如SEQ ID NO:26、SEQ ID NO:28、SEQ ID  NO:29、SEQ ID NO:30的核苷酸序列和编码抗CTLA-4抗体或其抗原结合片段的轻链可变区如SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35的核苷酸序列。在另一个具体的实施方案中，根据本发明的分离的核酸，其包含编码抗PD-1抗体或其抗原结合片段的重链可变区如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50的核苷酸序列和编码抗PD-1抗体或其抗原结合片段的轻链可变区如SEQ ID NO:44、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53的核苷酸序列。
本发明的另一方面提供表达载体。在一些实施方案中，本发明的表达载体表达本发明的抗CTLA-4/抗PD-1双特异性抗体。在一些实施方案中，本发明的表达载体表达本发明的抗CTLA-4抗体或其抗原结合片段。在另一些实施方案中，本发明的表达载体表达本发明的抗PD-1抗体或其抗原结合片段。在一些实施方案中，根据本发明的表达载体，表达本发明的抗CTLA-4抗体或其抗原结合片段的载体和表达本发明的抗PD-1抗体或其抗原结合片段的载体是同种表达载体。根据本发明的表达载体其包含本发明的分离的核酸分子。
本发明的另一方面提供一种嵌合抗原受体(CAR)融合蛋白，其包含本发明的抗CTLA-4抗体或其抗原结合片段和/或抗PD-1抗体或其抗原结合片段。在一些实施方案中，所述嵌合抗原受体融合蛋白包含本发明的抗CTLA-4抗体或其抗原结合片段，其为针对CTLA-4抗原的VH和VL的单链可变片段(scFv)。在另一些实施方案中，所述嵌合抗原受体融合蛋白包含本发明的抗PD-1抗体或其抗原结合片段，其为针对PD-1抗原的VH和VL的单链可变片段(scFv)。在另一些实施方案中，所述嵌合抗原受体融合蛋白包含针对CTLA-4抗原的VH和VL的第一单链可变片段(scFv)和针对PD-1抗原的VH和VL的第二单链可变片段(scFv)。所述针对CTLA-4抗原的VH和VL的第一scFv具有以上实施方案中描述的重链可变区的H1CDR1、H1CDR2和H1CDR3和轻链可变区的L1CDR1、L1CDR2和L1CDR3。所述针对PD-1抗原的VH和VL的第二scFv具有以上实施方案中描述的重链可变区的H2CDR1、H2CDR2和H2CDR3和轻链可变区的L2CDR1、L2CDR2和L2CDR3。
在一些具体的实施方案中，本发明提供一种抗CTLA-4/抗PD-1双特异性抗 体或其抗原结合片段是scFv结构的双特异性抗体或其抗原结合片段，其包含C端为CTLA-4结合部分和N端为PD-1结合部分，其中
(1)所述C端CTLA-4结合部分氨基酸序列选自：
(b1)如SEQ ID NO:55、SEQ ID NO:56所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述N端PD-1结合部分的氨基酸序列选自：
(b4)如SEQ ID NO:54所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4和PD-1的双特异性抗体或其抗原结合片段，其中所述N端PD-1结合部分氨基酸序列为SEQ ID NO:54，SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:54功能相同的氨基酸序列或与SEQ ID NO:54具有至少85％序列同一性的氨基酸序列，且所述C端CTLA-4结合部分氨基酸序列为SEQ ID NO:55，SEQ ID NO:55经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:55功能相同的氨基酸序列或与SEQ ID NO:55具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗CTLA-4和PD-1的双特异性抗体或其抗原结合片段，其中所述N端PD-1结合部分氨基酸序列为SEQ ID NO:54，SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:54功能相同的氨基酸序列或与SEQ ID NO:54具有至少85％序列同一性的氨基酸序列，且所述C端CTLA-4结合部分氨基酸序列为SEQ ID NO:56，SEQ ID NO:56经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:56功能相同的氨基酸序列或与SEQ ID NO:56具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明的抗CTLA-4/抗PD-1双特异性抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区， 和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗CTLA-4/抗PD-1双特异性抗体或其抗原结合片段还包含人源IgG1或IgG2或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗CTLA-4/抗PD-1双特异性抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv或F(ab)2。
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。
本发明的另一方面提供制备本发明的抗CTLA-4/抗PD-1双特异性抗体或其抗原结合片段的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。
本发明的另一方面提供一种药物组合物，其包含本发明的抗CTLA-4/抗PD-1双特异性人源化抗体或其抗原结合片段和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗CTLA-4/抗PD-1双特异性人源化抗体或其抗原结合片段，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。
可以将本发明的抗CTLA-4/抗PD-1双特异性人源化抗体或其抗原结合片段与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
本发明的另一方面提供抑制CTLA-4和/或PD-1活性的方法，所述方法包括向有此需要的个体施用本发明的抗CTLA-4/抗PD-1双特异性抗体或其抗原结合 片段或本发明的药物组合物。
本发明的另一方面提供本发明的抗CTLA-4/抗PD-1双特异性抗体或其抗原结合片段或本发明的药物组合物在制备用于抑制CTLA-4和/或PD-1活性的药物中的应用。在一些实施方案中，所述抑制CTLA-4和/或PD-1活性的药物用于治疗白血病、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、***、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素瘤。在一些实施方案中，本发明提供上述抗B7-H3抗体或其抗原结合片段或本发明的药物组合物在制备抗肿瘤的药物中的应用，优选地，所述肿瘤选自白血病、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、***、肾癌、膀胱癌、胰腺癌、神经胶质瘤和黑素瘤。
本发明提供的抗CTLA-4/抗PD-1双特异性抗体或其抗原结合片段具有更高的亲和力和稳定性，抗肿瘤作用显著，毒性低，可在制备用于治疗各类肿瘤疾病的药物中应用，具有广阔的市场前景。
定义
除非另有定义，本文中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。本文中所述的细胞和组织培养、分子生物学以及蛋白质和寡或多核苷酸化学及杂交中使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和组织培养与转化(如电穿孔、脂质转染)，使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等，Molecular Cloning:A Laboratory Manual)(第2版，Cold Spring Harbor Laboratory Press，纽约冷泉港(1989))。本文所述的分析化学、合成有机化学以及医学和药学化学中使用的命名以及实验室方法和技术是本领域公知且普遍使用的。
在本发明中，术语“至少80％序列同一性”是指至少80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在本发明中，术语“至少85％序列同一性”是指至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在一些优选的实施方案中，本发 明所述的序列同一性可以至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Instutute网站上的BLASTN/BLASTP算法来进行。
在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中以相对彼此的位置排列以形成抗原结合表面。抗原结合表面与所结合抗原的三维表面互补，且每条重链和轻链的三个高变区均被称作“互补决定区”或“CDR”。氨基酸向每个结构域的分配是根据Kabat《免疫学感兴趣的蛋白质的序列》(国立卫生研究院，马里兰州贝塞斯达(1987和1991))或Chothia和Lesk，J.Mol.Biol.196:901-917(1987)，Chothia等，Nature 342:878-883(1989)定义。
本发明的“抗体”是指特异性地识别并结合抗原的多肽或多肽复合物。抗体可以是完整抗体和其任何抗原结合片段或单链。本发明的“抗体”包括含有Ig分子的具有结合抗原的生物活性的至少一部分的任何蛋白质或肽。本发明“抗体”的实例包括但不限于重链或轻链的CDR或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分。
本发明所述的“抗原结合片段”包括具有抗原结合活性的Fab片段、Fab’片段、F(ab’)2片段及与人CTLA-4或PD-1结合的Fv片段、scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体或单链Fv(scFv)。本发明的抗CTLA-4或抗PD-1抗体可以是单链可变区片段(scFv)，其源自抗体的单链多肽，保留了结合抗原的能力。scFv的实例包括通过重组DNA技术形成的抗体多肽，其中免疫球蛋白重链(H链)和轻链(L链)片段的Fv区经由间隔序列连接。制备scFv的各种方法是本领域技术人员所熟知的。
本发明所述的抗体指免疫球蛋白分子或其免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10 -6)结合其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、 Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。
本发明所述的“鼠源抗体”为根据本领域知识和技能制备的对人CTLA-4的单克隆抗体。制备时用CTLA-4抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
本发明所述的“嵌合抗体”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。
本发明所述的“人源化抗体”也称为CDR移植抗体，是将小鼠的CDR序列移植到人的抗体可变区框架(FR)中产生的抗体。此类可变区框架序列可以从公共的DNA数据库或公开的参考文献获得，例如从ImMunoGeneTics(IMGT)网站http://imgt.cines.fr得到或从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明所述的“双特异性抗体”指对至少两种不同抗原具有结合特异性的单克隆抗体。
本发明所述的“肽接头”可以是包括1至10，特别是2至50个任何氨基酸的那些，并且可以包括任何种类的氨基酸而没有任何限制。
附图说明
图1是抗CTLA-4/抗PD-1双特异性抗体的肽链1的结构示意图。
图2是抗CTLA-4/抗PD-1双特异性抗体的肽链2的结构示意图。
图3是抗CTLA-4人源化抗体阻断CTLA-4结合CD80活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图4是抗CTLA-4人源化抗体阻断CTLA-4结合CD86活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图5是本发明的ScFv结构的双特异性抗CTLA-4/PD-1抗体结构示意图。
图6是双特异性抗体与CTLA-4结合实验结果(ELISA)，其中横坐标是抗体浓度(nM)，纵坐标是OD450处的吸光值。
图7是双特异性抗体与PD-1结合实验结果(ELISA)，其中横坐标是抗体浓度(nM)，纵坐标是OD450处的吸光值。
图8是双特异性抗体与细胞表面CTLA-4结合实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图9是双特异性抗体与细胞表面PD-1结合实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图10是双特异性抗体阻断CTLA-4结合CD80活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图11是双特异性抗体阻断CTLA-4结合CD86活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图12是双特异性抗体阻断PD-1结合PD-L1活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图13是双特异性抗体在双稳定转染细胞株上CD80阻断活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图14是双特异性抗体在双稳定转染细胞株上CD86阻断活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
图15是双特异性抗体在双稳定转染细胞株上PD-1阻断活性实验结果(FACS)，其中横坐标是抗体浓度(nM)，纵坐标是荧光强度。
具体实施方式
下面代表性的实施例是为了更好地说明本发明，而非用于限制本发明的保护范围。以下实施例中未注明条件的实验方法通常按照常规条件，如冷泉港的抗体技术实验手册、分子克隆手册等，或按照原料或商品制造厂商所建议的条件进行。实施例中使用的材料、试剂如无特殊说明均为商购获得。
实施例1：CTLA-4抗原蛋白的制备
1、抗原蛋白的表达载体构建
合成编码人源CTLA-4蛋白胞外区全长的基因片段，氨基酸序列设计如SEQ ID NO:14所示。将其核苷酸序列克隆至真核表达质粒pTargetT上，获得其表达质粒pTargetT-hCTLA-4。
合成编码猴源CTLA-4蛋白胞外区全长的基因片段，氨基酸序列设计如SEQ ID NO:15所示。将其核苷酸序列克隆至真核表达质粒pTargetT上，获得其表达质粒pTargetT-cynoCTLA-4。
融合的人源CTLA-4蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:16和SEQ ID NO:17所示，对上述氨基酸序列进行密码子优化后合成带有标签的CTLA-4-hFc和CTLA-4-His的核苷酸序列，并将其分别克隆至真核表达质粒pHR上，获得其表达质粒pHR-CTLA-4-hFc、pHR-CTLA-4-His。
融合的人源CTLA-4蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQIDNO:18所示。对人源CTLA-4蛋白胞外区序列进行密码子优化后，合成带有标签的CTLA-4-mFc的核苷酸序列，并将克隆至真核表达质粒pHR上，获得其表达质粒pHR-CTLA-4-mFc。
融合的猴源CTLA-4蛋白胞外区和hIgG1-Fc标签的氨基酸序列如SEQ ID NO:19所示，对上述氨基酸序列进行密码子优化后合成带有标签的cynoCTLA-4-hFc的核苷酸序列，并将其克隆至真核表达质粒pHR上，获得其表达质粒pHR-cynoCTLA-4-hIgG1-Fc。
合成编码人源CD80蛋白胞外区全长的基因片段，氨基酸序列设计如SEQ ID NO:20所示。将其核苷酸序列克隆至真核表达质粒pTargetT上，获得其表达质粒pTargetT-CD80。
合成编码人源CD86蛋白胞外区全长的基因片段，氨基酸序列设计如SEQ ID NO:21所示。将其核苷酸序列克隆至真核表达质粒pTargetT上，获得其表达质粒pTargetT-CD86。
融合的人源CD80蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:22所示，对上述氨基酸序列进行密码子优化后合成带有标签的CD80-mFc的核苷酸序列。将其克隆至真核表达质粒pHR上，获得其表达质粒pHR-CD80-mFc。
融合的人源CD86蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID  NO:23所示，对上述氨基酸序列进行密码子优化后合成带有标签的CD86-mFc的核苷酸序列。将其克隆至真核表达质粒pHR上，获得其表达质粒pHR-CD86-mFc。
融合的人源CD80蛋白胞外区和hIgG1-Fc标签的氨基酸序列如SEQ ID NO:24，对上述氨基酸序列进行密码子优化后合成带有标签的CD80-hFc核苷酸序列，并将其克隆至真核表达质粒pHR上，获得其表达质粒pHR-CD80-hFc。
融合的人源CD86蛋白胞外区和hIgG1-Fc标签的氨基酸序列如SEQ ID NO:25，对上述氨基酸序列进行密码子优化后合成带有标签的CD86-hFc核苷酸序列，并将其克隆至真核表达质粒pHR上，获得其表达质粒pHR-CD86-hFc。
2、抗原蛋白的表达与纯化
(1)表达抗原蛋白的稳定转染细胞株构建
将真核表达质粒pTargeT-hCTLA-4在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置于37℃，5％CO 2浓度的培养箱中培养。24h后采用含1000μg/ml G418(Gibco，#10131-027)的培养基进行加压培养。转染16天后采用流式细胞术检测转染pool的阳性率，将阳性率较高的pool的细胞进行铺板(按照1x10 6个/ml的细胞密度，100μl/孔，铺96孔板)，采用Ipilimumab抗体(商品名Yervoy，购于Bristol-Myers Squibb Company)和Goat pAb to Hu IgG(PE)(Abcam,ab98596)抗体与细胞孵育，以流式细胞仪(ACEABIO,Novocyte 2060R)检测585nm波长下mean值，使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1细胞株，该细胞株高水平表达CTLA-4分子，命名为CHO-K1-CTLA-4。
分别合成编码人源CTLA-4蛋白(SEQ ID NO:14)和编码人源PD1蛋白胞外区全长(SEQ ID NO:41)的基因片段。然后将其克隆至真核质粒pTargetT上，获得其表达质粒pTargetT-hPD-1-hCTLA-4。将pTargeT-hPD1-hCTLA-4在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置于37℃，5％CO 2浓度的培养箱中培养。24h后采用含1000μg/ml G418(Gibco，#10131-027)及500μg/ml潮霉素B的培养基进行加压培养。转染16天后采用流式细胞术检测转染pool的阳性率，将阳性率较高的pool的细胞进行铺板(按照1x10 6个/ml的细胞密度，100μl/孔，铺96孔板)，采用 Ipilimumab抗体和Nivolumab抗体(购于Bristol-Myers Squibb Company)和Goat pAb to Hu IgG(PE)(Abcam,ab98596)抗体与细胞孵育，以流式细胞仪(ACEABIO,Novocyte 2060R)检测585nm波长下mean值，使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1细胞株，该细胞株高水平表达PD-1和CTLA-4分子，命名为CHO-K1-PD1-CTLA-4。
(2)标签抗原蛋白的表达
在1L细胞培养瓶中接种密度为0.5 x 10 6个细胞/ml的293E细胞(来源于ATCC)，加入新鲜的预热的FreeStyle 293表达培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的CO 2培养箱中培养过夜。取8.5mL FreeStyle 293表达培养基，加入1mg/ml的PEI溶液500μl，混合均匀，取250μg待转染质粒加入8.5ml FreeStyle 293表达培养基中，混合均匀，其中标签抗原蛋白质粒pHR-CTLA-4-hFc、pHR-CTLA-4-His、pHR-cynoCTLA-4-hFc、pHR-CD80-hFc、pHR-CD86-hFc、pHR-CD80-mFc、pHR-CD86-mFc分别转染。。将PEI与FreeStyle293表达培养基的混合溶液加入到质粒中，混合均匀，然后加入细胞培养物中，置37℃，8％CO 2，加湿的CO 2培养箱中培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入2.5ml的谷氨酰胺(母液浓度为200mM)和5ml的葡萄糖(母液浓度为180g/L)。当细胞活力降至65％～75％时，收集细胞上清。将细胞培养物1500rpm离心5min，收集上清，再8000rpm离心20min，收集上清。
(3)亲和层析柱纯化
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用亲和层析柱进行纯化。
实施例2：抗CTLA-4单克隆抗体的制备
1、杂交瘤单克隆的制备
(1)动物免疫
采用不同标签的hCTLA-4抗原蛋白与佐剂共同免疫实验动物，实验动物包括Balb/c品系小鼠和SD大鼠。免疫动物按照首次免疫使用50μg抗原免疫一只动物，后期均使用25μg抗原免疫一只动物。免疫佐剂可以是弗氏佐剂(Sigma)或Quick Antibody-Mouse5W(北京博奥龙免疫技术有限公司)。采用弗氏佐剂乳化抗原，将不同标签的hCTLA-4抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状 后免疫小鼠。采用Quick Antibody-Mouse5W作为佐剂，将不同标签的hCTLA-4抗原蛋白样品与Quick Antibody-Mouse5W按照1:1的体积比进行混合，混匀后即采用肌肉注射的方式，免疫SD大鼠。免疫方案如表1所示。
表1动物免疫方案
Figure PCTCN2022079170-appb-000001
*i.m.肌内注射；s.c.皮下注射；i.p.腹腔注射。
(2)杂交瘤融合
脾细胞的获取和制备：将加强免疫后的小鼠/大鼠处死后浸泡75％的酒精中。解剖取出脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min，弃上清。加入2mL红细胞裂解液，室温裂解红细胞2min，加入PBS至20mL，1500rpm离心7min，弃上清，重悬后进行活细胞计数。收集培养瓶中的Sp2/0细胞，1000rpm离心5min后弃上清，重悬后进行活细胞计数。按脾细胞：Sp2/0细胞＝1:1的比例混合细胞，1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞，1500rpm离心7min。弃上清，重复一次。分别用适量电转缓冲液重悬细胞，保证细胞浓度2×10 7个细胞/mL左右。把细胞悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20％FBS的15mL RPMI 1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。
(3)杂交瘤及亚克隆筛选
融合一周后，取杂交瘤母克隆的细胞培养上清，通过ELISA筛选结合人 hCTLA-4蛋白和食蟹猴CTLA-4蛋白的杂交瘤母克隆，进一步通过流式细胞术筛选出能结合CHO-K1-CTLA-4稳定转染细胞株的母克隆。
利用有限稀释法将阳性母克隆进行亚克隆，培养一周后利用ELISA检测亚克隆上清与hCTLA-4分子的结合活性，筛选分泌抗hCTLA-4抗体的单克隆细胞株。获得多个较好的单克隆细胞株。其中一个抗hCTLA-4单克隆抗体标记为SHS010-C92。
2、单克隆抗体的制备
根据亚克隆上清活性分析结果确定单克隆抗体母克隆株，将其扩大培养。培养条件是含有10％胎牛血清、1x NAEE、1x丙酮酸钠、1％青链霉素双抗的1640培养基，待细胞汇合度大于80％时，将细胞传代扩培，待培养至约50ml时收集上清，纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。
3、单克隆抗体测序
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据小鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得小鼠抗体轻链与重链可变区的基因片段；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester et al.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55；2.SimonKorenMiha
Figure PCTCN2022079170-appb-000002
AnjaColjaVenturinietal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列。SHS010-C92测序结果见表2。
表2抗CTLA-4鼠源单克隆抗体序列
抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHS010-C92 SEQ ID NO:26 SEQ ID NO:27
抗体SHS010-C92的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:1、2、3，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为 SEQ ID NO:5、6、7。
实施例3：抗CTLA-4嵌合抗体的构建
将纯化后(纯化步骤见实施例1)的小鼠抗体轻链与重链可变区基因片段分别与线性化的含有人抗体轻链或重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将混合液均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序；将测序正确的嵌合抗体标记为SHS010-C92-CHI。
将嵌合抗体重轻链质粒共转染HEK293E细胞，表达纯化获得抗体，然后进行纯度检测、活性分析及亲和力的检测。
利用定点突变的方法，将嵌合抗体SHS010-C92-CHI进行基因突变，以筛选更优的抗体。SHS010-C92-CHI重链CDR第24位G突变为E，抗体稳定性提高，标记为SHS010-C92-G24E-CHI。嵌合抗体测序结果见表3。
表3抗CTLA-4嵌合抗体序列
嵌合抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHS010-C92-CHI SEQ ID NO:26 SEQ ID NO:27
SHS010-C92-CHI-G24E SEQ ID NO:28 SEQ ID NO:27
抗体SHS010-C92-CHI的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:1、2、3，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:5、6、7。抗体SHS010-C92-CHI-G24E的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:1、2、4，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:5、6、7。
实施例4：抗CTLA-4人源化抗体的构建及生产
根据免疫活性分析，选择多个活性好的嵌合抗体进行人源化抗体改造。
抗体的人源化改造，首先是通过与免疫基因数据库(IMGT)中的小鼠抗体序列进行比对，确认SHS010-C92-CHI抗体可变区的鼠源种系，经过同源比对，SHS010-C92-CHI抗体的重链可变区序列的FR区与小鼠抗体种系基因IGHV3-48*01最为相似；抗体轻链可变区的FR序列则与小鼠抗体IGKV3-11*01最为相似。以SHS010-C92-CHI抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代SHS010-C92-CHI的 FR1-FR3序列，重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，最终选择SHS010-C92-CHI的2条人源化重链可变区(参见SEQ ID NO:29、30)和5条人源化轻链可变区(参见SEQ ID NO:31、32、33、34、35)进行下一步优化。SHS010-C92-CHI的人源化抗体非CDR区序列达到95％以上人源化。
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有人K轻链恒定区的真核表达质粒(pHR-hK)共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与人IgG4重链恒定区的真核表达质粒(pHR-IgG4)共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
挑选纯度、活性和亲和力均较好的人源化抗体，标记为huC92-11，序列见表4。
表4抗CTLA-4人源化抗体序列
Figure PCTCN2022079170-appb-000003
抗体HuC92-11的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:1、2、4，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:5、6、7。
实施例5：抗CTLA-4抗体与人CTLA-4结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。将人CTLA-4-His蛋白(1μg/孔，实施例1、2中制得)包被到96孔酶标板，于4℃条件下孵育过夜。用1xPBST清洗3次后用5％的脱脂牛奶37℃封闭2h。用1xPBST清洗3次后，本发明的抗CTLA-4抗体作为一抗从10μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL，37℃孵育2h，对照抗体为Ipilimumab(购于Bristol-Myers Squibb Company)；用1xPBST清洗5次后，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:5000)，37℃孵育1h。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50。
实验结果显示，本发明的人源化抗CTLA-4抗体HuC92-11的EC 50为0.01nM，与对照抗体相当Ipilimumab(EC 50：0.01nM)相当，具有较好的与人CTLA-4结合的能力。
实施例6：抗CTLA-4抗体与食蟹猴CTLA-4结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。食蟹猴CTLA-4-His(1μg/孔，实施例1、2中制得)包被96孔酶标板。本发明的抗CTLA-4抗体作为一抗从10μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL，37℃孵育2h，对照抗体为Ipilimumab。二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，加入显色液TMB(3,3',5,5'-四甲基联苯胺)，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50，结果如表5所示。
表5抗CTLA-4抗体与食蟹猴CTLA-4结合活性
Figure PCTCN2022079170-appb-000004
实验结果显示，本发明的人源化抗CTLA-4抗体HuC92-11具有较好的与食蟹猴CTLA-4结合能力。
实施例7：抗CTLA-4抗体对CTLA-4与其配体的阻断活性测定(ELISA)
采用ELISA法分析抗体对CTLA-4与其配体的阻断活性。将人CTLA-4-His蛋白(1μg/孔，实施例1、2中制得)包被到96孔酶标板，于4℃条件下孵育过夜。用1xPBST清洗3次后用5％的脱脂牛奶37℃封闭2h。用1xPBST清洗3次后，配置浓度为1μg/mL的CD80-mFc以及5μg/mL的CD86-mFc配体溶液，以上述配体溶液为稀释液，本发明的抗CTLA-4抗体作为一抗从25μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为25000ng/mL、5000ng/mL、1000ng/mL、200ng/mL、40ng/mL、8ng/mL、1.6ng/mL、0.32ng/mL，37℃孵育2h，对照抗体为Ipilimumab；用1xPBST清洗5次后，二抗使用Anti-mouse IgG HRP(Jackson，109-035-003，1:5000)，37℃孵育1h。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成IC 50，结果如表6、表7所示。
表6抗CTLA-4人源化抗体阻断CTLA-4与CD80结合
Figure PCTCN2022079170-appb-000005
表7抗CTLA-4人源化抗体阻断CTLA-4与CD86结合
Figure PCTCN2022079170-appb-000006
实验结果显示，本发明的人源化抗CTLA-4抗体HuC92-11能够阻断CTLA-4与其配体结合，且阻断活性强于对照抗体Ipilimumab。
实施例8：抗CTLA-4抗体与细胞表面hCTLA-4结合的测定(FACS)
采用FACS分析抗体与CHO-K1-CTLA-4细胞表面的CTLA-4的结合活性。CHO-K1-CTLA-4细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明的抗CTLA-4抗体作为一抗从20μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL，4℃条件下孵育1h，对照抗体为Ipilimumab；二抗使用PE-Anti-Human IgG (Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测抗体与细胞表面结合产生的荧光强度，结果如表8所示。
表8抗CTLA-4抗体与细胞表面hCTLA-4结合活性
Figure PCTCN2022079170-appb-000007
实验结果显示，本发明的人源化抗CTLA-4抗体HuC92-11具有较好的与细胞表面CTLA-4结合能力。
实施例9：抗CTLA-4抗体对CTLA-4与其配体的阻断活性测定(FACS)
采用FACS分析抗体阻断CHO-K1-CTLA-4细胞表面的CTLA-4结合配体的能力。CHO-K1-CTLA-4细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明的抗CTLA-4抗体作为一抗从20μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL，将稀释好的抗体与1μg/mL CD80-mFc或者1μg/mL CD86-mFc 1:1混合后，4℃条件下孵育1h，对照抗体为Ipilimumab；二抗使用PE-Anti-mouse IgG(Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测配体与细胞表面结合产生的荧光强度，结果如图3、4所示。
实验结果显示，本发明的人源化抗CTLA-4抗体HuC92-11能够阻断CTLA-4与其配体结合，且阻断活性强于对照抗体Ipilimumab。
实施例10：抗PD-1抗体相关抗原蛋白的制备
1、抗原蛋白的表达载体构建
1)PD-1抗原蛋白的表达载体构建
设计人源PD-1蛋白胞外区氨基酸序列与hIgG1-Fc或mIgG1-Fc或His Tag标签氨基酸序列，氨基酸序列设计分别如SEQ ID NO:36、SEQ ID NO:37和SEQ ID NO:38所示。对上述氨基酸序列进行密码子优化后合成带有标签的PD-1蛋白胞外区基因片段PD-1-hFc、PD-1-mFc和PD-1-His，并将其分别克隆至真核表达质粒pHR中，获得其表达质粒pHR-PD-1-hFc、pHR-PD-1-mFc和pHR-PD-1-His。
2)PD-L1配体蛋白的表达载体构建
设计人源PD-L1蛋白胞外区氨基酸序列与hIgG1-Fc或mIgG1-Fc标签氨基酸序列，氨基酸序列设计分别如SEQ ID NO:39和SEQ ID NO:40所示。对上述氨基酸序列进行密码子优化后合成带有标签的PD-L1蛋白胞外区基因片段PD-L1-hFc、PD-L1-mFc和PD-L1-His，并将其分别克隆至真核表达质粒pHR中，获得其表达质粒pHR-PD-L1-hFc、pHR-PD-L1-mFc。
2、抗原蛋白的表达与纯化
1)抗原蛋白的稳定转染细胞株的构建
合成编码PD-1蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:41所示，然后克隆到真核表达质粒pTargeT上，获得其表达质粒pTargeT-PD-1。
将真核表达质粒pTargeT-PD-1在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置于37℃，5％CO2的培养箱中培养。24h后采用含1000μg/ml G418的DME/F12完全培养基加压培养。16天后采用FACS检测pool阳性率，并计算细胞活率。采用PE anti-human PD-1抗体(Biolegend，Inc.,621607)与细胞孵育，以流式细胞仪(ACEA，Novocyte)读取mean值和parent％值，使用GraphPad生成进行数据分析。将细胞活率在90％以上的阳性pool细胞进行亚克隆铺板(0.5个/孔的细胞密度，200μl/孔)，置于37℃，5％CO 2的培养箱中培养。待亚克隆生长12天后，挑选出克隆化的CHO-K1细胞株，FACS筛选PD-1高表达亚克隆，并将其命名为hPD-1-CHO-K1。
2)配体蛋白的稳定转染细胞株的构建
合成编码PD-L1蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:42所示，然后克隆到真核表达质粒pTargeT上，获得其表达质粒pTargeT-PD-L1。
将真核表达质粒pTargeT-PD-L1在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置于37℃，5％CO 2的培养箱中培养。24h后采用含1000μg/ml G418的DME/F12完全培养基加压培养。16天后采用FACS检测pool阳性率，并计算细胞活率。采用PE anti-human PD-L1抗体(Sino Biological Inc.，10084-R312-P)与细胞孵育，以流式细胞仪(ACEA，Novocyte)读取mean值和parent％值，使用GraphPad生成进行数据分析。将细胞活率在90％以上的阳性pool细胞进行亚克隆铺板(0.5个/孔的细胞密度，200μl/孔)，置于37℃，5％CO 2的培养箱中培养。待亚克隆生 长12天后，挑选出克隆化的CHO-K1细胞株，FACS筛选PD-L1高表达亚克隆，并将其命名为hPD-L1-CHO-K1。
3)标签蛋白的表达
在1L细胞培养瓶中接种密度为0.5×10 6个/ml的293E细胞，加入新鲜的预热的FreeStyle 293表达培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的CO 2培养箱中培养过夜。取8.5mL FreeStyle 293表达培养基，加入1mg/ml的PEI溶液500μl，混合均匀，取250μg待转染质粒加入8.5ml FreeStyle 293表达培养基中，混合均匀，其中标签抗原蛋白质粒pHR-PD-1-hFc、pHR-PD-1-mFc、pHR-PD-1-His分别转染；标签配体蛋白质粒pHR-PD-L1-hFc、pHR-PD-L1-mFc分别转染。将PEI与FreeStyle 293表达培养基的混合溶液加入到质粒中，混合均匀，然后加入细胞培养物中，置37℃，8％CO 2，加湿的CO 2培养箱中培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入2.5ml的谷氨酰胺(母液浓度为200mM)和5ml的葡萄糖(母液浓度为180g/L)。当细胞细胞活力降至65％～75％时，收集细胞上清。将细胞培养物1500rpm离心5min，收集上清，再8000rpm离心20min，收集上清。
4)亲和层析柱纯化
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用亲和层析柱进行纯化。
实施例11：抗PD-1单克隆抗体的制备
1、杂交瘤单克隆的制备
(1)动物免疫
采用不同标签的抗PD-1抗原蛋白(PD-1-His，购于Sino Biological Inc.，10377-H08H)、PD-1-hFc、PD-1-mFc)与佐剂共同免疫的方法免疫C57、SJL品系的实验小鼠，首次免疫使用50μg抗原，后期使用25μg抗原免疫；采用不同标签的抗PD-1抗原蛋白与佐剂共同免疫的方法免疫SD品系的实验大鼠，首次抗原使用100μg抗原，后期使用50μg抗原免疫。
免疫佐剂可以是Quick Antibody-Mouse5W(北京博奥龙免疫技术有限公司)或Titer Max(Sigma)与CpG(金斯瑞生物科技有限合成)/Alum(thermo)佐剂间隔。将不同标签的PD-1抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免 疫小鼠及大鼠。
高水平表达PD-1分子的细胞系如hPD-1-CHO-K1也用来免疫大鼠，使之产生抗体。用胰蛋白酶消化处理正在培养的实施例1中获得的hPD-1-CHO-K1阳性单细胞，1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，取样用细胞计数仪计数，剩余样品1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，加入适量的PBS以获得1×10 8个细胞/ml的细胞悬液。实验组小鼠每只免疫1×10 7个细胞。
免疫方案如表9、表10所示：
表9小鼠免疫方案
Figure PCTCN2022079170-appb-000008
表10大鼠免疫方案
Figure PCTCN2022079170-appb-000009
*i.m.肌内注射；s.c.皮下注射；i.p.腹腔注射。
(2)杂交瘤融合
脾细胞的获取和制备：将加强免疫后的小/大鼠处死后浸泡75％的酒精中。解剖取出脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min，弃上清。加入2mL红细胞裂解液，室温裂解红细胞2min，加入PBS至20mL，1500rpm离心7min，弃上清，重悬后进行活细胞计 数。收集培养瓶中的Sp2/0细胞，1000rpm离心5min后弃上清，重悬后进行活细胞计数。按脾细胞：Sp2/0细胞＝1.5:1的比例混合细胞，1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞，1500rpm离心7min。弃上清，重复一次。分别用适量电转缓冲液重悬细胞，保证细胞浓度2×10 7个细胞/mL左右。把细胞悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20％FBS的15mL H-SFM完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC-3、2％FBS的H-SFM培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔H-SFM完全培养基(含2％FBS，1×HAT，1×BIOMYC-3)。
(3)杂交瘤及亚克隆上清的筛选
初筛：融合一周后，取细胞上清，通过ELISA筛选出能结合PD-1-His蛋白或细胞表面PD-1的杂交瘤上清，利用PD-1-His筛选针对PD-1而非hFc、mFc的抗体。然后利用ELISA分析杂交瘤上清阻断PD-1和PD-L1相互作用的能力。具体方法为：包被PD-L1-mFc于酶标板上，加入重组人源蛋白PD-1-hFc与杂交瘤上清的混合物孵育2h，加入HRP标记的anti-human IgG Fc特异性抗体(Jackson Immuno Research)孵育1h，利用酶标仪检测450nm处的吸光值。
复测：将筛选获得的具有结合能力及阻断能力的杂交瘤母克隆扩大培养，进行ELISA结合活性的复测；通过FACS筛选出能结合hPD-1-CHO-K1细胞表面PD-1的杂交瘤上清；再通过ELISA筛选出能结合cyno-PD-1-His蛋白的杂交瘤上清；通过三次实验阳***叉的杂交瘤上清作为候选阳性克隆。
利用有限稀释法将阳性细胞株进行亚克隆，培养一周后利用ELISA检测亚克隆上清与PD-1分子的结合活性以及阻断PD-1和PD-L1相互作用的活性，筛选双阳性细胞株。其中一个抗PD-1单克隆抗体标记为SHS006-P01。
2、单克隆抗体的制备
将单克隆抗体母克隆株扩大培养。培养条件是含有10％胎牛血清、1×NAEE、1×丙酮酸钠、1％青链霉素双抗的1640培养基，待细胞汇合度大于>80％时，进将细胞传代扩培，待培养至约50ml时收集上清，纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。
3、单克隆抗体测序
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据大鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得大鼠抗体轻链与重链可变区的基因片段；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester etal.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55；2.Simon KorenMiha
Figure PCTCN2022079170-appb-000010
Colja Venturini etal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列。SHS006-P01测序结果见表11。
表11抗PD-1鼠源单克隆抗体序列
Figure PCTCN2022079170-appb-000011
抗体SHS006-P01的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:8、9、10，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:11、12、13。
实施例12：抗PD-1嵌合抗体的构建
将纯化后(纯化步骤见实施例1)的鼠抗轻链与重链可变区基因片段分别与线性化的含有人抗体轻链或重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将混合液均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序；将测序正确的嵌合抗体标记为SHS006-P01CHI。
嵌合抗体SHS006-P01CHI的重链可变区和轻链可变区与鼠源抗体SHS006-P01的相同。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于 37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得嵌合抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将嵌合抗体转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
实施例13：抗PD-1人源化抗体的构建及生产
根据活性分析、亲和力等结果，选择多个活性好的嵌合抗体进行人源化抗体改造。
抗体的人源化改造，首先是通过与免疫基因数据库(IMGT)中的大鼠抗体序列进行比对，确认SHS006-P01CHI抗体可变区的鼠源种系，经过同源比对，SHS006-P01CHI抗体的重链可变区序列的FR区与大鼠抗体种系基因IGHV2-26*01最为相似；抗体轻链可变区的FR序列则与大鼠抗体IGKV2-28*01最为相似。以SHS006-P01CHI抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代SHS006-P01CHI的FR1-FR3序列，重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，并将在结构模拟中显示对抗体结构稳定起到关键作用的的氨基酸位点回突变为鼠源性氨基酸残基。最终选择SHS006-P01CHI的6条人源化重链可变区(参见SEQ ID NO：45、46、47、48、49、50)和3条人源化轻链可变区(参见SEQ ID NO:51、52、53)进行下一步优化。SHS006-P01CHI人源化抗体非CDR区序列达到95％以上人源化。
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有IgG4轻链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与含S228P/L235E突变的IgG4重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
挑选纯度、活性和亲和力均较好的人源化抗体，其中之一标记为SHS006-HuP01-22(本文中简称HuP01-22)，序列见表12。
表12抗PD-1人源化抗体序列
Figure PCTCN2022079170-appb-000012
抗体SHS006-HuP01-22的VH的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:8、9、10，VL的CDR序列：CDR1、CDR2、CDR3的序列分别为SEQ ID NO:11、12、13。
实施例14：抗PD-1抗体与猴PD-1结合活性测定(ELISA)
采用Protein based ELISA分析抗体的结合活性。食蟹猴PD-1-His(0.1μg/孔，SB，Cat.No.90311-C08H)包被96孔酶标板，4℃过夜孵育。本发明的抗PD-1抗体作为一抗从10μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.13ng/mL，37℃孵育1.5h。二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，加入显色液TMB(3,3',5,5'-四甲基联苯胺)，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50。
本发明的人源化抗PD-1抗体SHS006-HuP01-22的EC 50为0.088nM，具有较好的与食蟹猴PD-1结合的能力。
实施例15：抗PD-1抗体与人PD-1结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。将人PD-1-His蛋白(2μg/ml，自产)包被到96孔酶标板，4℃过夜孵育。用1×PBST清洗3次后用5％的脱脂牛奶4℃封闭过夜。用1×PBST清洗3次后，本发明的抗PD-1抗体作为一抗从1μg/mL 开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为1000ng/mL、200ng/mL、40ng/mL、8ng/mL、1.6ng/mL、0.32ng/mL、0.064ng/mL、0ng/mL，37℃孵育2h，对照抗体为Nivolumab；用1×PBST清洗3次后，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，37℃孵育40min。用1×PBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50。
本发明的人源化抗PD-1抗体SHS006-HuP01-22与人PD-1结合的EC 50为0.18nM，具有较好的与人PD-1结合的能力。
实施例16：抗PD-1抗体与细胞表面PD-1结合活性测定(FACS)
采用FACS分析抗体的结合活性。hPD-1-CHO-K1细胞以每孔1×10 5个细胞的方式铺入96孔圆底细胞培养板，本发明的抗PD-1抗体作为一抗从3.3μg/mL开始，梯度稀释加入细胞板，共8个浓度，浓度分别为3333ng/mL、1111ng/mL、370ng/mL、123ng/mL、41ng/mL、13.7ng/mL、4.57ng/mL、1.52ng/mL，对照抗体为Nivolumab，37℃孵育1h；Cell stanining buffer洗涤3遍；二抗使用PE anti-human IgG Fc(Biolegend，409304，0.8μl/孔)，4℃避光孵育30min；Cell stanining buffer洗涤3遍后利用流式细胞仪(ACEABIO，Novocyte)测定585nm波长下mean值。使用GraphPad生成EC 50。
本发明的人源化抗PD-1抗体SHS006-HuP01-22与细胞表面PD-1结合的EC 50为0.11nM，具有较好的与细胞表面PD-1结合能力。
实施例17：抗PD-1抗体与人PD-1蛋白的亲和力测定
利用Fortebio Octet对以上实施例中制得的人源化抗PD-1抗体结合抗原PD-1-His(Sino Biological，10377-H08H)的亲和力进行测定。将所述人源化抗PD-1抗体用SD缓冲液(PBS+0.02％Tween20+0.1％BSA)稀释到浓度10μg/ml，抗原PD-1-His用SD缓冲液5倍浓度梯度稀释，使其浓度为5μg/ml、1μg/ml、0.2μg/ml、0μg/ml，选用AHC传感器固化抗体，按Fortebio Octet RED96的操作规程进行亲和力测定，具体参数及实验结果如表13所示。
表13抗PD-1抗体与人PD-1蛋白的亲和力测定
Figure PCTCN2022079170-appb-000013
Figure PCTCN2022079170-appb-000014
实验结果显示，本发明的人源化抗PD-1抗体SHS006-HuP01-22具有较好的与人PD-1蛋白结合亲和力。
实施例18：抗PD-1抗体阻断PD-1与PD-L1结合的检测(FACS)
采用FACS检测本发明的抗PD-1抗体阻断PD-1结合到细胞表面PD-L1的能力。hPD-L1-CHO-K1阳性细胞株作为PD-L1提供者，在梯度稀释的抗PD-1抗体存在的情况下，观察PD-1-hFc与PD-L1-CHO-K1的结合能力。二抗使用PE Goat anti-human IgG(Biolegend，405307，0.8μl/孔)来监测PD-1-hFc的变化。流式细胞仪(ACEABIO，Novocyte)读取585nm波长下mean值，使用GraphPad生成IC 50。
实验结果显示，本发明的人源化抗PD-1抗体SHS006-HuP01-22阻断人PD-1与PD-L1结合的EC 50为0.77nM，具有较好的阻断能力。
实施例19：抗PD-1抗体的体外药效检测
采用SEB两次刺激PBMC的方法来检测抗PD-1抗体的体外药效。
按所需细胞量复苏PBMC，加到8-9ml的IMDM完全培养基中，1200rpm离心10min，弃上清；用适量培养基重悬，用血球计数板计数，加入到6孔板中，同时加入终浓度为100ng/ml的SEB溶液，孵育48h；48h后，1200rpm离心10min，弃上清，使用IMDM完全培养基洗涤1-2次，用适量培养基重悬，用血球计数板计数，并重悬为1M/mL，100μL/孔加入96孔板中；按4倍浓度(即40μg/mL)，50μL/孔，用IMDM完全培养基配制Nivolumab和Isotype，做好标记，涡旋；将抗体溶液加入对应孔中，对照组加入50μL/孔的培养基，96孔板置于37℃孵箱中，细胞和抗体孵育1h；1h后，按4倍浓度(400ng/ml)，50μL/孔用量，用IMDM完全培养基配制SEB溶液，加入对应孔中；96孔板置于37℃，5％CO2孵箱中孵育72h后，离心去除上清收集无细胞上清150μL，按一定比例稀释后，按照hIFN-γ(R&D system Cat：DY285B)和hIL-2(R&D system Cat：DY202)ELISA检测试剂盒说明书检测上清中IFN-γ和IL-2的浓度。
实验结果显示，SHS006-HuP01-22具有较好的促IFN-γ释放和促IL-2释放能力。
本发明还采用荧光素酶报告基因法(NFAT)检测嵌合抗体的体外促细胞因 子IL-2的释放，结果如表14所示。
表14抗PD-1嵌合抗体体外促细胞因子的释放
Figure PCTCN2022079170-appb-000015
结果显示，SHS006-P01CHI能够很好地阻断PD-1与PD-L1的结合，引起下游NFAT信号响应。
实施例20：抗CTLA-4/抗PD-1双特异性抗体表达载体的构建及蛋白表达纯化
本发明所用抗PD-1抗体是通过用人PD-1-his蛋白免疫小鼠后筛选得到的小鼠单克隆抗体SHS006-P01(重链可变区：SEQ ID NO：43，轻链可变区：SEQ ID NO：44)，将其人源化后筛选获得人源化单克隆抗体SHS006-HuP01-22(重链可变区：SEQ ID NO：46，轻链可变区：SEQ ID NO：52)。
本发明所用抗CTLA-4抗体是通过用人CTLA-4-his蛋白免疫小鼠后筛选得到的小鼠单克隆抗体，之后获得嵌合抗体SHS010-C92-CHI-G24E(重链可变区序列为SEQ ID NO:28，轻链可变区序列为SEQ ID NO:27)，将其人源化后筛选获得人源化单克隆抗体HuC92-11(重链可变区序列为SEQ ID NO:30，轻链可变区序列为SEQ ID NO:32)。
本发明利用上述抗PD-1和抗CTLA-4的人源化抗体的可变区序列，构建了抗CTLA-4/抗PD-1双特异性抗体。此双特异性抗体为包含scFv的对称结构的双特异性抗体，如图5所示。具体地，本发明的双特异性抗体是将抗CTLA-4抗体的scFv通过一段柔性连接肽连接至完整的抗PD-1抗体重链C端，该连接肽包含GGGGS重复序列。
本发明的双特异性抗体序列示意结构：
肽链1的结构见图1所示；
肽链2的结构见图2所示；
其中：
抗PD-1抗体VH：为人源化抗PD-1单抗SHS006-HuP01-22重链可变区；
连接肽1：为3个GGGGS的重复序列的柔性连接肽；
抗CTLA-4抗体VH：为人源化抗CTLA-4单抗HuC92-11重链可变区；
连接肽2：为4个GGGGS的重复序列的柔性连接肽；
抗CTLA-4抗体VL：为人源化抗CTLA-4单抗HuC92-11轻链可变区；
抗PD-1抗体VL-CL：为人源化抗PD-1单抗SHS006-HuP01-22轻链；
根据上述对称结构形式，利用抗PD-1抗体SHS006-HuP01-22和抗CTLA-4抗体SHS010-HuC92-11的可变区序列，构建双特异性抗体ScFv-[P01-22-C92-11]，其肽链1的氨基酸序列如SEQ ID NO:55所示，肽链2的氨基酸序列如SEQ ID NO:54所示。此外，还构建了双特异性抗体ScFv-[P01-22-C92-12]，其肽链1的氨基酸序列如SEQ ID NO:56所示，肽链2的氨基酸序列如SEQ ID NO:54所示。双特异性抗体ScFv-[P01-22-C92-11]、ScFv-[P01-22-C92-12]的肽链1包含的CDR序列：H1CDR1、H1CDR2、H1CDR3、L1CDR1、L1CDR2、L1CDR3、H2CDR1、H2CDR2、H2CDR3的序列分别为SEQ ID NO:1、2、4、5、6、7、8、9、10，肽链2包含的CDR序列：L2CDR1、L2CDR2、L2CDR3的序列分别为SEQ ID NO:11、12、13。
将构建好并测序正确的双抗肽链1和肽链2阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。挑选纯度、活性和亲和力均较好的双特异性抗体，标记为BsAB0192-1(即抗体ScFv-[P01-22-C92-11])。
实施例21:与人CTLA-4和PD-1结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。将人CTLA-4-His蛋白(1μg/孔，实施例1、2中制得)和PD-1-His蛋白(1μg/孔，购于北京义翘神州生物科技，货号：50124-M08H)包被到96孔酶标板，于4℃条件下孵育过夜。用1xPBST清洗3次后用5％的脱脂牛奶37℃封闭2h。用1xPBST清洗3次后，本发明提供的双特异性抗体作为一抗从10μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL，37℃孵育2h，对照抗体分别为Ipilimumab及Nivolumab； 用1xPBST清洗5次后，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:5000)，37℃孵育1h。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC 50，结果如图6、7所示。
实验结果显示，本发明的双特异性抗体BsAB0192-1具有较好的与人源CTLA-4和人源PD-1结合的能力。
实施例22:抗体与细胞表面hCTLA-4和hPD-1结合的测定(FACS)
采用FACS分析抗体与CHO-K1-CTLA-4表面的CTLA-4以及CHO-K1-PD-1表面的PD-1的结合能力。CHO-K1-CTLA-4细胞以及CHO-K1-PD-1细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明提供的双特异性抗体作为一抗从20μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL，4℃条件下孵育1h，对照抗体分别为Ipilimumab及Nivolumab；二抗使用PE-Anti-Human IgG(Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测抗体与细胞表面结合产生的荧光强度，使用GraphPad生成EC 50，结果如图8、9所示。
实验结果显示，本发明的双特异性抗体BsAB0192-1具有较好的与细胞表面CTLA-4以及PD-1结合的能力。
实施例23:抗体对CTLA-4与其配体的阻断活性测定(FACS)
采用FACS分析抗体阻断CHO-K1-CTLA-4表面的CTLA-4结合配体的能力。CHO-K1-CTLA-4细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明提供的双特异性抗体作为一抗从20μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL，将稀释好的抗体与1μg/mL CD80-mFc或者1μg/mL CD86-mFc 1:1混合后，4℃条件下孵育1h，对照抗体为Ipilimumab；二抗使用PE-Anti-mouse IgG(Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测配体与细胞表面结合产生的荧光强度，结果如图10、11所示。
实验结果显示，本发明的双特异性抗体BsAB0192-1能够较好地阻断细胞表 面的CTLA-4与其配体CD80和CD86的结合。
实施例24:抗体对PD-1与其配体的阻断活性测定(FACS)
采用FACS分析抗体阻断CHO-K1-PD-1表面的PD-1结合配体的能力。CHO-K1-PD-1细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明提供的双特异性抗体作为一抗从10μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为10000ng/mL、3333.33ng/mL、1111.11ng/mL、370.37ng/mL、123.45ng/mL、41.15ng/mL、13.71ng/mL、4.57ng/mL，将稀释好的抗体与10μg/mL PD-L1-mFc 1:1混合后，4℃条件下孵育1h，对照抗体为Nivolumab；二抗使用PE-Anti-mouse IgG(Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测配体与细胞表面结合产生的荧光强度，结果如图12所示。
实验结果显示，本发明的双特异性抗体BsAB0192-1能够较好地阻断细胞表面的PD-1与其配体PD-L1的结合。
实施例25:抗体在共表达CTLA-4及PD-1的细胞上的阻断活性(FACS)
利用共表达CTLA-4以及PD-1的CHO-K1细胞，测定双特异性抗体阻断CTLA-4与其配体结合的能力。CHO-K1-PD-1-CTLA-4细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明提供的双特异性抗体作为一抗从20μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL，将稀释好的抗体与1μg/mL CD80-mFc或者1μg/mL CD86-mFc 1:1混合后，4℃条件下孵育1h，对照抗体为Ipilimumab；二抗使用PE-Anti-mouse IgG(Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测配体与细胞表面结合产生的荧光强度，结果如图13、14所示。
利用共表达CTLA-4以及PD-1的CHO-K1细胞，测定双特异性抗体阻断PD-1与其配体结合的能力。CHO-K1-PD-1-CTLA-4细胞消化后，将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明的双特异性抗体作为一抗从10μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为10000ng/mL、3333.33ng/mL、1111.11ng/mL、370.37ng/mL、123.45ng/mL、41.15ng/mL、13.71ng/mL、4.57ng/mL， 将稀释好的抗体与10μg/mL PD-L1-mFc 1:1混合后，4℃条件下孵育1h，对照抗体为Nivolumab；二抗使用PE-Anti-mouse IgG(Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测配体与细胞表面结合产生的荧光强度，结果如图15所示。
实验结果显示，本发明的双特异性抗体BsAB0192-1在双稳定转染细胞株上能够较好地阻断CTLA-4以及PD-1和其配体的结合能力，阻断活性优于相应的单抗HuC92-11、HuP01-22，且优于对照抗体Ipilimumab以及Nivolumab。
实施例26:双特异性抗体与人CTLA-4和人PD-1亲和力测定
利用Fortebio Octet对所获得的双特异性抗体结合抗原CTLA-4-His以及PD-1-His的亲和力进行测定。使用SD缓冲液PBS+0.02％Tween20+0.1％BSA现将抗体稀释成10μg/ml浓度，之后使用AHC传感器固化抗体，将抗原(CTLA-4-His、PD-1-His)使用SD缓冲液配置1.5μg/ml、0.3μg/ml、0.06μg/ml这三个不同的浓度，对照抗体选用Ipilimumab以及Nivolumab，按fortebio Octet RED96的操作规程进行亲和力测定，具体参数及实验结果如表15所示。
表15抗体与CTLA-4及PD-1亲和力的测定结果
Figure PCTCN2022079170-appb-000016
实验结果显示，与对照抗体相比，本发明的双特异性抗体与人CTLA-4和人PD-1蛋白结合具有更高的亲和力。
实施例27:MC38移植瘤模型抗肿瘤实验
1、实验材料
(1)实验细胞及动物
MC38细胞购自美国典型培养物保藏中心(ATCC)；
B6-hPD1/hCTLA-4小鼠，雌性，6-8周龄，体重18-20克，购自南京集萃药康生物科技；
(2)供试品及对照品
对照品Ipilimumab购自北京义翘神州生物科技(货号68052-H001)，用作阳性对照；对照品Nivolumab购于Bristol-Myers Squibb Company，用作阳性对照；试验前，将本发明的PD-1抗体SHS006-P01-22用PBS配制为1mg/mL。
2、实验方法
MC38移植瘤模型
将人肿瘤细胞MC38细胞接种于小鼠的右后边，接种细胞数目为5×10 6/只。待肿瘤生长至平均体积达140mm 3时开始分组，尾静脉给药每周2次，每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径，计算相对肿瘤增殖率(T/C)。记录连续给药24天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线，结果如表16所示。
表16 MC38移植瘤模型抗肿瘤试验结果
Figure PCTCN2022079170-appb-000017
实验结果显示，本发明的抗PD-1抗体能够较好地抑制肿瘤生长。
实施例28:人淋巴瘤Raji-PDL1移植瘤模型抗肿瘤试验
1、实验材料
(1)实验细胞及动物
Raji人淋巴瘤细胞购自南京科佰生物科技有限公司。Raji-PDL1细胞株通过常规方法构建。
NOD-Scid小鼠，雌性，5-8周龄，体重18-20克，购自百奥赛图(江苏)基 因生物技术有限公司；
(2)供试品及对照品
Nivolumab抗体购自百时美施贵宝公司，ipilimumab购自北京义翘神州科技股份有限公司。
实验前，将本发明的抗CTLA-4/PD-1双抗BsAB0192-1用PBS配制为2.66mg/mL，将本发明的Nivolumab和ipilimumab用PBS配制成2mg/mL，4℃保存。
(3)实验方法
用含有10％胎牛血清，100U/mL的青霉素和100μg/mL的链霉素的RMPI1640培养基在37℃、5％CO 2的培养箱中培养人淋巴瘤细胞Raji-PDL1。当细胞饱和度为80％-90％时，收取细胞，计数，接种。将含有5×106细胞的50μl的PBS同50uL的Matrigel混合(终体积100μL)接种于小鼠的右后边，接种细胞数目为5×106/只。待肿瘤生长至体积达100mm 3时开始分组，腹腔给药每周3次，每周三次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。抗体的抑瘤疗效用相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。RTV＝V21/V0，其中V0是分组给药时(即d0)测量所得肿瘤体积，V25为给药25天测量时的肿瘤体积。将给药组与溶媒组最后一天(Day25)瘤体积用T-test进行分析，用GraphPad Prism进行。结果如表17所示。
表17人淋巴瘤Raji-PDL1移植瘤模型抗肿瘤试验结果
Figure PCTCN2022079170-appb-000018
以上四组，Nivolumab 20mg/kg组、Ipilimumab 20mg/kg组、Nivolumab和Ipilimumab 10mg/kg+10mg/kg组、BsAB0192-1 26.6mg/kg组，具有相同的摩尔浓度。实验结果显示，在相同摩尔浓度下，本发明的双抗BsAB0192-1具有较好的抑制肿瘤生长的作用，效果优于Nivolumab、Ipilimumab以及Nivolumab和Ipilimumab联合给药。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。

Claims (18)

  1. 一种抗CTLA-4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
    (a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
    (a2)如SEQ ID NO:1、2和4所示的氨基酸序列；和
    (a3)与SEQ ID NO:1、2和3或SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    (2)所述轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
    (a4)如SEQ ID NO:5、6和7所示的氨基酸序列；和
    (a5)与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
  2. 如权利要求1所述的抗CTLA-4抗体或其抗原结合片段，其具有：
    所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；或
    所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和4或与SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
  3. 如权利要求1或2所述的抗CTLA-4抗体或其抗原结合片段，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (b1)如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30所示的氨基酸序列；
    (b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、 且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (b4)如SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；
    (b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  4. 如权利要求1-3之任一项所述的抗CTLA-4抗体或其抗原结合片段，其中
    所述重链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列；或者
    所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或 与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列。
  5. 一种抗CTLA-4/抗PD-1抗体，其包括抗CTLA-4抗体或其抗原结合片段和抗PD-1抗体或其抗原结合片段，其中：
    所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区包含选自如下组的H1CDR1、H1CDR2和H1CDR3：
    (a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
    (a2)如SEQ ID NO:1、2和4所示的氨基酸序列；和
    (a3)与(a1)或(a2)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    (2)所述轻链可变区包含选自如下组的L1CDR1、L1CDR2和L1CDR3：
    (a4)如SEQ ID NO:5、6和7所示的氨基酸序列；
    (a5)如SEQ ID NO:5、6和7所示的氨基酸序列；和
    (a6)与(a4)或(a5)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    所述抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区包含选自如下组的H2CDR1、H2CDR2和H2CDR3：
    (A1)如SEQ ID NO:8、9和10所示的氨基酸序列；
    (A2)与(A1)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和
    (2)所述轻链可变区包含选自如下组的L2CDR1、L2CDR2和L2CDR3：
    (A3)如SEQ ID NO:11、12和13所示的氨基酸序列；
    (A4)与(A3)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。
  6. 如权利要求5所述的抗CTLA-4/抗PD-1抗体，其中：
    所述抗CTLA-4抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所 示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区；或者
    所述抗CTLA-4抗体或其抗原结合片段包含所述H1CDR1、H1CDR2和H1CDR3分别为SEQ ID NO:1、2和4或与SEQ ID NO:1、2和4所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的重链可变区，和所述L1CDR1、L1CDR2和L1CDR3分别为SEQ ID NO:5、6和7或与SEQ ID NO:5、6和7所示的氨基酸序列具有至少85％序列同一性的氨基酸序列的轻链可变区。
  7. 如权利要求5或6所述的抗CTLA-4/抗PD-1抗体，其中：
    所述抗CTLA-4抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (b1)如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30所示的氨基酸序列；
    (b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (b4)如SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35所示的氨基酸序列；
    (b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    所述抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (B1)如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50所示的氨基酸序列；
    (B2)(B1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (B3)与(B1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (B4)如SEQ ID NO:44、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53所示的氨基酸序列；
    (B5)(B4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(B4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (B6)与(B4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  8. 如权利要求5-7之任一项所述的抗CTLA-4/抗PD-1抗体，其中：
    所述抗CTLA-4抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和4所示的氨基酸序列，且所述抗CTLA-4抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:5、6和7所示的氨基酸序列；或者
    所述抗CTLA-4抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性且所述H1CDR1、H1CDR2和H1CDR3如SEQ ID NO:1、2和4所示的氨基酸序列，且所述抗CTLA-4抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性且所述L1CDR1、L1CDR2和L1CDR3如SEQ ID NO:5、6和7所示的氨基酸序列。
  9. 如权利要求5-8之任一项所述的抗CTLA-4/抗PD-1抗体，其中：
    所述抗PD-1抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:43所示的氨基酸序列，SEQ ID NO:43经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:43功能相同的氨基酸序列或与SEQ ID NO:43具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:8、9 和10所示的氨基酸序列，且所述抗PD-1抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:44，SEQ ID NO:44经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:44功能相同的氨基酸序列或与SEQ ID NO:44具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:11、12和13所示的氨基酸序列；或者
    所述抗PD-1抗体或其抗原结合片段的重链可变区的氨基酸序列为SEQ ID NO:46所示的氨基酸序列，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性且所述H2CDR1、H2CDR2和H2CDR3如SEQ ID NO:8、9和10所示的氨基酸序列，且所述抗PD-1抗体或其抗原结合片段的轻链可变区的氨基酸序列为SEQ ID NO:52，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性且所述L2CDR1、L2CDR2和L2CDR3如SEQ ID NO:11、12和13所示的氨基酸序列。
  10. 如权利要求5-9之任一项所述的抗CTLA-4/抗PD-1抗体，其中所述抗体是双特异性抗体。
  11. 如权利要求1-4之任一项所述的抗CTLA-4抗体或其抗原结合片段或如权利要求5-10之任一项所述的抗CTLA-4/抗PD-1抗体，其中所述抗体是人源化抗体或完全人抗体。
  12. 一种分离的核酸，其编码如权利要求1-4之任一项所述的抗CTLA-4抗体或其抗原结合片段或如权利要求5-10之任一项所述的抗CTLA-4/抗PD-1抗体。
  13. 如权利要求12所述的核酸，其包含：
    (1)编码抗CTLA-4抗体或其抗原结合片段的重链可变区如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30的核苷酸序列；和
    (2)编码抗CTLA-4抗体或其抗原结合片段的轻链可变区如SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35的核苷酸序列；和/或
    (3)编码抗PD-1抗体或其抗原结合片段的重链可变区如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、 SEQ ID NO:50的核苷酸序列；和
    (4)编码抗PD-1抗体或其抗原结合片段的轻链可变区SEQ ID NO:44、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53的核苷酸序列。
  14. 一种表达载体，其包含如权利要求12或13所述的核酸。
  15. 一种宿主细胞，其转化如权利要求14所述的表达载体，所述宿主细胞选自原核细胞和真核细胞，优先为哺乳动物细胞。
  16. 制备权利要求1-4之任一项所述的抗CTLA-4抗体或其抗原结合片段或权利要求5-10之任一项所述的抗CTLA-4/抗PD-1抗体的方法，包括在如权利要求15所述的宿主细胞中表达抗体，以及从宿主细胞中分离所述抗体的步骤。
  17. 一种药物组合物，其包含权利要求1-4之任一项所述的抗CTLA-4抗体或其抗原结合片段或权利要求5-10之任一项所述的抗CTLA-4/抗PD-1抗体和药学可接受的载体。
  18. 如权利要求1-4之任一项所述的抗CTLA-4抗体或其抗原结合片段或权利要求5-10之任一项所述的抗CTLA-4/抗PD-1抗体或如权利要求17所述的药物组合物在制备用于抑制CTLA-4和/或PD-1活性的药物中的应用，优选地，所述抑制CTLA-4和/或PD-1活性的药物用于***。
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