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WO2022171104A1 - 抗pd-1抗体及其用途 - Google Patents

抗pd-1抗体及其用途 Download PDF

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Publication number
WO2022171104A1
WO2022171104A1 PCT/CN2022/075593 CN2022075593W WO2022171104A1 WO 2022171104 A1 WO2022171104 A1 WO 2022171104A1 CN 2022075593 W CN2022075593 W CN 2022075593W WO 2022171104 A1 WO2022171104 A1 WO 2022171104A1
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Prior art keywords
seq
amino acid
acid sequence
binding protein
isolated antigen
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PCT/CN2022/075593
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English (en)
French (fr)
Inventor
杨欣秀
王宗达
刘小五
顾春银
曹晓丹
邓俗俊
潘忠宗
王学萍
Original Assignee
上海济煜医药科技有限公司
江西济民可信集团有限公司
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Application filed by 上海济煜医药科技有限公司, 江西济民可信集团有限公司 filed Critical 上海济煜医药科技有限公司
Priority to CN202280014220.2A priority Critical patent/CN116761822A/zh
Priority to EP22752264.6A priority patent/EP4293046A1/en
Publication of WO2022171104A1 publication Critical patent/WO2022171104A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—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
    • C07K2317/76—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to the field of biomedicine, in particular to an anti-PD-1 antibody and its use in the preparation of medicines.
  • Malignant tumor is a disease that seriously threatens human health on a global scale, and is the main type of disease that causes human death.
  • the incidence of tumors continues to increase, and effective therapeutic drugs need to be developed urgently.
  • the development of immune checkpoint drugs has become a research hotspot in recent years.
  • Programmed death 1 (programmed death 1) referred to as PD-1 is widely expressed in immune cells and is an important immunosuppressive molecule.
  • the main ligand of PD-1 is PD-L1, which is mainly expressed on the surface of tumor cells. After the ligand PD-L1 binds to the receptor PD-1, it inhibits the activation of T cells in the tumor microenvironment, so that the immune system such as T cells cannot normally kill tumor cells, thereby achieving immune escape.
  • the mechanism of action of PD-1 or PD-L1 immunotherapy is to design specific monoclonal antibodies against PD-1 or PD-L1, prevent the recognition of PD-1 and PD-L1, restore the normal function of T cells, and make T cells effective Kill tumor cells.
  • a number of therapeutic antibodies targeting this signaling pathway such as Pembrolizumab and Nivolumab, have been developed, but in the course of treatment, the response rate is generally low and resistance is likely to develop sex, etc. Therefore, there is an urgent need for anti-tumor PD-1 antibodies with stable structure, good efficacy and suitable for large-scale industrial production.
  • the present application provides an isolated antigen-binding protein having one or more of the following properties: (1) capable of binding primate-derived PD with a KD value of 1 ⁇ 10 8 M or lower -1 protein; (2) capable of blocking the binding between PD-1 protein and PD-L1 protein; (3) capable of blocking the binding between PD-1 protein and PD-L2 protein; and (4) capable of stimulating Immune cells secrete cytokines, for example, to stimulate lymphocytes to secrete IL-2.
  • the primate includes humans and/or monkeys.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen binding fragment comprises Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein is capable of competing with a reference antibody for binding to the PD-1 protein, wherein the reference antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein
  • the VH of the reference antibody comprises HCDR1, HCDR2 and HCDR3, the VL of the reference antibody comprises LCDR1, LCDR2 and LCDR3, and the reference antibody comprises any group of amino acid sequences selected from the following: (1) HCDR1: SEQ ID NO: 1, HCDR2: SEQ ID NO: 2, HCDR3: SEQ ID NO: 3, LCDR1: SEQ ID NO: 8, LCDR2: SEQ ID NO: 9, and LCDR3: SEQ ID NO: 10; (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 28, HCDR3: SEQ ID NO: 29, LCDR1: SEQ ID NO: 34, LCDR2: SEQ ID NO: 35, and LCDR3: SEQ ID NO: 36 (3 ) HCDR1: SEQ ID NO:
  • the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 3 or 29.
  • the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, 28 or 85.
  • the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1 or 27.
  • the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:27, and the HCDR2 comprises SEQ ID NO: :2, the amino acid sequence shown in SEQ ID NO: 28 or SEQ ID NO: 85, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 29.
  • the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 are selected from any of the following group of amino acid sequences: (1) HCDR1: SEQ ID NO: 1, HCDR2 : SEQ ID NO: 2 and HCDR3: SEQ ID NO: 3; (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 28 and HCDR3: SEQ ID NO: 29; and (3) HCDR1: SEQ ID NO: 1, HCDR2: SEQ ID NO: 85 and HCDR3: SEQ ID NO: 3.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH comprises a framework region H-FR1, the C-terminus of the H-FR1 being directly or the N-terminus of the HCDR1 are indirectly linked, and the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:57 or SEQ ID NO:58.
  • the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO:30, and SEQ ID NO:43.
  • the VH comprises a framework region H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the H-FR2 set forth in SEQ ID NO: 59 or 60 amino acid sequence.
  • the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:18, SEQ ID NO:31, and SEQ ID NO:44.
  • the VH comprises a framework region H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the H-FR3 set forth in SEQ ID NO: 61 or 62 amino acid sequence.
  • the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:32, and SEQ ID NO:45.
  • the VH comprises a framework region H-FR4, the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO: 63 or 64 amino acid sequence.
  • the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:33, and SEQ ID NO:46.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises SEQ ID NO:4, SEQ ID NO:17, The amino acid sequence shown in any one of SEQ ID NO:30 and SEQ ID NO:43, the H-FR2 comprises SEQ ID NO:5, SEQ ID NO:18, SEQ ID NO:31 and SEQ ID NO:44
  • the amino acid sequence shown in any one the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:32 and SEQ ID NO:45 and the Said H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:20, SEQ ID NO:33 and SEQ ID NO:46.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from from any of the following sets of amino acid sequences:
  • H-FR1 SEQ ID NO: 4
  • H-FR2 SEQ ID NO: 5
  • H-FR3 SEQ ID NO: 6
  • H-FR4 SEQ ID NO: 7;
  • H-FR1 SEQ ID NO: 17
  • H-FR2 SEQ ID NO: 18
  • H-FR3 SEQ ID NO: 19
  • H-FR4 SEQ ID NO: 20;
  • H-FR1 SEQ ID NO:30
  • H-FR2 SEQ ID NO:31
  • H-FR3 SEQ ID NO:32
  • H-FR4 SEQ ID NO:33;
  • H-FR1 SEQ ID NO: 43
  • H-FR2 SEQ ID NO: 44
  • H-FR3 SEQ ID NO: 45
  • H-FR4 SEQ ID NO: 46.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 65 or 66.
  • the VH comprises the amino acid sequence set forth in any one of SEQ ID NO:15, SEQ ID NO:25, SEQ ID NO:41, SEQ ID NO:51, and SEQ ID NO:86.
  • the isolated antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region is derived from a human IgG constant region.
  • the heavy chain constant region is derived from a human IgG4 constant region, and the human IgG4 constant region comprises the amino acid sequence set forth in SEQ ID NO:77 or SEQ ID NO:81.
  • the isolated antigen binding protein comprises an antibody heavy chain HC
  • the HC comprises the amino acid sequence set forth in SEQ ID NO:79, SEQ ID NO:83, or SEQ ID NO:87.
  • the isolated antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:36.
  • the isolated antigen binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:35.
  • the isolated antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:34.
  • the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:8 and SEQ ID NO:34, the LCDR2 comprise the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:35, and the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:10 and SEQ ID NO:36.
  • the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, and the LCDR1, LCDR2, and LCDR3 are selected from any of the following group of amino acid sequences: (1) LCDR1: SEQ ID NO: 34, LCDR2 : SEQ ID NO: 35 and LCDR3: SEQ ID NO: 36; and (2) LCDR1: SEQ ID NO: 8, LCDR2: SEQ ID NO: 9 and LCDR3: SEQ ID NO: 10.
  • the isolated antigen binding protein comprises a light chain variable region VL, wherein the VL comprises a framework region L-FR1, the C-terminus of L-FR1 being directly or N-terminus of the LCDR1 are indirectly linked, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:69 or SEQ ID NO:70.
  • the L-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:37, and SEQ ID NO:47.
  • the VL comprises a framework region L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises SEQ ID NO: 71 or SEQ ID NO: The amino acid sequence shown in 72.
  • the L-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:38, and SEQ ID NO:48.
  • the VL comprises a framework region L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises SEQ ID NO:73 or SEQ ID NO: The amino acid sequence shown in 74.
  • the L-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:13, SEQ ID NO:23, SEQ ID NO:39, and SEQ ID NO:49.
  • the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence set forth in SEQ ID NO:75 or SEQ ID NO:76.
  • the L-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:40, and SEQ ID NO:50.
  • the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1 comprises SEQ ID NO:11, SEQ ID NO:21, The amino acid sequence shown in any one of SEQ ID NO:37 and SEQ ID NO:47, the L-FR2 comprises SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:38 and SEQ ID NO:48
  • the amino acid sequence shown in any one, the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 39 and SEQ ID NO: 49 and wherein Said L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:40 and SEQ ID NO:50.
  • the isolated antigen binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, and the L-FR1, L-FR2, L-FR3 and L-FR4 are selected from from any of the following sets of amino acid sequences:
  • L-FR1 SEQ ID NO: 11
  • L-FR2 SEQ ID NO: 12
  • L-FR3 SEQ ID NO: 13
  • L-FR4 SEQ ID NO: 14;
  • L-FR1 SEQ ID NO: 21, L-FR2: SEQ ID NO: 22, L-FR3: SEQ ID NO: 23 and L-FR4: SEQ ID NO: 24;
  • L-FR1 SEQ ID NO: 37
  • L-FR2 SEQ ID NO: 38
  • L-FR3 SEQ ID NO: 39
  • L-FR4 SEQ ID NO: 40;
  • L-FR1 SEQ ID NO: 47
  • L-FR2 SEQ ID NO: 48
  • L-FR3 SEQ ID NO: 49
  • L-FR4 SEQ ID NO: 50.
  • the isolated antigen binding protein comprises a light chain variable region VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO:67 or SEQ ID NO:68.
  • the VL comprises the amino acid sequence set forth in any one of SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:42, and SEQ ID NO:52.
  • the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:78 or SEQ ID NO:82.
  • the isolated antigen binding protein comprises an antibody light chain LC
  • the LC comprises the amino acid sequence set forth in SEQ ID NO:80 or SEQ ID NO:84.
  • the application provides a polypeptide comprising the isolated antigen binding protein.
  • the application provides an immunoconjugate comprising the isolated antigen binding protein or the polypeptide.
  • the application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins.
  • the application provides a vector comprising the nucleic acid molecule.
  • the present application provides a cell comprising the nucleic acid molecule or the vector.
  • the application provides a method of preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the present application provides the use of the isolated antigen-binding proteins, polypeptides, immunoconjugates, nucleic acid molecule carriers, cells and/or pharmaceutical compositions in the preparation of medicaments for prophylaxis, Relieve and/or treat tumors.
  • the application provides a method of preventing, ameliorating or treating a tumor, the method comprising administering to a subject in need thereof the isolated antigen binding protein, polypeptide, immunoconjugate, nucleic acid molecule carrier, Cells and/or pharmaceutical compositions.
  • the present application provides the isolated antigen binding protein for use in preventing, alleviating or treating tumors.
  • the tumor comprises a tumor with high expression of PD-1 or PD-L1.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises melanoma, lung cancer, kidney cancer, esophageal cancer, head and neck cancer, lymphoma, liver cancer, and/or gastric cancer.
  • the application provides a method of inhibiting the binding of PD-1 protein to PD-L1 protein, the method comprising administering the isolated antigen binding protein, polypeptide, immunoconjugate, nucleic acid molecule, vector, cell and/or or pharmaceutical compositions.
  • the present application provides a method for inhibiting the binding of PD-1 protein to PD-L2 protein, comprising administering said isolated antigen binding protein, polypeptide, immunoconjugate, nucleic acid molecule, vector, cell and/or or pharmaceutical compositions.
  • the application provides methods of stimulating immune cells to secrete cytokines, comprising administering said isolated antigen binding proteins, polypeptides, immunoconjugates, nucleic acid molecules, vectors, cells and/or pharmaceutical compositions.
  • the present application provides methods for detecting the presence and/or content of PD-1 protein, comprising administering the isolated antigen binding proteins, polypeptides, immunoconjugates, nucleic acid molecules, vectors, cells and/or or pharmaceutical compositions.
  • the application provides a kit comprising the isolated antigen binding protein, the polypeptide, the immunoconjugate, the nucleic acid molecule, the carrier, the cell and/or the drug combination.
  • Figure 1 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application bind to the human PD-1 protein on the surface of CHOK1 cells.
  • Figure 2 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in the present application bind to the cynomolgus monkey PD-1 protein on the surface of CHOK1 cells.
  • Figure 3 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application block the binding of human PD-L1 to the human PD-1 protein on the surface of CHOK1.
  • Figure 4 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application block the human PD-1 protein on the surface of human PD-L2 and CHOK1 cells.
  • Figure 5 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described herein stimulate cytokine IL-2 secretion in a mixed lymphocyte reaction.
  • Figure 6 shows the results of binding of candidate antibodies to cell surface human PD-1.
  • Figure 7 shows the results of binding of candidate antibodies to monkey PD-1 on the cell surface.
  • Figure 8 shows the results of candidate antibodies activating mixed lymphocytes to release IL-2.
  • PD-1 generally refers to the programmed death 1 receptor, which may also be referred to as “programmed death 1", “CD279", “cluster of differentiation 279", “PD1", “PDCD1” or “CD297”.
  • PD-1 proteins typically include an extracellular IgV domain, a transmembrane region, and an intracellular tail.
  • PD-1 is commonly expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs).
  • DCs dendritic cells
  • PD-1 can bind to its ligands PD-L1 and PD-L2.
  • PD-1 encompasses any native PD-1 or modified PD-1 from any vertebrate source, including mammals, such as primates (eg, humans or monkeys) and rodents ( For example, mice or rats).
  • the term encompasses "full length", unprocessed PD-1 and any form of PD-1 produced by processing in a cell.
  • PD-1 can exist as a transmembrane protein or as a soluble protein.
  • PD-1 includes complete PD-1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-1, as well as functional variants, isoforms, species homologues, derivatives, analogs, and Epitope analogs.
  • PD-1 sequences are known in the art.
  • an exemplary full-length human PD-1 protein sequence can be found under NCBI accession number NP_005009.2 and an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found under NCBI accession number NP_001271065 or Uniprot accession number BOLAJ3 turn up.
  • PD-L1 generally refers to the programmed death ligand 1 protein.
  • PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by (in humans) the CD274 gene.
  • CD274 cluster of differentiation 274
  • B7-H1 B7 homolog 1
  • PD-L1 can bind to its receptors, such as programmed death 1 (PD-1).
  • PD-1 and PD-1 exerts immunosuppressive effects by inhibiting T cell proliferation and production of cytokines IL-2 and IFN- ⁇ .
  • PD-L1 encompasses any native PD-L1 or modified PD-1 from any vertebrate source, including mammals, such as primates (eg, humans or monkeys) and rodents ( For example, mice or rats).
  • the term encompasses "full-length", unprocessed PD-L1 as well as any form of PD-L1 produced by processing in a cell.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • the term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants.
  • the basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • PD-L1 sequences are known in the art. Information on the human PD-L1 gene (including genomic DNA sequence) can be found, for example, under NCBI Gene ID No. 29126.
  • the amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI Accession No. NP_054862 or UniProt Accession No. Q9NZQ7.
  • the term “PD-L2” generally refers to the programmed death ligand 2 protein, which may also be referred to as “programmed death 2", “CD273", “cluster of differentiation 273", “B7-DC” or " PDCD1LG2”.
  • the term “PD-L2” encompasses any native PD-L2 or modified PD-L2 from any vertebrate source, including mammals, such as primates (eg, humans or monkeys) and rodents ( For example, mice or rats).
  • the term encompasses "full-length”, unprocessed PD-1 and any form of PD-L2 produced by processing in a cell.
  • PD-L2 can exist as a transmembrane protein or as a soluble protein.
  • PD-L2 includes complete PD-L2 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-L2, and functional variants, isoforms, derivatives, and analogs of PD-L2, Epitope analogs.
  • PD-L2 sequences are known in the art. For example, an exemplary full-length human PD-L2 protein sequence can be found under NCBI Accession No. NP_079515.2.
  • antigen-binding protein generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone moiety that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen.
  • Antigen binding proteins may typically comprise antibody light chain variable regions (VL), antibody heavy chain variable regions (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., so long as they show The desired antigen-binding activity can be obtained.
  • antibody generally refers to an immunoglobulin reactive against a specified protein or peptide or fragment thereof.
  • Antibodies can be antibodies from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (eg, IgGl, IgG2, IgG3, and IgG4).
  • the antibody may have a heavy chain constant region selected from, eg, IgGl, IgG2, IgG3, or IgG4.
  • the antibody may also have a light chain selected from, for example, kappa ( ⁇ ) or lambda ( ⁇ ).
  • the antibodies of the present application can be derived from any species.
  • antigen-binding fragment generally refers to a portion of an antibody molecule comprising amino acid residues that interact with and confer specificity and affinity for the antigen to the antibody.
  • antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAbs.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain
  • Fab' generally refers to a fragment that differs from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain
  • F(ab"') 2 generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.
  • Fv generally refers to the smallest antibody fragment containing the entire antigen recognition and binding site.
  • the fragment may consist of a heavy chain variable region and a light chain variable region in a tightly non-covalently bound dimer;
  • dsFv generally refers to disulfide-stabilized Fv fragments, The bond between its single light chain variable region and single heavy chain variable region is a disulfide bond.
  • dAb fragment generally refers to antibody fragments consisting of VH domains.
  • scFv generally refers to a monovalent molecule formed by covalently linking and pairing one heavy chain variable domain and one light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have a general Structure: NH2 -VL-Linker-VH-COOH or NH2 -VH-Linker-VL-COOH.
  • variable region or “variable domain” generally refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen.
  • variable generally refers to certain portions of the sequence of the variable domains of antibodies that vary strongly, resulting in the binding and specificity of each particular antibody for its particular antigen. Variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved portions of the variable domains are referred to as framework regions (FRs).
  • CDRs complementarity determining regions
  • HVRs hypervariable regions
  • FRs framework regions
  • variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) , mostly adopting a ⁇ -sheet configuration, connected by three loop regions of the CDR structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions, and together with the CDRs from the other chain form the antigen-binding site of the antibody.
  • variable regions of an antibody or the CDRs of an antibody can be encoded by a variety of methods, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or In addition to post-translational modifications (eg, isomerization, amidation). Monoclonal antibodies are highly specific, directed against a single antigenic site.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. In the CDR regions, additions, deletions, insertions, substitutions or modifications of amino acids are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody.
  • the term "fully human antibody” generally refers to an antibody in which all parts, including the variable and constant regions of the antibody, are encoded by genes of human origin.
  • Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.
  • binding generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can be determined in the presence of a molecule
  • a target in the context of a heterogeneous population (including biological molecules).
  • an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody that specifically binds a target is an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets.
  • An antibody is said to "specifically bind" to an antigen when it binds to an epitope more readily through its antigen-binding domain than it would bind to a random, unrelated epitope.
  • KD KD
  • KD KD
  • KD the equilibrium dissociation constant
  • kdis the dissociation rate constant
  • koff the dissociation rate constant
  • kon the association rate constant
  • KD equilibrium dissociation constant
  • association and dissociation rate constants are well known in the art and include, but are not limited to, Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , co-immunoprecipitation (Co-IP) and protein chip technology.
  • BBI Biofilm Interferometry
  • RIA Radioimmunoassay
  • SPR Surface Plasmon Resonance
  • FRET Fluorescence Resonance Energy Transfer
  • Co-IP co-immunoprecipitation
  • the measured affinity for a particular protein-protein interaction can vary if measured under different conditions (eg, salt concentration, pH).
  • the term "primate” generally refers to monkey and ape species, and includes monkey species such as those from the genus Macaque (eg, Macaca fascicularis and or rhesus monkey (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (from tamarinds ( Saguinus), and ape species, such as chimpanzees (Pan troglodytes), and also including Homo sapiens.
  • monkey species such as those from the genus Macaque (eg, Macaca fascicularis and or rhesus monkey (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri
  • polypeptide or “protein” are used interchangeably and generally refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers.
  • the term may also include modified amino acid polymers, eg, by addition of sugar residues to form glycoproteins or modified by phosphorylation.
  • Polypeptides and proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of the native protein, or deletions, additions, or deletions of one or more amino acids of the native sequence and/or substituted molecules.
  • polypeptide and “protein” specifically include deleted, added and/or substituted sequences of one or more amino acids of the antigen binding proteins described herein.
  • isolated generally refers to biological material (eg, viruses, nucleic acids, or proteins) that is substantially free of components that normally accompany or interact with its naturally occurring environment.
  • the isolated biological material optionally contains additional material that the biological material is not found to have in its natural environment (eg, nucleic acids or proteins).
  • isolated when referring to a protein, “isolated” generally refers to the separation and separation of the molecule in question from the entire organism in which the molecule is found to occur naturally, or the substantial absence of other biological macromolecules of the same type.
  • nucleic acid molecule it is completely or partially separated from the sequence with which it is naturally associated, or the nucleic acid has a heterologous sequence associated with it, or the nucleic acid is separated from the chromosome.
  • immunoconjugate generally refers to a substance formed by linking an antigen-binding protein with other active agents, which can be small molecule active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes or spectral probes.
  • nucleic acid generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from their natural environment or artificially synthesized, or analogs thereof.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • the term "cell” generally refers to a plasmid or vector that may contain or already contains a nucleic acid molecule described herein, or an individual cell, cell line or cell culture capable of expressing an antigen binding protein described herein thing.
  • the cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein.
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be mammalian cells.
  • the mammalian cells can be CHO-K1 cells.
  • the term "pharmaceutical composition” generally refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain substances that are unacceptably toxic to the subject to whom the composition is to be administered. additional ingredients.
  • treatment generally refers to the desire to alter the natural course of the disease in the individual being treated, and may be a clinical intervention to achieve prevention or during the course of a clinical disease.
  • Desirable therapeutic effects include, but are not limited to, preventing disease occurrence or recurrence, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or ameliorating the disease state, and alleviating or improving prognosis.
  • antibodies eg, anti-PD-1 antibodies
  • administration generally refers to the administration of a dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition comprising an anticancer therapeutic agent) to a subject (eg, a patient).
  • a pharmaceutical composition eg, a pharmaceutical composition comprising an anticancer therapeutic agent
  • Administration can be by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired for topical treatment, intralesional administration.
  • Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • tumor generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • the tumor can be a tumor with high expression of PD-1 or PD-L1 in cells and tissues.
  • Tumors can include solid tumors and/or non-solid tumors (eg, hematological tumors, lymphomas).
  • homologue generally refers to an amino acid sequence or nucleotide sequence that has some homology to a wild-type amino acid sequence and a wild-type nucleotide sequence.
  • the term “homology” may be equivalent to sequence "identity”.
  • homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will contain the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • the term "between” generally means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and its N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment.
  • indirect connection In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR3.
  • first amino acid fragment" and "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an isolated antigen-binding protein.
  • the isolated antigen-binding protein is capable of binding PD-derived primate-derived PD- 1.
  • the binding affinity of the primate PD-1 antigen-binding protein to PD-1 can be determined by any method known in the art. In certain instances, binding affinity can be determined by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), bound antigen precipitation, equilibrium dialysis, biofilm interference (BLI). In certain instances, the binding affinity and KD value of the PD-1 antigen binding protein for PD-1 can be determined by biofilm interference (BLI). For example, the ForteBio Octet Molecular Interaction Analyzer can be used to analyze the binding kinetics between antigen and antibody.
  • the isolated antigen-binding protein is capable of binding primate-derived PD-1 with a KD value of 1 ⁇ 10 ⁇ 8 M or lower.
  • the value of the KD can be about 1 x 10-8 M or less, about 9 x 10-9 M or less, about 8 x 10-9 M or less, about 7 x 10-9 M or less, about 6 x 10 -9 M or less, about 5 x 10 -9 M or less, about 4 x 10 -9 M or less, about 3 x 10 -9 M or less, about 2 x 10 -9 M or less, about A value of 1 ⁇ 10 ⁇ 9 M or less binds human-derived PD-L1, eg, as detected using the FortieBio Octet Molecular Interaction Analyzer.
  • the binding activity of the PD-1 antigen-binding protein described herein to PD-1 can be detected using flow cytometry or enzyme-linked immunosorbent assay.
  • the PD-1 antigen binding protein binds PD-1 with an EC50 value of between about 0.0001 nM and about 100 nM, eg, Between about 0.001 nM and about 10 nM, between about 0.001 nM and about 5 nM, between about 0.001 nM and about 1 nM, between about 0.01 nM and about 1 nM, between about 0.01 nM and about 1.2 nM, or, about 0.1 Between nM to about 1.5 nM.
  • the PD-L1 antigen binding protein binds PD-L1 with an EC50 value of between about 0.0001 nM and about 100 nM, eg, Between about 0.001 nM and about 10 nM, between about 0.001 nM and about 5 nM, between about 0.01 nM and about 1 nM, between about 0.02 nM and about 1.0 nM, between about 0.1 nM and about 1.0 nM.
  • the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L1.
  • the antigen binding protein blocks the binding of PD-1 to PD-L1 can be determined by flow cytometry FACS, enzyme-linked immunosorbent assay ELISA.
  • host cells stably expressing human PD-1 are first incubated with decreasing amounts of unlabeled said antigen-binding protein, followed by incubation with biotin-labeled PD-L1 protein. Cells were then analyzed using FACS to confirm that the antigen binding protein blocked PD-1 binding to PD-L1.
  • IC50 values are between about 0.001 nM and about 10 nM, between about 0.001 nM and about 5 nM, between about 0.01 nM and about 5 nM, between about 0.1 nM and about 1.0 nM, between about 0.5 nM and about 1.5 nM .
  • host cells stably expressing human PD-1 are first incubated with decreasing amounts of unlabeled said antigen-binding protein, followed by incubation with biotin-labeled PD-L2 protein. The cells were then analyzed using FACS to confirm that the antigen binding protein blocked the binding of PD-1 to PD-L2.
  • PD-1 eg, CHOK1 cells
  • FACS FACS to confirm that the antigen binding protein blocked the binding of PD-1 to PD-L2.
  • nM and about 10 nM between about 0.001 nM and about 5 nM, between about 0.1 nM and about 2.5 nM, between about 0.1 nM and about 3 nM.
  • the isolated antigen binding protein may comprise HCDR3, and the HCDR3 may comprise the CDR3 of VH whose amino acid sequence is as shown in SEQ ID NO:65.
  • the isolated antigen binding protein may comprise a HCDR3, and the HCDR3 may comprise a CDR3 of a VH whose amino acid sequence is set forth in SEQ ID NO: 15 or 25.
  • the isolated antigen binding protein can comprise HCDR3, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:3.
  • the isolated antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the CDR2 of VH whose amino acid sequence is as shown in SEQ ID NO:65.
  • the isolated antigen binding protein may comprise a HCDR2, and the HCDR2 may comprise a CDR2 of a VH having an amino acid sequence as shown in SEQ ID NO: 15 or 25.
  • the isolated antigen binding protein can comprise HCDR2, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:2.
  • the isolated antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the CDR2 of VH whose amino acid sequence is shown in SEQ ID NO:86.
  • the isolated antigen binding protein can comprise HCDR2, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:85.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is shown in SEQ ID NO:65.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is shown in SEQ ID NO: 15 or 25.
  • the isolated antigen binding protein can comprise HCDR1, which can comprise the amino acid sequence set forth in SEQ ID NO:1.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH having the amino acid sequence as shown in SEQ ID NO: 65, and the HCDR2 may comprise the amino acid sequence as shown in SEQ ID The CDR2 of the VH shown in NO:65, the HCDR3 may comprise the CDR3 of the VH whose amino acid sequence is shown in SEQ ID NO:65.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is as shown in SEQ ID NO: 15 or 25, and the HCDR2 may comprise the amino acid sequence as shown in SEQ ID NO: 15 or 25.
  • the CDR2 of the VH set forth in SEQ ID NO: 15 or 25, the HCDR3 may comprise the CDR3 of the VH set forth in the amino acid sequence of SEQ ID NO: 15 or 25.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH having the amino acid sequence as shown in SEQ ID NO: 86, and the HCDR2 may comprise the amino acid sequence as shown in SEQ ID The CDR2 of the VH shown in NO:86, the HCDR3 may comprise the CDR3 of the VH whose amino acid sequence is shown in SEQ ID NO:86.
  • the isolated antigen binding protein can comprise the CDR1 of the VH having the amino acid sequence set forth in SEQ ID NO: 15, can comprise the CDR2 of the VH having the amino acid sequence set forth in SEQ ID NO: 15, and can comprise the amino acid sequence set forth as SEQ ID NO: 15 CDR3 of VH shown in NO:15.
  • the isolated antigen binding protein can comprise the CDR1 of the VH having the amino acid sequence set forth in SEQ ID NO:25, can comprise the CDR2 of the VH having the amino acid sequence set forth in SEQ ID NO:25, and can comprise the amino acid sequence set forth as SEQ ID NO:25 CDR3 of VH shown in NO:25.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2 and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:1, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:2 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:1, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:85 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the isolated antigen binding protein package may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 57: EVX 3 LVESGGGLVKPGGSLX 19 LX 21 CAAS (SEQ ID NO: 57), wherein X 3 is K or Q, X 19 is K or R, and X 21 is A or S.
  • SEQ ID NO: 57 EVX 3 LVESGGGLVKPGGSLX 19 LX 21 CAAS
  • X 3 is K or Q
  • X 19 is K or R
  • X 21 is A or S.
  • it can be divided according to the Chothia rule.
  • the H-FR1 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 3 , X 19 and/or X 21 compared to the amino acid sequence shown in SEQ ID NO: 4 amino acid substitutions.
  • the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:4 or 17.
  • the isolated antigen binding protein may comprise H-FR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 59: DMSWVRQX 8 PX 10 KX 12 LEWVX 17 TI (SEQ ID NO:59), wherein X 8 is A or T, X 10 is E or G, X 12 is G or R, and X 17 is A or S.
  • SEQ ID NO:59 DMSWVRQX 8 PX 10 KX 12 LEWVX 17 TI
  • X 8 is A or T
  • X 10 is E or G
  • X 12 is G or R
  • X 17 is A or S.
  • it can be divided according to the Chothia rule.
  • the H-FR2 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 8 , X 10 , X 12 and/or compared to the amino acid sequence shown in SEQ ID NO: 5 or amino acid substitution at X17.
  • the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 5 or 18.
  • the isolated antigen-binding protein may comprise H-FR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 61:
  • TYYX4DSVKGRFTISRDNAKNX21LYLQMX27SLRX31EDTAX36YYCVS ( SEQ ID NO :61), wherein X4 is A or P, X21 is S or N, X27 is N or S, and X31 is A or S, and X 36 is L or V.
  • X4 is A or P
  • X21 is S or N
  • X27 is N or S
  • X31 is A or S
  • X 36 is L or V.
  • it can be divided according to the Chothia rule.
  • the H-FR3 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 4 , X 21 , X 27 , X compared to the amino acid sequence shown in SEQ ID NO: 6 Amino acid substitutions at 31 and/or X36 .
  • the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 6 or 19.
  • the isolated antigen-binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:63: WGQGTX 6 VTVSS (SEQ ID NO:63), wherein , X 6 is L or S.
  • SEQ ID NO:63 the amino acid sequence shown in SEQ ID NO:63: WGQGTX 6 VTVSS (SEQ ID NO:63), wherein , X 6 is L or S.
  • it can be divided according to the Chothia rule.
  • the H - FR3 may comprise at least an amino acid substitution at X6 compared to the amino acid sequence set forth in SEQ ID NO:7.
  • the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 7 or 20.
  • the isolated antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 65:
  • the VH may comprise at least an amino acid substitution at a position selected from the group consisting of: X 3 , X 19 , X 21 , X 40 , as compared to the amino acid sequence shown in SEQ ID NO: 15. Amino acid substitutions at X 42 , X 44 , X 49 , X 61 , X 78 , X 84 , X 88 , X 93 and/or X 111 .
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO: 15 or 25.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO:86.
  • the isolated antigen binding protein of the present application can comprise LCDR3, and the LCDR3 can comprise the CDR3 of VL whose amino acid sequence is set forth in SEQ ID NO:67.
  • the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise the CDR3 of VL whose amino acid sequence is as shown in SEQ ID NO: 16 or 26.
  • the isolated antigen binding protein can comprise LCDR3, which can comprise the amino acid sequence set forth in SEQ ID NO:10.
  • the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the CDR2 of VL whose amino acid sequence is set forth in SEQ ID NO:67.
  • the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the CDR2 of VL whose amino acid sequence is set forth in SEQ ID NO: 16 or 26.
  • the isolated antigen binding protein can comprise LCDR2, which can comprise the amino acid sequence set forth in SEQ ID NO:9.
  • the isolated antigen binding protein may comprise LCDR1, and the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is set forth in SEQ ID NO:67.
  • the isolated antigen binding protein may comprise LCDR1, and the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is set forth in SEQ ID NO: 16 or 26.
  • the isolated antigen binding protein can comprise LCDR1, which can comprise the amino acid sequence set forth in SEQ ID NO:8.
  • the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is as shown in SEQ ID NO: 67, and the LCDR2 may comprise the amino acid sequence as shown in SEQ ID The CDR2 of the VL shown in NO:67, the LCDR3 may comprise the CDR3 of the VL whose amino acid sequence is shown in SEQ ID NO:67.
  • the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the CDR1 of the VL whose amino acid sequence is as shown in SEQ ID NO: 16 or 26, and the LCDR2 may comprise the amino acid sequence such as The CDR2 of the VL set forth in SEQ ID NO: 16 or 26, the LCDR3 may comprise the CDR3 of the VL set forth in the amino acid sequence of SEQ ID NO: 16 or 26.
  • the isolated antigen binding protein can comprise the CDR1 of VL having the amino acid sequence set forth in SEQ ID NO: 16, can comprise the CDR2 of the VL having the amino acid sequence set forth in SEQ ID NO: 16, and can comprise the amino acid sequence set forth as SEQ ID NO: 16 CDR3 of VL shown in NO:16.
  • the isolated antigen binding protein can comprise the CDR1 of VL having the amino acid sequence as set forth in SEQ ID NO: 26, can comprise the CDR2 of the VL having the amino acid sequence set forth in SEQ ID NO: 26, and can comprise the amino acid sequence as set forth in SEQ ID NO: 26 CDR3 of VL shown in NO:26.
  • the isolated antigen binding proteins described herein can comprise LCDR1, LCDR2, and LCDR3, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:8, and the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:9 amino acid sequence, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 10.
  • the isolated antigen-binding protein may comprise L-FR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 69: DIX 3 X 4 TQSX 8 X 9 FX 11 SX 13 SVGDRVX 20 TC (SEQ ID NO: 69), wherein X 3 is Q or V, X 4 is L or M, X 8 is P or H, X 9 is K or S, X 11 is L or M, X 13 is A or T, and X 20 is S or T.
  • SEQ ID NO: 69 DIX 3 X 4 TQSX 8 X 9 FX 11 SX 13 SVGDRVX 20 TC (SEQ ID NO: 69), wherein X 3 is Q or V, X 4 is L or M, X 8 is P or H, X 9 is K or S, X 11 is L or M, X 13 is A or T, and X 20 is S or T.
  • it can be divided according to the Chothia rule.
  • the L-FR1 may comprise at least amino acid substitutions at positions selected from the group consisting of: at X 3 , X 4 , X 8 , X compared to the amino acid sequence shown in SEQ ID NO: 11 Amino acid substitutions at 9 , X 11 , X 13 and/or X 20 .
  • the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 11 or 21.
  • the isolated antigen binding protein package may comprise L-FR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 71: WYQQKPGX 8 X 9 PKLLIY (SEQ ID NO: 71) , where X8 is K or Q, and X9 is A or S.
  • SEQ ID NO: 71 WYQQKPGX 8 X 9 PKLLIY (SEQ ID NO: 71) , where X8 is K or Q, and X9 is A or S.
  • X8 is K or Q
  • X9 is A or S.
  • it can be divided according to the Chothia rule.
  • the L-FR2 may comprise at least amino acid substitutions at positions selected from the group consisting of amino acids at X8 and/or X9 compared to the amino acid sequence shown in SEQ ID NO: 12 replace.
  • the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 12 or 22.
  • the isolated antigen-binding protein may comprise L-FR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:73:
  • GVPX 4 RFX 7 GSGSGTX 14 FTLTISX 21 X 22 QX 24 EDX 27 AX 29 YFC (SEQ ID NO: 73), wherein X 4 is D or S, X 7 is S or T, X 14 is D or E, X 21 is N or S, X22 is L or V, X24 is P or S, X27 is F or L, and X29 is D or T.
  • X 4 is D or S
  • X 7 is S or T
  • X 14 is D or E
  • X 21 is N or S
  • X22 is L or V
  • X24 P or S
  • X27 is F or L
  • X29 is D or T.
  • it can be divided according to the Chothia rule.
  • the L-FR3 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 4 , X 7 , X 14 , X compared to the amino acid sequence shown in SEQ ID NO: 13 Amino acid substitutions at 21 , X 22 , X 24 , X 27 and/or X 29 .
  • the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13 or 23.
  • the isolated antigen-binding protein may comprise L-FR4, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:75: FGGGTKX7EX9K (SEQ ID NO:75), wherein X 7 is L or V, and X 9 is I or V.
  • SEQ ID NO:75 FGGGTKX7EX9K
  • X 7 is L or V
  • X 9 is I or V.
  • it can be divided according to the Chothia rule.
  • the L-FR4 may comprise at least an amino acid substitution at a position selected from the group consisting of amino acids at X7 and/or X9 compared to the amino acid sequence shown in SEQ ID NO: 14 replace.
  • the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 14 or 24.
  • the isolated antigen binding protein may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:67:
  • the VL may comprise at least an amino acid substitution at a position selected from the group consisting of: at X3 , X4 , X8 , X9 , as compared to the amino acid sequence shown in SEQ ID NO: 16. Amino acid substitutions at X 11 , X 13 , X 20 , X 42 , X 43 , X 60 , X 63 , X 70 , X 77 , X 78 , X 80 , X 83 , X 85 , X 104 and/or X 106 .
  • the VL may comprise the amino acid sequence set forth in SEQ ID NO: 16 or 26.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 1
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:8
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:8
  • the amino acid sequence shown in SEQ ID NO:9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 1
  • the amino acid sequence shown in SEQ ID NO:85, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:8, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:8
  • the amino acid sequence shown in SEQ ID NO:9, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the isolated antigen binding protein may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:65, and the VL may comprise the amino acid sequence shown in SEQ ID NO:67 .
  • the isolated antigen binding protein may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 15 or 25, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16 or 26 amino acid sequence shown.
  • the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:15, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:16.
  • the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:25, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:26.
  • the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:86, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:26.
  • the isolated antigen binding protein may comprise a heavy chain constant region, which may be derived from IgG.
  • the heavy chain constant region can be from human IgG.
  • the heavy chain constant regions can be from IgGl, IgG2, IgG3 and IgG4.
  • the heavy chain constant region can be derived from human IgG4.
  • the heavy chain constant region may be amino acid mutated compared to native human IgG4.
  • the heavy chain constant region may be a sequence obtained by point mutation of the S228P amino acid in IgG4.
  • the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 77 or 81.
  • the isolated antigen binding protein may comprise a heavy chain, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO:79.
  • the isolated antigen binding protein may comprise a heavy chain, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO:87.
  • the isolated antigen binding protein may comprise a light chain constant region, which may be derived from light chain lambda and light chain kappa.
  • the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 78 or 82.
  • the isolated antigen binding protein may comprise a light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 80 or 84.
  • the isolated antigen binding protein can comprise a heavy chain and a light chain
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:79
  • the light chain can comprise the amino acid sequence set forth in SEQ ID NO:80 .
  • the isolated antigen binding protein can comprise a heavy chain and a light chain
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:83
  • the light chain can comprise the amino acid sequence set forth in SEQ ID NO:84 .
  • the isolated antigen binding protein can comprise a heavy chain and a light chain
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:87
  • the light chain can comprise the amino acid sequence set forth in SEQ ID NO:84 .
  • the isolated antigen binding protein of the present application can comprise HCDR3, and the HCDR3 can comprise the CDR3 of VH whose amino acid sequence is set forth in SEQ ID NO:66.
  • the isolated antigen binding protein may comprise HCDR3, and the HCDR3 may comprise the CDR3 of VH whose amino acid sequence is as shown in SEQ ID NO: 41 or 51.
  • the isolated antigen binding protein can comprise HCDR3, and the HCDR3 can comprise the amino acid sequence set forth in SEQ ID NO:29.
  • the isolated antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the CDR2 of VH whose amino acid sequence is as shown in SEQ ID NO:66.
  • the isolated antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the CDR2 of VH whose amino acid sequence is as shown in SEQ ID NO: 41 or 51.
  • the isolated antigen binding protein can comprise HCDR2, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:28.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the CDR1 of VH whose amino acid sequence is shown in SEQ ID NO:66.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the CDR1 of VH whose amino acid sequence is as shown in SEQ ID NO: 41 or 51.
  • the isolated antigen binding protein can comprise HCDR1, which can comprise the amino acid sequence set forth in SEQ ID NO:27.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH having the amino acid sequence as shown in SEQ ID NO:66, and the HCDR2 may comprise the amino acid sequence as shown in SEQ ID The CDR2 of the VH shown in NO:66, the HCDR3 may comprise the CDR3 of the VH whose amino acid sequence is shown in SEQ ID NO:66.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH having an amino acid sequence as shown in SEQ ID NO: 41 or 51, and the HCDR2 may comprise an amino acid sequence such as The CDR2 of the VH set forth in SEQ ID NO: 41 or 51, the HCDR3 may comprise the CDR3 of the VH set forth in the amino acid sequence of SEQ ID NO: 41 or 51.
  • the isolated antigen binding protein may comprise the CDR1 of the VH having the amino acid sequence as set forth in SEQ ID NO:41, may comprise the CDR2 of the VH having the amino acid sequence set forth as SEQ ID NO:41, and may comprise the amino acid sequence as set forth in SEQ ID NO:41 CDR3 of VH shown in NO:41.
  • the isolated antigen binding protein can comprise the CDR1 of the VH having the amino acid sequence set forth in SEQ ID NO:51, can comprise the CDR2 of the VH having the amino acid sequence set forth in SEQ ID NO:51, and can comprise the amino acid sequence set forth as SEQ ID NO:51 CDR3 of VH shown in NO:51.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2 and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:27, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:28 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the isolated antigen binding protein package may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 58: QVQLX 5 QSGAEX 11 X 12 X 13 PGASVKX 20 SCKAX 25 GFTFI (SEQ ID NO:58), wherein X5 is V or Q, X11 is L or V, X12 is K or V, X13 is K or R, X20 is L or V, and X25 is L or S.
  • SEQ ID NO:58 the amino acid sequence shown in SEQ ID NO: 58: QVQLX 5 QSGAEX 11 X 12 X 13 PGASVKX 20 SCKAX 25 GFTFI (SEQ ID NO:58), wherein X5 is V or Q, X11 is L or V, X12 is K or V, X13 is K or R, X20 is L or V, and X25 is L or S.
  • X5 is V or Q
  • X11 is L or V
  • the H-FR1 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 5 , X 11 , X 12 , X compared to the amino acid sequence set forth in SEQ ID NO: 30 Amino acid substitutions at 13 , X20 and/or X25 .
  • the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 30 or 43.
  • the isolated antigen-binding protein may comprise H-FR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 60: WVRQX5PX7X8GLEWIG (SEQ ID NO: 60), wherein X 5 is A or T, X7 is V or G, and X8 is H or Q.
  • SEQ ID NO: 60 WVRQX5PX7X8GLEWIG
  • X 5 is A or T
  • X7 is V or G
  • X8 is H or Q.
  • it can be divided according to the Chothia rule.
  • the H-FR2 may comprise at least an amino acid substitution at a position selected from the group consisting of amino acids at X5 and/or X8 compared to the amino acid sequence shown in SEQ ID NO: 31 replace.
  • the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 31 or 44.
  • the isolated antigen-binding protein may comprise H-FR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 62:
  • X 1 ATLX 5 ADISX 10 X 11 TAX 14 MEX 17 SX 19 LX 21 SX 23 DX 25 AVYYCTR (SEQ ID NO: 62), wherein X 1 is K or R, X 5 is I or T, and X 10 is T or I, X11 is N or S, X14 is S or Y, X17 is L or V, X19 is R or S, X21 is R or T, X23 is D or E, and X25 is S or T.
  • it can be divided according to the Chothia rule.
  • the H-FR3 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 1 , X 5 , X 10 , X compared to the amino acid sequence shown in SEQ ID NO: 32 Amino acid substitutions at 11 , X 14 , X 17 , X 19 , X 21 , X 23 and/or X 25 .
  • the H-FR3 can comprise the amino acid sequence set forth in SEQ ID NO: 32 or 45.
  • the isolated antigen-binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:64: WGX3GTTVTVSS (SEQ ID NO:64), wherein X 3 is A or Q.
  • SEQ ID NO:64 the amino acid sequence shown in SEQ ID NO:64: WGX3GTTVTVSS (SEQ ID NO:64), wherein X 3 is A or Q.
  • X 3 is A or Q.
  • it can be divided according to the Chothia rule.
  • the H-FR4 may comprise at least an amino acid substitution at a position selected from the group consisting of an amino acid substitution at X 3 compared to the amino acid sequence set forth in SEQ ID NO: 33.
  • the H-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 33 or 46.
  • the isolated antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 66:
  • the VH may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 5 , X 11 , X 12 , X 13 , compared to the amino acid sequence shown in SEQ ID NO: 41 , X 20 , X 25 , X 40 , X 42 , X 43 , X 67 , X 71 , X 76 , X 77 , X 80 , X 83 , X 85 , X 87 , X 89 , X 91 and/or X 112 amino acid substitutions.
  • the VH may comprise the amino acid sequence set forth in SEQ ID NO: 41 or 51.
  • the isolated antigen binding protein of the present application can comprise LCDR3, and the LCDR3 can comprise the CDR3 of VL whose amino acid sequence is set forth in SEQ ID NO:68.
  • the isolated antigen binding protein may comprise LCDR3, and the LCDR3 may comprise the CDR3 of VL whose amino acid sequence is as shown in SEQ ID NO: 52 or 42.
  • the isolated antigen binding protein can comprise LCDR3, which can comprise the amino acid sequence set forth in SEQ ID NO:36.
  • the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the CDR2 of VL whose amino acid sequence is set forth in SEQ ID NO:68.
  • the isolated antigen binding protein may comprise LCDR2, and the LCDR2 may comprise the CDR2 of VL whose amino acid sequence is set forth in SEQ ID NO: 52 or 42.
  • the isolated antigen binding protein can comprise LCDR2, which can comprise the amino acid sequence set forth in SEQ ID NO:35.
  • the isolated antigen binding protein may comprise LCDR1, and the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is as shown in SEQ ID NO:68.
  • the isolated antigen binding protein may comprise LCDR1, and the LCDR1 may comprise the CDR1 of VL whose amino acid sequence is set forth in SEQ ID NO: 52 or 42.
  • the isolated antigen binding protein can comprise LCDR1, which can comprise the amino acid sequence set forth in SEQ ID NO:34.
  • the isolated antigen binding proteins described herein can comprise LCDR1, LCDR2, and LCDR3, the LCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:34, and the LCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:35 amino acid sequence, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the isolated antigen binding protein package may comprise L-FR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 70: DVX 3 MTQX 7 PLSLPVX 14 LGX 17 X 18 ASISC ( SEQ ID NO: 70), wherein X 3 is L or V, X 7 is S or T, X 14 is S or T, X 17 is D or Q, and X 18 is Q or P.
  • SEQ ID NO: 70 SEQ ID NO: 70
  • X 3 is L or V
  • X 7 is S or T
  • X 14 is S or T
  • X 17 is D or Q
  • X 18 is Q or P.
  • it can be divided according to the Chothia rule.
  • the L-FR1 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 3 , X 7 , X 14 , X compared to the amino acid sequence shown in SEQ ID NO: 37 Amino acid substitutions at 17 and/or X 18 .
  • the L-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 37 or 47.
  • the isolated antigen binding protein package may comprise L-FR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 72: WYX 3 QX 5 PGQSPX 11 LLIY (SEQ ID NO: 72), wherein X 3 is L or Q, X 5 is K or R, and X 11 is K or R.
  • SEQ ID NO: 72 WYX 3 QX 5 PGQSPX 11 LLIY
  • X 3 is L or Q
  • X 5 is K or R
  • X 11 is K or R.
  • it can be divided according to the Chothia rule.
  • the L-FR2 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 3 , X 5 and/or X 11 compared to the amino acid sequence shown in SEQ ID NO: 38 amino acid substitutions.
  • the L-FR2 may comprise the amino acid sequence set forth in SEQ ID NO: 38 or 48.
  • the isolated antigen binding protein may comprise L-FR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:74:
  • GVPDRFSGSGSGTDFTLKISRVEX 24 EDX 27 GVYYC (SEQ ID NO: 74), wherein X 24 is A or Q and X 27 is L or V.
  • X 24 is A or Q
  • X 27 is L or V.
  • it can be divided according to the Chothia rule.
  • the L-FR3 may comprise at least an amino acid substitution at a position selected from the group consisting of amino acids at X 24 and/or X 27 compared to the amino acid sequence shown in SEQ ID NO: 39 replace.
  • the L-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 39 or 49.
  • the isolated antigen-binding protein may comprise L-FR4, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:76: FGX 3 GTKLEIK (SEQ ID NO: 76), wherein , X 3 is G or Q.
  • SEQ ID NO: 76 FGX 3 GTKLEIK
  • X 3 is G or Q.
  • it can be divided according to the Chothia rule.
  • the L-FR4 may comprise at least an amino acid substitution at X3 compared to the amino acid sequence set forth in SEQ ID NO:40.
  • the L-FR4 may comprise the amino acid sequence set forth in SEQ ID NO: 40 or 50.
  • the isolated antigen binding protein may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:68:
  • the VL may comprise at least an amino acid substitution at a position selected from the group consisting of: at X3 , X7 , X14 , X17 , compared to the amino acid sequence set forth in SEQ ID NO:42, Amino acid substitutions at X 18 , X 42 , X 44 , X 50 , X 85 , X 88 and/or X 105 .
  • the VL may comprise the amino acid sequence set forth in SEQ ID NO: 42 or 52.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 27.
  • the amino acid sequence shown in SEQ ID NO: 28 the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 34
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the isolated antigen binding protein may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO:66, and the VL may comprise the amino acid sequence shown in SEQ ID NO:68 .
  • the isolated antigen binding protein may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 41 or 51, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 42 or 52 amino acid sequence shown.
  • the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:41 and the VL can comprise the amino acid sequence set forth in SEQ ID NO:42.
  • the isolated antigen binding protein can comprise VH and VL, and the VH can comprise the amino acid sequence set forth in SEQ ID NO:51, and the VL can comprise the amino acid sequence set forth in SEQ ID NO:52.
  • the isolated antigen binding protein may comprise a heavy chain constant region, which may be derived from IgG.
  • the heavy chain constant region can be from human IgG.
  • the heavy chain constant regions can be from IgGl, IgG2, IgG3 and IgG4.
  • the heavy chain constant region can be from human IgG4.
  • the heavy chain constant region may be amino acid mutated compared to native human IgG4.
  • the heavy chain constant region may be a sequence obtained by point mutation of the S228P amino acid in IgG4.
  • the heavy chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 77 or 81.
  • the isolated antigen binding protein may comprise a heavy chain, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO:79.
  • the isolated antigen binding protein may comprise a heavy chain, and the heavy chain may comprise the amino acid sequence shown in SEQ ID NO:87.
  • the isolated antigen binding protein may comprise a light chain constant region, which may be derived from light chain lambda and light chain kappa.
  • the light chain constant region may comprise the amino acid sequence set forth in SEQ ID NO: 78 or 82.
  • the isolated antigen binding protein may comprise a light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO: 80 or 84.
  • the isolated antigen binding protein can comprise a heavy chain and a light chain
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:79
  • the light chain can comprise the amino acid sequence set forth in SEQ ID NO:80 .
  • the isolated antigen binding protein can comprise a heavy chain and a light chain
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:83
  • the light chain can comprise the amino acid sequence set forth in SEQ ID NO:84 .
  • the isolated antigen binding protein can comprise a heavy chain and a light chain
  • the heavy chain can comprise the amino acid sequence set forth in SEQ ID NO:87
  • the light chain can comprise the amino acid sequence set forth in SEQ ID NO:84 .
  • each heavy or light chain amino acid sequence of the antigen binding protein is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class.
  • the variable and constant portions of the light and heavy chains are derived from the variable and constant regions of antibodies of one animal species (eg, human).
  • the homologue may be at least about 85% of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to the PD-L1 protein) (eg, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) Proteins or polypeptides with sequence homology.
  • the polypeptide eg, an antibody or fragment thereof that specifically binds to the PD-L1 protein
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl.
  • the physical/chemical properties and/or biological activities of the PD-1 antigen binding proteins described herein can be identified, screened or characterized by various assays known in the art.
  • the present application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FACS), immunohistochemistry, immunofluorescence, and the like Antigen-binding activity of antigen-binding proteins.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting eg, Western blot
  • flow cytometry eg, FACS
  • immunohistochemistry eg, immunofluorescence, and the like
  • Antigen-binding activity of antigen-binding proteins e.g, antigen-binding proteins.
  • the application also provides isolated one or more nucleic acid molecules.
  • the one or more nucleic acid molecules can encode the antigen binding proteins described herein.
  • each of the one or more nucleic acid molecules can encode the entire antigen binding protein or a portion thereof (eg, HCDR1-3, LCDR1-3, VL, VH, light chain or one or more of the heavy chains).
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • nucleic acids encoding the antibodies, antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including, but not limited to, manipulation using restriction fragments or using synthetic oligonucleotides. Overlap extension PCR.
  • the application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • One or more of the nucleic acid molecules may be included in each vector.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • the vector is an expression vector.
  • the application provides host cells that may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein
  • the present application provides methods of making said antibodies or antigen-binding fragments thereof.
  • the method may comprise culturing the host cell described herein under conditions such that the antibody or antigen-binding fragment thereof is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • the application provides a pharmaceutical composition, which can comprise the antigen binding protein described in the application, the polypeptide, the nucleic acid molecule, the vector, the host cell, and any A pharmaceutically acceptable carrier is selected.
  • the pharmaceutically acceptable adjuvants are non-toxic to recipients at the doses and concentrations employed, and the pharmaceutical compositions herein may also contain more than one active compound, usually one that does not adversely affect each other's properties. those active compounds with complementary activities.
  • the type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.
  • the pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical compositions of the present application can inhibit or delay the development or progression of a disease, can reduce tumor size (or even substantially eliminate a tumor), and/or can alleviate and/or stabilize a disease state.
  • compositions described herein may comprise a prophylactically and/or therapeutically effective amount of the antibody, antigen-binding fragment thereof.
  • the prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) a disease or disorder and/or any complications thereof in a subject having or at risk of developing it.
  • the present application provides the use of the antigen binding protein and/or the fusion protein in the preparation of medicine.
  • the medicament is used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the tumor or cancer is a tumor or cancer with abnormal PD-L1 expression and/or abnormal PD-1 expression.
  • the tumor comprises a tumor with high expression of PD-1 or PD-L1.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises melanoma, lung cancer, kidney cancer, esophageal cancer, head and neck cancer, lymphoma, liver cancer, and/or gastric cancer.
  • the present application provides a method for inhibiting the binding of PD-L1 to PD-1, comprising administering the antigen binding protein and/or the polypeptide described herein.
  • the method can be an ex vivo or in vitro method.
  • the method may be a non-therapeutic method.
  • the method can include contacting a biological sample with an antigen binding protein and/or PD-1 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind PD-L1 , detecting whether a complex is formed between the antigen binding protein and PD-L1, and detecting whether a complex is formed between PD-1 and PD-L1.
  • the present application provides a method for inhibiting the binding of PD-L2 to PD-1, comprising administering the antigen binding protein and/or the polypeptide described herein.
  • the method can be an ex vivo or in vitro method.
  • the method may be a non-therapeutic method.
  • the method can include contacting a biological sample with an antigen binding protein and/or PD-1 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind PD-L2 , detecting whether a complex is formed between the antigen binding protein and PD-L2, and detecting whether a complex is formed between PD-1 and PD-L2.
  • the present application provides a method of stimulating immune cells to secrete cytokines, comprising administering the isolated antigen binding protein and/or the polypeptide.
  • the cytokine may be IL-2.
  • the immune cells can be lymphocytes.
  • T lymphocytes can be lymphocytes.
  • the method can be an ex vivo or in vitro method.
  • the method may be a non-therapeutic method.
  • the present application provides a method for detecting the presence and/or content of PD-1 protein, comprising administering the isolated antigen binding protein and/or the polypeptide.
  • the method can be an ex vivo or in vitro method.
  • the method may be a non-therapeutic method.
  • the present application also provides the use of an antigen binding protein in a method of diagnosing a subject with a tumor or cancer, the method comprising: determining by contacting a sample with the antigen binding protein of the present application and detecting the presence of bound antibody The presence or expression level of PD-1 in a sample obtained from a subject.
  • the present application provides a chimeric antigen receptor (CAR), which may comprise the nucleic acid molecule described herein or the antigen binding protein described herein.
  • CAR chimeric antigen receptor
  • an antibody drug conjugate which may comprise a cytotoxic agent, and an antigen-binding fragment as described herein.
  • Antibody drug conjugates usually refer to the use of specific linkers to connect antibodies and small molecule cytotoxic drugs, and its main components can include antibodies, linkers and small molecule cytotoxic drugs.
  • the present application provides a kit, which may comprise the antigen binding protein described in the present application, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate, and/or the antigen binding protein described in the present application.
  • pharmaceutical composition may include the antigen binding proteins, chimeric antigen receptors, genetically modified cells, and/or antibody drug conjugates described herein, optionally together with one or more therapeutic agents, in a single conventional container. Combinations, optionally formulated together in pharmaceutical compositions.
  • the present application provides a drug delivery device, which can be used to administer the antigen binding protein or the pharmaceutical composition thereof described herein.
  • mice Five Balb/c and SJL mice were immunized with Human PD-1, Mouse IgG2a Fc Tag fusion protein (Acrobiosystem, PD1-H5255a) and pcDNA3.4-hPD-1 plasmid DNA, 2 to 3 weeks apart each time. After the first booster immunization was performed 1-3 times with Human PD-1, Mouse IgG2a Fc Tag fusion protein or CHOK1-hPD-1 stable transfection cell line (GenScript, M00529), and blood was collected to measure the titer.
  • Human PD-1 Mouse IgG2a Fc Tag fusion protein
  • CHOK1-hPD-1 stable transfection cell line GenScript, M00529
  • the titer was detected by flow cytometry to determine the serum binding to CHOK1 or 293T cells overexpressing human PD-1 protein, and the mouse splenocytes with high binding titers were fused with myeloma (Sp2/0) cells.
  • the clones that specifically bind to CHOK1-hPD-1 cells and simultaneously block the binding of ligands PD-L1 and PD-L2 to CHOK1-hPD1 cells were screened from the fusion plate by flow cytometry. Cloning, and finally screened to a monoclonal that can secrete specific antibodies. Serum-free medium was used for small-scale production and purification to obtain monoclonal antibodies for subsequent identification, including detection of the binding ability of the antibody to CHOK1-hPD1, the blocking function of the binding of PD-L1, PD-L2 and CHOK1-hPD-1, and mixing The lymphocyte experiment finally obtained candidate anti-human PD-1 monoclonal antibodies 41D2-2C3D7 and 46H3A8.
  • the variable region sequences of mouse hybridoma clones 41D2-2C3D7 and 46H3A8 obtained by monoclonal antibody sequencing are as follows:
  • the most homologous human Germline antibody (data source: IMGT) was selected by sequence alignment as the humanization design framework (light chain with IGKV1-9*01, IGKJ4*01 as the framework, heavy chain with IGHV3-21*01, IGHJ5 *01, is the framework), Chothia numbering of antibody light and heavy chain variable regions [Chothia & Lesk, 1987], antibody CDR regions are defined: LCDR1 (L24-L34), LCDR2 (L50-L56), LCDR3 (L89-L97), HCDR1 (H26-H32), HCDR2 (H52-H56), HCDR3 (H95-H97), humanized and mutated the amino acids of the variable region of antibody light and heavy chain according to sequence alignment and variable region structure information; design expression vector, gene synthesis , Mammalian cells express and purify recombinant antibodies, compare the activities and physical and chemical properties of humanized antibodies and chimeric antibodies, and carry out 1-2 rounds of humanization optimization.
  • the optimized design of the following light and heavy chains are all humanized sequences grafted with the above Germline antibody as the framework CDR (41D2HzL0 and 41D2HzH0 are the light and heavy chains of the chimeric antibody, and 41D2HzL4 and 41D2HzH3 are the light and heavy chains of the humanized antibody).
  • the 46H3A8 humanization scheme is the same as 1.2.1.
  • the optimized design of the following light and heavy chains is based on the humanized sequence of the Germline antibody as the framework CDR grafting (1910h3hzL0 and 1910h3hzH0 are the light and heavy chains of the chimeric antibody, 1910h3hzL3 and 1910h3hzH4 are the humanized antibody sequences. light and heavy chains).
  • Each cycle consists of the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of antigen to the sensor; 3) regeneration with 10 mM glycine solution pH 1.7; 4) immersion in buffer for 60 seconds; 5) antibody immobilization On the sensor, the time was 20 seconds; 6) the sensor was immersed in the buffer for 180 seconds; 7) the antigen-antibody binding time was 180 seconds; 8) the dissociation of the antigen-antibody, the time was 10 minutes; 9) the sensor regeneration.
  • Example 4 Binding activity of the antigen-binding protein of the present application to human or cynomolgus monkey PD-1 on the cell surface
  • Figure 1 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application bind to the human PD-1 protein on the surface of CHOK1 cells.
  • Figure 2 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in the present application bind to the cynomolgus monkey PD-1 protein on the surface of CHOK1 cells.
  • the results showed that the binding activity of 1910h3hzL3H4 and 41D2HzL4H3 to cell surface human or cynomolgus monkey PD-1 was comparable to that of Pembrolizumab.
  • Example 5 The antigen-binding protein of the present application blocks the binding activity of human PD-1 on the cell surface to PD-L1 or PD-L2
  • the CHOK1-hPD-1 cells were collected, washed with FACS buffer and then resuspended. The cells were dispensed into a 96-well U-shaped plate at 1E5/50ul/well.
  • the antibody was diluted and added to the above 96-well plate, 25ul/well, biotinylated PD-L1 (Sinobiological, Cat. No. 10084-H02H-B) (or PD-L2, Acrobiosystem, Cat. No. PD2-H82F6) was diluted and added to the above In a 96-well plate, 25ul/well, incubate at 4°C for 1 hour after mixing.
  • FIG. 3 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application block the binding of human PD-L1 to the human PD-1 protein on the surface of CHOK1.
  • Figure 4 shows that the antigen binding proteins 1910h3hzL3H4 and 41D2HzL4H3 described in this application block the human PD-1 protein on the surface of human PD-L2 and CHOK1 cells.
  • the results showed that the binding activity of 1910h3hzL3H4 and 41D2HzL4H3 in blocking cell surface human PD-1 to PD-L1 or PD-L2 was comparable to that of the control antibody Pembrolizumab.
  • the antigen-binding protein of the present application has good activity in blocking the binding of PD-1 to PD-L1 or PD-L2.
  • Example 6 Mixed lymphocyte reaction: secretion of cytokine IL2
  • each CDR mutation library and the yeast display plasmid were respectively transferred into Saccharomyces cerevisiae strain EBY100 (purchased from ATCC), so that each CDR mutation library was displayed on the surface of yeast in the form of Fab.
  • the parental sequence of 41D2HzL4H3 was displayed on the surface of yeast in the form of Fab as a control.
  • mutant sequences of 10 clones such as YC208B3, YC208E2, YC208G3, YC209C5, YC209D6, YC215D1, YC215F3, YC215H3, YC216D5, YC216F2 were selected.
  • An expression plasmid was constructed with IgG1 LALA D265S subtype, and the antibody number was named Ab2005Am01-10.
  • the heavy chain variable region sequence of each clone was fused with IgG1 LALA D265S, and the light chain variable region was fused with the Kappa chain constant region; after gene synthesis, it was loaded into the expression vector pcDNA3.4 (Life Technologies).
  • the light and heavy expression plasmids were transferred into ExpiCHO cells (ThermoFisher Scientific, A29133), and the antibody was transiently expressed according to the method of the supplier's ExpiCHO expression system.
  • Expi CHO cells were grown to a density of 6 ⁇ 10 6 /mL, and 10 ⁇ g each of the antibody light and heavy chain expression plasmids were transfected with ExpiFectamine transfection reagent; one day after transfection, 150 ⁇ L and 4 mL of ExpiCHO enhancer and ExpiCHO supplement were added to the cultured cells. , continue to culture to 9 days, 4 degrees, 3500 rpm centrifugation to take the supernatant.
  • the antibody Ab2005Am01-10 was used as a candidate molecule to continue the physicochemical drugability evaluation, as follows:
  • MC methylcellulose
  • urea 5M 80 ⁇ L urea 5M 80 ⁇ L
  • ampholyte Pharmalyte pH 3-10 8 ⁇ L pI marker 5.5 and 2 ⁇ L each of 9.5.
  • the physicochemical properties of the candidate antibody Ab2005Am01-10 are summarized in Table 6. The results show that the physicochemical indicators such as purity, thermal stability and hydrophilicity of the candidate antibody are relatively ideal, and the in vitro activity of the candidate antibody is continued to be evaluated.
  • Octet RED96e (Fortebio) was used to determine the affinity of the candidate antibody with human PD-1 (Acro, Cat. No.: PD-1-H5221), monkey PD-1 (Acro, Product No.: PD-1-C5223) affinity
  • both antigen and antibody were diluted with 1 ⁇ PBST (1 ⁇ PBS: Sanko, B548117-0500; 0.02% Tween 20: sigma, P1379), the concentration of antigen was 100nM, antibody A concentration of 50 nM was used.
  • Each cycle consists of the following steps: 1) immersion in buffer for 60 s; 2) detection of non-specific binding of the antigen to the sensor; 3) regeneration with 10 mM glycine solution at pH 1.7; 4) immersion in buffer for 60 s; 5) antibody immobilization on the sensor 6)
  • the sensor is immersed in the buffer for 180s; 7) The antigen is combined with the antibody, the time is 180s; 8) The dissociation of the antigen and the antibody is 10 minutes; 9) The sensor is regenerated.
  • Figure 6 shows the results of binding of candidate antibodies to cell surface human PD-1.
  • Figure 7 shows the results of binding of candidate antibodies to monkey PD-1 on the cell surface.
  • PBMC and mitomycin C-treated DC cells adjust the DC cell density to 2 ⁇ 10 5 cells/mL, and then add the cells to the culture plate, 50 ⁇ L per well, that is, the number of DC cells is 1 ⁇ 10 4 cells/well; adjust the density of PBMC cells to 2 ⁇ 10 6 cells/mL, and then add cells to the culture plate, 100 ⁇ L per well, that is, the number of PBMC cells is 2 ⁇ 10 5 cells/well.
  • HCDR2 is SGGGRY, as shown in SEQ ID NO: 85; VH is

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Abstract

提供了一种分离的抗原结合蛋白，其能够结合PD-1蛋白，能够阻断PD-1蛋白与PD-L1和/或PD-L2蛋白之间的结合，和/或，能够刺激免疫细胞分泌细胞因子。还提供了所述分离的抗原结合蛋白在制备药物中的用途。

Description

抗PD-1抗体及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种抗PD-1抗体及其在制备药物中的用途。
背景技术
恶性肿瘤是目前全球范围内严重威胁人类健康的疾病，是疾病导致人类死亡的主要类型。随着国内人口老龄化的日渐加剧，肿瘤发生率不断提高，有效的治疗药物亟待开发，其中免疫检查点药物的开发为近年来研究热点。
程序性死亡受体1(programmed death 1)简称PD-1广泛表达于免疫细胞，是一种重要的免疫抑制分子。PD-1的主要配体为PD-L1，PD-L1主要表达于肿瘤细胞表面。配体PD-L1和受体PD-1结合后，在肿瘤微环境中抑制T细胞活化，导致T细胞等免疫系统无法正常杀伤肿瘤细胞，进而实现免疫逃逸。
PD-1或PD-L1免疫疗法的作用机制是针对PD-1或PD-L1设计特定的单克隆抗体，阻止PD-1和PD-L1的识别，恢复T细胞正常功能，从而使T细胞有效杀伤肿瘤细胞。目前已开发出多款针对这一信号通路的治疗性抗体，如帕博利珠单抗(Pembrolizumab)和纳武利尤单抗(Nivolumab)，但在治疗过程中普遍存在响应率低，易产生耐药性等现象。因此，亟需结构稳定、疗效好且适合大规模工业化生产的抗肿瘤PD-1抗体。
发明内容
本申请提供了一种分离的抗原结合蛋白，其具有下述性质中的一种或多种：(1)能够以1×10 8M或更低的KD值结合源自灵长类动物的PD-1蛋白；(2)能够阻断PD-1蛋白与PD-L1蛋白之间的结合；(3)能够阻断PD-1蛋白与PD-L2蛋白之间的结合；和(4)能够刺激免疫细胞分泌细胞因子，例如，刺激淋巴细胞分泌IL-2。
在某些实施方式中，所述灵长类动物包括人和/或猴。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白能够与参比抗体竞争结合所述PD-1蛋白，其中所述参比抗体包含重链可变区VH和轻链可变区VL，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，所述参比抗体的VL包含LCDR1、LCDR2和LCDR3，且所述参比抗体包含选自下述的任意一组氨基酸序列：(1)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，HCDR3：SEQ ID NO:3，LCDR1：SEQ ID NO:8，LCDR2：SEQ ID NO:9，和LCDR3：SEQ ID NO:10；(2)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:28，HCDR3：SEQ ID NO:29，LCDR1：SEQ ID NO:34，LCDR2：SEQ ID NO:35，和LCDR3：SEQ ID NO:36(3)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:85，HCDR3：SEQ ID NO:3，LCDR1：SEQ ID NO:8，LCDR2：SEQ ID NO:9，和LCDR3：SEQ ID NO:10。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，且所述HCDR3包含SEQ ID NO：3或29所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO：2、28或85所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，且所述HCDR1包含SEQ ID NO：1或27所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:1或SEQ ID NO:27所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、SEQ ID NO:28或SEQ ID NO:85所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:3或SEQ ID NO:29所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3选自以下任一组氨基酸序列：(1)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2和HCDR3：SEQ ID NO:3；(2)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:28和HCDR3：SEQ ID NO:29；和(3)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:85和HCDR3：SEQ ID NO:3。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:57或SEQ ID NO:58所示的氨基酸序列。
在某些实施方式中，所述H-FR1包含SEQ ID NO:4、SEQ ID NO:17、SEQ ID NO:30和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述 HCDR2之间，且所述H-FR2包含SEQ ID NO:59或60所示的氨基酸序列。
在某些实施方式中，所述H-FR2包含SEQ ID NO:5、SEQ ID NO:18、SEQ ID NO:31和SEQ ID NO:44中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:61或62所示的氨基酸序列。
在某些实施方式中，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:19、SEQ ID NO:32和SEQ ID NO:45中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:63或64所示的氨基酸序列。
在某些实施方式中，所述H-FR4包含SEQ ID NO:7、SEQ ID NO:20、SEQ ID NO:33和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:4、SEQ ID NO:17、SEQ ID NO:30和SEQ ID NO:43中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5、SEQ ID NO:18、SEQ ID NO:31和SEQ ID NO:44中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:19、SEQ ID NO:32和SEQ ID NO:45中任一项所示的氨基酸序列且所述H-FR4包含SEQ ID NO:7、SEQ ID NO:20、SEQ ID NO:33和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4选自以下任一组氨基酸序列：
(1)H-FR1：SEQ ID NO:4，H-FR2：SEQ ID NO:5，H-FR3：SEQ ID NO:6和H-FR4：SEQ ID NO:7；
(2)H-FR1：SEQ ID NO:17，H-FR2：SEQ ID NO:18，H-FR3：SEQ ID NO:19和H-FR4：SEQ ID NO:20；
(3)H-FR1：SEQ ID NO:30，H-FR2：SEQ ID NO:31，H-FR3：SEQ ID NO:32和H-FR4：SEQ ID NO:33；和
(4)H-FR1：SEQ ID NO:43，H-FR2：SEQ ID NO:44，H-FR3：SEQ ID NO:45和H-FR4：SEQ ID NO:46。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，且所述VH包含SEQ ID NO:65或66所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:15、SEQ ID NO:25、SEQ ID NO:41、 SEQ ID NO:51和SEQ ID NO:86中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括重链恒定区，且所述重链恒定区源自人IgG恒定区。
在某些实施方式中，所述重链恒定区源自人IgG4恒定区，且所述人IgG4恒定区包含SEQ ID NO:77或SEQ ID NO:81所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链HC，且所述HC包含SEQ ID NO:79、SEQ ID NO:83或SEQ ID NO:87所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR3，且所述LCDR3包含SEQ ID NO:10或SEQ ID NO:36所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR2，且所述LCDR2包含SEQ ID NO:9或SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1，且所述LCDR1包含SEQ ID NO:8或SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1、LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:8和SEQ ID NO:34中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:9和SEQ ID NO:35中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:10和SEQ ID NO:36中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3选自以下任一组氨基酸序列：(1)LCDR1：SEQ ID NO:34，LCDR2：SEQ ID NO:35和LCDR3：SEQ ID NO:36；和(2)LCDR1：SEQ ID NO:8，LCDR2：SEQ ID NO:9和LCDR3：SEQ ID NO:10。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL，其中所述VL包括框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:69或SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述L-FR1包含SEQ ID NO:11、SEQ ID NO:21、SEQ ID NO:37和SEQ ID NO:47中任一项所示的氨基酸序列。
在某些实施方式中，所述VL包括框架区L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:71或SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述L-FR2包含SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:38和SEQ ID NO:48中任一项所示的氨基酸序列。
在某些实施方式中，所述VL包括框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:73或SEQ ID NO:74所示的氨基酸序列。
在某些实施方式中，所述L-FR3包含SEQ ID NO:13、SEQ ID NO:23、SEQ ID NO:39和SEQ ID NO:49中任一项所示的氨基酸序列。
在某些实施方式中，所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:75或SEQ ID NO:76所示的氨基酸序列。
在某些实施方式中，所述L-FR4包含SEQ ID NO:14、SEQ ID NO:24、SEQ ID NO:40和SEQ ID NO:50中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1、L-FR2、L-FR3和L-FR4，其中所述L-FR1包含SEQ ID NO:11、SEQ ID NO:21、SEQ ID NO:37和SEQ ID NO:47中任一项所示的氨基酸序列，所述L-FR2包含SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:38和SEQ ID NO:48中任一项所示的氨基酸序列，所述L-FR3包含SEQ ID NO:13、SEQ ID NO:23、SEQ ID NO:39和SEQ ID NO:49中任一项所示的氨基酸序列且所述L-FR4包含SEQ ID NO:14、SEQ ID NO:24、SEQ ID NO:40和SEQ ID NO:50中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1、L-FR2、L-FR3和L-FR4选自以下任一组氨基酸序列：
(1)L-FR1：SEQ ID NO:11，L-FR2：SEQ ID NO:12，L-FR3：SEQ ID NO:13和L-FR4：SEQ ID NO:14；
(2)L-FR1：SEQ ID NO:21，L-FR2：SEQ ID NO:22，L-FR3：SEQ ID NO:23和L-FR4：SEQ ID NO:24；
(3)L-FR1：SEQ ID NO:37，L-FR2：SEQ ID NO:38，L-FR3：SEQ ID NO:39和L-FR4：SEQ ID NO:40；和
(4)L-FR1：SEQ ID NO:47，L-FR2：SEQ ID NO:48，L-FR3：SEQ ID NO:49和L-FR4：SEQ ID NO:50。
在某些实施方式中，所述分离的抗原结合蛋白包含轻链可变区VL，且所述VL包含SEQ ID NO:67或SEQ ID NO:68所示的氨基酸序列。
在某些实施方式中，所述VL包含SEQ ID NO:16、SEQ ID NO:26、SEQ ID NO:42和SEQ ID NO:52中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体轻链恒定区，且所述抗体轻链恒定区包含SEQ ID NO:78或SEQ ID NO:82所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链LC，且所述LC包含SEQ ID NO:80或SEQ ID NO:84所示的氨基酸序列。
另一方面，本申请提供了一种多肽，其包含所述分离的抗原结合蛋白。
另一方面，本申请提供了一种免疫缀合物，其包含所述分离的抗原结合蛋白或所述多肽。
另一方面，本申请提供了分离的一种或多种核酸分子，其编码所述分离的抗原结合蛋白。
另一方面，本申请提供了一种载体，其包含所述核酸分子。
另一方面，本申请提供了一种细胞，其包含所述核酸分子或所述载体。
另一方面，本申请提供了制备所述分离的抗原结合蛋白的方法，所述方法包括在使得所述分离的抗原结合蛋白表达的条件下，培养所述细胞。
另一方面，本申请提供了药物组合物，其包含所述分离的抗原结合蛋白、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的载体。
另一方面，本申请提供了所述述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子载体、细胞和/或药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
另一方面，本申请提供了预防、缓解或治疗肿瘤的方法，所述方法包括向有需要的受试者施用所述述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子载体、细胞和/或药物组合物。
另一方面，本申请提供了所述分离的抗原结合蛋白，用于预防、缓解或治疗肿瘤。
在某些实施方式中，所述肿瘤包括PD-1或PD-L1高表达的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括黑色素瘤、肺癌、肾癌、食管癌、头颈癌、淋巴瘤、肝癌和/或胃癌。
另一方面，本申请提供了抑制PD-1蛋白与PD-L1蛋白结合的方法，所述方法包括施用述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物。
另一方面，本申请提供了抑制PD-1蛋白与PD-L2蛋白结合的方法，其包括施用所述述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物。
另一方面，本申请提供了刺激免疫细胞分泌细胞因子的方法，其包括施用所述述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物。
另一方面，本申请提供了用于检测PD-1蛋白的存在和/或含量的方法，其包括施用述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子、载体、细胞和/或药物组合物。
另一方面，本申请提供了试剂盒，其包含所述分离的抗原结合蛋白、所述多肽、所述免 疫缀合物、所述核酸分子、所述载体、所述细胞和/或所述药物组合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3结合CHOK1细胞表面的人PD-1蛋白。
图2显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3结合CHOK1细胞表面的食蟹猴PD-1蛋白。
图3显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3阻断人PD-L1与CHOK1表面的人PD-1蛋白结合。
图4显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3阻断人PD-L2与CHOK1细胞表面的人PD-1蛋白。
图5显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3在混合淋巴细胞反应中刺激细胞因子IL-2分泌。
图6显示的是候选抗体与细胞表面人PD-1的结合结果图。
图7显示的是候选抗体与细胞表面猴PD-1的结合结果图。
图8显示的是候选抗体激活混合淋巴细胞释放IL-2的结果图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“PD-1”通常是指程序性死亡1受体，也可称为“程序性死亡1”、 “CD279”、“分化簇279”、“PD1”、“PDCD1”或“CD297”。PD-1蛋白通常包括胞外IgV域、跨膜区和胞内尾。PD-1通常在T细胞、B细胞、自然杀伤T细胞、活化的单核细胞和树突细胞(DC)上表达。PD-1可以结合其配体PD-L1和PD-L2。术语“PD-1”涵盖任何脊椎动物来源的任何天然PD-1或经修饰的PD-1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人或猴)和啮齿类(例如，小鼠或大鼠)。所述术语涵盖“全长”、未加工的PD-1以及由细胞中的加工所产生的任何形式的PD-1。PD-1可作为跨膜蛋白或作为可溶性蛋白存在。“PD-1”包括完整的PD-1及其片段，还包括PD-1的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与PD-1共同表位的类似物。PD-1序列是本领域已知的。例如，示例性的全长人PD-1蛋白序列可在NCBI登录号NP_005009.2下找到，示例性的全长食蟹猴的PD-1蛋白序列可在NCBI登录号NP_001271065或Uniprot登录号B0LAJ3下找到。
在本申请中，术语“PD-L1”通常是指程序性死亡配体1蛋白。PD-L1也称为分化簇274(CD274)或B7同源物1(B7-H1)，并且是由(人类中)CD274基因编码的蛋白。PD-L1可以结合其受体，例如程序性死亡1(PD-1)。PD-L1和PD-1的络合通过抑制T细胞增殖和产生细胞因子IL-2和IFN-γ发挥免疫抑制作用。术语“PD-L1”涵盖任何脊椎动物来源的任何天然PD-L1或经修饰的PD-1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人或猴)和啮齿类(例如，小鼠或大鼠)。所述术语涵盖“全长”、未加工的PD-L1以及由细胞中的加工所产生的任何形式的PD-L1。PD-L1可作为跨膜蛋白或作为可溶性蛋白存在。所述术语还涵盖天然存在的PD-L1的变体，例如剪接变体或等位基因变体。PD-L1的基本结构包括4个结构域：胞外Ig样V型结构域和Ig样C2型结构域、跨膜结构域以及细胞质结构域。PD-L1序列是本领域已知的。例如可在NCBI Gene ID No.29126下找到关于人PD-L1基因(包括基因组DNA序列)的信息。示例性的全长人PD-L1蛋白的氨基酸序列可在NCBI登录号NP_054862或UniProt登录号Q9NZQ7下找到。
在本申请中，术语“PD-L2”通常是指程序性死亡配体2蛋白，也可称为“程序性死亡2”、“CD273”、“分化簇273”、“B7-DC”或“PDCD1LG2”。术语“PD-L2”涵盖任何脊椎动物来源的任何天然PD-L2或经修饰的PD-L2，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人或猴)和啮齿类(例如，小鼠或大鼠)。所述术语涵盖“全长”、未加工的PD-1以及由细胞中的加工所产生的任何形式的PD-L2。PD-L2可作为跨膜蛋白或作为可溶性蛋白存在。“PD-L2”包括完整的PD-L2及其片段，还包括PD-L2的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与PD-L2共同表位的类似物。PD-L2序列是本领域已知的。例如，示例性的全长人PD-L2蛋白序列可在NCBI登录号NP_079515.2下找到。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者，及其功能性片段。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“抗体”通常是指对指定蛋白质或肽或其片段有反应性的免疫球蛋白。抗体可以是来自任何类的抗体，包括但不限于IgG、IgA、IgM、IgD和IgE，及来自任何亚类(例如IgG1、IgG2、IgG3、和IgG4)的抗体。抗体可具有选自例如IgG1、IgG2、IgG3、或IgG4的重链恒定区。抗体还可具有选自例如kappa(κ)或lambda(λ)的轻链。本申请的抗体可衍生自任何物种。
在本申请中，术语“抗原结合片段”通常是指抗体分子的某部分，该部分包含氨基酸残基，该氨基酸残基与抗原相互作用并赋予抗体对于抗原的特异性和亲和力。抗原结合片段的实例可包括但不限于Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。
在本申请中，术语“可变区”或“可变结构域”通常是指参与抗体与抗原的结合的抗体重链或轻链的结构域。在本申请中，术语“可变”通常是指，抗体的可变结构域的序列的某些部分变化强烈，形成各种特定抗体对其特定抗原的结合和特异性。变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链可变区和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)，分别为LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和 HCDR3。可变域中更高度保守的部分被称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDR结构环区连接。每条链中的CDR通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。
在本领域中，可以通过多种方法来编码抗体的可变区或划分抗体的CDR，例如基于序列可变性的Kabat编号方案和定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))，基于结构环区域位置的Chothia编号方案和定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)，efranc等人的基于种系V基因的氨基酸序列比对的IMGT编号方案和定义规则，还有Honneger’s编号方案(AHo’s)，Martin编号方案，Gelfand编号方案等，可参见Mathieu Dondelinger等人，Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition,Front.Immunol.,16 October 2018.
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即构成群体的各个抗体相同，除了可能以极小量存在的可能的天然存在突变和/或翻译后修饰(例如异构化、酰胺化)外。单克隆抗体是高度特异性的，针对单一抗原性位点。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。
在本申请中，术语“全人源抗体”通常是指所有部分(包括抗体的可变区和恒定区)均 由人类来源的基因所编码的抗体。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“结合”、“特异性结合”或“对…特异性的”通常是指可测量且可再现的相互作用，诸如抗原和抗体之间的结合，其可以确定在存在分子(包括生物学分子)的异质群体的情况中靶物的存在。例如，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更大的亲和力、亲合力、更容易和/或以更大的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。
在本申请中，术语“KD”、“K D”可互换地使用，通常是指平衡解离常数，“KD”是解离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。可使用结合速率常数(kon)、解离速率常数(kdis)和平衡解离常数(KD)表示抗原结合蛋白(例如抗体)对抗原的结合亲和力。确定结合和解离速率常数的方法为本领域熟知，包括但不限于生物膜干涉技术(BLI)、放射免疫法(RIA)、平衡透析法、表面等离子共振(SPR)、荧光共振能量迁移(FRET)、免疫共沉淀(Co-IP)以及蛋白质芯片技术。如果在不同的条件(例如盐浓度、pH)下测量，则所测得的某种特定蛋白-蛋白相互作用的亲和力可不同。
在本申请中，术语“灵长类动物”通常是指猴和猿物种，并包括猴物种，诸如来自弥猴属(例如，食蟹猴(Macaca fascicularis)和或恒河猴(Macaca mulatta))和狒狒(豚尾狒狒(Papio ursinus))的猴，以及狨猴(来自狨(Callithrix)属的物种)，松鼠猴(来自松鼠猴(Saimiri)属的物种)和绢毛猴(来自柽柳猴(Saguinus)属的物种)，以及猿物种，诸如黑猩猩(Pan troglodytes)，并且还包括智人(Homo sapiens)。
在本申请中，术语“多肽”或“蛋白质”可互换地使用，通常是指氨基酸残基的聚合物。该术语也适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的类似物或模拟物的氨基酸聚合物、以及天然存在的氨基酸聚合物。该术语也可包括修饰的氨基酸聚合物，例如，通过添加糖残基以形成糖蛋白或被磷酸化修饰。多肽和蛋白质可由天然存在的和非重组的细胞或由遗传工程改造的或重组的细胞产生，并且可包含具有天然蛋白质的氨基酸序列的分子、或具有天然序列的一个或多个氨基酸的缺失、添加和/或取代的分子。术语“多肽”和“蛋白质”特别包括本申请所述的抗原结合蛋白的一个或多个氨基酸的缺失、添加和/或取代的序列。
在本申请中，术语“分离的”通常是指大体上不含其天然存在的环境中通常伴随或与之相互作用的组分的生物材料(例如病毒、核酸或蛋白质)。所述分离的生物材料任选地包含在其天然环境(例如，核酸或蛋白质)中所述生物材料未发现具有的另外的材料。在本申请中，当涉及蛋白质时，“分离”通常是指所述的分子从发现该分子天然存在的整个生物体中分离和分开，或基本不存在其它相同类型的生物大分子。当涉及核酸分子时，它与天然与其结合的序列完全或部分分离，或该核酸具有与其结合的异源序列，或该核算从染色体分离。
在本申请中，术语“免疫缀合物”通常是指抗原结合蛋白与其它活性剂连接形成的物质，其他活性剂可以是小分子活性剂，例如化疗剂、毒素、免疫治疗剂、成像探针或光谱探针。
在本申请中，术语“核酸”分子通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以包含或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗原结合蛋白的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。
在本申请中，术语“药物组合物”通常是指这样的制剂，其以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。
在本申请中，术语“治疗”通常是指期望改变所治疗个体的天然病程，且可为实现防治或在临床病变过程中进行的临床介入。合乎需要的治疗效果包括但不限于防止疾病发生或复 发性、减轻症状、减弱疾病的任何直接或间接病理学后果、防止转移、降低疾病进展速率、改善或缓解疾病状态以及缓和或改善预后。在一些情形中，抗体(例如，抗PD-1抗体)可用来延迟疾病发展或减缓疾病进展。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的化合物(例如，抗癌治疗剂)或药物组合物(例如，包含抗癌治疗剂的药物组合物)的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。胃肠外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。
在本申请中，术语“肿瘤”通常是指所有赘生性细胞生长和增殖(无论恶性还是良性)以及所有癌前和癌性细胞和组织。在本申请中，所述肿瘤可以为细胞和组织的PD-1或PD-L1高表达的肿瘤。肿瘤可包括实体瘤和/或非实体瘤(例如，血液瘤、淋巴瘤)。
在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
在本申请中，术语“在……之间”通常是指某种氨基酸片段的C端与第一氨基酸片段的N端直接或间接连接，并且其N端与第二氨基酸片段的C端直接或间接连接。在轻链中，例如，所述L-FR2的N末端与所述LCDR1的C末端直接或间接相连，且所述L-FR2的C末端与所述LCDR2的N末端直接或间接相连。又例如，所述L-FR3的N末端与所述LCDR2的C末端直接或间接相连，且所述L-FR3的C末端与所述LCDR3的N末端直接或间接相连。在重链中，例如，所述H-FR2的N末端与所述HCDR1的C末端直接或间接相连，且所述H-FR2的C末端与所述HCDR2的N末端直接或间接相连。又例如，所述H-FR3的N末端与所述HCDR2的C末端直接或间接相连，且所述H-FR3的C末端与所述HCDR3的N末端直接或间接相连。在本申请中，“第一氨基酸片段”和“第二氨基酸片段”可以为相同或不同的任意一段氨基酸片段。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白，本申请中，所述的分离的抗原结合蛋白能够以1×10 -8M或更低的KD值结合源自灵长类动物的PD-1。所述灵长类动物PD-1抗原结合蛋白对PD-1的结合亲和力可通过本领域已知的任何方法测定。在某些情形中，结合亲和力可通过表面等离子共振法(SPR)、酶联免疫法(ELISA)、结合抗原沉淀法、平衡透析法、生物膜干涉(BLI)测定。在某些情形中，PD-1抗原结合蛋白对PD-1的结合亲和力和KD值可通过生物膜干涉(BLI)测定。例如，可使用ForteBio Octet分子相互作用分析仪，来进行抗原抗体之间的结合动力学分析。
本申请中，所述分离的抗原结合蛋白能够以1×10 -8M或更低的KD值结合源自灵长类动物的PD-1。例如，所述KD的值可以以约1×10 -8M或以下、约9×10 -9M或以下、约8×10 -9M或以下、约7×10 -9M或以下、约6×10 -9M或以下、约5×10 -9M或以下、约4×10 -9M或以下、约3×10 -9M或以下、约2×10 -9M或以下、约1×10 -9M或以下的值结合源自人的PD-L1，例如，使用FortieBio Octet分子相互作用分析仪所检测的。
在另一情形中，本申请所述PD-1抗原结合蛋白与PD-1的结合活性可使用流式细胞术或酶联免疫法测定进行检测。
例如，在FACS检测中，使用稳定表达人PD-1的宿主细胞(如CHOK1细胞)，所述PD-1抗原结合蛋白结合PD-1的EC50值在约0.0001nM至约100nM之间，例如，约0.001nM至约10nM之间，约0.001nM至约5nM之间，约0.001nM至约1nM之间，约0.01nM至约1nM之间，约0.01nM至约1.2nM之间，或，约0.1nM至约1.5nM之间。
例如，在FACS检测中，使用稳定表达猴PD-L1的宿主细胞(如CHOK1细胞)，所述PD-L1抗原结合蛋白结合PD-L1的EC50值在约0.0001nM至约100nM之间，例如，约0.001nM至约10nM之间，约0.001nM至约5nM之间，约0.01nM至约1nM之间，约0.02nM至约1.0nM之间，约0.1nM至约1.0nM之间。
在另一个方面，本申请所述的抗原结合蛋白能够阻断PD-1与PD-L1的结合。在某些情形中，所述的抗原结合蛋白阻断PD-1与PD-L1的结合可通过流式细胞技术FACS、酶联免疫 法ELISA测定。
例如，首先将稳定表达人PD-1的宿主细胞(如HEK293细胞)与递减量的未标记的所述抗原结合蛋白孵育，随后用生物素标记的PD-L1蛋白孵育。然后，使用FACS分析细胞，以证实所述抗原结合蛋白阻断PD-1与PD-L1结合。例如，IC50值约0.001nM至约10nM之间，约0.001nM至约5nM之间，约0.01nM至约5nM之间，约0.1nM至约1.0nM之间，约0.5nM至约1.5nM之间。
例如，首先将稳定表达人PD-1的宿主细胞(如CHOK1细胞)与递减量的未标记的所述抗原结合蛋白孵育，随后用生物素标记的PD-L2蛋白孵育。然后，使用FACS分析细胞，以证实所述抗原结合蛋白阻断PD-1与PD-L2结合。例如，约0.001nM至约10nM之间，约0.001nM至约5nM之间，约0.1nM至约2.5nM之间，约0.1nM至约3nM之间。
在本申请中，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含氨基酸序列如SEQ ID NO:65所示的VH的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含氨基酸序列如SEQ ID NO:15或25所示的VH的CDR3。例如，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含如SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含氨基酸序列如SEQ ID NO:65所示的VH的CDR2。
在本申请中，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含氨基酸序列如SEQ ID NO:15或25所示的VH的CDR2。例如，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含如SEQ ID NO:2所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含氨基酸序列如SEQ ID NO:86所示的VH的CDR2。例如，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含如SEQ ID NO:85所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含氨基酸序列如SEQ ID NO:65所示的VH的CDR1。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含氨基酸序列如SEQ ID NO:15或25所示的VH的CDR1。例如，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含如SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含氨基酸序列如SEQ ID NO:65所示的VH的CDR1，所述HCDR2可包含氨基酸序列如 SEQ ID NO:65所示的VH的CDR2，所述HCDR3可包含氨基酸序列如SEQ ID NO:65所示的VH的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含氨基酸序列如SEQ ID NO:15或25所示的VH的CDR1，所述HCDR2可包含氨基酸序列如SEQ ID NO:15或25所示的VH的CDR2，所述HCDR3可包含氨基酸序列如SEQ ID NO:15或25所示的VH的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含氨基酸序列如SEQ ID NO:86所示的VH的CDR1，所述HCDR2可包含氨基酸序列如SEQ ID NO:86所示的VH的CDR2，所述HCDR3可包含氨基酸序列如SEQ ID NO:86所示的VH的CDR3。
例如，所述分离的抗原结合蛋白可包含氨基酸序列如SEQ ID NO:15所示的VH的CDR1，可包含氨基酸序列如SEQ ID NO:15所示的VH的CDR2，且包含氨基酸序列如SEQ ID NO:15所示的VH的CDR3。
例如，所述分离的抗原结合蛋白可包含氨基酸序列如SEQ ID NO:25所示的VH的CDR1，可包含氨基酸序列如SEQ ID NO:25所示的VH的CDR2，且包含氨基酸序列如SEQ ID NO:25所示的VH的CDR3。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:2所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:3所示的氨基酸序列。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:85所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR1，所述H-FR1可包含如SEQ ID NO:57所示的氨基酸序列：EVX 3LVESGGGLVKPGGSLX 19LX 21CAAS(SEQ ID NO:57)，其中，X 3为K或Q，X 19为K或R，且X 21为A或S。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:4所示的氨基酸序列相比，所述H-FR1可至少包含在选自下组位置处的氨基酸取代：在X 3、X 19和/或X 21处的氨基酸取代。例如，所述H-FR1可包含如SEQ ID NO:4或17所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR2，所述H-FR2可包含如SEQ ID NO:59所示的氨基酸序列：DMSWVRQX 8PX 10KX 12LEWVX 17TI(SEQ ID NO:59)，其中，X 8为 A或T，X 10为E或G，X 12为G或R，且X 17为A或S。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:5所示的氨基酸序列相比，所述H-FR2可至少包含在选自下组位置处的氨基酸取代：在X 8、X 10、X 12和/或X 17处的氨基酸取代。例如，所述H-FR2可包含如SEQ ID NO:5或18所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR3，所述H-FR3可包含如SEQ ID NO:61所示的氨基酸序列：
TYYX 4DSVKGRFTISRDNAKNX 21LYLQMX 27SLRX 31EDTAX 36YYCVS(SEQ ID NO:61)，其中，X 4为A或P，X 21为S或N，X 27为N或S，X 31为A或S，且X 36为L或V。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:6所示的氨基酸序列相比，所述H-FR3可至少包含在选自下组位置处的氨基酸取代：在X 4、X 21、X 27、X 31和/或X 36处的氨基酸取代。
例如，所述H-FR3可包含如SEQ ID NO:6或19所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR4，所述H-FR4可包含如SEQ ID NO:63所示的氨基酸序列：WGQGTX 6VTVSS(SEQ ID NO:63)，其中，X 6为L或S。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:7所示的氨基酸序列相比，所述H-FR3可至少包含在X 6处的氨基酸取代。例如，所述H-FR4可包含如SEQ ID NO:7或20所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH，所述VH可包含SEQ ID NO:65所示的氨基酸序列：
EVX 3LVESGGGLVKPGGSLX 19LX 21CAASGFTFSNYDMSWVRQX 40PX 42KX 44LEWVX 49TISGGGSYTYYX 61DSVKGRFTISRDNAKNX 78LYLQMX 84SLRX 88EDTAX 93YYCVSPYYGMEYWGQGTX 111VTVSS(SEQ ID NO:65)，其中，X 3为K或Q，X 19为K或R，X 21为A或S，X 40为A或T，X 42为E或G，X 44为G或R，X 49为A或S，X 61为A或P，X 78为N或S，X 84为S或N，X 88为A或S，X 93为L或V，且X 111为L或S。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:15所示的氨基酸序列相比，所述VH可至少包含在选自下组位置处的氨基酸取代：在X 3、X 19、X 21、X 40、X 42、X 44、X 49、X 61、X 78、X 84、X 88、X 93和/或X 111处的氨基酸取代。
例如，所述VH可包含SEQ ID NO:15或25所示的氨基酸序列。例如，所述VH可包含SEQ ID NO:86所示的氨基酸序列。
本申请的分离的抗原结合蛋白可包含LCDR3，所述LCDR3可包含氨基酸序列如SEQ ID NO:67所示的VL的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含LCDR3，所述LCDR3可包含氨基酸序列如SEQ ID NO:16或26所示的VL的CDR3。例如，所述分离的抗原结合蛋白可包含LCDR3，所述LCDR3可包含如SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR2，所述LCDR2可包含氨基酸序列如SEQ ID NO:67所示的VL的CDR2。
在本申请中，所述分离的抗原结合蛋白可包含LCDR2，所述LCDR2可包含氨基酸序列如SEQ ID NO:16或26所示的VL的CDR2。例如，所述分离的抗原结合蛋白可包含LCDR2，所述LCDR2可包含如SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1，所述LCDR1可包含氨基酸序列如SEQ ID NO:67所示的VL的CDR1。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1，所述LCDR1可包含氨基酸序列如SEQ ID NO:16或26所示的VL的CDR1。例如，所述分离的抗原结合蛋白可包含LCDR1，所述LCDR1可包含如SEQ ID NO:8所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含氨基酸序列如SEQ ID NO:67所示的VL的CDR1，所述LCDR2可包含氨基酸序列如SEQ ID NO:67所示的VL的CDR2，所述LCDR3可包含氨基酸序列如SEQ ID NO:67所示的VL的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含氨基酸序列如SEQ ID NO:16或26所示的VL的CDR1，所述LCDR2可包含氨基酸序列如SEQ ID NO:16或26所示的VL的CDR2，所述LCDR3可包含氨基酸序列如SEQ ID NO:16或26所示的VL的CDR3。
例如，所述分离的抗原结合蛋白可包含氨基酸序列如SEQ ID NO:16所示的VL的CDR1，可包含氨基酸序列如SEQ ID NO:16所示的VL的CDR2，且包含氨基酸序列如SEQ ID NO:16所示的VL的CDR3。
例如，所述分离的抗原结合蛋白可包含氨基酸序列如SEQ ID NO:26所示的VL的CDR1，可包含氨基酸序列如SEQ ID NO:26所示的VL的CDR2，且包含氨基酸序列如SEQ ID NO:26所示的VL的CDR3。
例如，本申请所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1 可包含如SEQ ID NO:8所示的氨基酸序列，所述LCDR2可包含如SEQ ID NO:9所示的氨基酸序列，且所述LCDR3可包含如SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR1，所述L-FR1可包含如SEQ ID NO:69所示的氨基酸序列：DIX 3X 4TQSX 8X 9FX 11SX 13SVGDRVX 20TC(SEQ ID NO:69)，其中，X 3为Q或V，X 4为L或M，X 8为P或H，X 9为K或S，X 11为L或M，X 13为A或T，且X 20为S或T。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:11所示的氨基酸序列相比，所述L-FR1可至少包含在选自下组位置处的氨基酸取代：在X 3、X 4、X 8、X 9、X 11、X 13和/或X 20处的氨基酸取代。
例如，所述L-FR1可包含如SEQ ID NO:11或21所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR2，所述L-FR2可包含如SEQ ID NO:71所示的氨基酸序列：WYQQKPGX 8X 9PKLLIY(SEQ ID NO:71)，其中，X 8为K或Q，且X 9为A或S。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:12所示的氨基酸序列相比，所述L-FR2可至少包含在选自下组位置处的氨基酸取代：在X 8和/或X 9处的氨基酸取代。
例如，所述L-FR2可包含如SEQ ID NO:12或22所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR3，所述L-FR3可包含如SEQ ID NO:73所示的氨基酸序列：
GVPX 4RFX 7GSGSGTX 14FTLTISX 21X 22QX 24EDX 27AX 29YFC(SEQ ID NO:73)，其中，X 4为D或S，X 7为S或T，X 14为D或E，X 21为N或S，X 22为L或V，X 24为P或S，X 27为F或L，且X 29为D或T。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:13所示的氨基酸序列相比，所述L-FR3可至少包含在选自下组位置处的氨基酸取代：在X 4、X 7、X 14、X 21、X 22、X 24、X 27和/或X 29处的氨基酸取代。
例如，所述L-FR3可包含如SEQ ID NO:13或23所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR4，所述L-FR4可包含如SEQ ID NO:75所示的氨基酸序列：FGGGTKX7EX9K(SEQ ID NO:75)，其中，X 7为L或V，且X 9为I或V。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:14所示的氨基酸序列相比，所述L-FR4可至少包含在选自下组位置处的氨基酸取代：在X 7和/或X 9处的氨基酸取代。
例如，所述L-FR4可包含如SEQ ID NO:14或24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL，所述VL可包含SEQ ID  NO:67所示的氨基酸序列：
DIX 3X 4TQSX 8X 9FX 11SX 13SVGDRVX 20ITCKASQDVGTAVAWYQQKPGX 42X 43PKLLIYWASTRHTGVPX 60RFX 60GSGSGTX 70FTLTISX 77X 78QX 80EDX 83AX 85YFCQQYSSYPWTFGGGTKX 104EX 106K(SEQ ID NO:67)，其中，X 3为Q或V，X 4为L或M，X 8为P或H，X 9为K或S，X 11为L或M，X 13为A或T，且X 20为S或T，X 42为K或Q，X 43为A或S，X 60为D或S，X 63为S或T，X 70为D或E，X 77为N或S，X 78为L或V，X 80为P或S，X 83为F或L，且X 85为D或T，X 104为L或V，且X 106为I或V。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:16所示的氨基酸序列相比，所述VL可至少包含在选自下组位置处的氨基酸取代：在X 3、X 4、X 8、X 9、X 11、X 13、X 20、X 42、X 43、X 60、X 63、X 70、X 77、X 78、X 80、X 83、X 85、X 104和/或X 106处的氨基酸取代。
例如，所述VL可包含SEQ ID NO:16或26所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1可包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:2所示的氨基酸序列，所述HCDR3可包含如SEQ ID NO:3所示的氨基酸序列，所述LCDR1可包含如SEQ ID NO:8所示的氨基酸序列，所述LCDR2可包含如SEQ ID NO:9所示的氨基酸序列，且所述LCDR3可包含如SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1可包含如SEQ ID NO:1所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:85所示的氨基酸序列，所述HCDR3可包含如SEQ ID NO:3所示的氨基酸序列，所述LCDR1可包含如SEQ ID NO:8所示的氨基酸序列，所述LCDR2可包含如SEQ ID NO:9所示的氨基酸序列，且所述LCDR3可包含如SEQ ID NO:10所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:65所示的氨基酸序列，所述VL可包含SEQ ID NO:67所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:15或25所示的氨基酸序列，所述VL可包含SEQ ID NO:16或26所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:15所示的氨基酸序列，所述VL可包含SEQ ID NO:16所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:25所示的氨基酸序列，所述VL可包含SEQ ID NO:26所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:86所 示的氨基酸序列，所述VL可包含SEQ ID NO:26所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链恒定区，所述重链恒定区可以来自IgG。例如，所述重链恒定区可以来自人IgG。所述重链恒定区可以来自IgG1、IgG2、IgG3和IgG4。例如，所述重链恒定区可以来自人IgG4。与天然的人IgG4相比，所述重链恒定区可以是氨基酸突变的。例如，所述重链恒定区可以是IgG4进行S228P氨基酸点突变后的序列。例如，所述重链恒定区可以包含SEQ ID NO:77或81所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链，所述重链可包含SEQ ID NO:79所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链，所述重链可包含SEQ ID NO:87所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链恒定区，所述轻链恒定区可以来自轻链λ和轻链κ。例如，所述轻链恒定区可以包含SEQ ID NO:78或82所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链，所述轻链可包含SEQ ID NO:80或84所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:79所示的氨基酸序列，所述轻链可包含SEQ ID NO:80所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:83所示的氨基酸序列，所述轻链可包含SEQ ID NO:84所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:87所示的氨基酸序列，所述轻链可包含SEQ ID NO:84所示的氨基酸序列。
本申请的分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含氨基酸序列如SEQ ID NO:66所示的VH的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含氨基酸序列如SEQ ID NO:41或51所示的VH的CDR3。例如，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含如SEQ ID NO:29所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含氨基酸序列如SEQ ID NO:66所示的VH的CDR2。
在本申请中，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含氨基酸序列如SEQ ID NO:41或51所示的VH的CDR2。例如，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含如SEQ ID NO:28所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含氨基酸序列如SEQ ID NO:66所示的VH的CDR1。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含氨基酸序列如SEQ ID NO:41或51所示的VH的CDR1。例如，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含如SEQ ID NO:27所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含氨基酸序列如SEQ ID NO:66所示的VH的CDR1，所述HCDR2可包含氨基酸序列如SEQ ID NO:66所示的VH的CDR2，所述HCDR3可包含氨基酸序列如SEQ ID NO:66所示的VH的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含氨基酸序列如SEQ ID NO:41或51所示的VH的CDR1，所述HCDR2可包含氨基酸序列如SEQ ID NO:41或51所示的VH的CDR2，所述HCDR3可包含氨基酸序列如SEQ ID NO:41或51所示的VH的CDR3。
例如，所述分离的抗原结合蛋白可包含氨基酸序列如SEQ ID NO:41所示的VH的CDR1，可包含氨基酸序列如SEQ ID NO:41所示的VH的CDR2，且包含氨基酸序列如SEQ ID NO:41所示的VH的CDR3。
例如，所述分离的抗原结合蛋白可包含氨基酸序列如SEQ ID NO:51所示的VH的CDR1，可包含氨基酸序列如SEQ ID NO:51所示的VH的CDR2，且包含氨基酸序列如SEQ ID NO:51所示的VH的CDR3。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:27所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:28所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:29所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR1，所述H-FR1可包含如SEQ ID NO:58所示的氨基酸序列：QVQLX 5QSGAEX 11X 12X 13PGASVKX 20SCKAX 25GFTFI(SEQ ID NO:58)，其中，X 5为V或Q，X 11为L或V，X 12为K或V，X 13为K或R，X 20为L或V，且X 25为L或S。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:30所示的氨基酸序列相比，所述H-FR1可至少包含在选自下组位置处的氨基酸取代：在X 5、X 11、X 12、X 13、X 20和/或X 25处的氨基酸取代。
例如，所述H-FR1可包含如SEQ ID NO:30或43所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR2，所述H-FR2可包含如SEQ ID NO: 60所示的氨基酸序列：WVRQX5PX7X8GLEWIG(SEQ ID NO:60)，其中，X 5为A或T，X 7为V或G，且X 8为H或Q。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:31所示的氨基酸序列相比，所述H-FR2可至少包含在选自下组位置处的氨基酸取代：在X 5和/或X 8处的氨基酸取代。
例如，所述H-FR2可包含如SEQ ID NO:31或44所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR3，所述H-FR3可包含如SEQ ID NO:62所示的氨基酸序列：
X 1ATLX 5ADISX 10X 11TAX 14MEX 17SX 19LX 21SX 23DX 25AVYYCTR(SEQ ID NO:62)，其中，X 1为K或R，X 5为I或T，X 10为T或I，X 11为N或S，X 14为S或Y，X 17为L或V，X 19为R或S，X 21为R或T，X 23为D或E，且X 25为S或T。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:32所示的氨基酸序列相比，所述H-FR3可至少包含在选自下组位置处的氨基酸取代：在X 1、X 5、X 10、X 11、X 14、X 17、X 19、X 21、X 23和/或X 25处的氨基酸取代。
例如，所述H-FR3可包含如SEQ ID NO:32或45所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR4，所述H-FR4可包含如SEQ ID NO:64所示的氨基酸序列：WGX3GTTVTVSS(SEQ ID NO:64)，其中，X 3为A或Q。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:33所示的氨基酸序列相比，所述H-FR4可至少包含在选自下组位置处的氨基酸取代：在X 3处的氨基酸取代。
例如，所述H-FR4可包含如SEQ ID NO:33或46所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH，所述VH可包含SEQ ID NO:66所示的氨基酸序列：
QVQLX 5QSGAEX 11X 12X 13PGASVKX 20SCKAX 25GFTFIDYEMHWVRQX 40PX 42X 43GLEWIGGIHPGSGGTAYNQKFKGX 67ATLX 71ADISX 76X 77TAX 80MEX 83SX 85LX 87SX 89DX 91AVYYCTREGFDVGWYFDVWGX 112GTTVTVSS(SEQ ID NO:66)，其中，X 5为V或Q，X 11为L或V，X 12为K或V，X 13为K或R，X 20为L或V，X 25为L或S，X 40为A或T，X 42为V或G，X 43为H或Q，X 67为K或R，X 71为I或T，X 76为T或I，X 77为N或S，X 80为S或Y，X 83为L或V，X 85为R或S，X 87为R或T，X 89为D或E，X 91为S或T，且X 112为A或Q。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:41所示的氨基酸序列相比，所述VH可至少包含在选自 下组位置处的氨基酸取代：在X 5、X 11、X 12、X 13、X 20、X 25、X 40、X 42、X 43、X 67、X 71、X 76、X 77、X 80、X 83、X 85、X 87、X 89、X 91和/或X 112处的氨基酸取代。
例如，所述VH可包含SEQ ID NO:41或51所示的氨基酸序列。
本申请的分离的抗原结合蛋白可包含LCDR3，所述LCDR3可包含氨基酸序列如SEQ ID NO:68所示的VL的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含LCDR3，所述LCDR3可包含氨基酸序列如SEQ ID NO:52或42所示的VL的CDR3。例如，所述分离的抗原结合蛋白可包含LCDR3，所述LCDR3可包含如SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR2，所述LCDR2可包含氨基酸序列如SEQ ID NO:68所示的VL的CDR2。
在本申请中，所述分离的抗原结合蛋白可包含LCDR2，所述LCDR2可包含氨基酸序列如SEQ ID NO:52或42所示的VL的CDR2。例如，所述分离的抗原结合蛋白可包含LCDR2，所述LCDR2可包含如SEQ ID NO:35所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1，所述LCDR1可包含氨基酸序列如SEQ ID NO:68所示的VL的CDR1。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1，所述LCDR1可包含氨基酸序列如SEQ ID NO:52或42所示的VL的CDR1。例如，所述分离的抗原结合蛋白可包含LCDR1，所述LCDR1可包含如SEQ ID NO:34所示的氨基酸序列。
例如，本申请所述分离的抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含如SEQ ID NO:34所示的氨基酸序列，所述LCDR2可包含如SEQ ID NO:35所示的氨基酸序列，且所述LCDR3可包含如SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR1，所述L-FR1可包含如SEQ ID NO:70所示的氨基酸序列：DVX 3MTQX 7PLSLPVX 14LGX 17X 18ASISC(SEQ ID NO:70)，其中，X 3为L或V，X 7为S或T，X 14为S或T，X 17为D或Q，且X 18为Q或P。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:37所示的氨基酸序列相比，所述L-FR1可至少包含在选自下组位置处的氨基酸取代：在X 3、X 7、X 14、X 17和/或X 18处的氨基酸取代。
例如，所述L-FR1可包含如SEQ ID NO:37或47所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR2，所述L-FR2可包含如SEQ ID NO:72所示的氨基酸序列：WYX 3QX 5PGQSPX 11LLIY(SEQ ID NO:72)，其中，X 3为L或Q，X 5 为K或R，且X 11为K或R。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:38所示的氨基酸序列相比，所述L-FR2可至少包含在选自下组位置处的氨基酸取代：在X 3、X 5和/或X 11处的氨基酸取代。
例如，所述L-FR2可包含如SEQ ID NO:38或48所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR3，所述L-FR3可包含如SEQ ID NO:74所示的氨基酸序列：
GVPDRFSGSGSGTDFTLKISRVEX 24EDX 27GVYYC(SEQ ID NO:74)，其中，X 24为A或Q，且X 27为L或V。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:39所示的氨基酸序列相比，所述L-FR3可至少包含在选自下组位置处的氨基酸取代：在X 24和/或X 27处的氨基酸取代。
例如，所述L-FR3可包含如SEQ ID NO:39或49所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含L-FR4，所述L-FR4可包含如SEQ ID NO:76所示的氨基酸序列：FGX 3GTKLEIK(SEQ ID NO:76)，其中，X 3为G或Q。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:40所示的氨基酸序列相比，所述L-FR4可至少包含在X 3处的氨基酸取代。
例如，所述L-FR4可包含如SEQ ID NO:40或50所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL，所述VL可包含SEQ ID NO:68所示的氨基酸序列：
DVX 3MTQX 7PLSLPVX 14LGX 17X 18ASISCRSSQSIVHSDGDTFLEWYX 42QX 44PGQSPX 50LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEX 85EDX 88GVYYCFQGSHVPYTFGX 105GTKLEIK(SEQ ID NO:68)，其中X 3为L或V，X 7为S或T，X 14为S或T，X 17为D或Q，X 18为Q或P，X 42为L或Q，X 44为K或R，且X 50为K或R，X 85为A或Q，X 88为L或V，且X 105为G或Q。例如，可根据Chothia规则划分。
在某些情形中，与SEQ ID NO:42所示的氨基酸序列相比，所述VL可至少包含在选自下组位置处的氨基酸取代：在X 3、X 7、X 14、X 17、X 18、X 42、X 44、X 50、X 85、X 88和/或X 105处的氨基酸取代。
例如，所述VL可包含SEQ ID NO:42或52所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1可包含如SEQ ID NO:27所示的氨基酸序列，所述HCDR2可包 含如SEQ ID NO:28所示的氨基酸序列，所述HCDR3可包含如SEQ ID NO:29所示的氨基酸序列，所述LCDR1可包含如SEQ ID NO:34所示的氨基酸序列，所述LCDR2可包含如SEQ ID NO:35所示的氨基酸序列，且所述LCDR3可包含如SEQ ID NO:36所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:66所示的氨基酸序列，所述VL可包含SEQ ID NO:68所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:41或51所示的氨基酸序列，所述VL可包含SEQ ID NO:42或52所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:41所示的氨基酸序列，所述VL可包含SEQ ID NO:42所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VH和VL，且所述VH可包含SEQ ID NO:51所示的氨基酸序列，所述VL可包含SEQ ID NO:52所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链恒定区，所述重链恒定区可以来自IgG。例如，所述重链恒定区可以来自人IgG。所述重链恒定区可以来自IgG1、IgG2、IgG3和IgG4。例如，所述重链恒定区可以来自人IgG4。与天然的人IgG4相比，所述重链恒定区可以是氨基酸突变的。例如，所述重链恒定区可以是IgG4进行S228P氨基酸点突变后的序列。例如，所述重链恒定区可以包含SEQ ID NO:77或81所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链，所述重链可包含SEQ ID NO:79所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链，所述重链可包含SEQ ID NO:87所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链恒定区，所述轻链恒定区可以来自轻链λ和轻链κ。例如，所述轻链恒定区可以包含SEQ ID NO:78或82所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含轻链，所述轻链可包含SEQ ID NO:80或84所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:79所示的氨基酸序列，所述轻链可包含SEQ ID NO:80所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:83所示的氨基酸序列，所述轻链可包含SEQ ID NO:84所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含重链和轻链，所述重链可包含SEQ ID NO:87所 示的氨基酸序列，所述轻链可包含SEQ ID NO:84所示的氨基酸序列。
在本申请中，所述抗原结合蛋白每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别。例如，轻链和重链的可变区及恒定部分均来自一个动物物种(如人)的抗体的可变区及恒定区。在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，特异性结合PD-L1蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
检测方法
可通过本领域已知的各种测定鉴别、筛选或表征本申请所述的PD-1抗原结合蛋白的物理/化学特性和/或生物活性。
在一个方面，例如可通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请抗原结合蛋白的抗原结合活性。
核酸、载体、宿主细胞和制备方法
在另一个方面，本申请还提供了分离的一种或多种核酸分子。所述一种或多种核酸分子可编码本申请所述的抗原结合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体、其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。例如，所述载体为表达载体。
在另一个方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体
在另一个方面，本申请提供了制备所述的抗体或其抗原结合片段的方法。所述方法可包括，在使得所述的抗体或其抗原结合片段表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
药物组合物、方法、用途
在另一个方面，本申请提供了一种药物组合物，其可包含本申请所述的抗原结合蛋白，所述多肽，所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的载体。所述药学上可接受的佐剂在所采用的剂量和浓度下对接受者无毒性，本申请中的药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。
所述药物组合物可以用于抑制肿瘤生长。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述抗体、其抗原结合片段。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试 者中的疾病或病症和/或其任何并发症而所需的剂量。
另一方面，本申请提供了所述抗原结合蛋白和/或所述融合蛋白在制备药物中的用途。所述药物用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。在某些实施方式中，所述肿瘤或癌症为PD-L1表达异常和/或PD-1表达异常的肿瘤或癌症。在某些实施方式中，所述肿瘤包括PD-1或PD-L1高表达的肿瘤。在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。在某些实施方式中，所述肿瘤包括黑色素瘤、肺癌、肾癌、食管癌、头颈癌、淋巴瘤、肝癌和/或胃癌。
另一方面，本申请提供了抑制PD-L1与PD-1结合的方法，包括施用本申请所述的抗原结合蛋白和/或所述多肽。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。在某些情形中，所述方法可包括使生物样品与本申请所述的抗原结合蛋白和/或PD-1在容许所述抗原结合蛋白和/或PD-1结合PD-L1的条件下接触，检测在所述抗原结合蛋白与PD-L1之间是否形成复合物，和检测PD-1与PD-L1之间是否形成复合物。
另一方面，本申请提供了抑制PD-L2与PD-1结合的方法，包括施用本申请所述的抗原结合蛋白和/或所述多肽。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。在某些情形中，所述方法可包括使生物样品与本申请所述的抗原结合蛋白和/或PD-1在容许所述抗原结合蛋白和/或PD-1结合PD-L2的条件下接触，检测在所述抗原结合蛋白与PD-L2之间是否形成复合物，和检测PD-1与PD-L2之间是否形成复合物。
另一方面，本申请提供了刺激免疫细胞分泌细胞因子的方法，其包括施用所述分离的抗原结合蛋白和/或所述的多肽。所述细胞因子可以是IL-2。所述免疫细胞可以是淋巴细胞。例如，T淋巴细胞。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。
另一方面，本申请提供了一种用于检测PD-1蛋白的存在和/或含量的方法，其包括施用所述分离的抗原结合蛋白和/或所述的多肽。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。
本申请还提供了抗原结合蛋白在诊断患有肿瘤或癌症的受试者的方法中的用途，所述方法包括：通过使样品与本申请的抗原结合蛋白接触并检测结合的抗体的存在来确定获自受试者的样品中PD-1的存在或表达水平。
另一方面，本申请提供了一种嵌合抗原受体(CAR)，其可以包含本申请所述的核酸分子或本申请所述的抗原结合蛋白。
另一方面，本申请提供了一种抗体药物偶联物，其可以包含细胞毒性剂，以及本申请所 述的抗原结合片段。抗体药物偶联物通常是指采用特定的连接子将抗体和小分子细胞毒药物连接起来，其主要组成成分可以包括抗体、连接子和小分子细胞毒药物。
另一方面，本申请提供了一种试剂盒，其可以包含本申请所述的抗原结合蛋白，嵌合抗原受体，基因修饰的细胞，抗体药物偶联物，和/或本申请所述的药物组合物。其可在单一常用容器中包括本申请所述的抗原结合蛋白，嵌合抗原受体，基因修饰的细胞，和/或抗体药物偶联物，也可任选地与一种或多种治疗剂组合，任选地一起配制于药物组合物中。
另一方面，本申请提供了一种给药装置，它可以用来施用本申请所述的抗原结合蛋白或其药物组合物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1 抗PD-1的人源化抗体制备与检测
1.1杂交瘤抗体发现
用Human PD-1,Mouse IgG2a Fc Tag融合蛋白(Acrobiosystem，PD1-H5255a)与pcDNA3.4-hPD-1质粒DNA免疫Balb/c和SJL小鼠各5只，每次间隔2～3周，3次后用Human PD-1,Mouse IgG2a Fc Tag融合蛋白或CHOK1-hPD-1稳转细胞株(金斯瑞，M00529)做加强免疫1-3次，采血测效价。效价检测采用流式细胞仪测定血清与过表达人PD-1蛋白的CHOK1或293T细胞结合，取结合效价高的小鼠脾细胞与骨髓瘤(Sp2/0)细胞进行融合。
利用流式细胞仪从融合板中筛选出与CHOK1-hPD-1细胞特异性结合并能同时阻断配体PD-L1、PD-L2与细胞CHOK1-hPD1结合的克隆，利用有限稀释法进行亚克隆，最终筛选到可分泌特异性抗体的单克隆。采用无血清培养基进行小规模生产纯化得到单克隆抗体做后续鉴定，包含检测抗体与CHOK1-hPD1的结合能力、对PD-L1、PD-L2与CHOK1-hPD-1结合的阻断功能以及混合淋巴细胞实验最终得到候选抗人PD-1单克隆抗体41D2-2C3D7与46H3A8。经单克隆抗体测序获得鼠源杂交瘤克隆41D2-2C3D7与46H3A8的可变区序列如下：
>41D2-2C3D7重链可变区序列
Figure PCTCN2022075593-appb-000001
>41D2-2C3D7轻链可变区序列
Figure PCTCN2022075593-appb-000002
>46H3A8重链可变区序列
Figure PCTCN2022075593-appb-000003
>46H3A8轻链可变区序列
Figure PCTCN2022075593-appb-000004
表1 鼠源杂交瘤克隆41D2-2C3D7重链及轻链CDR区序列
Figure PCTCN2022075593-appb-000005
表2 鼠源杂交瘤克隆46H3A8重链及轻链CDR区序列
Figure PCTCN2022075593-appb-000006
实施例2 单克隆抗体人源化
2.1鼠源杂交瘤抗体人源化
通过序列比对挑选最同源的人Germline抗体(数据来源：IMGT)做为人源化设计框架(轻链以IGKV1-9*01，IGKJ4*01为框架，重链以IGHV3-21*01，IGHJ5*01，为框架)，对抗体轻重链可变区进行Chothia编号[Chothia&Lesk,1987]，定义抗体CDR区：LCDR1(L24-L34)，LCDR2(L50-L56)，LCDR3(L89-L97)，HCDR1(H26-H32)，HCDR2(H52-H56)，HCDR3(H95-H97)，根据序列比对和可变区结构信息对抗体轻重链可变区氨基酸进行人源化 突变；设计表达载体，基因合成，哺乳细胞表达纯化重组抗体，比较人源化抗体和嵌合抗体活性，理化性质的差异，进行1-2轮人源化优化。
Germline抗体序列信息：
IGKV1-9*01
Figure PCTCN2022075593-appb-000007
IGKJ4*01
Figure PCTCN2022075593-appb-000008
IGHV3-21*01
Figure PCTCN2022075593-appb-000009
IGHJ5*01
Figure PCTCN2022075593-appb-000010
以下轻重链的优化设计都是以上面Germline抗体为框架CDR移植的人源化序列(41D2HzL0和41D2HzH0为嵌合抗体的轻重链，41D2HzL4和41D2HzH3为人源化抗体的轻重链)。
41D2HzL0轻链可变区序列：
Figure PCTCN2022075593-appb-000011
41D2HzL4轻链可变区序列
Figure PCTCN2022075593-appb-000012
41D2HzH0重链可变区序列
Figure PCTCN2022075593-appb-000013
41D2HzH3重链可变区序列
Figure PCTCN2022075593-appb-000014
2.2鼠源杂交瘤抗体46H3A8人源化
46H3A8人源化方案同1.2.1，以下轻重链的优化设计都是以Germline抗体为框架CDR移植的人源化序列(1910h3hzL0和1910h3hzH0为嵌合抗体的轻重链，1910h3hzL3和1910h3hzH4为人源化抗体的轻重链)。
1910h3hzL0轻链可变区序列
Figure PCTCN2022075593-appb-000015
1910h3hzH0重链可变区序列
Figure PCTCN2022075593-appb-000016
1910h3hzL3轻链可变区序列
Figure PCTCN2022075593-appb-000017
1910h3hzH4重链可变区序列
Figure PCTCN2022075593-appb-000018
实施例3 人源化抗体动力学参数测定
(1)采用Octet RED96e(Fortebio)测定人源化抗体1910h3hzL3H4及41D2HzL4H3与人PD-1(Sino Biological,货号：10377-H08H)的亲和力，抗原及抗体均用1xPBST(1xPBS：生工，B548117-0500；0.02％吐温20：sigma-alorich，P1379)稀释，抗原使用浓度为100nM，抗体使用浓度为50nM。
(2)样品上机检测(Octet Data Acquisition 11.1.0.11)：首先，将样品加入96孔板(Greiner bio-one，655209)，体系为200μL/well。然后设置软件参数，板温设定为30℃，收集标准动力学信号的频率为5.0HZ。接着，用1xPBST预湿AHC传感器(Fortébio,货号：18-0015)10分钟，然后上机检测。每个循环包含以下步骤：1)浸入缓冲液60秒；2)检测抗原是否与传感器有非特异性结合；3)10mM pH1.7的甘氨酸溶液再生；4)浸入缓冲液60秒；5)抗 体固化在传感器上，时间为20秒；6)传感器浸入缓冲液180秒；7)抗原与抗体结合，时间180秒；8)抗原抗体的解离，时间10分钟；9)传感器再生。
(3)数据分析
采用Fortebio的Data Analysis 11.0软件，对抗原-抗体以1:1的结合方式，测定结合速率(Ka)和解离速率(Kd)，以此计算抗体的平衡解离常数(KD)。结果如下表：
表3 人源化抗体与人PD-1的亲和力
候选抗体 K D(M) kon(1/Ms) kdis(1/s)
1910h3hzL3H4 8.038E-09 1.47E+05 1.18E-03
41D2HzL4H3 1.919E-09 2.83E+05 5.42E-04
Pembrolizumab 7.693E-09 5.45E+05 4.19E-03
从表3得出，1910h3hzL3H4与Pembrolizumab比较，抗体与人PD-1的亲和力相当；41D2HzL4H3与人PD-1的亲和力较Pembrolizumab提高约4.2倍。说明本申请抗原结合蛋白与PD-1具有较高的结合亲和力。
实施例4 本申请抗原结合蛋白与细胞表面人或食蟹猴PD-1的结合活性
收集CHOK-hPD-1(金斯瑞，M00529)细胞或CHOK1-cynoPD-1(金斯瑞，M00572),用FACS缓冲液(含有2％FBS的PBS)洗一遍后重悬，按1E5/50ul/孔，分液到96孔板(Corning 3799)中。抗体加到上述96孔U型板中，50ul/孔，混匀后4℃孵育1小时。2000rpm离心5分钟去掉上清，每孔加200ul FACS缓冲液洗一遍。每孔加入1∶1000的Alexa Fluor488二抗(Invitrogen)，100ul/孔，混匀4℃孵育1小时。2000rpm离心5分钟去掉上清，每孔加200ul FACS buffer洗一遍，30ul FACS buffer重悬上机检测。实验结果如下图1和图2所示。图1显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3结合CHOK1细胞表面的人PD-1蛋白。图2显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3结合CHOK1细胞表面的食蟹猴PD-1蛋白。结果表明1910h3hzL3H4和41D2HzL4H3与细胞表面人或食蟹猴PD-1的结合活性同Pembrolizumab相当。
实施例5 本申请抗原结合蛋白阻断细胞表面人PD-1与PD-L1或PD-L2的结合活性
收集CHOK1-hPD-1细胞，用FACS buffer洗一遍后重悬，按1E5/50ul/孔，分液到96孔U型板中。抗体稀释后加到上述96孔板中，25ul/孔，生物素化的PD-L1(Sinobiological，货号10084-H02H-B)(或PD-L2，Acrobiosystem,货号PD2-H82F6)稀释后加到上述96孔板中，25ul/孔，混匀后4℃孵育1小时。2000rpm离心5分钟去掉上清，每孔加200ul FACS buffer 洗一遍。每孔加入1∶1000的SA-Alexa Fluor 488(Invitrogen cat：S32354)，100ul/孔，混匀4℃孵育30分钟。2000rpm离心5分钟去掉上清，每孔加200ul FACS buffer洗一遍，30ul FACS buffer重悬上机检测。图3显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3阻断人PD-L1与CHOK1表面的人PD-1蛋白结合。图4显示的是本申请所述抗原结合蛋白1910h3hzL3H4和41D2HzL4H3阻断人PD-L2与CHOK1细胞表面的人PD-1蛋白。结果表明1910h3hzL3H4和41D2HzL4H3阻断细胞表面人PD-1与PD-L1或PD-L2的结合活性与对照抗体Pembrolizumab相当。说明本申请抗原结合蛋白阻断PD-1与PD-L1或PD-L2结合的活性良好。
实施例6 混合淋巴细胞反应：细胞因子IL2的分泌
使用细胞培养基(1640+2％FBS)复苏人树突状DC细胞，调整DC细胞密度至1*10 5-1*10 7细胞/mL，然后加入终浓度为50μg/ml丝裂霉素C，37度避光处理30分钟后加入10ml培养基终止，400g离心10分钟，随后用10mL培养基清洗一遍。梯度稀释anti-PD1抗体：抗体最高终浓度为2.5μg/mL(配制浓度为10μg/mL)，10倍梯度稀释(5个浓度点+1个0浓度)，然后相应细胞培养板(康宁，货号：3599)中加入50μL配制好的anti-PD1抗体。收集人外周血淋巴细胞PBMC和丝裂霉素C处理好的DC细胞，调整DC细胞密度至2*10 5细胞/mL，随后将细胞加入培养板中，每孔50μL，即每孔DC细胞数为1*10 4细胞/孔；调整PBMC细胞密度至2*10 6细胞/mL，随后将细胞加入培养板中，每孔100μL，即每孔PBMC细胞数为2*10 5细胞/孔。将细胞培养板置于37℃、5％二氧化碳细胞培养箱孵育5天，5天后，300g离心5分钟，收集上清用Human IL-2ELISA试剂盒(BD，货号：550611)检测IL-2含量，检测方法严格按照试剂盒说明书进行，数据用GraphPad Prism软件进行处理。结果如图5所示，结果表明，人源化抗体1910h3hzL3H4和41D2HzL4H3都能刺激淋巴细胞分泌IL2，活性与Pembrolizumab相似。说明本申请抗原结合蛋白刺激免疫细胞分泌细胞因子的能力较高。
实施例7 抗原结合蛋白的亲和力成熟
7.1酵母展示突变文库的构建、筛选与单克隆鉴定
酵母展示突变文库的构建与筛选
基于41D2HzL4H3，对其抗原结合决定簇(CDR)位点的氨基酸进行随机突变，构建各CDR区的突变文库，利用酵母展示技术高通量筛选与抗原特异性结合力强的序列。将41D2HzL4H3的重链可变区氨基酸序列按Chothia编码规则进行编码，CDR区按Chothia定义。对各分子重链可变区CDR1、CDR2、CDR3，设计NNK突变引物进行聚合酶链式反应 (PCR)扩增各CDR突变文库基因片段。将各CDR突变文库基因片段与酵母展示质粒分别转入酿酒酵母菌株EBY100(购自ATCC)，使各CDR突变文库以Fab形式展示于酵母表面。同时将41D2HzL4H3的亲本序列以Fab形式展示于酵母表面，作为对照使用。
文库经过培养、诱导后，按1OD为1.0×10 7细胞数计算，各取1.5×10 9细胞，用磁珠分选系统进行第一轮富集。各文库细胞重悬于含5nM生物素标记的人PD-1(Acro，货号：PD1-H82E4，简称Bitoin hPD-1)的1×PBSA(1×PBS+1％BSA)中孵育30分钟，洗涤后加入Anti biotin beads(miltenyi，货号：130-090-485)混匀孵育10分钟，过磁力柱(Quadro MACS Starting Kit)收集阳性细胞。阳性细胞经过再次培养、诱导后，各取3.0×10 7细胞，用1nM Bitoin hPD-1进行第二轮流式分选，收集展示水平高且与抗原结合力强的细胞群；阳性细胞经过再次培养、诱导后，各取2.0×10 7细胞，用10nM生物素标记的猴PD-1(Acro，货号：PD1-C82E6,简称Bitoin cyno PD-1)进行第三轮流式分选，分选后取细胞测序分析，获得重链各个CDR区突变的单一序列。
突变单克隆的鉴定
经测序分析，共计获得42个新的突变序列，对相应单克隆进行流式染色鉴定，分别与1nM Bitoin hPD-1以及1nM Bitoin cyno PD-1孵育染色，比较不同克隆与抗原结合的平均荧光信号强度(MFI)，MFI越高说明与抗原的亲和力越强。根据荧光信号的强度，筛选出YC208B3、YC208E2、YC208G3、YC210H2、YC215D1、YC215F3、YC215H3、YC215H5、YC216D5、YC216F1、YC216F2、YC208E1、YC209C5、YC209D4、YC209D6、YC209E6、YC214E2等17个克隆进一步测定与人、猴PD-1的结合曲线Ag 50。
按列分布各克隆，1.0×10 5细胞/孔平铺96孔板，1×PBSA洗涤离心后加入梯度稀释的人、猴PD-1抗原室温孵育30分钟；离心弃上清，加入100μL二抗荧光抗体，避光孵育30分钟；洗涤后重悬于30μL 1×PBSA，上流式细胞仪读取相应荧光信号，数据用GraphPad Prism 8软件进行拟合，计算各克隆与人、猴PD-1的结合曲线Ag 50，结果见表4。
表4 突变克隆与人、猴PD-1的结合测定
Figure PCTCN2022075593-appb-000019
Figure PCTCN2022075593-appb-000020
根据各克隆的序列及与人、猴PD-1的Ag 50值等综合考虑，选取YC208B3、YC208E2、YC208G3、YC209C5、YC209D6、YC215D1、YC215F3、YC215H3、YC216D5、YC216F2等10个克隆的突变序列，选用IgG1 LALA D265S亚型构建表达质粒，抗体编号命名为Ab2005Am01-10。
7.2候选抗体表达
将各克隆的重链可变区序列与IgG1 LALA D265S融合，轻链可变区与Kappa链恒定区融合；基因合成后装入表达载体pcDNA3.4(Life Technologies)。轻、重表达质粒转入ExpiCHO细胞(ThermoFisher Scientific，A29133)，根据供应商ExpiCHO表达系统方法进行抗体瞬转表达，过程如下：在培养总体积25mL培养基中，36.5℃，8％二氧化碳浓度下培养Expi CHO细胞到密度6×10 6/mL，使用ExpiFectamine转染试剂化转各10μg抗体轻重链表达质粒到细胞；转染一天后，各取150μL和4mL ExpiCHO增强剂和ExpiCHO辅料添加到培养细胞中，继续培养至9天，4度，3500转离心取上清。混合AmMagTM Protein A磁珠(Genscript，L00695)和抗体表达上清，室温孵育2小时，PBS洗涤两次弃上清，加入适量洗脱缓冲液Protein G or A SefinoseTM Elution buffer(Sangon,C600481)，充分混匀后置于试管架上静止孵育5分钟，孵育期间重悬磁珠2-3次，重复洗脱2次，洗脱后，立即加入适量中和液1M Tris-HCl，pH7.5(Sangon,B548124)中和备用，得到纯化的抗体见表5。
表5 候选抗体表达、纯化数据
Figure PCTCN2022075593-appb-000021
7.3候选抗体理化性质评估
将抗体Ab2005Am01-10作为候选分子继续进行理化成药性评估，具体如下：
SEC-HPLC纯度分析
(1)将样品浓度调整至1mg/mL，混匀，12000rpm离心5min，取上清转至样品瓶，放入HPLC样品盘。设置色谱条件如下：
Figure PCTCN2022075593-appb-000022
(2)色谱柱采用流动相(200mM磷酸盐缓冲液，pH6.8)平衡后，进样分析，用色谱软件进行数据分析，峰面积归一化法计算各个峰的峰面积百分比，百分比越高说明抗体纯度越高。
HIC-HPLC分析
(1)将样品浓度调整至1mg/ml，离心取上清待测。设置色谱条件如下：
Figure PCTCN2022075593-appb-000023
(2)用流动相A(50mM磷酸盐缓冲液/1M硫酸铵，pH 7.0)和流动相B(50mM磷酸盐缓冲液，pH 7.0)进行梯度洗脱，记录主峰保留时间，出峰时间短则抗体亲水性强。
熔解温度(Tm)值分析
将样品浓度调整至1mg/mL，然后按照Protein Thermal Shift TM Starter Kit说明书，取供试品溶液13μL加入至PCR管内，加入5μL Protein Thermal shift  TM Buffer，加入2μL10×染色液，使反应体积为20μL，混匀后，12000rpm离心5min以去除气泡。将检测样品置于PCR仪内，进行样品分析，记录样品的Tm值，Tm值越高表示抗体的热稳定性越好。
iCIEF分析
取样品溶液加入到已经充分混匀的以下体系中：1％的甲基纤维素(MC)70μL，尿素5M80μL，两性电解质Pharmalyte pH 3-10 8μL，pI marker 5.5和9.5各2μL。补加适当体积超纯水至200μL，混匀。离心取上清进样分析。分析结束后，将结果文件导入ChromPerfect软件进行图谱积分处理并计算各峰的等电点以及各峰百分比。
将候选抗体Ab2005Am01-10的理化性质汇总如表6，结果显示，候选抗体的纯度、热稳定性、亲水性等理化指标较为理想，继续评估候选抗体的体外活性。
表6 候选抗体理化性质分析结果
Figure PCTCN2022075593-appb-000024
Figure PCTCN2022075593-appb-000025
7.4候选抗体亲和力测定
为了测定候选抗体Ab2005Am01-10与人、猴PD-1的亲和力，采用Octet RED96e(Fortebio)测定候选抗体与人PD-1(Acro,货号：PD-1-H5221)、猴PD-1(Acro,货号：PD-1-C5223)的亲和力，抗原及抗体均用1×PBST(1×PBS：生工，B548117-0500；0.02％吐温20：sigma，P1379)稀释，抗原使用浓度为100nM，抗体使用浓度为50nM。将候选抗体样品按200μL/孔加入96孔板(Greiner bio-one，655209)，设置软件参数，温度30℃、收集标准动力学信号的频率为5.0Hz；用1×PBST预湿AHC传感器(Fortébio，货号：18-0015)10分钟，然后上机检测。每个循环包含以下步骤：1)浸入缓冲液60s；2)检测抗原是否与传感器有非特异性结合；3)10mM pH1.7的甘氨酸溶液再生；4)浸入缓冲液60s；5)抗体固化在传感器上，时间为13s；6)传感器浸入缓冲液180s；7)抗原与抗体结合，时间180s；8)抗原抗体的解离，时间10分钟；9)传感器再生。采用Fortebio的Data Analysis 12.0软件，对抗原-抗体以1:1的结合方式，测定结合速率(K on)和解离速率(K off)，以此计算抗体的平衡解离常数(K D)，结果如表7、8。
表7 候选抗体与人PD-1的亲和力测定
抗体编号 响应值 K D(M) kon(1/Ms) kdis(1/s)
41D2HzL4H3 0.15 1.27E-09 4.64E+05 5.89E-04
Ab2005Am01 0.13 7.11E-10 4.87E+05 3.46E-04
Ab2005Am02 0.15 1.10E-09 4.27E+05 4.70E-04
Ab2005Am03 0.16 9.07E-10 4.02E+05 3.65E-04
Ab2005Am04 0.17 8.36E-10 4.47E+05 3.74E-04
Ab2005Am05 0.16 5.29E-10 4.93E+05 2.61E-04
Ab2005Am06 0.15 7.79E-10 4.17E+05 3.25E-04
Ab2005Am07 0.15 1.31E-09 4.93E+05 6.46E-04
Ab2005Am08 0.17 5.04E-10 4.57E+05 2.30E-04
Ab2005Am09 0.15 4.63E-10 4.79E+05 2.22E-04
Ab2005Am10 0.15 6.48E-10 4.86E+05 3.15E-04
表8 候选抗体与猴PD-1的亲和力测定
抗体编号 响应值 K D(M) kon(1/Ms) kdis(1/s)
41D2HzL4H3 0.14 5.20E-09 2.95E+05 1.53E-03
Ab2005Am01 0.14 2.19E-09 3.04E+05 6.63E-04
Ab2005Am02 0.15 4.14E-09 2.65E+05 1.10E-03
Ab2005Am03 0.17 4.24E-09 2.77E+05 1.18E-03
Ab2005Am04 0.17 7.54E-09 3.17E+05 2.39E-03
Ab2005Am05 0.13 1.81E-08 2.55E+05 4.62E-03
Ab2005Am06 0.14 2.80E-09 2.34E+05 6.55E-04
Ab2005Am07 0.15 3.62E-09 3.10E+05 1.12E-03
Ab2005Am08 0.17 1.52E-09 3.20E+05 4.86E-04
Ab2005Am09 0.16 2.06E-09 3.82E+05 7.88E-04
Ab2005Am10 0.16 2.63E-09 3.48E+05 9.16E-04
由结果可知，候选抗体Ab2005Am06、08、09与人、猴PD-1的亲和力比亲和力成熟之前的亲本抗体强2倍左右，继续进行体外结合与功能实验评估。
7.5候选抗体与细胞表面人、猴PD-1的结合
为测定候选抗体Ab2005Am06、08、09与细胞表面人、猴PD-1的结合，收集CHOK-hPD-1(金斯瑞，M00529)细胞或CHOK-cynoPD-1(金斯瑞，M00572)，用FACS缓冲液(含有2％FBS的PBS)洗一遍后重悬，按1E5/50μL/孔，分液到96孔板中；候选抗体Ab2005Am06、08、09从10μg/mL的起始浓度，4倍梯度稀释；将稀释后的抗体溶液加到上述96孔U型板中，50μL/孔，混匀后4℃孵育1小时；2000rpm离心5分钟去除上清；每孔加入200μL FACS缓冲液洗一遍，加入1:1000稀释的Alexa Fluor 488二抗(Invitrogen，A-11013)，100μL/孔，混匀4℃孵育1小时；2000rpm离心5分钟去除上清；每孔加200μL FACS buffer洗一遍，30μL FACS buffer重悬上机检测。数据用GraphPad Prism软件进行处理，实验结果显示候选抗体可以与细胞表面人、猴PD-1的结合。图6显示的是候选抗体与细胞表面人PD-1的结合结果图。图7显示的是候选抗体与细胞表面猴PD-1的结合结果图。
7.6候选抗体激活混合淋巴细胞释放IL-2功能实验
为比较候选抗体Ab2005Am06、08、09与亲本抗体41D2HzL4H3及对标抗体Pembrolizumab的体外活性，在同一次激活混合淋巴细胞释放IL-2的实验中进行，具体如下：使用细胞培养基(1640+2％FBS)复苏人树突状DC细胞，调整DC细胞密度至1×10 5- 1×10 7cells/mL，然后加入终浓度为50μg/mL丝裂霉素C，37度避光处理30分钟后加入10mL培养基终止，400g离心10分钟，随后用10mL培养基清洗一遍。梯度稀释抗PD1抗体：抗体最高终浓度为2.5μg/mL(配制浓度为10μg/mL)，10倍梯度稀释(5个浓度点+1个0浓度点)，然后相应细胞培养板(康宁，货号：3599)中加入50μL配制好的抗PD1抗体。收集人外周血淋巴细胞PBMC和丝裂霉素C处理好的DC细胞，调整DC细胞密度至2×10 5cells/mL，随后将细胞加入培养板中，每孔50μL，即DC细胞数为1×10 4cells/孔；调整PBMC细胞密度至2×10 6cells/mL，随后将细胞加入培养板中，每孔100μL，即PBMC细胞数为2×10 5cells/孔。将细胞培养板置于37℃、5％二氧化碳细胞培养箱孵育3天，3天后，300g离心5分钟，收集上清用Human IL-2ELISA试剂盒(Novus，货号：VAL110)检测IL-2含量，检测方法严格按照试剂盒说明书进行，数据用GraphPad Prism软件进行处理，结果显示候选抗体可以激活混合淋巴细胞释放IL-2。图8显示的是候选抗体激活混合淋巴细胞释放IL-2的结果图。
综上可知，候选抗体Ab2005Am08(HCDR2为SGGGRY，如SEQ ID NO:85所示；VH为
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYDMSWVRQAPGKGLEWVSTISGGGRYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVSPYYGMEYWGQGTLVTVSS，如SEQ ID NO:86所示)经过亲和力成熟后，相比亲本抗体41D2HzL4H3具有更强的人、猴PD-1结合能力，在体外激活混合淋巴细胞释放IL-2的实验中优于亲本抗体及对标抗体Pembrolizumab；同时，候选抗体Ab2005Am08在表达量、理化性质等方面均符合成药性标准，具有继续开发的潜力。

Claims (69)

  1. 分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    (1)能够以1×10 8M或更低的K D值结合源自灵长类动物的PD-1蛋白；
    (2)能够阻断PD-1蛋白与PD-L1蛋白之间的结合；
    (3)能够阻断PD-1蛋白与PD-L2蛋白之间的结合；和
    (4)能够刺激免疫细胞分泌细胞因子。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其中所述灵长类动物包括人和/或猴。
  3. 根据权利要求1或2所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  4. 根据权利要求3所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。
  5. 根据权利要求3-4中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其能够与参比抗体竞争结合所述PD-1蛋白，其中所述参比抗体包含重链可变区VH和轻链可变区VL，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，所述参比抗体的VL包含LCDR1、LCDR2和LCDR3，且所述参比抗体包含选自下述的任意一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2，HCDR3：SEQ ID NO:3，LCDR1：SEQ ID NO:8，LCDR2：SEQ ID NO:9，和LCDR3：SEQ ID NO:10；
    (2)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:28，HCDR3：SEQ ID NO:29，LCDR1：SEQ ID NO:34，LCDR2：SEQ ID NO:35，和LCDR3：SEQ ID NO:36；以及
    (3)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:85，HCDR3：SEQ ID NO:3，LCDR1：SEQ ID NO:8，LCDR2：SEQ ID NO:9，和LCDR3：SEQ ID NO:10。
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其包含HCDR3，且所述HCDR3包含SEQ ID NO:3或SEQ ID NO:29所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其包含HCDR2，且所述HCDR2包含SEQ ID NO:2、SEQ ID NO:28或SEQ ID NO:85所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其包含HCDR1，且所述HCDR1包含SEQ ID NO:1或SEQ ID NO:27所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:1和SEQ ID NO:27中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2、SEQ ID NO:28和SEQ ID NO:85中任一项所示 的氨基酸序列，且所述HCDR3包含SEQ ID NO:3和SEQ ID NO:29中任一项所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3选自以下任一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:2和HCDR3：SEQ ID NO:3；
    (2)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:28和HCDR3：SEQ ID NO:29；和
    (3)HCDR1：SEQ ID NO:1，HCDR2：SEQ ID NO:85和HCDR3：SEQ ID NO:3。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:57或SEQ ID NO:58所示的氨基酸序列。
  13. 根据权利要求12所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:4、SEQ ID NO:17、SEQ ID NO:30和SEQ ID NO:43中任一项所示的氨基酸序列。
  14. 根据权利要求12-13中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:59或SEQ ID NO:60所示的氨基酸序列。
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:5、SEQ ID NO:18、SEQ ID NO:31和SEQ ID NO:44中任一项所示的氨基酸序列。
  16. 根据权利要求12-15中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:61或SEQ ID NO:62所示的氨基酸序列。
  17. 根据权利要求16所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:6、SEQ ID NO:19、SEQ ID NO:32和SEQ ID NO:45中任一项所示的氨基酸序列。
  18. 根据权利要求12-17中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:63或SEQ ID NO:64所示的氨基酸序列。
  19. 根据权利要求18所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:7、SEQ ID NO:20、SEQ ID NO:33和SEQ ID NO:46中任一项所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的分离的抗原结合蛋白，其包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:4、SEQ ID NO:17、SEQ ID NO:30和SEQ  ID NO:43中任一项所示的氨基酸序列，所述H-FR2包含SEQ ID NO:5、SEQ ID NO:18、SEQ ID NO:31和SEQ ID NO:44中任一项所示的氨基酸序列，所述H-FR3包含SEQ ID NO:6、SEQ ID NO:19、SEQ ID NO:32和SEQ ID NO:45中任一项所示的氨基酸序列且所述H-FR4包含SEQ ID NO:7、SEQ ID NO:20、SEQ ID NO:33和SEQ ID NO:46中任一项所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4选自以下任一组氨基酸序列：
    (1)H-FR1：SEQ ID NO:4，H-FR2：SEQ ID NO:5，H-FR3：SEQ ID NO:6和H-FR4：SEQ ID NO:7；
    (2)H-FR1：SEQ ID NO:17，H-FR2：SEQ ID NO:18，H-FR3：SEQ ID NO:19和H-FR4：SEQ ID NO:20；
    (3)H-FR1：SEQ ID NO:30，H-FR2：SEQ ID NO:31，H-FR3：SEQ ID NO:32和H-FR4：SEQ ID NO:33；和
    (4)H-FR1：SEQ ID NO:43，H-FR2：SEQ ID NO:44，H-FR3：SEQ ID NO:45和H-FR4：SEQ ID NO:46。
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，且所述VH包含SEQ ID NO:65或SEQ ID NO:66所示的氨基酸序列。
  23. 根据权利要求22中任一项所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:15、SEQ ID NO:25、SEQ ID NO:41、SEQ ID NO:51和SEQ ID NO:86中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包括重链恒定区，且所述重链恒定区源自人IgG恒定区。
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG4恒定区，且所述人IgG4恒定区包含SEQ ID NO:77或81所示的氨基酸序列。
  26. 根据权利要求1-22中任一项所述的分离的抗原结合蛋白，其包含抗体重链HC，且所述HC包含SEQ ID NO:79、83或87所示的氨基酸序列。
  27. 根据权利要求1-26中任一项所述的分离的抗原结合蛋白，其包含LCDR3，且所述LCDR3包含SEQ ID NO:10或SEQ ID NO:36所示的氨基酸序列。
  28. 根据权利要求1-27中任一项所述的分离的抗原结合蛋白，其包含LCDR2，且所述LCDR2包含SEQ ID NO:9或SEQ ID NO:35所示的氨基酸序列。
  29. 根据权利要求1-28中任一项所述的分离的抗原结合蛋白，其包含LCDR1，且所述LCDR1包含SEQ ID NO:8或SEQ ID NO:34所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的分离的抗原结合蛋白，其包含LCDR1、LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:8和SEQ ID NO:34中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:9和SEQ ID NO:35中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:10和SEQ ID NO:36中任一项所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的分离的抗原结合蛋白，其包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3选自以下任一组氨基酸序列：
    (1)LCDR1：SEQ ID NO:34，LCDR2：SEQ ID NO:35和LCDR3：SEQ ID NO:36；和
    (2)LCDR1：SEQ ID NO:8，LCDR2：SEQ ID NO:9和LCDR3：SEQ ID NO:10。
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL，其中所述VL包括框架区L-FR1，所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:69或SEQ ID NO:70所示的氨基酸序列。
  33. 根据权利要求32所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NO:11、SEQ ID NO:21、SEQ ID NO:37和SEQ ID NO:47中任一项所示的氨基酸序列。
  34. 根据权利要求32-33中任一项所述的分离的抗原结合蛋白，其中所述VL包括框架区L-FR2，所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:71或SEQ ID NO:72所示的氨基酸序列。
  35. 根据权利要求34所述的分离的抗原结合蛋白，其中所述L-FR2包含SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:38和SEQ ID NO:48中任一项所示的氨基酸序列。
  36. 根据权利要求32-35所述的分离的抗原结合蛋白，其中所述VL包括框架区L-FR3，所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:73或SEQ ID NO:74所示的氨基酸序列。
  37. 根据权利要求36所述的分离的抗原结合蛋白，其中所述L-FR3包含SEQ ID NO:13、SEQ ID NO:23、SEQ ID NO:39和SEQ ID NO:49中任一项所示的氨基酸序列。
  38. 根据权利要求32-37中任一项所述的分离的抗原结合蛋白，其中所述L-FR4的N末端与所述LCDR3的C末端相连，且所述L-FR4包含SEQ ID NO:75或SEQ ID NO:76所示的氨基酸序列。
  39. 根据权利要求38所述的分离的抗原结合蛋白，其中所述L-FR4包含SEQ ID NO:14、SEQ ID NO:24、SEQ ID NO:40和SEQ ID NO:50中任一项所示的氨基酸序列。
  40. 根据权利要求1-39中任一项所述的分离的抗原结合蛋白，其包含L-FR1、L-FR2、L-FR3和L-FR4，其中所述L-FR1包含SEQ ID NO:11、SEQ ID NO:21、SEQ ID NO:37和SEQ ID NO:47中任一项所示的氨基酸序列，所述L-FR2包含SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:38和SEQ ID NO:48中任一项所示的氨基酸序列，所述L-FR3包含SEQ ID NO:13、SEQ ID NO:23、SEQ ID NO:39和SEQ ID NO:49中任一项所示的氨基酸序列且所述L-FR4包含SEQ ID NO:14、SEQ ID NO:24、SEQ ID NO:40和SEQ ID NO:50中任一项所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的分离的抗原结合蛋白，其包含L-FR1、L-FR2、L-FR3和L-FR4，且所述L-FR1、L-FR2、L-FR3和L-FR4选自以下任一组氨基酸序列：
    (1)L-FR1：SEQ ID NO:11，L-FR2：SEQ ID NO:12，L-FR3：SEQ ID NO:13和L-FR4：SEQ ID NO:14；
    (2)L-FR1：SEQ ID NO:21，L-FR2：SEQ ID NO:22，L-FR3：SEQ ID NO:23和L-FR4：SEQ ID NO:24；
    (3)L-FR1：SEQ ID NO:37，L-FR2：SEQ ID NO:38，L-FR3：SEQ ID NO:39和L-FR4：SEQ ID NO:40；和
    (4)L-FR1：SEQ ID NO:47，L-FR2：SEQ ID NO:48，L-FR3：SEQ ID NO:49和L-FR4：SEQ ID NO:50。
  42. 根据权利要求1-41中任一项所述的分离的抗原结合蛋白，其包含轻链可变区VL，且所述VL包含SEQ ID NO:67或SEQ ID NO:68所示的氨基酸序列。
  43. 根据权利要求1-42中任一项所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:16、SEQ ID NO:26、SEQ ID NO:42和SEQ ID NO:52中任一项所示的氨基酸序列。
  44. 根据权利要求1-43中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区，且所述抗体轻链恒定区包含SEQ ID NO:78或82所示的氨基酸序列。
  45. 根据权利要求1-38中任一项所述的分离的抗原结合蛋白，其包含抗体轻链LC，且所述LC包含SEQ ID NO:80或84所示的氨基酸序列。
  46. 多肽，其包含权利要求1-45中任一项所述的分离的抗原结合蛋白。
  47. 免疫缀合物，其包含权利要求1-45中任一项所述的分离的抗原结合蛋白或权利要求46所述的多肽。
  48. 分离的一种或多种核酸分子，其编码权利要求1-45中任一项所述的分离的抗原结合蛋白。
  49. 载体，其包含根据权利要求48所述的核酸分子。
  50. 细胞，其包含根据权利要求48所述的核酸分子或根据权利要求49所述的载体。
  51. 制备权利要求1-45中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-45中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求50所述的细胞。
  52. 药物组合物，其包含权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体和/或权利要求50所述的细胞，以及任选地药学上可接受的载体。
  53. 权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
  54. 根据权利要求53所述的用途，其中所述肿瘤包括PD-1或PD-L1高表达的肿瘤。
  55. 根据权利要求53-54中任一项所述的用途，其中所述肿瘤包括实体瘤和/或非实体瘤。
  56. 根据权利要求53-55中任一项所述的用途，其中所述肿瘤包括黑色素瘤、肺癌、肾癌、食管癌、头颈癌、淋巴瘤、肝癌和/或胃癌。
  57. 预防、缓解或治疗肿瘤的方法，所述方法包括向有需要的受试者施用权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物。
  58. 根据权利要求57所述的方法，其中所述肿瘤包括PD-1或PD-L1高表达的肿瘤。
  59. 根据权利要求57-58中任一项所述的方法，其中所述肿瘤包括实体瘤和/或非实体瘤。
  60. 根据权利要求57-59中任一项所述的方法，其中所述肿瘤包括黑色素瘤、肺癌、肾癌、食管癌、头颈癌、淋巴瘤、肝癌和/或胃癌。
  61. 权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物，用于预防、缓解或治疗肿瘤。
  62. 根据权利要求61所述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子载体、细胞和/或药物组合物，其中所述肿瘤包括PD-1或PD-L1高表达的肿瘤。
  63. 根据权利要求61-62中任一项所述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子载体、细胞和/或药物组合物，其中所述肿瘤包括实体瘤和/或非实体瘤。
  64. 根据权利要求61-63中任一项所述的分离的抗原结合蛋白、多肽、免疫缀合物、核酸分子载体、细胞和/或药物组合物，其中所述肿瘤包括黑色素瘤、肺癌、肾癌、食管癌、头颈癌、淋巴瘤、肝癌和/或胃癌。
  65. 抑制PD-1蛋白与PD-L1蛋白结合的方法，所述方法包括施用权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物。
  66. 抑制PD-1蛋白与PD-L2蛋白结合的方法，其包括施用权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物。
  67. 刺激免疫细胞分泌细胞因子的方法，其包括施用权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物。
  68. 一种用于检测PD-1蛋白的存在和/或含量的方法，其包括权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物。
  69. 试剂盒，其包含权利要求1-45中任一项所述的分离的抗原结合蛋白、权利要求46所述的多肽、权利要求47所述的免疫缀合物、权利要求48所述的核酸分子、权利要求49所述的载体、权利要求50所述的细胞和/或权利要求52所述的药物组合物。
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