WO2021063352A1 - 一种抗pd-l1抗原结合蛋白及其应用 - Google Patents

一种抗pd-l1抗原结合蛋白及其应用 Download PDF

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WO2021063352A1
WO2021063352A1 PCT/CN2020/118811 CN2020118811W WO2021063352A1 WO 2021063352 A1 WO2021063352 A1 WO 2021063352A1 CN 2020118811 W CN2020118811 W CN 2020118811W WO 2021063352 A1 WO2021063352 A1 WO 2021063352A1
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seq
amino acid
acid sequence
sequence shown
antibody
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PCT/CN2020/118811
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French (fr)
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何云
任舒文
刘斌
甘馨
李东海
陈飞
王玲
李瑾
戎一平
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和铂医药(上海)有限责任公司
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Priority to KR1020227014733A priority Critical patent/KR20220071264A/ko
Priority to CN202080005996.9A priority patent/CN113039208B/zh
Priority to US17/765,270 priority patent/US20220411513A1/en
Priority to EP20872778.4A priority patent/EP4023672A1/en
Priority to JP2022520093A priority patent/JP2022550420A/ja
Publication of WO2021063352A1 publication Critical patent/WO2021063352A1/zh

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    • 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
    • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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    • C07—ORGANIC CHEMISTRY
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    • 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]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • 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
    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • This application relates to the field of biomedicine, in particular to an anti-PD-L1 antigen binding protein and its application, and an anti-PD-L1 and anti-TGFB fusion protein and its application.
  • P-L1 Programmed death receptor ligand 1
  • CD274 cluster of differentiation 274
  • B7 homologous protein 1 B7-H1
  • PD-L1 is a 40KDa type I transmembrane protein, usually in activated T It is expressed on cells, B cells, monocytes, dendritic cells, macrophages and many non-hematopoietic cells.
  • PD-L1 can bind to programmed death receptor 1 (PD-1) and B7-1 (CD80).
  • PD-L1/PD-1 signal pathway is a very important co-suppressive signal pathway in the immune response, which negatively regulates the T cell immune response, inhibits T cell activity, and weakens the secretion of cytokines.
  • Transforming growth factor-B is a powerful cytokine that has a significant impact on the immune system. It participates in many proliferative and non-proliferative cell processes, such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, and wounds Healing, hematopoiesis and immune and inflammatory response. Whether the existing treatment technology is chemotherapy or tumor-targeted therapy, an important bottleneck that affects its efficacy is that tumor cells develop immune tolerance. Tumor cells use a variety of immunosuppressive mechanisms in the tumor microenvironment to evade recognition and attack by the body's immune system. These immunosuppressive mechanisms include immunosuppressive cytokines (such as TGFB), regulatory T cells (Tregs), and co-suppressive signaling pathways Molecules, myeloid inhibitory cells, etc.
  • immunosuppressive cytokines such as TGFB
  • Tregs regulatory T cells
  • This application provides a PD-L1 antigen binding protein that can bind to primate-derived PD-L1 with a KD value of 1 ⁇ 10 -8 M or lower.
  • the antigen binding protein can block the binding of PD-1 and CD80 to PD-L1, stimulate the secretion of IFN- ⁇ and/or IL2 in immune cells, and can inhibit tumor growth and/or tumor cell proliferation.
  • the application also provides a fusion protein, which includes human TGFBRII or a fragment thereof and the antigen binding protein.
  • the application also provides the application of the antigen binding protein and/or the fusion protein in the prevention and treatment of tumors or cancers.
  • the present application provides an isolated antigen binding protein that binds to PD-L1, which comprises at least one CDR in the VH of the variable region of the antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO: 193;
  • the VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 90-93, 95 and 97-104.
  • the isolated antigen binding protein comprises at least one CDR in the variable region VL of the antibody light chain, and the VL comprises the amino acid sequence shown in SEQ ID NO: 194; preferably, the VL Contains the amino acid sequence shown in any one of SEQ ID NOs: 108-114, 116, and 118.
  • the isolated antigen-binding protein includes an antibody or an antigen-binding fragment thereof; preferably, the antibody wherein the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, and humanized antibodies And fully human antibodies; preferably, the antigen-binding fragments include Fab, Fab', Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the isolated antigen binding protein includes a light chain variable region VL and a heavy chain variable region VH
  • the light chain variable region VL includes LCDR1, LCDR2, and LCDR3, and the LCDR1 includes SEQ ID
  • the LCDR1 includes the amino acid sequence shown in any one of SEQ ID NOs: 181, 48-51; the LCDR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 182, 63-64 Amino acid sequence; the LCDR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 191, 77-79; HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5; HCDR2 includes SEQ ID NOs: 179, 14, The amino acid sequence shown in any one of 16 and 17; HCDR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 180, 29-33.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 48; the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 63; the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5; the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14; the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29; or,
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 49
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 48
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 30; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 50
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 63
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 50
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 63
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 79
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 31; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 63
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 32; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 50
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 48
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 30; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 48
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 30; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 63
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 32; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 63
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 32; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 50
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 50
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33.
  • the antibody heavy chain variable region VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4; preferably, the C-terminus of the H-FR1 is connected to the The N-terminus of HCDR1 is connected and includes the amino acid sequence shown in SEQ ID NO: 1; and/or the H-FR2 is located between the HCDR1 and the HCDR2 and includes the amino acid sequence shown in SEQ ID NO: 9; and / Or the H-FR3 is located between the HCDR2 and the HCDR3 and includes the amino acid sequence shown in SEQ ID NO: 192; and/or the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, It includes the amino acid sequence shown in SEQ ID NO: 38; further preferably, the H-FR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 21, 22, and 24.
  • the antibody light chain variable region VL includes the framework regions L-FR1, L-FR2, L-FR3, and L-FR4; preferably, the C-terminus of the L-FR1 is connected to the LCDR1 is connected to the N-terminus and includes the amino acid sequence shown in SEQ ID NO: 185; and/or the L-FR2 is located between the LCDR1 and the LCDR2 and includes the amino acid sequence shown in SEQ ID NO: 186; and / Or the L-FR3 is located between the LCDR2 and the LCDR3 and includes the amino acid sequence shown in SEQ ID NO: 187; and/or the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, Contains the amino acid sequence shown in SEQ ID NO: 188.
  • the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 41-44; and/or the L-FR2 comprises any one of SEQ ID NOs: 56-59 And/or the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 69-72; and/or the L-FR4 comprises any one of SEQ ID NOs: 85-86 The amino acid sequence shown in one item.
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 193;
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 194;
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 90-93, 95 and 97-104;
  • the antibody light chain variable region VL comprises the amino acid sequence shown in any one of SEQ ID NOs: 108-114, 116 and 118.
  • the antibody heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 90
  • the antibody light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 108; or ,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 90
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 109; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 91
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 110; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 90
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 111; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 92
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 112; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 93
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 113; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 90
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 114; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 95
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 116; or
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 97
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 118; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 98
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 118; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 99
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 113; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 100
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 113; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 101
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 114; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 102
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 114; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 103
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 116; or,
  • the antibody heavy chain variable region VH comprises the amino acid sequence shown in SEQ ID NO: 104
  • the antibody light chain variable region VL comprises the amino acid sequence shown in SEQ ID NO: 116.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region; preferably, wherein the antibody heavy chain constant region includes a constant region such as SEQ ID NOs: the amino acid sequence shown in any one of 172-175.
  • the isolated antigen binding protein comprises an antibody heavy chain HC
  • the HC comprises an amino acid sequence as shown in any one of SEQ ID NOs: 122-125, 127, and 129-137.
  • the isolated antigen binding protein includes an antibody light chain constant region, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 170.
  • the isolated antigen binding protein comprises an antibody light chain LC
  • the LC comprises an amino acid sequence as shown in any one of SEQ ID NOs: 150-156, 158, and 160-163.
  • the isolated antigen binding protein has one or more of the following properties:
  • control antibody comprises LCDR1 shown in SEQ ID NO: 52 and LCDR2 shown in SEQ ID NO: 65 and LCDR3 shown in SEQ ID NO: 81, and the control antibody comprises the LCDR1 shown in SEQ ID NO: 6 HCDR1, HCDR2 shown in SEQ ID NO: 15 and HCDR3 shown in SEQ ID NO: 34;
  • control antibody comprises LCDR1 shown in SEQ ID NO:53, LCDR2 shown in SEQ ID NO:66, and LCDR3 shown in SEQ ID NO:82, and the control antibody comprises the LCDR1 shown in SEQ ID NO:5 HCDR1, HCDR2 shown in SEQ ID NO: 18 and HCDR3 shown in SEQ ID NO: 35;
  • the control antibody includes LCDR1 shown in SEQ ID NO: 55, LCDR2 shown in SEQ ID NO: 68, and LCDR3 shown in SEQ ID NO: 84, and the control antibody includes HCDR1 shown in SEQ ID NO: 8 , HCDR2 shown in SEQ ID NO: 20 and HCDR3 shown in SEQ ID NO: 37.
  • the primates include humans and/or monkeys.
  • the present application provides a fusion protein comprising: a) human TGFBRII or a fragment thereof; and b) the isolated antigen binding protein.
  • the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the heavy chain of the isolated antigen binding protein or a fragment thereof and the human TGFBRII or And the second polypeptide comprises the light chain of the isolated antigen binding protein or its fragment; preferably, the antibody heavy chain or its fragment is fused in frame with the human TGFBRII or its fragment to form the The first polypeptide; further preferably, wherein the C-terminus of the antibody heavy chain or a fragment thereof is directly or indirectly connected to the N-terminus of the human TGFBRII or a fragment thereof.
  • the heavy chain of the first polypeptide or a fragment thereof includes HCDR1, HCDR2, and HCDR3, and HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5; HCDR2 includes SEQ ID NOs: 179, 14 The amino acid sequence shown in any one of, 16 and 17; HCDR3 includes the amino acid sequence shown in any one of SEQ ID NOs: 180, 29-33; and/or the second polypeptide includes LCDR1, LCDR2, and LCDR3 , LCDR1 includes the amino acid sequence shown in any one of SEQ ID NOs: 181, 48-51; LCDR2 includes the amino acid sequence shown in any one of SEQ ID NOs: 182, 63-64; LCDR3 includes SEQ ID NOs: 191 , The amino acid sequence shown in any one of 77-79.
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 It includes the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29;
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33; or
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO: 33; or
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5.
  • the amino acid sequence shown, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 17, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29; or,
  • LCDR1 includes the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 includes the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 includes the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 includes the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29.
  • the antibody heavy chain or fragment thereof is connected to the human TGFBRII or fragment thereof through a linker; preferably, the linker is a peptide linker, and the peptide linker comprises SEQ ID NOs: the amino acid sequence shown in any one of 167-169.
  • the human TGFBRII or a fragment thereof comprises the extracellular domain of human TGFBRII; preferably, the human TGFBRII or a fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 176-178 .
  • the heavy chain or fragment of the first polypeptide is combined with the light chain or fragment of the second polypeptide to form an antigen binding portion that specifically binds to PD-L1.
  • the first polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 138, 139, 142, 143, and 145-148; wherein the second polypeptide comprises SEQ ID NOs : The amino acid sequence shown in any one of 162-163.
  • the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 138
  • the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 162; or
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 139, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 142
  • the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 143, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 145, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 163; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 146, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 163; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 147, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 148, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162.
  • the present application provides isolated one or more nucleic acid molecules, which encode the isolated antigen binding protein or the fusion protein.
  • the present application provides a vector, which contains the nucleic acid molecule.
  • the present application provides a cell, which contains the nucleic acid molecule or the vector.
  • the present application provides a method for preparing the isolated antigen binding protein or the fusion protein, the method comprising under the condition that the isolated antigen binding protein and the fusion protein are expressed, Culture the cells.
  • the present application provides a chimeric antigen receptor, which comprises the isolated antigen binding protein.
  • the present application provides genetically modified cells, which comprise the chimeric antigen receptor.
  • the present application provides an antibody drug conjugate, which includes a cytotoxic agent, and the isolated antigen binding protein.
  • the present application provides a pharmaceutical composition, which comprises the isolated antigen binding protein, the fusion protein, the nucleic acid molecule, the vector, the cell, the chimeric The antigen receptor, the genetically modified cell and/or the antibody drug conjugate, and optionally a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition also contains a hormone preparation, a target In the group consisting of small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutics, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines One or more of.
  • the present application provides the isolated antigen binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell, the chimeric antigen receptor, the Use of the genetically modified cells and/or the antibody-drug conjugate and/or the pharmaceutical composition in the preparation of a medicine for the prevention, alleviation and/or treatment of tumors or cancers, and inhibition of tumors Growth and/or inhibition of tumor cell proliferation; preferably, the tumor or cancer is a tumor or cancer with abnormal PD-L1 expression; preferably, the tumor or cancer includes colorectal cancer.
  • the present application provides a method for preventing, alleviating or treating tumors, inhibiting tumor growth and/or inhibiting tumor cell proliferation, the method comprising administering the isolated antigen binding protein to a subject in need ,
  • the fusion protein, the nucleic acid molecule, the vector, the cell, the chimeric antigen receptor, the genetically modified cell and/or the antibody drug conjugate And/or the pharmaceutical composition preferably, the tumor or cancer is a tumor or cancer with abnormal PD-L1 expression; preferably, the tumor or cancer includes colorectal cancer.
  • the present application provides the isolated antigen binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell, the chimeric antigen receptor,
  • the genetically modified cell and/or the antibody-drug conjugate and/or the pharmaceutical composition are used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the present application provides a method for inhibiting the binding of PD-L1 and or CD80 to PD-1, the method comprising administering the isolated antigen binding protein, the fusion protein, and the nucleic acid molecule , The carrier, the cell, the chimeric antigen receptor, the genetically modified cell and/or the antibody-drug conjugate and/or the pharmaceutical composition.
  • this application provides a kit, which includes the isolated antigen binding protein, the fusion protein, the nucleic acid molecule, the carrier, the cell, and the cell. Integrating antigen receptors, the genetically modified cells and/or the antibody-drug conjugate and/or the pharmaceutical composition; preferably, the kit further includes (i) an application device; and/ Or (ii) Instructions for use.
  • Figure 1 shows that the antigen binding protein described in this application binds to CHO-K1 cells overexpressing human PD-L1;
  • Figure 2 shows that the antigen binding protein described in this application binds to CHO-K1 cells overexpressing cynomolgus PD-L1.
  • Figure 3 shows that the antigen binding protein described in this application binds to human PD-L1 protein
  • Figure 4 shows that the antigen binding protein described in this application binds to the PD-L1 protein of cynomolgus monkey
  • Figure 5 shows that the antigen binding protein described in this application blocks the binding of human PD-1 to CHO-K1 cells overexpressing human PD-L1;
  • Figure 6 shows that the antigen binding protein described in this application blocks the binding of human PD-1 to CHO-K1 cells overexpressing human PD-L1;
  • Figure 7 shows that the antigen binding protein described in this application blocks the binding of biotinylated human PD-1 protein to human PD-L1 protein;
  • Figure 8 shows that the antigen binding protein described in this application blocks the binding of biotinylated human B7-1 (CD80) protein to human PD-L1 protein;
  • Figure 9 shows the inhibitory effect of the antigen binding protein of the present application on the PD-1 signaling pathway
  • Figure 10 shows the inhibitory effect of the antigen binding protein of the present application on the PD-1 signaling pathway
  • Figure 11 shows the affinity of the antigen binding protein described in this application to human PD-L1: (A) single concentration antigen binding and dissociation curve of PR000265, (B) single concentration antigen binding and dissociation curve of PR000266, (C) PR000265 Multi-concentration antigen binding and dissociation curve of (D) PR000151 multi-concentration antigen binding and dissociation curve, (E) PR001598 multi-concentration antigen binding and dissociation curve
  • Figure 12 shows that the antigen-binding protein of the present application stimulates cytokine secretion in the mixed lymphocyte reaction (MLR): in the first set of donor-paired MLR experiments, (A) IFN- in the supernatant after 120 hours ⁇ level, (B) IL-2 level in the supernatant after 72 hours; in the MLR experiment of the second group of donor pairing, (C) IFN- ⁇ level in the supernatant after 120 hours, (D) 72 hours The level of IL-2 in the supernatant;
  • MLR mixed lymphocyte reaction
  • Figure 13 shows the antibody-dependent cytotoxicity results of the antigen binding protein described in this application: (AC) the killing of tumor cells MDA-MB-231 by PBMC from three different donors; (DF) the results of three different donors The killing of tumor cells NCI-H292 by PBMC;
  • Figure 14 shows the in vivo tumor-inhibiting activity of the antigen-binding protein described in this application, which is the change in tumor volume (A) and the change in mouse body weight (B), respectively;
  • Figure 15 shows that the fusion protein described in this application binds to CHO-K1 cells overexpressing human PD-L1: (A) the fusion protein listed in Table 20-1, (B) the fusion protein listed in Table 20-2;
  • Figure 16 shows that the fusion protein described in this application blocks the binding of human PD-1 to CHO-K1 cells overexpressing human PD-L1: (A) the fusion protein listed in Table 21-1, (B) Table 21- 2 listed fusion proteins;
  • Figure 17 shows the inhibitory effect of the fusion protein of the present application on the PD-1 signaling pathway
  • Figure 18 shows that the fusion protein described in this application binds to TGFB1: (A) the fusion protein listed in Table 23-1, (B) the fusion protein listed in Table 23-2, and (C) the fusion protein listed in Table 23-3;
  • Figure 19 shows the inhibitory effect of the fusion protein of the present application on the activation of the Smad signaling pathway induced by TGFB1;
  • Figure 20 shows the fusion protein of the present application in the mixed lymphocyte reaction (MLR), without or with the addition of TGFB1, stimulated T lymphocytes to secrete cytokine levels: (A) 72 hours later in the supernatant IL-2 level, (B) IFN- ⁇ level in the supernatant after 120 hours;
  • MLR mixed lymphocyte reaction
  • Figure 21 shows the antibody-dependent cytotoxicity results of the fusion protein described in this application: (AC) the killing of tumor cells MDA-MB-231 by PBMC from three different donors; (DF) PBMC from three different donors Killing tumor cells NCI-H292;
  • Figure 22 shows the pharmacokinetic results of the fusion protein PR001902 described in this application.
  • Figure 23 shows the pharmacokinetic results of the fusion protein PR001488 described in this application.
  • Figure 24 shows the anti-tumor activity of the fusion protein described in the present application on an in vivo animal model of tumor, which is the change in tumor volume (A) and the change in mouse body weight (B), respectively.
  • the term "antigen-binding protein” generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen-binding fragments
  • Fv fragments F(ab') 2
  • scFv fragments
  • di-scFv and/or dAb immunoconjugates
  • immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • fusion protein generally refers to a protein composed of two or more polypeptides, which are usually not combined in the natural state, but the respective amino and carboxyl termini can be directly or indirectly combined to form one Continuous peptides.
  • fusion protein can be used interchangeably with "antibody” when referring to the fusion protein described in this application.
  • fusion protein can be used interchangeably with "antigen binding protein” when referring to the fusion protein described in this application.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' usually refers to a fragment that is different from Fab by adding a small number of residues (including one or more cysteine from the hinge region of an antibody) to the carboxyl end of the CH1 domain of the heavy chain;
  • F(ab ') 2 generally refers to Fab' dimer antibody fragments comprising two Fab fragments by a disulfide bridge at the hinge region.
  • Fv generally refers to the smallest antibody fragment that contains a complete antigen recognition and binding site.
  • the fragment may be composed of a dimer in which a heavy chain variable region and a light chain variable region are tightly non-covalently bound;
  • dsFv generally refers to a disulfide bond-stabilized Fv fragment, The bond between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
  • dAb fragment generally refers to an antibody fragment composed of VH domains.
  • scFv generally refers to a monovalent molecule formed by covalently connecting and pairing a heavy chain variable domain and a light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have general Structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • the more highly conserved parts of variable domains are called the framework (FR).
  • the variable domains of the natural heavy chain and light chain each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) Most of them adopt ⁇ -sheet configuration and are connected by three CDR structure loop regions.
  • the CDRs in each chain are close together through the FR region, and together with the CDRs from the other chain form the antigen binding site of the antibody.
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions. , Such as involved in antibody-dependent cytotoxicity.
  • the CDR of an antibody can be defined by a variety of methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Star, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997).
  • the combined definition rule including the Kabat definition and Chothia definition is also used to determine the amino acid residues in the variable domain sequence and the full-length antibody sequence. Refer to Table 1 for antibody CDR definition methods.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rules) to position bb (Chothia coding rules);
  • Haa-Hbb can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain , The amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule).
  • L24-L34 can refer to the amino acid sequence from the 24th to the 34th starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and following the Chothia coding rules The amino acid sequence from position 26 to position 32.
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, separated and/or recovered from a component of its production environment (for example, natural or recombinant).
  • the pollutant components of the environment are usually substances that interfere with its research, diagnostic or therapeutic use, and can include enzymes, hormones and other protein or non-protein solutes.
  • the isolated antigen binding protein or antibody will usually be prepared through at least one purification step.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the individual antibodies in the group are the same, except for a small number of natural mutations that may exist.
  • Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins.
  • the modifier "monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous antibody population, and is not interpreted as requiring the production of the antibody by any specific method.
  • the monoclonal antibody used according to the present invention can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • Fully human antibody generally refers to an antibody that is expressed by the animal by transferring a human antibody-encoding gene into a genetically engineered animal with a gene-deficient antibody gene. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies on the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.
  • the term "specific binding” generally means that the antibody binds to the epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, when an antibody binds to an epitope through its antigen binding domain more easily than it will bind to a random, unrelated epitope, an antibody is said to “specifically bind” to that antigen.
  • Epitope refers to a specific atomic group (for example, sugar side chain, phosphoryl, sulfonyl) or amino acid that binds to an antigen-binding protein (such as an antibody) on an antigen.
  • KD and “K D” are used interchangeably, usually referring to the equilibrium dissociation constant
  • KD is the dissociation rate constant (kdis, also known as the “off-rate ) (koff)” or “kd") and the ratio of the association rate constant (kon, also known as “combination rate (kon)” or “ka”).
  • the binding rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD) can be used to express the binding affinity of an antigen-binding protein (for example, an antibody) to an antigen.
  • Methods for determining the rate constants of association and dissociation are well known in the art, including but 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. If measured under different conditions (such as salt concentration, pH), the measured affinity of a specific protein-protein interaction may be different.
  • PD-L1 generally refers to the programmed death ligand 1 protein, its functional variants and/or its functional fragments.
  • PD-L1 is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), and is a protein encoded by the CD274 gene (in humans).
  • PD-L1 binds to its receptors, such as programmed death 1 (PD-1), which is expressed in activated T cells, B cells, and macrophages (Ishida et al., 1992 EMBO J, 11: 3887-3395; Okazaki et al., Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001; 291:319-22).
  • PD-1 programmed death 1
  • the complexation of PD-L1 and PD-1 exerts an immunosuppressive effect by inhibiting the proliferation of T cells and the production of cytokines IL-2 and IFN- ⁇ (Freeman et al., Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J.Exp.Med.2000,192:1027-1034; Carter et al., PD-1:PD-L inhibitory pathway affects both CD4(+)and CD8(+)T cells and is overcome by IL-2.Eur.J.Immunol.2002,32:634–643).
  • PD-L1 encompasses any natural PD-L1 of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • the term encompasses "full length", unprocessed PD-L1 and any form of PD-L1 produced by processing in the 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 4 domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • the PD-L1 sequence is known in the art. For example, you can find information about the human PD-L1 gene (including genomic DNA sequence) under NCBI Gene ID No. 29126. For another example, you can find information about mouse PD-L1 gene (including genomic DNA sequence) under NCBI Gene ID No. 60533. For another example, you can find information about the cynomolgus monkey PD-L1 gene (including genomic DNA sequence) under NCBI Gene ID No. 102145573.
  • the amino acid sequence of an exemplary full-length human PD-L1 protein can be found under NCBI accession number NP_054862 or UniProt accession number Q9NZQ7.
  • Exemplary full-length mouse PD-L1 protein sequences can be found under NCBI accession number NP_068693 or Uniprot accession number Q9EP73.
  • An exemplary full-length cynomolgus monkey PD-L1 protein sequence can be found under NCBI accession number XP_005581836 or Uniprot accession number G7PSE7.
  • PD-1 generally refers to the programmed death 1 receptor (also known as CD279), its functional variants and/or its functional fragments.
  • PD-1 is usually expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DC).
  • DC dendritic cells
  • PD-1 can bind to its ligands PD-L1 and PD-L2.
  • variants that differ in amino acid sequence from naturally-occurring PD-1 but retain the ability to specifically bind to PD-L1.
  • a variant that enhances the biological activity of PD-L1 is further included.
  • the PD-1 sequence is known in the art.
  • an exemplary full-length human PD-1 protein sequence can be found under NCBI accession number NP_005009
  • an exemplary full-length cynomolgus monkey PD-1 protein sequence can be found under NCBI accession number NP_001271065 or Uniprot accession number BOLAJ3.
  • CD80 generally refers to cluster of differentiation 80 (also known as B7-1), its functional variants and/or its functional fragments. CD80 is usually expressed on professional antigen presenting cells (APC). CD80 can bind to PD-L1. Also included within the definition of CD80 are variants that differ in amino acid sequence from naturally-occurring CD80 but retain the ability to specifically bind to PD-L1.
  • CD80 as used herein includes human CD80 (hCD80), variants, isomers and species homologs of hCD80, and analogs having at least one common epitope containing hCD80.
  • CD80 also encompasses CD80 from other species such as other mammals (e.g., rats, mice, rabbits, non-human primates, pigs, or cattle).
  • mammals e.g., rats, mice, rabbits, non-human primates, pigs, or cattle.
  • the complete human CD80 sequence can be found in Uniprot accession number P33681.
  • TGFBRII generally refers to a receptor for transforming growth factor B (TGFB) (also known as TGFBR2).
  • TGFBR transforming growth factor B
  • the TGFBR on the cell surface can be combined and activated by transforming growth factor (TGFB), signal transmission through the SMAD pathway, and has the activity of regulating growth, anti-inflammatory and immune regulation.
  • TGFBRII sequence can be found in Uniprot accession number P37173.
  • the term "specific anti-PD-L1 antibody” generally refers to an antibody that can compete with the antibody of the present application for binding to PD-L1.
  • the antigen binding protein of the present application may include those antibodies that have the same amino acid sequence as the specific anti-PD-L1 antibody.
  • control antibody generally refers to the PD-L1 antibody used in the examples of the present application to provide a standard against which to evaluate experimental results.
  • control antibody may refer to the positive control in the examples of this application, such as PR000151, PR001598, and PR002466.
  • the term "primate” generally refers to monkey and ape species, and includes monkey species, such as from the genus Macaca, such as, and specifically, cynomolgus (Macaca fascicularis) and Or rhesus monkeys (Macaca mulatta) and baboons (Papio ursinus) monkeys, as well as marmosets (from species of the genus Callithrix), squirrel monkeys (from squirrel monkeys (Saimiri)) Species of the genus) and tamarins (species from the genus Saguinus), and ape species such as chimpanzees (Pan troglodytes), and also include Homo sapiens.
  • monkey species such as from the genus Macaca, such as, and specifically, cynomolgus (Macaca fascicularis) and Or rhesus monkeys (Macaca mulatta) and baboons (Papio ursinus
  • epitope generally refers to a certain region or region of an antigen to which an antigen binding protein (such as an antibody) specifically binds.
  • Epitopes usually consist of chemically active surface groups such as amino acids or carbohydrates or sugar side chain molecules, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • the epitope can be a "linear epitope” or a “configuration epitope”. In a linear epitope, all interaction points between a protein and an interacting molecule (such as an antibody) occur linearly along the protein's primary amino acid sequence. In a conformational epitope, the points of interaction crossover occur on amino acids on separate proteins.
  • epitope mapping Methods for which epitope a given antigen-binding protein (such as an antibody) binds (e.g., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, for example, testing for overlap or adjacency The reactivity of the peptide (e.g. from PD-L1) with a given antigen binding protein (e.g. anti-PD-L1 antibody).
  • Methods for determining the spatial conformation of an epitope include those in the art and those described in this application, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • epitope mapping methods such as x-ray analysis of crystals of antigen: antibody complexes, which provide atomic resolution of epitopes Rate, and hydrogen/deuterium exchange mass spectrometry (HDX-MS).
  • the term "subject” generally refers to a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as human and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). ).
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.
  • 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 vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the term "cell” generally refers to an individual cell, cell line, or cell line that can or already contains a plasmid or vector containing the nucleic acid molecule described in this application, or capable of expressing the antibody or antigen-binding fragment thereof described in this application.
  • the cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells and the original parent cells may not necessarily be identical in morphology or genome, but they can express the antibodies or antigen-binding fragments described in this application.
  • the cells can be obtained by transfecting cells in vitro using the vectors described in this application.
  • the cell may be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or Myeloma cells).
  • the cell may be a mammalian cell.
  • the mammalian cell may be a CHO-K1 cell.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector is introduced.
  • the recombinant host cell includes not only certain specific cells, but also the progeny of these cells.
  • the term "pharmaceutical composition” generally refers to a preparation that exists in a form that allows the biological activity of the active ingredient to be effective, and does not contain unacceptable toxicity to the subject to which the composition will be administered. Additional ingredients.
  • the composition is sterile.
  • “Sterile” compositions are sterile or free of all living microorganisms and their spores.
  • treatment generally refers to the desire to change the natural course of the individual to be treated, and can be a clinical intervention to achieve prevention and treatment or in the course of clinical pathology.
  • Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis.
  • antibodies e.g., anti-PD-L1 antibodies
  • administering generally refers to the subject (eg, patient) administering a certain dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition containing an anticancer therapeutic agent)
  • a compound eg, an anticancer therapeutic agent
  • a pharmaceutical composition eg, a pharmaceutical composition containing an anticancer therapeutic agent
  • Methods can be carried out by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if local treatment is required) intralesional administration.
  • Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the administration can be carried out by any suitable route, for example by injection (such as intravenous or subcutaneous injection).
  • Various administration procedures are covered herein, including but not limited to single administration or multiple administrations at various time points, bolus administration, and pulse infusion.
  • tumor generally refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all precancerous and cancerous cells and tissues.
  • the tumor may include colon cancer.
  • the term "between” usually 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 the N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment. Indirect connection.
  • the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1
  • the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly connected to the C-terminus of the LCDR2
  • the C-terminus of the L-FR3 is directly or indirectly connected to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3.
  • the "first amino acid fragment" and the "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%, above or below the specified value. Variation within the range of 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 antigen binding protein comprising at least one CDR in the variable region VL of an antibody light chain.
  • the antigen binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 181:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the LCDR1 may include at least amino acid substitutions selected from the following positions: amino acid substitutions at X 7 and/or X 8 .
  • the LCDR1 may include at least the amino acid substitutions at X 7 and/or X 8 , wherein the amino acid at X 7 may be substituted It is Y or F; the amino acid at X 8 can be substituted with S.
  • the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NOs: 48-51.
  • the antigen binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 182:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • LCDR2 may comprise at least the amino acid substitution at a position selected from: an amino acid substitution at the 7 X.
  • LCDR2 may comprise at least the amino acid substitution at X 7, wherein X 7 at amino acid may be substituted with T.
  • the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NOs: 63-64.
  • the antigen binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 183:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the LCDR3 may include at least amino acid substitutions selected from the following positions: at X 4 , X 5 and/or X 6 The amino acid substitutions.
  • the LCDR3 may include at least amino acid substitutions at X 4 , X 5 and/or X 6 , wherein the amino acid at X 4 It can be substituted with H; the amino acid at X 5 can be substituted with S or T; the amino acid at X 6 can be substituted with S.
  • the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 77-80.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 191:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the LCDR3 may include at least amino acid substitutions selected from the following positions: amino acids at X 5 and/or X 6 replace.
  • the LCDR3 may include at least the amino acid substitution at X 5 and/or X 6 , wherein the amino acid at X 5 may be Replaced with S; the amino acid at X 6 can be substituted with S.
  • the LCDR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 77-79.
  • the antigen binding protein may include the framework region L-FR1.
  • the C-terminus of the L-FR1 is connected to the N-terminus of the LCDR1, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 185:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the L-FR1 may include at least amino acid substitutions selected from the following positions: X 13 , X 17 and / Or X 21 amino acid substitution.
  • the L-FR1 may include at least amino acid substitutions at X 13 , X 17 and/or X 21, wherein ,
  • the amino acid at X 13 can be substituted with A; the amino acid at X 17 can be substituted with H; the amino acid at X 21 can be substituted with V.
  • the L-FR1 may include the amino acid sequence shown in any one of SEQ ID NOs: 41-44.
  • the antigen binding protein may comprise L-FR2.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 186:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the L-FR2 includes at least amino acid substitutions selected from the following positions: X 5 and/or X 11 Amino acid substitutions at.
  • the L-FR2 may include at least an amino acid substitution at X 5 and/or X 11 , wherein X 5
  • the amino acid at X may be substituted with H; the amino acid at X 11 may be substituted with D or N.
  • the L-FR2 may include the amino acid sequence shown in any one of SEQ ID NOs: 56-59.
  • the antigen binding protein may include L-FR3.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid shown in SEQ ID NO: 187 sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the L-FR3 may include at least amino acid substitutions selected from the following positions: at X 9 , X 28 and/or X 31 Amino acid substitutions at.
  • the L-FR3 may include at least amino acid substitutions at X 9 , X 28 and/or X 31, wherein ,
  • the amino acid at X 9 can be substituted with N; the amino acid at X 28 can be substituted with T; the amino acid at X 31 can be substituted with F.
  • the L-FR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 69-72.
  • the antigen binding protein may include L-FR4, the N-terminus of L-FR4 is connected to the C-terminus of LCDR3, and the L-FR4 may include SEQ ID NO: 188
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • L-FR4 comprises at least the amino acid substitution at a position selected from the following: amino acid substitution at X 10.
  • the L-FR4 may include at least the amino acid substitution at X 10 , wherein the amino acid at X 10 may be substituted with R.
  • the L-FR4 may include the amino acid sequence shown in any one of SEQ ID NOs: 85-86.
  • the antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 190:
  • the VL may include at least amino acid substitutions at positions selected from: X 13 , X 17 , X 21 , X 30 , X 31 , X 39 , X 45 , X 56 , X 65 , X 84 , X 87 , X 92 , X 93 , X 94 , X 107 amino acid substitutions.
  • the VL may be included in at least X 13 , X 17 , X 21 , X 30 , X 31 , X 39 , X 45 , X 56 , Amino acid substitutions at X 65 , X 84 , X 87 , X 92 , X 93 , X 94 , X 107 , wherein the amino acid at X 13 can be substituted with A, the amino acid at X 17 can be substituted with H, X The amino acid at 21 can be replaced with V, the amino acid at X 30 can be replaced with S or Y, the amino acid at X 31 can be replaced with S, the amino acid at X 39 can be replaced with H, the amino acid at X 45 can be replaced Can be substituted with N or D, X 56 can be substituted with T, X 65 can be substituted with N, X 84 can be substituted with T, X 84 , Amino acid substitutions at X 65 , X 84
  • the VL region may include the amino acid sequence shown in any one of SEQ ID NOs: 108-116 and 118.
  • VL described in this application may include the amino acid sequence shown in SEQ ID NO: 194:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the VL may include at least amino acid substitutions at positions selected from: X 13 , X 17 , X 21 , X 30 , X 31 , X 39 , X 45 , X 56 , X 65 , X 84 , X 87 , X 93 , X 94 , X 107 amino acid substitutions.
  • the VL may be included in at least X 13 , X 17 , X 21 , X 30 , X 31 , X 39 , X 45 , X 56 ,
  • the amino acid substitutions at X 65 , X 84 , X 87 , X 93 , X 94 , and X 107 wherein the amino acid at X 13 can be substituted with A, the amino acid at X 17 can be substituted with H, and the amino acid at X 21 Amino acids can be replaced with V, amino acids at X 30 can be replaced with S or Y, amino acids at X 31 can be replaced with S, amino acids at X 39 can be replaced with H, and amino acids at X 45 can be replaced Is N or D, X 56 can be substituted with T, X 65 can be substituted with N, X 84 can be substituted with T, X 87 can be substituted with F
  • the VL region may include the amino acid sequence shown in any one of SEQ ID NOs: 108-114, 116, and 118.
  • the antigen binding protein of the present application may include a light chain constant region CL, and the antibody light chain constant region may include a human Ig ⁇ constant region.
  • CL region may include the amino acid sequence shown below: SEQ ID NO: 170.
  • the antigen binding protein described in the present application may include an antibody light chain LC, and the antibody light chain may include the amino acid sequence shown in any one of SEQ ID NOs: 150-158 and 160-163.
  • the antibody light chain of the antigen binding protein described in the present application may comprise the amino acid sequence shown in any one of SEQ ID NOs: 150-156, 158, and 160-163.
  • the antigen binding protein described in the present application may comprise at least one CDR in the VH of the variable region of the antibody heavy chain.
  • the antigen binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antigen binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 179:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the HCDR2 may include at least amino acid substitutions selected from the following positions: amino acid substitutions at X 3 and/or X 4 .
  • the HCDR2 may include at least the amino acid substitution at X 3 and/or X 4 , wherein the amino acid at X 3 may be Substitution is E, X 4 can be substituted with A.
  • the HCDR2 may include the amino acid sequence shown in any one of SEQ ID NOs: 14, 16, and 17.
  • the antigen binding protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 180:
  • sequence may be a sequence determined according to Chothia's definition rules.
  • the HCDR3 may include at least amino acid substitutions selected from the following positions: at X 3 , X 8 , X 9 and/or the amino acid substitution at X 10.
  • the HCDR3 may include at least amino acid substitutions at X 3 , X 8 , X 9 and/or X 10 , wherein X 3
  • the amino acid at X may be substituted with P; the amino acid at X 8 may be substituted with S; the amino acid at X 9 may be substituted with A; the amino acid at X 10 may be substituted with F.
  • the HCDR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 29-33.
  • the antigen binding protein may include the framework region H-FR1.
  • the C-terminus of the H-FR1 is connected to the N-terminus of the HCDR1, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO:1:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antigen binding protein may include H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may include the sequence shown in SEQ ID NO: 9 Amino acid sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antigen binding protein may include H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may include the sequence shown in SEQ ID NO: 184 Amino acid sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the H-FR3 may include at least amino acid substitutions selected from the following positions: X 4 , X 22 , X 33 and/ Or an amino acid substitution at X 37.
  • the H-FR3 may include at least amino acid substitutions at X 4 , X 22 , X 33 and/or X 37, wherein, the amino acid at X 4 can be substituted with G; the amino acid at X 22 can be substituted with Q; the amino acid at X 33 can be substituted with E; the amino acid at X 37 can be substituted with F.
  • the H-FR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 21-24.
  • the H-FR3 described in the present application may include the amino acid sequence shown in SEQ ID NO: 192:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the H-FR3 may include at least amino acid substitutions selected from the following positions: at X 4 , X 33 and/or X 37 Amino acid substitutions at.
  • the H-FR3 may include at least amino acid substitutions at X 4 , X 33 and/or X 37, wherein X
  • the amino acid at 4 can be substituted with G; the amino acid at X 33 can be substituted with E; the amino acid at X 37 can be substituted with F.
  • the H-FR3 may include the amino acid sequence shown in any one of SEQ ID NOs: 21-22 and 24.
  • the antigen binding protein may include H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may include SEQ ID NO: 38
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the antigen binding protein may include the heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 189:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the VH may include at least amino acid substitutions at positions selected from: X 54 , X 55 , X 61 , X 79 , X 90 , X 94 , X 101 , X 106 , X 107 and/or X 108 amino acid substitutions.
  • the VH may be at least included in X 54 , X 55 , X 61 , X 79 , X 90 , X 94 , X 101 , X 106 , amino / or 108 X-107 and X-substituted, wherein the amino acid X 54 at may be substituted is E, amino acids 55 to X may be substituted with a, the amino acid X 61 is at may be substituted with G, X 79 at a
  • the amino acid at X can be replaced with Q, the amino acid at X 90 can be replaced with E, the amino acid at X 94 can be replaced with F, the amino acid at X 101 can be replaced with P, and the amino acid at X 106 can be replaced with S, the amino acid at X 107 may be substituted with A, and the amino acid at X 108 may be substituted with F.
  • the VH region may comprise the amino acid sequence shown in any one of SEQ ID NOs: 90-95 and 97-104.
  • the VH described in this application may include the amino acid sequence shown in SEQ ID NO: 193:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the VH may include at least amino acid substitutions selected from the following positions: X 54 , X 55 , X 61 , X 90 , X 94 , X 101 , X 106 , X 107 and/or X 108 amino acid substitutions.
  • the VH may be at least included in X 54 , X 55 , X 61 , X 90 , X 94 , X 101 , X 106 , X 107 and /Or the amino acid substitution at X 108 , wherein the amino acid at X 54 can be substituted with E, the amino acid at X 55 can be substituted with A, the amino acid at X 61 can be substituted with G, and the amino acid at X 90 can be substituted
  • the amino acid at X 94 can be replaced with F
  • the amino acid at X 101 can be replaced with P
  • the amino acid at X 106 can be replaced with S
  • the amino acid at X 107 can be replaced with A
  • X The amino acid at 108 can be substituted with F.
  • the VH region may comprise the amino acid sequence shown in any one of SEQ ID NOs: 90-93, 95, and 97-104.
  • the antigen binding protein of the present application may include a heavy chain constant region CH, and the antibody heavy chain constant region may include a human IgG constant region.
  • the human IgG constant region may comprise a human IgG1 constant region.
  • the human IgG1 constant region may include natural and artificially synthesized IgG1 constant regions or mutants thereof.
  • the mutation may include one or more of the following mutations: L234, L235, N297 or K447. For example, it may contain mutations at one, two or more positions. Mutations may include deletions, insertions or substitutions of amino acids.
  • the human IgG1 constant region may include the following mutations: 1) N297A, 2) K447 deletion, 3) N297A and K447 deletion, 4) L234A, L235A and K447 deletion.
  • the human IgG1 constant region of the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 172-175.
  • the isolated antigen binding protein may comprise LCDR1-3.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 181; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 182; and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 183 .
  • the LCDR1 of the antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 181; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 182; and the LCDR3 may include SEQ ID NO : 191 amino acid sequence.
  • the LCDR1 of the antigen binding protein described in the present application may include the amino acid sequence shown in any of the following: SEQ ID NO: 48-51; the LCDR2 may include any of the following The amino acid sequence shown: SEQ ID NO: 63-64; and the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 77-80.
  • the LCDR1 of the antigen binding protein described in the present application may include the amino acid sequence shown in any one of the following: SEQ ID NO: 48-51; the LCDR2 may include any of the following The amino acid sequence shown: SEQ ID NO: 63-64; and the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 77-79.
  • the antigen binding protein described in this application may include LCDR1-3 which is the same as PR000064.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 48
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as PR000065, where LCDR1 may include the amino acid sequence shown in SEQ ID NO: 49, and LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64, LCDR3 It may include the amino acid sequence shown in SEQ ID NO: 78.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as PR000066, PR000261, and PR000262.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 48
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as PR000067, where LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50, and LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63, LCDR3 It may include the amino acid sequence shown in SEQ ID NO: 77.
  • the antigen binding protein described in this application may include LCDR1-3 which is the same as PR000068.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 79.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as PR000069, PR000263, and PR000264, wherein LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51, and LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63 Amino acid sequence, LCDR3 may include the amino acid sequence shown in SEQ ID NO:77.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as PR000070, PR000265, and PR000266.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as PR000071, wherein LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50, and LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63, LCDR3 It may include the amino acid sequence shown in SEQ ID NO: 80.
  • the antigen binding protein described in the present application may include LCDR1-3 which is the same as PR000072, PR000267, and PR000268.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77.
  • the isolated antigen binding protein may comprise L-FR1-4.
  • the L-FR1 of the antigen binding protein of the present application may include the amino acid sequence shown in any one of the following: SEQ ID NO: 41-44; the L-FR2 may include the following The amino acid sequence shown in any item: SEQ ID NO: 56-59; the L-FR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 69-72; and the L- FR4 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 85-86.
  • the antigen binding protein described in the present application may contain the same L-FR1-4 as PR000064.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 41
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in the present application may contain the same L-FR1-4 as PR000065.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 57
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in this application may contain the same L-FR1-4 as PR000066.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 43
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 58
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 70
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in this application may contain the same L-FR1-4 as PR000067.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in this application may contain the same L-FR1-4 as PR000068.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 71
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in this application may contain the same L-FR1-4 as PR000071.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in the present application may include the same L-FR1-4 as PR000069, PR000263, and PR000264.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 59
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 72
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 86.
  • the antigen binding protein described in the present application may contain the same L-FR1-4 as PR000070, PR000265, and PR000266.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 44
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 57
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in the present application may include the same L-FR1-4 as PR000072, PR000267, and PR000268.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 57
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the antigen binding protein described in this application may contain the same L-FR1-4 as PR000261 and PR000262.
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 43
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 58
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO:85.
  • the isolated antigen binding protein may comprise HCDR1-3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5;
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 179;
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 180 .
  • the HCDR1 of the antigen binding protein described in the present application may include the amino acid sequence shown below: SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 14, 16 and 17; and the HCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 29-33.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000064, PR000065, PR000067, and PR000070.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000066.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000068.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 31.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000069.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000071 and PR000072.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000261.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein described in this application may include HCDR1-3 which is the same as PR000262.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000263.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the antigen binding protein described in this application may include HCDR1-3 which is the same as PR000264.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the antigen binding protein described in the present application may include the same HCDR1-3 as PR000265.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000266.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000267.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the antigen binding protein described in the present application may contain the same HCDR1-3 as PR000268.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the isolated antigen binding protein may comprise H-FR1-4.
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 1; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 9; the H-FR3 may include SEQ ID NO: 184 The amino acid sequence shown; and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38.
  • the H-FR1 may include the amino acid sequence shown in SEQ ID NO:1; the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 9; the H-FR3 may include SEQ ID NO: 192 The amino acid sequence shown; and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38.
  • the H-FR1 of the antigen binding protein of the present application may include the amino acid sequence shown below: SEQ ID NO: 1; the H-FR2 may include the amino acid sequence shown below: SEQ ID NO: 9; The H-FR3 may include the amino acid sequence shown in any of the following: SEQ ID NO: 21-24; and the H-FR4 may include the amino acid sequence shown in any of the following Amino acid sequence: SEQ ID NO: 38.
  • the H-FR1 of the antigen binding protein of the present application may include the amino acid sequence shown below: SEQ ID NO: 1; the H-FR2 may include the amino acid sequence shown below: SEQ ID NO: 9; The H-FR3 may include the amino acid sequence shown in any of the following: SEQ ID NOs: 21-22 and 24; and the H-FR4 may include any of the following The amino acid sequence shown: SEQ ID NO: 38.
  • the antigen binding protein described in the present application may include H-FR1-4, wherein the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 9.
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 21, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38.
  • the antigen binding protein described in the present application may include H-FR1-4, wherein the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 9.
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 22, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38.
  • the antigen binding protein described in the present application may include H-FR1-4, wherein the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 9.
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 23, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38.
  • the antigen binding protein described in the present application may include H-FR1-4, wherein the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 1, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 9.
  • the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 24, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 38.
  • the isolated antigen binding protein may comprise LCDR1-3 and HCDR1-3.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 181; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 182; and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 183
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 179; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 180.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 181; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 182; and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 191
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 179; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 180.
  • the LCDR1 of the antigen binding protein described in the present application may include the amino acid sequence shown in any of the following: SEQ ID NOs: 48-51; the LCDR2 may include any of the following The amino acid sequence shown: SEQ ID NOs: 63-64; and the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 77-80; the HCDR1 may include the following Amino acid sequence: SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 14, 16, and 17; and the HCDR3 may include any one of the following The amino acid sequence of: SEQ ID NOs: 29-33.
  • the LCDR1 of the antigen binding protein of the present application may include the amino acid sequence shown in any of the following: SEQ ID NOs: 48-51; the LCDR2 may include any of the following The amino acid sequence shown: SEQ ID NOs: 63-64; and the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 77-79; the HCDR1 may include the following Amino acid sequence: SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 14, 16, and 17; and the HCDR3 may include any one of the following The amino acid sequence of: SEQ ID NOs: 29-33.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000064.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 48
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in this application may include LCDR1-3 and HCDR1-3 which are the same as PR000065.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 49
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 may include SEQ ID NO The amino acid sequence shown in: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000066.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 48
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein described in this application may include LCDR1-3 and HCDR1-3 which are the same as PR000067.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50;
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63;
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77, wherein HCDR1 may include SEQ ID NO The amino acid sequence shown in: 5,
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000068.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 79
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 31.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000069.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 may include SEQ ID NO The amino acid sequence shown in: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000070.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000071.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 80
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000072.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 may include SEQ ID NO The amino acid sequence shown in: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 14
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000261.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 48
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000262.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 48
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 30.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000263.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 may include SEQ ID NO
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the antigen binding protein described in this application may include LCDR1-3 and HCDR1-3 which are the same as PR000264.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 63
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 may include SEQ ID NO The amino acid sequence shown in: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000265 and PR000416.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000266.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 78
  • HCDR1 may include SEQ ID
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000267.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 may include SEQ ID NO The amino acid sequence shown in: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 16
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the antigen binding protein described in the present application may include LCDR1-3 and HCDR1-3 which are the same as PR000268.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 50
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 64
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 77
  • HCDR1 may include SEQ ID NO The amino acid sequence shown in: 5
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 17
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the antigen binding protein may comprise a light chain variable region VL and a heavy chain variable region VH.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 194, and the VH may include the amino acid sequence shown in SEQ ID NO: 193.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 190, and the VH may include the amino acid sequence shown in SEQ ID NO: 189.
  • the VL of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 108-116 and 118, and the VH may include SEQ ID NOs: 90-95, The amino acid sequence shown in any one of 97-104.
  • the VL of the antigen binding protein may include the amino acid sequence shown in any one of SEQ ID NOs: 108-114, 116, and 118, and the VH may include SEQ ID NOs: 90- The amino acid sequence shown in any one of 93, 95, and 97-104.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000064, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 108, and the VH may include the amino acid sequence shown in SEQ ID NO: 90. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000065, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 109, and the VH may include the amino acid sequence shown in SEQ ID NO: 90. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000066, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 110, and the VH may include the amino acid sequence shown in SEQ ID NO: 91. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000067, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 111, and the VH may include the amino acid sequence shown in SEQ ID NO: 90. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000068, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 112, and the VH may include the amino acid sequence shown in SEQ ID NO: 92. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000069, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 113, and the VH may include the amino acid sequence shown in SEQ ID NO: 93. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000070, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 114, and the VH may include the amino acid sequence shown in SEQ ID NO: 90. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000071, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 115, and the VH may include the amino acid sequence shown in SEQ ID NO: 94 The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000072, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 116, and the VH may include the amino acid sequence shown in SEQ ID NO: 95. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000261, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 118, and the VH may include the amino acid sequence shown in SEQ ID NO: 97. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000262, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 118, and the VH may include the amino acid sequence shown in SEQ ID NO: 98. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000263, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 113, and the VH may include the amino acid sequence shown in SEQ ID NO: 99. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000264, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 113, and the VH may include the amino acid sequence shown in SEQ ID NO: 100. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000265 or PR000416, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 114, and the VH may include SEQ ID NO: The amino acid sequence shown in 101.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000266, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 114, and the VH may include the amino acid sequence shown in SEQ ID NO: 102. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000267, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 116, and the VH may include the amino acid sequence shown in SEQ ID NO: 103. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same VL and VH as PR000268, wherein the VL may include the amino acid sequence shown in SEQ ID NO: 116, and the VH may include the amino acid sequence shown in SEQ ID NO: 104. The amino acid sequence shown.
  • the antigen binding protein may include a light chain constant region and a heavy chain constant region, wherein the light chain constant region may include the amino acid sequence shown in SEQ ID NO: 170, and the heavy chain is constant
  • the region may include the amino acid sequence shown in any one of SEQ ID NOs: 172-175.
  • the antigen binding protein may comprise an antibody light chain LC and an antibody heavy chain HC, wherein the antibody light chain LC may comprise the amino acid sequence shown in any one of the following: SEQ ID NO: 150 -158 and 160-163, and the antibody heavy chain HC may comprise the amino acid sequence shown in any one of the following: SEQ ID NO: 122-127 and 129-137.
  • the antigen binding protein may comprise an antibody light chain LC and an antibody heavy chain HC, wherein the antibody light chain LC may comprise the amino acid sequence shown in any one of the following: SEQ ID NO: 150 -156, 158 and 160-163, and the antibody heavy chain HC may comprise the amino acid sequence shown in any one of the following: SEQ ID NO: 122-125, 127 and 129-137.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000064, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 150, and the HC may include the amino acid sequence shown in SEQ ID NO: 122. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000065, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 151, and the HC may include the amino acid sequence shown in SEQ ID NO: 122. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000066, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 152, and the HC may include the amino acid sequence shown in SEQ ID NO: 123. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000067, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 153, and the HC may include the amino acid sequence shown in SEQ ID NO: 122. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000068, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 154, and the HC may include the amino acid sequence shown in SEQ ID NO: 124. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same LC and HC as PR000069, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 155, and the HC may include the same as that shown in SEQ ID NO: 125. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000070, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 156, and the HC may include the amino acid sequence shown in SEQ ID NO: 122. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000071, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 157, and the HC may include the amino acid sequence shown in SEQ ID NO: 126. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same LC and HC as PR000072, where the LC may include the amino acid sequence shown in SEQ ID NO: 158, and the HC may include the same as that shown in SEQ ID NO: 127. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000261, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 160, and the HC may include the amino acid sequence shown in SEQ ID NO: 129. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same LC and HC as PR000262, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 160, and the HC may include the same as that shown in SEQ ID NO: 130.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000263, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 161, and the HC may include the amino acid sequence shown in SEQ ID NO: 131. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000264, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 161, and the HC may include the amino acid sequence shown in SEQ ID NO: 132. The amino acid sequence shown.
  • the antigen-binding protein described in the present application may include the same LC and HC as PR000265, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 162, and the HC may include the amino acid sequence shown in SEQ ID NO: 133. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same LC and HC as PR000266, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 162, and the HC may include the same as that shown in SEQ ID NO: 134.
  • the antigen binding protein described in the present application may include the same LC and HC as PR000267, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 163, and the HC may include the same as that shown in SEQ ID NO: 135. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same LC and HC as PR000268, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 163, and the HC may include the same as that shown in SEQ ID NO: 136. The amino acid sequence shown.
  • the antigen binding protein described in the present application may include the same LC and HC as PR000416, wherein the LC may include the amino acid sequence shown in SEQ ID NO: 162, and the HC may include the amino acid sequence shown in SEQ ID NO: 137. The amino acid sequence shown.
  • the isolated antigen binding protein described in this application can compete with a specific anti-PD-L1 antibody to bind to PD-L1.
  • the specific anti-PD-L1 antibody may comprise LCDR1-3.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 181;
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 182;
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 191 .
  • the LCDR1 of the specific anti-PD-L1 antibody described in this application may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 48-51; the LCDR2 may include any of the following The amino acid sequence shown in one item: SEQ ID NOs: 63-64; and the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 77-79.
  • the specific anti-PD-L1 antibody may comprise HCDR1-3.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 179; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 180 .
  • the HCDR1 of the specific anti-PD-L1 antibody described in this application may include the amino acid sequence shown below: SEQ ID NO: 5; the HCDR2 may include any of the following Amino acid sequence: SEQ ID NOs: 14, 16, and 17; and the HCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 29-33.
  • the specific anti-PD-L1 antibody may comprise LCDR1-3 and HCDR1-3.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 181; the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 182; and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 191
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 179; and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 180.
  • the LCDR1 of the specific anti-PD-L1 antibody described in this application may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 48-51; the LCDR2 may include any of the following The amino acid sequence shown in one item: SEQ ID NOs: 63-64; and the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 77-79; the HCDR1 may include the following The amino acid sequence shown: SEQ ID NO: 5; the HCDR2 may include the amino acid sequence shown in any of the following: SEQ ID NOs: 14, 16, and 17; and the HCDR3 may include any of the following The amino acid sequence shown in: SEQ ID NOs: 29-33.
  • the specific anti-PD-L1 antibody may comprise a light chain variable region VL and a heavy chain variable region VH.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 194, and the VH may include the amino acid sequence shown in SEQ ID NO: 193.
  • the VL of the specific anti-PD-L1 antibody may include the amino acid sequence shown in any one of SEQ ID NOs: 108-114, 116, and 118, and the VH may include SEQ ID NOs : The amino acid sequence shown in any one of 90-93, 95, 97-104.
  • the physical/chemical properties and/or biological activity of the PD-L1 antigen binding protein or fusion protein described in the present application can be identified, screened or characterized by various assays known in the art.
  • the application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (for example, Western blot), flow cytometry (for example, FACS), immunohistochemistry, immunofluorescence, etc.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting for example, Western blot
  • flow cytometry for example, FACS
  • immunohistochemistry immunofluorescence, etc.
  • the isolated antigen binding protein can bind to primate-derived PD-L1 with a KD value of 1 ⁇ 10 -8 M or lower.
  • the binding affinity of the primate PD-L1 antigen binding protein to PD-L1 can be determined by any method known in the art. In some cases, binding affinity can be measured by surface plasmon resonance (SPR), enzyme-linked immunoassay (ELISA), bound antigen precipitation, equilibrium dialysis, and biofilm interference (BLI). In some cases, the binding affinity of the PD-L1 antigen binding protein to PD-L1 and the KD value can be measured by biological membrane 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 can bind to primate-derived PD-L1 with a KD value of 1 ⁇ 10 -8 M or lower.
  • the value of KD may be about 1 ⁇ 10 -8 M or less, about 9 ⁇ 10 -9 M or less, about 8 ⁇ 10 -9 M or less, about 7 ⁇ 10 -9 M or less, about 6 ⁇ 10 -9 M or less, about 5 ⁇ 10 -9 M or less, about 4 ⁇ 10 -9 M or less, about 3 ⁇ 10 -9 M or less, about 2 ⁇ 10 -9 M or less, about A value of 1 ⁇ 10 -9 M or less binds to human-derived PD-L1, for example, using a FortieBio Octet molecular interaction analyzer detected.
  • the binding activity of the PD-L1 antigen binding protein described in this application to PD-L1 can be detected using flow cytometry or enzyme-linked immunoassay.
  • the EC50 value of the PD-L1 antigen binding protein and PD-L1 is between about 0.0001 nM to about 100 nM, for example, Between about 0.001 nM and about 10 nM, between about 0.01 nM and about 10 nM, between about 0.05 nM and about 5 nM, and between about 0.05 nM and about 1 nM.
  • human PD-L1 antigen protein is used, and the EC50 value of the PD-L1 antigen binding protein and PD-L1 is between about 0.0001 nM and about 100 nM, for example, 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 0.5 nM, between about 0.01 nM and about 0.1 nM.
  • a competition assay can be used to identify antibodies that compete with any of the antigen binding proteins described in this application for binding to PD-L1.
  • such competing antibodies bind to overlapping epitopes (e.g., linear or conformational epitopes) bound by any of the antigen binding proteins described in this application.
  • overlapping epitopes e.g., linear or conformational epitopes
  • Exemplary methods for mapping epitopes to which antigen binding proteins are bound include, but are not limited to, X-ray co-crystals and cryo-electron microscopy (cryo-EM), array-based oligopeptide scanning, site-directed mutagenesis.
  • Figure cross-linking coupled mass spectrometry and biofilm interferometry.
  • the antigen binding protein described in the present application can block the binding of PD-1 and PD-L1.
  • the blocking of the binding of PD-1 and PD-L1 by the antigen-binding protein can be determined by FACS and enzyme-linked immunoassay ELISA.
  • the host cells stably expressing PD-L1 such as CHOK1 cells
  • the cells were analyzed using FACS to confirm that the antigen binding protein blocked the binding of PD-1 to PD-L1.
  • the PD-L1 antigen protein is first coated on the plate, and a decreasing amount of the unlabeled antigen binding protein and the biotin-labeled PD-1 protein are mixed and incubated together. Then, the cells were analyzed using ELISA to confirm that the antigen binding protein can block the binding of PD-1 and PD-L1.
  • the antigen binding protein described in this application can block the binding of CD80 to PD-L1.
  • the antigen binding protein blocking the binding of CD80 and PD-L1 can be determined by flow cytometry FACS and enzyme-linked immunoassay ELISA.
  • the host cells stably expressing PD-L1 are first incubated with a decreasing amount of the unlabeled antigen binding protein, and then incubated with a biotin-labeled CD80 protein. Then, the cells were analyzed using FACS to confirm that the antigen binding protein blocked the binding of CD80 to PD-L1.
  • the PD-L1 antigen protein is first coated on the plate, and a decreasing amount of the unlabeled antigen binding protein and the biotin-labeled CD80 protein are mixed and incubated together. Then, the cells were analyzed using ELISA to confirm that the antigen binding protein can block the binding of CD80 and PD-L1.
  • the antigen binding protein described in this application can stimulate the secretion of IFN- ⁇ and/or IL2 in immune cells.
  • the immune cells may include lymphocytes, such as B cells, T cells, natural killer cells, myeloid cells, such as monocytes, macrophages, mast cells, basophils, and granulocytes. Any method known to those skilled in the art can be used to measure the secretion of cytokines in immune cells, for example, the proliferation of immune cells (such as T cells) or the cytokines produced by immune cells (such as IFN- ⁇ or IL-2 produced by T cells).
  • the antigen binding protein described in the present application can bind to an epitope that does not completely overlap with PD-L1 derived from a primate.
  • the control antibody may be the anti-PD-L1 antibody avelumab (avilumab), that is, the antibody PR001598 described in this application.
  • PR001598 may include a light chain variable region and a heavy chain variable region.
  • the light chain variable region of the control antibody may include LCDR1, LCDR2, and LCDR3.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 53
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 66
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 82
  • the heavy chain variable region of the control antibody includes HCDR1, HCDR2, and HCDR3, and the HCDR1 includes SEQ
  • the amino acid sequence shown in ID NO: 5 the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 18, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 35
  • the light chain variable region of the control antibody It includes the amino acid sequence shown in SEQ ID NO: 119
  • the heavy chain variable region of the control antibody includes the amino acid sequence shown in SEQ ID NO: 105.
  • control antibody may be the anti-PD-L1 antibody atezolizumab (atezolizumab), that is, the antibody PR000151 described in this application.
  • PR000151 may include a light chain variable region and a heavy chain variable region, the light chain variable region of the control antibody includes LCDR1, LCDR2, and LCDR3, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 52, the LCDR2 It includes the amino acid sequence shown in SEQ ID NO: 65, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 81; the heavy chain variable region of the control antibody includes HCDR1, HCDR2, and HCDR3, and the HCDR1 includes SEQ ID The amino acid sequence shown in NO: 6, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 15, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 34; the light chain variable region of the control antibody includes The amino acid sequence shown in SEQ ID NO: 117, and the heavy chain variable region of the control antibody includes the
  • the control antibody may be Hengrui fusion protein 9, ie, the antibody PR002466 described in this application.
  • PR002466 may include a light chain variable region and a heavy chain variable region, the light chain variable region of the control antibody includes LCDR1, LCDR2, and LCDR3, and the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 55; the LCDR2 It includes the amino acid sequence shown in SEQ ID NO: 68; the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 84, the heavy chain variable region of the control antibody includes HCDR1, HCDR2, and HCDR3, and the HCDR1 includes SEQ ID The amino acid sequence shown in NO: 8; the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 20; the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 37; the light chain variable region of the control antibody includes The amino acid sequence shown in SEQ ID NO: 121, and the heavy chain variable region of the control antibody includes the amino acid sequence shown in SEQ ID NO
  • Methods for determining epitopes in the art include, but are not limited to, synthetic peptide methods, immunoinformatics prediction methods, polypeptide activity determination, epitope peptide scanning technology, protein cleavage methods, phage display technology, X-ray diffraction and nuclear magnetic resonance analysis, and the use of computers
  • the software performs epitope prediction.
  • a biological membrane-based interferometry (BLI) Octet molecular interaction analysis platform can be used to perform competitive binding of the antigen-binding protein of the present application and the control antibody to an antigen epitope. For example, the first antibody is mixed with the antigen, and then the second antibody is added.
  • ForteBio Octet is used to determine the competitive inhibition rate of the second antibody against the first antibody.
  • the competitive inhibition rate is less than 85% (for example, (Less than 84%, less than 83%, less than 80% or less)
  • the present application provides a fusion protein, which may comprise: a) human TGFBRII or a fragment thereof; and b) the antigen binding protein described above in the present application.
  • the human TGFBRII or fragments thereof of the fusion protein of the present application may comprise the extracellular domain of human TGFBRII.
  • the human TGFBRII or a fragment thereof may comprise the amino acid sequence shown in any one of SEQ ID NOs: 176-178.
  • the fusion protein may comprise the isolated antigen binding protein described in this application.
  • the isolated antigen binding protein may comprise a light chain variable region VL and a heavy chain variable region VH.
  • the VL may include the amino acid sequence shown in SEQ ID NO: 190
  • the VH may include the amino acid sequence shown in SEQ ID NO: 189.
  • the isolated antigen binding protein may also include an antibody heavy chain constant region, and the heavy chain constant region may include a human IgG constant region.
  • the human IgG constant region may comprise a human IgG1 constant region.
  • the human IgG1 constant region may include natural and artificially synthesized IgG1 constant regions or mutants thereof.
  • the mutation may include one or more of the following mutations: L234, L235, N297 or K447. For example, it may contain mutations at one, two or more positions. Mutations may include deletions, insertions or substitutions of amino acids.
  • the human IgG1 constant region may include the following mutations: 1) N297A, 2) K447 deletion, 3) N297A and K447 deletion, 4) L234A, L235A and K447 deletion.
  • the human IgG1 constant region of the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 172-175.
  • the isolated antigen binding protein of the fusion protein described in this application may comprise an antibody heavy chain or a fragment thereof.
  • the antibody heavy chain or fragments thereof may comprise the amino acid sequence shown in any one of the following or a part thereof: SEQ ID NOs: 122-127 and 129-137.
  • the antibody heavy chain or a fragment thereof may comprise the amino acid sequence shown in any one of the following or a part thereof: SEQ ID NOs: 122 and 133-137.
  • the isolated antigen binding protein of the fusion protein described herein may comprise an antibody light chain or a fragment thereof.
  • the antibody light chain or fragments thereof may comprise the amino acid sequence shown in any one of the following or a part thereof: SEQ ID NOs: 150-158 and 160-163.
  • the antibody light chain or a fragment thereof may comprise the amino acid sequence shown in any one of the following or a part thereof: SEQ ID NOs: 156 and 162-163.
  • the heavy chain or fragment thereof of the fusion protein and the light chain or fragment thereof can form an antigen binding portion that specifically binds to PD-L1 after being combined.
  • the antibody heavy chain or its fragment of the antigen binding protein contained in the fusion protein of the present application is fused in frame with the human TGFBRII or its fragment to form a fusion polypeptide.
  • the N-terminus of the antibody heavy chain or fragments thereof may be directly or indirectly connected to the C-terminus of the human TGFBRII or fragments thereof.
  • the C-terminus of the antibody heavy chain or fragments thereof may be directly or indirectly connected to the N-terminus of the human TGFBRII or fragments thereof.
  • the C-terminus of the antibody heavy chain or its fragment can be directly connected to the N-terminus of the human TGFBRII or its fragment.
  • the antibody heavy chain or fragments thereof may be connected to the human TGFBRII or fragments thereof through a linker.
  • the peptide linker may include any one of SEQ ID NOs: 167-169. The amino acid sequence.
  • the fusion protein may comprise a first polypeptide and a second polypeptide.
  • the first polypeptide may comprise the heavy chain of the isolated antigen binding protein or a fragment thereof and the human TGFBRII or a fragment thereof.
  • the first polypeptide may include the heavy chain of the antigen-binding fragment or a fragment thereof, the peptide linker, and the human TGFBRII or a fragment thereof from the N-terminus to the C-terminus.
  • the first polypeptide may comprise the amino acid sequence shown in any one of SEQ ID NOs: 138-139, 142-143, and 145-148.
  • the second polypeptide may comprise the light chain of the isolated antigen binding protein or a fragment thereof, for example, the second polypeptide may comprise the amino acid sequence shown in any one of SEQ ID NOs: 162-163.
  • the first polypeptide of the fusion protein may include the amino acid sequence shown in SEQ ID NO: 138
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 162
  • the The fusion protein may comprise the same first polypeptide and second polypeptide as PR001487;
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 139
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 162
  • the fusion protein may include the same as PR001488 The first polypeptide and the second polypeptide;
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 142
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 162
  • the fusion protein may include the same as PR001901
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 143
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 162
  • the fusion protein may include the same as PR001902
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 145
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 163.
  • the fusion protein may include the same as PR002247
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 146
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 163.
  • the fusion protein may include the same as PR002248 The first polypeptide and the second polypeptide;
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 147
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 162.
  • the fusion protein may include the same as PR002249 The first polypeptide and the second polypeptide;
  • the first polypeptide may include the amino acid sequence shown in SEQ ID NO: 148
  • the second polypeptide may include the amino acid sequence shown in SEQ ID NO: 162.
  • the fusion protein may include the same as PR002251 The first polypeptide and the second polypeptide.
  • the fusion protein of the present application may include two of the first polypeptides and two of the second polypeptides.
  • a part of the amino acid sequence of each heavy or light chain of the antibody binding protein is homologous to the corresponding amino acid sequence of an antibody from a specific species, or belongs to a specific category.
  • the variable regions and constant parts of the light and heavy chains are all derived from the variable and constant regions of an antibody of an animal species (such as human).
  • the homologue may be at least about 85% (for example, the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or a fragment thereof that specifically binds to the PD-L1 protein)).
  • the homologue may be 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 more) Proteins or polypeptides of sequence homology.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology" can be calculated in the following way: the two sequences to be aligned are compared in the comparison window to determine the presence of the same nucleic acid base (for example, A, T, C, G) or Positions of the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to generate the sequence homology percentage.
  • the same nucleic acid base for example, A, T, C, G
  • Positions of the same amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu,
  • the alignment in order to determine the percentage of sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA for a description of the FASTA algorithm, please refer to WRPearson and DJ Lipman's "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad.
  • the application also provides isolated one or more nucleic acid molecules.
  • the one or more nucleic acid molecules may encode the antigen binding protein described in this application.
  • each nucleic acid molecule in the one or more nucleic acid molecules may encode the entire antigen binding protein, or may encode part of it (for example, HCDR1-3, LCDR1-3, VL, VH, light chain Or one or more of the heavy chain).
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesis, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the antibody and its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides. Overlapping extension PCR. For specific operations, please refer to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York NY, 1993.
  • this application provides one or more vectors, which comprise one or more nucleic acid molecules described in this application.
  • Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters can include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part can be fused with a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), and it may or may not contain additional introns.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application.
  • each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application.
  • each or each host cell may contain multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in this application can be introduced into the host cell, such as a eukaryotic cell, such as a plant-derived cell, fungus, or yeast cell.
  • the vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the application provides a method for preparing the antibody or antigen-binding fragment thereof.
  • the method may include culturing the host cell described in the present application under conditions such that the antibody or antigen-binding fragment thereof is expressed.
  • culturing the host cell described in the present application under conditions such that the antibody or antigen-binding fragment thereof is expressed.
  • the method may further include the step of isolating and/or purifying the antibody or antigen-binding fragment thereof.
  • protein G-Sepharose or Protein A-Sepharose can be used for affinity chromatography, and gel electrophoresis and/or high performance liquid chromatography can also be used to purify and separate the antibodies or antigen-binding fragments described in this application. .
  • the application provides a pharmaceutical composition, which may comprise the antigen binding protein and/or the fusion protein described in the application, the nucleic acid molecule, the vector, the host cell , And optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvant is non-toxic to the recipient at the dose and concentration used, and may include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine Acid; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride) chloride), phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol ); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gel, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamyl acid, Aspartic acid, his
  • the pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the tumor size (or even substantially eliminate the tumor), and/or can alleviate and/or stabilize the disease state.
  • the pharmaceutical composition described in the present application may include a preventive and/or therapeutically effective amount of the antibody or antigen-binding fragment thereof.
  • the prophylactic and/or therapeutically effective amount is a dose required to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications thereof in a subject suffering from or at risk of development.
  • the application provides the use of the antigen binding protein and/or the fusion protein in the preparation of medicines.
  • the medicine is used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the tumor or cancer comprises colorectal tumor or cancer.
  • the tumor or cancer is a tumor or cancer in which PD-L1 expression is abnormal. This application also provides the method for detecting PD-L1 expression in biological samples as described below.
  • the method includes contacting a biological sample with the antigen binding protein and/or fusion protein described in the present application under conditions that allow the antigen binding protein and/or fusion protein to bind PD-L1, and detecting Whether a complex is formed between the antigen binding protein and/or fusion protein and PD-L1.
  • the tumor or cancer is a tumor or cancer in which PD-L1 expression is increased compared to a non-tumor or cancer sample.
  • Such methods can be in vitro or in vivo methods.
  • the antigen binding protein and/or fusion protein described in the present application can be used in, for example, immunoassays, including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (for example, western blot), flow cytometry Technology (e.g. FAGS) and enzyme-linked immunosorbent assay (ELISA).
  • immunoassays including, for example, immunohistochemistry (IHC), immunofluorescence (IF), immunoblotting (for example, western blot), flow cytometry Technology (e.g. FAGS) and enzyme-linked immunosorbent assay (ELISA).
  • IHC immunohistochemistry
  • IF immunofluorescence
  • IF immunoblotting
  • flow cytometry Technology e.g. FAGS
  • ELISA enzyme-linked immunosorbent assay
  • the application also provides the use of the antigen-binding protein and/or fusion protein in a method for diagnosing a subject suffering from a disorder (for example, cancer or immune dysfunction), the method comprising: contacting a sample with the present invention
  • the antigen binding protein and/or fusion protein is contacted with and detected for the presence of the bound antigen binding protein and/or fusion protein to determine the presence or expression level of PD-L1 in a sample obtained from the subject.
  • the present application provides a method for inhibiting the binding of PD-L1 to PD-1, including administering the antigen binding protein described in the present application.
  • the method can be an ex vivo or in vitro method.
  • the method may include contacting a biological sample with the antigen binding protein and/or PD-1 described in the present application 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-L1 to CD80, including administering the antigen binding protein described in the present application.
  • the method can be an ex vivo or in vitro method.
  • the method includes contacting a biological sample with the antigen binding protein and/or CD80 described in this application under conditions that allow the antigen binding protein and/or CD80 to bind PD-L1, and detecting Whether a complex is formed between the antigen binding protein and PD-L1, and whether a complex is formed between CD80 and PD-L1.
  • the present application provides a method for inhibiting the binding of TGFB to TGFBRII, including administering the fusion protein described in the present application.
  • the method can be an ex vivo or in vitro method.
  • the method includes allowing the fusion protein and/or the reporter gene cell expressing TGFBRII to bind TGFB to the biological sample under conditions that allow the fusion protein and/or reporter gene cell expressing TGFBRII to bind to TGFB.
  • tissue or cell samples include tissue or cell samples.
  • a biological sample may include cells or tissues from normal or cancer patients.
  • the source of the tissue or cell sample can be, for example, solid tissue from fresh, frozen and/or preserved organ or tissue samples or biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, Amniotic fluid, peritoneal fluid, or interstitial fluid; cells from the subject at any time during pregnancy or development.
  • the biological sample is obtained from an in vitro tissue or cell culture.
  • biological samples in this application include, but are not limited to, tumor biopsy, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and Preserved tumor samples, such as formalin-fixed paraffin-embedded tumor samples or frozen tumor samples.
  • the application also provides the use of an antigen binding protein in a method for diagnosing a subject suffering from a tumor or cancer, the method comprising: contacting a sample with the antigen binding protein of the application and detecting the presence of bound antibody The presence or expression level of PD-L1 in a sample obtained from a subject.
  • the sample can be selected from the group consisting of a tissue sample, a whole blood sample, a serum sample, and a plasma sample.
  • the tissue sample may be a tumor sample.
  • the tumor sample may include tumor infiltrating immune cells, tumor cells, stromal cells, and any combination thereof.
  • the present application provides a method for treating cancer in a subject, inhibiting tumor growth in a subject, and/or inhibiting tumor cell proliferation, including administering to a subject in need or said tumor cell the method of The antigen binding fragment and/or the fusion protein, the molecular nucleic acid, the vector, the host cell and/or the pharmaceutical composition. It can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, transcutaneously, intraarterially, and intraperitoneally.
  • Intra-injury, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, and rectal Locally, intratumorally, peritoneally, subconjunctivally, intracapsular, mucosal, intrapericardial, intraumbilical, intraocular, intraorbital, orally Way, by topical way, by transdermal way, by intravitreal way (for example, by intravitreal injection), by eye drops, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by direct Bathe the local perfusion of target cells, through a catheter, through lavage, in the form of a cream or in the form of a lipid composition.
  • composition used in the methods described herein can also be administered systemically or locally.
  • the method of administration may vary depending on various factors (for example, the compound or composition being administered and the severity of the condition, disease, or disorder being treated).
  • intravenous, intramuscular, subcutaneous, topical, oral, transdermal, intraperitoneal, intraorbital, implantation, inhalation Intrathecal, intraventricular, or intranasal administration of anti-cancer therapy (e.g., anti-PD-L1 antibody).
  • anti-cancer therapy e.g., anti-PD-L1 antibody
  • the administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection.
  • Various dosing schedules are covered herein, including but not limited to a single administration or multiple administrations at various time points, bolus administrations, and pulse infusions.
  • the antigen binding protein or pharmaceutical composition described herein can be formulated, administered, and administered in a manner consistent with good medical practice.
  • the considerations in this situation include the specific condition being treated, the specific mammal being treated, the clinical condition of a single patient, the cause of the condition, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner .
  • the therapeutic agent e.g., anti-PD-L1 antibody
  • the effective amount of such other agents depends on the amount of therapeutic agent (e.g., anti-PD-L1 antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above.
  • These agents can generally be used in any dosage that is empirically/clinically determined to be appropriate and through any route that is empirically/clinically determined to be appropriate. Compared with a single treatment, the dose of the antibody administered in the combination treatment can be reduced. It is easy to monitor the progress of this therapy by conventional techniques.
  • the present application provides a chimeric antigen receptor (CAR), which may comprise the nucleic acid molecule described in this application or the antigen binding protein described in this application. In some embodiments, it may comprise an extracellular domain capable of binding antigen (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region), and a polypeptide that allows cytoplasmic signals to be transmitted to the domain (ie Intracellular signal domain).
  • the hinge domain can be considered as a part for providing flexibility to the extracellular antigen binding region.
  • the intracellular signal domain refers to a protein that transmits information into the cell to regulate cell activity by generating a second messenger through a certain signal transduction pathway, or a protein that functions as an effector by corresponding to such a messenger, and produces a protein that can promote CAR Of the immune effector function of the cells (such as CAR-T cells).
  • the intracellular signal domain may include a signal transduction domain, and may also include a costimulatory intracellular domain derived from a costimulatory molecule.
  • the costimulatory molecule can be selected from 4-1BB, CD27, ICOS and/or CD28.
  • the present application provides genetically modified cells, which may contain the chimeric antigen receptor.
  • the genetically modified cells may include eukaryotic cells.
  • the genetically modified cells may include isolated human cells.
  • the genetically modified cells may include immune cells such as T cells, or NK cells.
  • an antibody drug conjugate which may include a cytotoxic agent and the antigen-binding fragment described in the present application.
  • Antibody-drug conjugates usually refer to the use of specific linkers to connect antibodies and small molecule cytotoxic drugs. Its main components can include antibodies, linkers and small molecule cytotoxic drugs.
  • the targeting of antibody-drug conjugates can come from the antibody part, most of the toxicity can come from the payload of small molecule drugs, and the antibody part can also have its own toxicity (ADCC and CDC).
  • this application provides a kit, which may contain the antigen binding protein, chimeric antigen receptor, genetically modified cell, antibody drug conjugate, and/or the antigen binding protein described in this application Pharmaceutical composition. It can include the antigen binding protein, chimeric antigen receptor, genetically modified cell, and/or antibody drug conjugate described in the present application in a single common container, and can also optionally be combined with one or more therapeutic agents The combination, optionally formulated together in a pharmaceutical composition.
  • the kit may also include a device for administering the antigen binding protein, chimeric antigen receptor, genetically modified cell, antibody-drug conjugate or pharmaceutical composition described in this application; for example, the device Depends on how the contents are administered.
  • the kit may include a package insert that includes information about the antigen binding protein, pharmaceutical composition, and dosage form in the kit. Generally, this type of information helps patients and physicians use encapsulated antigen binding proteins, pharmaceutical compositions, and dosage forms effectively and safely.
  • the container used in such kits may generally comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, in which one or more of the detection and/or therapeutic composition may be placed, and preferably appropriate Divide equally.
  • the kit may also contain a second different container in which the second detection and/or therapeutic composition can be placed.
  • a second container in which the second detection and/or therapeutic composition can be placed.
  • multiple compounds can be prepared as a single pharmaceutical composition, and can be packaged in a single container device such as a vial, flask, syringe, bottle, or other suitable single container.
  • the present application provides a drug delivery device (such as a plastic or vial, such as a hollow pin or a syringe cylinder), which can be used to administer the antigen binding protein described in this application or a pharmaceutical composition thereof.
  • the device can introduce a substance into the patient through parenteral routes (for example, intramuscular, subcutaneous, or intravenous).
  • the injection device may be a syringe (such as a pre-filled syringe with the antigen binding protein described in this application or a pharmaceutical composition thereof, such as an auto-injector), which may include a fluid for containing the fluid to be injected (such as the antigen described in this application).
  • Binding protein or its pharmaceutical composition syringes and needles (which can be used to pierce the skin and/or blood vessels).
  • the mode of administration can be changed.
  • the route of administration may include oral, intramuscular injection, subcutaneous injection, and rectal administration.
  • An isolated antigen binding protein comprising at least one CDR in the VH of the variable region of the antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO: 193.
  • VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 90-93, 95 and 97-104.
  • the isolated antigen binding protein according to any one of embodiments 1-2 which comprises at least one CDR in the variable region VL of the antibody light chain, the VL comprising the amino acid sequence shown in SEQ ID NO: 194 .
  • VL comprises the amino acid sequence shown in any one of SEQ ID NOs: 108-114, 116 and 118.
  • the isolated antigen-binding protein according to any one of embodiments 1-4, which comprises an antibody or an antigen-binding fragment thereof.
  • antigen binding protein comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the isolated antigen binding protein according to any one of embodiments 1-7, which competes with a specific anti-PD-L1 antibody for binding to PD-L1, wherein the specific anti-PD-L1 antibody comprises a light chain variable region And the heavy chain variable region, the light chain variable region of the specific anti-PD-L1 antibody includes LCDR1, LCDR2, and LCDR3, the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 181, and the LCDR2 includes SEQ ID NO The amino acid sequence shown in: 182, the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 191; the heavy chain variable region of the specific anti-PD-L1 antibody includes HCDR1, HCDR2, and HCDR3, and the HCDR1 includes SEQ ID The amino acid sequence shown in NO: 5, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 179, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 180.
  • VH comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 5.
  • HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 14, 16, and 17.
  • VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 21, 22, and 24.
  • the isolated antigen binding protein according to any one of embodiments 1-20 which comprises an antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 193.
  • VH comprises the amino acid sequence shown in any one of SEQ ID NOs: 90-93, 95, and 97-104.
  • VL includes framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the isolated antigen binding protein according to any one of embodiments 1-40 which comprises an antibody light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 194.
  • control antibody comprises LCDR1 shown in SEQ ID NO: 52 and LCDR2 shown in SEQ ID NO: 65 and LCDR3 shown in SEQ ID NO: 81, and the control antibody comprises the LCDR1 shown in SEQ ID NO: 6 HCDR1, HCDR2 shown in SEQ ID NO: 15 and HCDR3 shown in SEQ ID NO: 34;
  • control antibody comprises LCDR1 shown in SEQ ID NO:53, LCDR2 shown in SEQ ID NO:66, and LCDR3 shown in SEQ ID NO:82, and the control antibody comprises the LCDR1 shown in SEQ ID NO:5 HCDR1, HCDR2 shown in SEQ ID NO: 18 and HCDR3 shown in SEQ ID NO: 35;
  • the control antibody includes LCDR1 shown in SEQ ID NO: 55, LCDR2 shown in SEQ ID NO: 68, and LCDR3 shown in SEQ ID NO: 84, and the control antibody includes HCDR1 shown in SEQ ID NO: 8 , HCDR2 shown in SEQ ID NO: 20 and HCDR3 shown in SEQ ID NO: 37.
  • a fusion protein comprising: a) human TGFBRII or a fragment thereof; and b) the isolated antigen binding protein of any one of embodiments 1-46.
  • the fusion protein of embodiment 47 wherein the isolated antigen binding protein comprises an antibody heavy chain or a fragment thereof, and the antibody heavy chain or a fragment thereof is fused in frame with the human TGFBRII or a fragment thereof to form The fusion polypeptide.
  • fusion protein according to any one of embodiments 48-49, wherein the antibody heavy chain or fragment thereof is connected to the human TGFBRII or fragment thereof via a linker.
  • linker is a peptide linker
  • the peptide linker comprises the amino acid sequence shown in any one of SEQ ID NOs: 167-169.
  • fusion protein according to any one of embodiments 47-52, wherein the human TGFBRII or a fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 176-178.
  • fusion protein according to any one of embodiments 47-53, which comprises a first polypeptide and a second polypeptide,
  • the first polypeptide comprises the heavy chain of the isolated antigen binding protein or a fragment thereof and the human TGFBRII or a fragment thereof;
  • the second polypeptide comprises the light chain of the isolated antigen binding protein or a fragment thereof.
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 138, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 139, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 142
  • the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 143, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 145, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 163; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 146, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 163; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 147, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162; or,
  • the first polypeptide includes the amino acid sequence shown in SEQ ID NO: 148, and the second polypeptide includes the amino acid sequence shown in SEQ ID NO: 162.
  • fusion protein according to any one of embodiments 54-58, which comprises two of said first polypeptides and two of said second polypeptides.
  • One or more isolated nucleic acid molecules that encode the isolated antigen binding protein of any one of embodiments 1-46, and/or the fusion protein of any one of embodiments 47-59.
  • a vector comprising the nucleic acid molecule according to embodiment 60.
  • a cell comprising the nucleic acid molecule according to embodiment 60 or the vector according to embodiment 61.
  • a method for preparing the isolated antigen binding protein according to any one of the embodiments 1-46 and/or the fusion protein according to any one of the embodiments 47-59 which method comprises the following steps: The isolated antigen binding protein of any one of 46 is expressed under the condition of the fusion protein of any one of embodiments 47-59, and the cell according to embodiment 49 is cultured.
  • a chimeric antigen receptor comprising the isolated antigen binding protein of any one of embodiments 1-46.
  • a genetically modified cell comprising the chimeric antigen receptor of embodiment 64.
  • An antibody drug conjugate comprising a cytotoxic agent and the isolated antigen binding protein of any one of embodiments 1-46.
  • a pharmaceutical composition comprising the isolated antigen binding protein of any one of embodiments 1-46, the fusion protein of any one of embodiments 47-59, the nucleic acid molecule of embodiment 60, The vector of embodiment 61, the cell of embodiment 62, the chimeric antigen receptor of embodiment 64, the genetically modified cell of embodiment 65, and/or the antibody drug couple of embodiment 66 Conjugate, and optionally a pharmaceutically acceptable carrier.
  • the chimeric antigen receptor of embodiment 65, the genetically modified cell of embodiment 65, the antibody-drug conjugate of embodiment 66 and/or the use of the pharmaceutical composition of embodiment 67 in the preparation of a medicine the The medicine is used to prevent, alleviate and/or treat tumors or cancers, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • embodiment 68 wherein the tumor or cancer is a tumor or cancer with abnormal expression of PD-L1.
  • a method for preventing, alleviating or treating tumors, inhibiting tumor growth and/or inhibiting tumor cell proliferation comprising administering the isolated antigen binding of any one of embodiments 1-46 to a subject in need Protein, the nucleic acid molecule of embodiment 60, the vector of embodiment 61, the cell of embodiment 62, the chimeric antigen receptor of embodiment 64, the genetically modified cell of embodiment 65, The antibody drug conjugate of embodiment 66 and/or the pharmaceutical composition of embodiment 67.
  • the isolated antigen binding protein of any one of the embodiments 1-46, the nucleic acid molecule of the embodiment 60, the vector of the embodiment 61, the cell of the embodiment 62, the embodiment 64 The chimeric antigen receptor of embodiment 65, the genetically modified cell of embodiment 65, the antibody-drug conjugate of embodiment 66 and/or the pharmaceutical composition of embodiment 67, which are used to treat cancer and inhibit tumors Growth and/or inhibition of tumor cell proliferation.
  • a method for inhibiting the binding of PD-L1 to PD-1 comprising administering the isolated antigen binding protein according to any one of embodiments 1-46, the nucleic acid molecule according to embodiment 60, and the method according to embodiment 61.
  • the vector, the cell of embodiment 62, the chimeric antigen receptor of embodiment 64, the genetically modified cell of embodiment 65, the antibody-drug conjugate of embodiment 66, and/or the implementation The pharmaceutical composition described in Scheme 67.
  • a method for inhibiting the binding of CD80 to PD-L1 comprising administering the isolated antigen binding protein of any one of embodiments 1-46, the nucleic acid molecule of embodiment 60, or the method of embodiment 61 A vector, the cell of embodiment 62, the chimeric antigen receptor of embodiment 64, the genetically modified cell of embodiment 65, the antibody-drug conjugate of embodiment 66, and/or the embodiment 67 The pharmaceutical composition.
  • a kit comprising the isolated antigen binding protein of any one of embodiments 1-46, the nucleic acid molecule of embodiment 60, the vector of embodiment 61, the cell of embodiment 62, The chimeric antigen receptor of embodiment 64, the genetically modified cell of embodiment 65, the antibody drug conjugate of embodiment 66, the pharmaceutical composition of embodiment 67, and/or (i ) Applicator.
  • the PD-L1 antigen can be used to immunize experimental animals to obtain antibody molecules that specifically bind to PD-L1.
  • the experimental animals can be mice, rats, rabbits, sheep, camels, and the like.
  • the antibody molecules obtained are of non-human origin. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability.
  • the process of humanization of antibodies has its technical complexity, and molecules that have been humanized will often reduce their affinity for antigens.
  • transgenic technology have made it possible to breed genetically engineered mice that carry human immunoglobulin immune libraries and have their endogenous mouse immune libraries missing.
  • the antibodies produced by the transgenic mice have fully human sequences, so there is no need for further humanization modification, which greatly improves the efficiency of therapeutic antibody development.
  • the Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin immune library.
  • the antibodies produced by it have complete human antibody variable domains and rat constant domains.
  • the soluble recombinant human PD-L1-mFc fusion protein (Novo Protein Inc. catalog number CM06, lot number 0330837) was used for multiple rounds of immunization against Harbour H2L2 mice.
  • the antigen protein is mixed with the immune adjuvant to form the immunogenic reagent, and then injected subcutaneously through the inguinal or injected through the abdominal cavity. In each round of immunization, the total injection dose received by each mouse is 100 microliters.
  • each mouse was immunized with an immunogen reagent prepared by mixing 50 ⁇ g antigen protein (human PD-L1-mFc) and complete Freund's adjuvant (Sigma, catalog number F5881) at a volume ratio of 1:1.
  • each mouse was immunized with immunogenic reagents mixed with 25 ⁇ g antigen protein and Sigma Adjuvant System adjuvant (Sigma, catalog number S6322).
  • the interval between each round of booster immunity is at least two weeks, usually no more than five rounds of booster immunity.
  • the immunization time was 0, 14, 28, 42, 56, and 70 days; and on the 49th and 77th days, the mouse serum antibody titers were detected. Three days before the cell fusion, the final booster immunization was performed at a dose of 25 ⁇ g antigen protein per mouse.
  • the mouse spleen cells are taken out and fused with the myeloma cell line to obtain hybridoma cells; after multiple rounds of screening and cloning of hybridoma cells Several hybridoma cells expressing anti-PD-L1 monoclonal antibody molecules are isolated.
  • the isolated hybridoma cells and the monoclonal antibodies expressed are indicated by the corresponding clone numbers, for example: 63G11H3G9, 91G3H5H3 and so on.
  • the isolated hybridoma cells express antibody molecules with intact human variable domains and rat constant domains of heavy and light chains.
  • the above-mentioned monoclonal antibodies were further identified, and several hybridomas were selected according to their binding ability to human PD-L1, the binding ability of cynomolgus PD-L1, and the ability to inhibit the binding of PD-L1 to PD-1.
  • Clones are sequenced. Conventional hybridoma sequencing methods are used to obtain the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence.
  • the sequence of the variable domain of the anti-PD-L1 monoclonal antibody molecule obtained from the immunized Harbour H2L2 mouse is a human antibody sequence.
  • the CDR sequences of antibody variable domains can be analyzed by Kabat or Chothia or other CDR definition rules (such as Combined definition rules) (see Table 1 for CDR definitions).
  • Table 2 lists the variable region sequences of hybridoma cells and recombinant antibodies and the SEQ ID NO numbers corresponding to the CDR sequences.
  • the CDR uses the Chothia definition rule.
  • the antibody heavy chain variable domain sequence (VH) was synthesized by gene and cloned into the human IgG1 antibody heavy chain constant domain sequence (SEQ ID NO: 172) or its variant sequence containing amino acid mutations ( SEQ ID NO: 173-175) in the mammalian cell expression plasmid vector to encode the full-length heavy chain (or its variant) of human IgG antibody;
  • the antibody light chain variable domain sequence (VL) is synthesized through gene synthesis Clone into the mammalian cell expression plasmid vector encoding the human antibody ⁇ light chain constant domain sequence (SEQ ID NO: 170) to encode the full-length ⁇ light chain of the antibody; or synthesize and clone VL into the coding human
  • the antibody lambda light chain constant domain sequence (SEQ ID NO: 171) is expressed in a mammalian cell expression plasmid vector to encode the full-length lambda light chain for antibody production.
  • Table 2 lists the amino acid sequences of the light and heavy chain variable domains of the PD-L1 antibody in this example, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG1) and the CDRs defined according to Chothia's definition rules. The amino acid sequence.
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, A1383504). Before the start of transient transfection, adjust the cell concentration to 6-8x10 5 cells/ml, and incubate at 37°C in an 8% CO 2 shaker for 24 hours, and the cell concentration is 1.2x10 6 cells/ml.
  • the sequence of the variable domain of the heavy chain of the antibody is derived from events such as the gene rearrangement of the germline gene V, D, and J gene fragments of the heavy chain gene group on the chromosome and somatic high frequency mutation; the sequence of the variable domain of the light chain is derived from Gene rearrangement of germline genes V and J gene fragments of light chain gene group and somatic high frequency mutations and other events. Gene rearrangement and somatic mutations are the main factors that increase the diversity of antibodies. Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is relatively high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • Protein or polypeptide amino acid chains sometimes introduce chemical modifications after translation and synthesis in cells, called post-translational modifications (PTM).
  • PTM post-translational modifications
  • some PTM sites are very conservative.
  • EU numbering conserved amino acid asparagine Asn at position 297 (EU numbering) of the constant domain of human IgG1 antibodies usually undergoes glycosylation modification.
  • the sugar chain, and the structure of the sugar chain is critical to the structure of the antibody and related effector functions.
  • PTMs are present in the variable domains of antibodies, especially the antigen-binding regions (such as CDRs), the presence of these PTMs may have a greater impact on the binding of the antigen, and may also affect the physicochemical properties of the antibody. To change.
  • glycosylation, deamidation, isomerization, oxidation, etc. may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding some potential PTMs is very important for the development of therapeutic antibodies.
  • some PTMs are highly correlated with the composition of the amino acid sequence, especially the "pattern" of the adjacent amino acid composition, so that potential PTMs can be predicted from the primary amino acid sequence of the protein.
  • the sequence pattern of N-x-S/T (the first position is asparagine, the second position is any amino acid other than proline, and the third position is serine or threonine) sequence pattern predicts an N-linked glycosylation site.
  • the amino acid sequence pattern that causes PTM may be derived from the germline gene sequence.
  • the human germline gene fragment IGHV3-33 naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic high-frequency mutations.
  • Table 3 lists the predicted PTM of the variable domains VH and VL of the antibody of Example 1.3.
  • NGS or NLT may be glycosylation sites, and DG may cause the isomerization of aspartic acid.
  • Amino acid mutations can be used to disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of specific PTMs.
  • mutation design methods One method is to replace "hot spot" amino acids (such as N or S in the NS pattern) with amino acids with similar physical and chemical properties (such as mutating N to Q). If the PTM sequence pattern is derived from somatic high-frequency mutations and does not exist in the germline gene sequence, then another method can be to replace the sequence pattern with the corresponding germline gene sequence. In actual operation, multiple mutation design methods may be used for the same PTM sequence pattern.
  • Table 4 lists new antibody molecules (referred to as PTM variants) obtained by amino acid mutations on the sequences of six antibodies with potential PTM sites from Example 1.3.
  • Table 5-1 lists the amino acid sequences of the light and heavy chain variable domains of these PTM variants in this example, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG1 (N297A)), and the Chothia defines the amino acid sequence of the CDR defined by the rules. All the designed PTM variants obtained purified recombinant antibodies according to the method described in Example 1.3, and were further verified in the subsequent functional experiments.
  • VH PTMs Variant VL PTMs PR000066 PR000261 H:D54E; L:N65S / / PR000066 PR000262 H:G55A; L:N65S / / PR000069 PR000263 H:D54E / / PR000069 PR000264 H:G55A / / PR000070 PR000265 H:D54E / / PR000070 PR000266 H:G55A / / PR000072 PR000267 H:D54E / / PR000072 PR000268 H:G55A / /
  • Table 5-2 lists the molecular sequence numbers of the Fc-type conversion antibody PR000416 of the control antibodies PR000151, PR001598, and PR000265 appearing in the examples of the application, where PR000151 is Roche's anti-PD-L1 antibody atezolizumab ( ⁇ In this article, PR000151 and atezolizumab are used interchangeably; PR001598 is Merck KGaA's anti-PD-L1 antibody avelumab (avilumab) analogue, in this article, PR001598 and avelumab are interchangeable Change the ground to use.
  • the above antibody was prepared and purified by the method of Example 1.3.
  • Antibody description Antibody number Light chain Heavy chain VL VH Constant region type atezolizumab analogs PR000151 159 128 117 96 hIgG1(N297A) avelumab analogs PR001598 164 140 119 105 hIgG1 Fc type conversion of PR000265 PR000416 162 137 114 101 hIgG1
  • the CHOK1 cell line overexpressing human PD-L1 (CHO-K1/hPD-L1, Nanjing GenScript, M00543 )
  • the CHOK1 cell line overexpressing cynomolgus PD-L1 (CHOK1/cynoPD-L1, Nanjing GenScript, M00573) for cell-level binding experiments.
  • PD-L1 cells were digested and resuspended in F-12K complete medium, and the cell density was adjusted to 1 ⁇ 10 6 cells/mL.
  • the highest antigen-binding protein is the highest final concentration.
  • the concentration was 100 nM, a total of 8 concentrations, and the isotype antibody hIgG1 iso was used as a control. Place the cells at 4°C and incubate for 1 hour in the dark. After that, 100 ⁇ L/well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • enzyme-linked immunosorbent assay was used to study the activity of PD-L1 antigen binding protein in binding to human/cynomolgus PD-L1 protein in vitro.
  • Human PD-L1 (Acrobiosystems, PD1-H5229) was diluted with PBS to 2 ⁇ g/ml, added to a 96-well plate (Corning, 9018), 100 ⁇ l per well, incubated overnight at 4°C. After discarding the liquid, wash the plate 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), add 250 ⁇ l 2% BSA blocking solution, and incubate at 37°C for 1 hour. Discard the blocking solution and wash the plate 3 times with PBST buffer (pH 7.4, containing 0.05% Tween-20).
  • the antigen-binding protein to be tested will be diluted by 5 times successively starting from the concentration of 100 nM, totaling 8 concentrations Gradient, add 100 ⁇ l to each well, incubate at 37°C for 1 hour, and the isotype antibody hIgG1iso as a control. After washing with PBST buffer (pH7.4, containing 0.05% Tween-20) for 3 times, goat anti-human HRP secondary antibody (Invitrogen, A18805) diluted 4000 times was added, and incubated for 1 hour at 37°C in the dark.
  • PBST buffer pH7.4, containing 0.05% Tween-20
  • Figure 3 and Table 8 show that all the PD-L1 antigen binding proteins described in this application can bind to human PD-L1 protein, and the PTM variants still retain the binding activity of the parent antibody.
  • Antigen binding protein EC50(nM) PR000151 0.0259 PR000066 0.0189 PR000261 0.0385 PR000262 0.0338 PR000069 0.0637 PR000263 0.0321
  • the PD-L1 protein (Acrobiosystems, PD1-C52H4) of Cynomolgus (Cynomolgus) was diluted with PBS to 2 ⁇ g/ml, added to a 96-well plate (Corning, 9018), 100 ⁇ l per well, incubated overnight at 4°C. After the liquid was discarded, the plate was washed 3 times with PBST, 250 ⁇ l of 2% BSA was added to block, and the plate was incubated for 1 hour at room temperature.
  • the blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), the concentration of the antigen-binding protein to be tested was diluted to 5 ⁇ g/ml, 100 ⁇ l/well was added, and the plate was incubated for 1 hour at 37°C. After washing 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), goat anti-human HRP secondary antibody (Invitrogen, A18805) diluted 4000 times was added, and incubated at 37°C for 1 hour.
  • PBST buffer pH 7.4, containing 0.05% tween-20
  • goat anti-human HRP secondary antibody Invitrogen, A18805
  • TMB Biopanda, TMB-S-003
  • stop solution BBI Life Sciences, E661006-0200
  • Example 4 Antigen binding protein blocks the binding of human PD-1 to CHO-K1 cells overexpressing human PD-L1
  • the CHOK1 cell line overexpressing human PD-L1 (CHO-K1/hPD-L1, Nanjing GenScript, M00543 ) Perform a cell-level human PD-1/human PD-L1 binding and blocking experiment.
  • CHO-K1/hPD-L1 cells were digested, and resuspended in F-12K complete medium, and the cell density was adjusted to 1 ⁇ 10 6 cells/mL.
  • the highest antigen-binding protein is the highest final concentration.
  • the concentration was 100 nM, a total of 8 concentrations, and the isotype antibody hIgG1 iso was used as a control. Place the cells at 4°C and incubate for 1 hour in the dark.
  • Figure 5 and Table 9 show that the antigen binding proteins before PTM mutation described in this application can block the binding of human PD-1 to human PD-L1 on the cell surface;
  • Figure 6 and Table 10 show the antigens of the PTM variants described in this application
  • the binding protein can block the binding of human PD-1 to human PD-L1 on the cell surface; the blocking ability is equivalent to that of atezolizumab analogs.
  • PR000266 91.54 0.838 PR000267 91.04 1.040 PR000268 91.34 1.160
  • Example 5 Antigen binding protein blocks the binding of human PD-L1 protein to its ligand protein
  • enzyme-linked immunosorbent assay was used to study the ability of PD-L1 antigen binding protein to block the binding of human PD-L1 protein to its ligand protein PD-1 or CD80 in vitro.
  • Antigen binding protein blocks the binding of human PD-L1 and human PD-1 (ELISA)
  • Human PD-L1 (Acrobiosystems, EP-101-96tests, A001-214) was diluted with PBS to 2 ⁇ g/ml and added to a 96-well plate (Corning, 9018), 100 ⁇ l per well, coated overnight at 4°C. After the liquid was discarded, the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), 250 ⁇ l 2% BSA blocking solution was added, and the plate was incubated at 37° C. for 1 hour. Discard the blocking solution and wash the plate 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20).
  • Figure 7 and Table 11 show that the antigen binding protein of the present application can block the binding of human PD-1 to human PD-L1 protein; the blocking ability is equivalent to that of atezolizumab analogs.
  • Antigen binding protein blocks the binding of human PD-L1 to human B7-1 (CD80) (ELISA)
  • the human PD-L1-hFc protein (Acrobiosystems, PD1-H5258) was diluted with PBS to 1 ⁇ g/ml and added to a 96-well plate (Corning, 9018), 100 ⁇ l per well, coated overnight at 4°C. After the liquid was discarded, the plate was washed 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20), 250 ⁇ l 2% BSA blocking solution was added, and the plate was incubated at 37° C. for 1.5 hours. Discard the blocking solution and wash the plate 3 times with PBST buffer (pH 7.4, containing 0.05% tween-20).
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve and IC50 value are obtained.
  • Figure 8 and Table 12 show that the antigen binding proteins described in this application can block the binding of human B7-1 (CD80) to human PD-L1 protein.
  • Example 6 Using reporter gene cell line to detect the inhibitory effect of antigen binding protein on PD-1 signaling pathway
  • Hep3B (built by Ruizhi Chemical (Shanghai)) cells co-expressing human PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) were seeded on a 96-well plate with a cell mass of 1.25 ⁇ 10 4 /well, 100 ⁇ L/well. Incubate overnight at 37°C under 5% CO 2 environment. Remove the supernatant, add 50 ⁇ L/well of the antigen-binding protein dilution to be tested, the initial concentration is 100nM, diluted by 5 times the concentration, and the isotype antibody hlgG1 iso is used as the control group.
  • Figure 9 and Table 13 and Figure 10 and Table 14 show that the antigen binding protein described in this application has an inhibitory effect on the PD-1 signaling pathway; except for PR000071, the inhibitory effect of other molecules on the PD-1 signaling pathway is similar to that of atezolizumab analogs. quite.
  • Example 7 Using the BLI method to determine the affinity of the antigen-binding protein to human PD-L1
  • the human PD-L1 protein with histidine tag was purchased from the manufacturer NovoProtein (Cat. No. C315); the test buffer is 1x Kinetic Buffer (diluted from 10x Kinetic Buffer (ForteBio, Article No. 18-1105)) for Affinity test and dilution of antigen and antibody; Biomembrane Interference (BLI) technology and Octet Molecular Interaction Analyzer (ForteBio, model Octet Red96e) are used to analyze the binding kinetics between antigen and antibody.
  • the test buffer is 1x Kinetic Buffer (diluted from 10x Kinetic Buffer (ForteBio, Article No. 18-1105)) for Affinity test and dilution of antigen and antibody
  • Biomembrane Interference (BLI) technology and Octet Molecular Interaction Analyzer (ForteBio, model Octet Red96e) are used to analyze the binding kinetics between antigen and antibody.
  • the sensor rotation speed is 1000 revolutions per minute.
  • equilibrate the two AHC sensors in a row in the test buffer for 10 minutes then use the AHC sensor to capture the antigen-binding protein to be tested with a height of 0.9-1.1 nm, and then equilibrate the AHC sensor in the test buffer for 2 minutes.
  • the AHC sensor was bound to the antigen (60 nM and 0 nM) for 3 minutes, and finally dissociated for 15 minutes.
  • the AHC sensor is immersed in a 10 mM glycine (pH 1.5) solution for regeneration to elute the protein bound to the sensor.
  • the affinity of the antigen (multi-concentration) and antibody was determined.
  • the sensor rotation speed is 1000 rpm.
  • two rows of AHC sensors (8 sensors in each row; the first row is called the reference AHC sensor, and the second row is called the test AHC sensor) equilibrate in the test buffer for 10 minutes.
  • test AHC sensors were used to capture the test antigen binding protein (concentration 40nM), the capture time was 30 seconds, and the capture height was about 0.7nm; the 8 test AHC sensors were equilibrated in the test buffer for 2 minutes and then diluted with the antigen protein in gradient (For example, the antigen concentration can be a two-fold dilution of 50-1.56nM and 0nM) binding, 8 sensors can be respectively immersed in up to 8 different concentrations of antigen; bind to the antigen for 5 minutes, then dissociate for 15 minutes; and finally The AHC sensor is immersed in a 10mM glycine (pH 1.5) solution for regeneration to elute the protein bound to the sensor.
  • the experimental procedure of the reference AHC sensor is the same as that of the test AHC sensor. The only difference is that in the first step, the 8 reference AHC sensors are immersed in the test buffer without the molecule to be tested for 30 seconds.
  • Example 8 Using BLI method to measure epitope competition of antigen-binding protein binding PD-L1
  • the human PD-L1 protein (NovoProtein, C315) was first biotinylated using a biotinylation kit (EZ-Link Sulfo-NHS-LC-Biotin, ThermoFisher, A39257) according to the instructions. Then use the ForteBio Octet platform to perform epitope competition experiments on the obtained antigen binding protein, PR000151 (atezolizumab analog) and PR001598 (avelumab analog). The first step is to obtain the 100% signal of the antibody: use the SA sensor to capture the biotinylated PD-L1 protein with a capture height of 0.25 nm.
  • the sensor was immersed in the antibody (50 nM) for 500 seconds, and the final signal of the antibody binding to PD-L1 was recorded as the 100% signal of the antibody.
  • the inhibition rate is calculated by the following formula,
  • A 100% signal of a certain antibody (obtained from the first step)
  • B signal of the antibody as a second antibody (obtained from the second step).
  • the obtained inhibition rate is greater than 85 (%), it means that the epitopes of the two antibodies completely overlap; if the inhibition rate is less than 85 (%), it means that the epitopes bound by the two antibodies do not completely overlap.
  • the antibody PR000416 is an antibody obtained by replacing the constant region of the heavy chain of the antigen-binding protein PR000265 described in the present application with the constant region of human IgG1 without mutation.
  • Example 9 Antigen binding protein stimulates cytokine secretion in mixed lymphocyte reaction (MLR)
  • Ficoll-Paque Plus (GE Healthcare, 17144002) was used to isolate PBMC cells from the first donor's whole blood, and recombinant human IL-4 (R&D Systems, 204-GMP) and recombinant human GM-CSF (R&D Systems, 215) were added.
  • -GM/CF 6 days after induction, obtain immature human CD14 + dendritic cells (iDC cells); continue to add 1 ⁇ g/ml lipopolysaccharide (Lipopolysaccharide, LPS; Sigma, L2630), induce 24 hours, obtain mature Dendritic cells (mDC cells).
  • T cell isolation kit (StemCell, 17951) was used to isolate T lymphocytes from the PBMC cells of the second donor. Inoculate 1 ⁇ 10 5 /well T lymphocytes and 1 ⁇ 10 4 /well mDC cells into a 96-well plate at a ratio of 10:1, add 10 ⁇ g/ml of each antigen binding protein and control antibody, and do 10 times or Corresponding to multiple dilutions. Incubate for 5 days in a 37°C, 5% CO 2 incubator. The supernatants after 72 hours and 120 hours were collected respectively.
  • the IL-2 ELISA kit (Thermo, 88-7025-88) was used to detect the level of IL-2 in the supernatant after 72 hours; the IFN- ⁇ ELISA kit (Thermo, 88-7316-88) was used to detect the supernatant after 120 hours.
  • the level of IFN- ⁇ in the product refer to the reagent instructions for specific operations.
  • PBMCs from four donors were divided into two groups of donor pairs for mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • A-D the antigen binding protein can enhance the secretion of cytokines IL-2 and IFN- ⁇ by activated T lymphocytes.
  • This example measures antibody-dependent cytotoxicity (ADCC).
  • PBMC was used as effector cells, and multiple tumor cell lines with high PD-L1 expression were used as target cells (such as MDA-MB-231 cells (ATCC, HTB-26), NCI-H292 cells (ATCC, CRL-1848). )).
  • target cells such as MDA-MB-231 cells (ATCC, HTB-26), NCI-H292 cells (ATCC, CRL-1848).
  • the fresh PBMC was centrifuged to discard the supernatant, resuspended in 10% FBS-RPMI1640 medium, and incubated overnight in a 37° incubator.
  • PBMC cells collect PBMC cells, centrifuge to discard the supernatant, resuspend in 2% FBS-RPMI1640 medium, count, adjust the cell density to 1 ⁇ 10 7 /ml, add PBMC to a U-shaped bottom 96-well plate, 50 ⁇ l/well .
  • Collect the target cells centrifuge to discard the supernatant, resuspend in 2% FBS-RPMI1640 medium, count, adjust the cell density to 4 ⁇ 10 5 /ml, add the target cells to the U-shaped bottom 96-well plate, 25 ⁇ l/ hole.
  • the ratio of effector cells to target cells is 50:1. Dilute the antibody to an intermediate concentration.
  • the starting concentration is 1.6 nM, and it will be diluted 5 times in a gradient for a total of 3 concentrations.
  • Cytotoxicity (%) ((ER-CMB)-(ESR-CMB)-(TSR-CMB))/(TMR-VCC) ⁇ 100%
  • ESR effector cell natural release hole, effector cell + culture medium
  • TSR natural release hole of target cell, target cell + culture medium
  • TMR Maximum release of target cells, target cells+medium+lysate
  • VCC volume reference well, medium + lysate
  • PR000416 is an antibody obtained by replacing PR000265 with human IgG1 constant region without mutation
  • PR001598 avelumab analog
  • MC38-hPD-L1 5 ⁇ 10 5 colon cancer cells/0.1mL of MC38-hPD-L1 (MC38 cells overexpressing human PD-L1) were inoculated subcutaneously on the right side of female B-hPD-1 humanized mice (Biocytometer), When the tumor grows to about 118mm 3 , the tumor will be randomly divided into groups according to the tumor volume, 10 in each group, 5 groups in total, namely: Human IgG1 (3mg/kg, human IgG1 isotype control group), PR000151 (3mg/kg, Atezolizumab low-dose group ), PR000151 (10mg/kg, Atezolizumab high-dose group), PR000265 (3mg/kg, low-dose antibody group of this application) and PR000265 (10mg/kg, high-dose antibody of this application group).
  • the route of administration is intraperitoneal injection, once every 2 days, for a total of 13 administrations (q2dx13).
  • the tumor volume and body weight were measured twice
  • the tumor volume of the hIgG1 control group was 1359 ⁇ 171 mm 3 .
  • Anti-PD-L1 antibody PR000151 at 3mg / kg and 10mg / kg dose of tumor volumes were 898 ⁇ 233mm 3 and 741 ⁇ 203mm 3, TGI of 33.9% and 45.5%, respectively.
  • the tumor volume of the anti-PD-L1 antibody PR000265 was 881 ⁇ 169 mm 3 and 1035 ⁇ 472 mm 3 at the doses of 3 mg/kg and 10 mg/kg, respectively, and the TGI was 35.1% and 23.8%, respectively.
  • the tumor volume of the human IgG1 control group was 1876 ⁇ 203 mm 3 .
  • Anti-PD-L1 antibody PR000151 at 3mg / kg and 10mg / kg dose of tumor volumes were 1349 ⁇ 306mm 3 and 949 ⁇ 266mm 3, TGI of 28.1% and 49.4%, respectively.
  • the tumor volume of the anti-PD-L1 antibody PR000265 was 1228 ⁇ 187 mm 3 and 937 ⁇ 225 mm 3 at the doses of 3 mg/kg and 10 mg/kg, respectively, and the TGI was 34.5% and 50.1%, respectively.
  • PR000151 and PR000265 had obvious tumor suppressive effects at the doses of 3mg/kg and 10mg/kg, and compared with hIgG1, there was a statistical difference (P ⁇ 0.05).
  • the efficacy of PR000151 and PR000265 were similar, and there was no statistical difference between the groups (P>0.05).
  • a bifunctional fusion protein molecule was constructed by fusing the sequence of the extracellular region of the human TGFB receptor (TGFBRII) with a flexible peptide (Linker) of appropriate length.
  • the plasmid encoding the polypeptide sequence is transfected into mammalian host cells (such as HEK293 or ExpiCHO), and the fusion protein is recombinantly expressed and purified using the method described in Example 1.3 to obtain the fusion protein PR001487, PR001488, PR001901, PR001902, PR002247, PR002248, PR002249 and PR002251, the structure of each part is shown in Table 17, and the sequence numbers of the antibody light chain, heavy chain and each domain are shown in Table 18.
  • the protein molecule M7824 also known as PR001599, which is a bintrafusp alfa analog
  • the protein molecule Hengrui fusion protein 9 also known as PR002466, whose sequence is derived from patent WO2018205985A1
  • TGFBRII Trap also known as PR002040
  • the sequence of the anti-chicken lysozyme antibody clone TEL16 was fused with the truncated sequence of the extracellular region of TGFBRII) as a control in the example.
  • the structure and sequence number of each part are shown in Table 17 and Table 18.
  • Analytical size exclusion chromatography was used to analyze the purity and aggregate form of protein samples. Connect the analytical column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 ⁇ m, 7.8mm x 30cm) to a high pressure liquid chromatograph (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II), equilibrate with PBS buffer at room temperature for at least 1 hour .
  • HPLC high pressure liquid chromatograph
  • An appropriate amount of protein sample (at least 10 ⁇ g, the sample concentration adjusted to 1mg/ml) is filtered with a 0.22 ⁇ m filter membrane and injected into the system, and the HPLC program is set: the sample is flowed at a flow rate of 1.0ml/min with PBS (pH 7.4) buffer The longest time for passing the chromatographic column is 20 minutes; the detection wavelength is 280nm.
  • the ChemStation software uses the ChemStation software to integrate the chromatogram and calculate the relevant data, generate an analysis report, and report the residence time of the components of different molecular sizes in the sample.
  • HIC hydrophobic interaction chromatography
  • the setting method is a linear gradient from 100% mobile phase A (20mM histidine, 1.8M ammonium sulfate, pH 6.0) to 100% mobile phase B (20mM histidine, pH 6.0) within 16 minutes, and the flow rate is set It is 0.7ml/min, the protein sample concentration is 1mg/ml, the injection volume is 20 ⁇ l, and the detection wavelength is 280nm.
  • ChemStation software uses ChemStation software to integrate the chromatogram and calculate the relevant data, generate an analysis report, and report the residence time of the components of different molecular sizes in the sample.
  • DSF Differential Scanning Fluorimetry
  • Example 12.1 The expression and physicochemical properties of the fusion protein obtained in Example 12.1 are shown in Table 19. The results showed that PR001488 and PR001902 had higher yields than the control proteins PR002466 and PR001599, and PR001488 and PR001902 had better hydrophilicity than the control protein PR001599 (represented by a shorter retention time on HPLC-HIC). In general, PR001488 and PR001902 show more stable physical and chemical substances than PR001599.
  • Example 13 Fusion protein binds to CHO-K1 cells overexpressing human PD-L1
  • Figure 15(A) and Table 20-1 and Figure 15(B) and Table 20-2 show that the fusion protein described in the present application can bind to CHO-K1 cells overexpressing human PD-L1.
  • Example 14 Fusion protein blocks the binding of human PD-1 to CHO-K1 cells overexpressing human PD-L1
  • Example 4 According to the method of Example 4, the activity of the fusion protein described in this application to block the binding of human PD-1 and human PD-L1 in vitro was tested.
  • Figure 16 (A) and Table 21-1 and Figure 16 (B) and Table 21-2 show that the fusion protein described in the present application can block the interaction of human PD-1 and CHO-K1 cells overexpressing human PD-L1. Combine.
  • Fusion protein IC50(nM) PR001487 0.2157 PR001488 0.2661 PR001598 0.4416 PR001599 0.3225 PR001901 0.2598 PR001902 0.3107 PR000416 0.1271
  • Example 15 Detecting the inhibitory effect of the fusion protein on the PD-1 signaling pathway using reporter gene cell lines
  • the reporter gene cell line was used to detect the inhibitory effect of the fusion protein described in this application on the PD-1 signal pathway.
  • Figure 17 and Table 22 show that the fusion protein described in this application has an inhibitory effect on the PD-1 signaling pathway.
  • Example 16 Enzyme-linked immunosorbent assay method to detect the in vitro binding effect of fusion protein to TGFB1
  • TGFB1 Novoprotein, CA59
  • a 96-well plate was coated at 100 ⁇ l/well at 4°C overnight. Wash 3 times with 200 ⁇ l/well 1 ⁇ PBST, add 200 ⁇ l/well 2% BSA, and block at 37°C for 2 hours. Add 200 ⁇ l/well of 1xPBST to wash 3 times, add 5-fold concentration gradient antibody, where the highest final concentration of antibody is 100nM, and incubate at 37°C for 1 hour.
  • Figure 18 (A) and Table 23-1 and Figure 18 (B) and Table 23-2 and Figure 18 (C) and Table 23-3 show that the fusion protein described in this application can effectively bind to TGFB1, and has Drug concentration and dose-dependent effects.
  • HEK-Blue TM TGFB cells (Invivogen, hkb-tgfb,) that highly express human TGFBRI and the Smad3/4 gene fused with the SEAP reporter gene were used to study the effect of the fusion protein on the Smad signaling pathway induced by TGFB1 at the cellular level. Inhibition of activation.
  • 10 ⁇ l/well of human TGFB1 protein R&D Systems, 240-B/F
  • 10 ⁇ l/well of human TGFB1 protein R&D Systems, 240-B/F
  • TGFB1 antibody Biointron, B5484
  • TGFBRII-Fc fusion protein Novoprotein, CC10
  • the software GraphPad Prism 8 is used for data processing and graphing analysis, through four-parameter nonlinear fitting, the binding curve and IC50 value and other parameters are obtained.
  • the results are shown in Figure 19 and Table 24.
  • the fusion protein inhibited the reporter activity of pSMAD3 induced by TGFB1 in a dose-dependent manner.
  • PR001902 has comparable efficacy and IC50 to the positive control PR001599 (M7824); its inhibitory activity and IC50 are 8 times stronger than the positive control PR002466 (Hengrui fusion protein 9) and TGFB antibody.
  • Example 18 Using BLI to determine the affinity of the fusion protein to human PD-L1
  • Example 7.2 The method described in Example 7.2 was used to test the affinity of the fusion protein with human PD-L1 protein.
  • Example 19 Using the BLI method to determine the affinity of the fusion protein to human TGFB1
  • the human TGFB1 protein (NovoProtein, CA59) is first biotinylated using a biotinylation kit (EZ-Link Sulfo-NHS-LC-Biotin, ThermoFisher, A39257) according to the instructions. Then, the affinity of the fusion protein with human TGFB1 was determined according to the method similar to that described in Example 7.2. In particular, first put two rows of SA sensors (8 sensors in each row; the first row is called the reference SA sensor, and the second row is called the test SA sensor) in a test buffer containing 0.02% Tween and 1 ⁇ PBS Equilibrate for 10 minutes.
  • SA sensors 8 sensors in each row; the first row is called the reference SA sensor, and the second row is called the test SA sensor
  • the test SA sensor captures the biotinylated human TGFB1, the capture height is set to 0.3 nm, and the reference SA sensor is immersed in the buffer for 30 seconds.
  • the two rows of sensors are then combined with the fusion protein to be tested in a gradient dilution (for example, the concentration of the fusion protein to be tested can be a two-fold dilution of 6.25 to 0.39 nM and 0 nM); bind for 3 minutes, and then dissociate for 10 minutes.
  • the results are shown in Table 26.
  • the K D value of the fusion protein of this application and human TGFB1 is less than 1 ⁇ 10 9 M, and shows higher affinity than the control molecules PR001599 and PR002466.
  • Example 20 Using BLI method to measure epitope competition of fusion protein binding PD-L1
  • the epitope competition experiment was performed on the fusion protein and the control molecule according to the method of Example 8. The results are shown in Table 27-1 and Table 27-2. The epitopes of PR001488 and PR001902 and the control molecules PR001599 or PR002466 do not completely overlap.
  • Example 21 MLR method in vitro detection of fusion protein activation effect on T cells
  • PBMC peripheral blood mononuclear cells
  • monocytes were isolated (Meltenyi, 130-050-201), and complete medium (RPI1640) was used.
  • the cultured cells were collected by centrifugation, washed 4 times with PBS containing 2% FBS, resuspended in a fresh medium, adjusted to a density of 2 ⁇ 10 5 cells/mL, and inoculated to a 96-well cell culture plate with 100 ⁇ l/well.
  • Pan T cells (Meltenyi, 130-096-535) were isolated from fresh allogeneic PBMC and resuspended in fresh medium, adjusted the cell density to 1 ⁇ 10 6 cells/ml, and inoculated 100 ⁇ l/well to a 96-well cell culture plate.
  • IL-2 ELISA kit (Thermo, 88-7025-88) was used to detect the level of IL-2 in the supernatant after 72 hours; the IFN- ⁇ ELISA kit (Thermo, 88-7316-88) was used to detect the supernatant after 120 hours.
  • the level of IFN- ⁇ in the product refer to the reagent instructions for specific operations.
  • the fusion protein can enhance the secretion of cytokines IL-2 ( Figure 20 (A)) and IFN- ⁇ ( Figure 20 (B)) by activated T lymphocytes, and has It has a stronger activation effect than the PD-L1 monoclonal antibody PR000416 and the positive controls PR001599 and PR002446.
  • Example 10 the tumor cell-mediated antibody-dependent cytotoxicity of the fusion protein described in this application was detected.
  • three donor PBMCs and two tumor cell lines were used for a total of six independent ADCC experiments.
  • the results are shown in Figure 21 (A-F).
  • PR001488 has a stronger ADCC effect than the positive control PR001599.
  • This example tests the pharmacokinetic properties of the fusion protein.
  • the method is as follows: select 3 female C57BL/6 mice weighing 18-22 grams, and administer the fusion protein drug by intravenous injection at a dose of 9.53 mg/kg PR001902 and 10 mg/kg PR001488; before and after administration 0.5 hour, 24 hours (1 day), day 2, day 4, day 7, day 10, and day 14 whole blood was collected, the whole blood was allowed to stand for 30 minutes to coagulate, and then at 4 °C Centrifuge at 2,000 rpm for 5 minutes and freeze the separated serum samples at -80°C until analysis.
  • two ELISA methods were used to quantitatively determine the drug concentration in mouse serum.
  • ELISA method one the Fc end detection method, captures the human Fc-containing fusion protein in mouse serum by goat anti-human Fc polyclonal antibody coated on a 96-well plate, and then adds HRP-labeled goat anti-human Fc second Antibody detection
  • ELISA method two the TGFBRII end detection method, captures the human TGFBRII domain-containing fusion protein in mouse serum through human TGFB1 protein coated on a 96-well plate, and then adds HRP-labeled goat anti-human Fc secondary antibody to detect.
  • NCA non-compartmental model
  • Figure 22 and Table 28 show the pharmacokinetic data of the fusion protein PR001902.
  • the results show that the half-life of PR001902 in mice is about 11 days under the Fc terminal detection method; the TGFBRII terminal detection method shows that PR001902 is in mice The half-life is about 8.5 days.
  • Figure 23 and Table 29 show the pharmacokinetic data of the fusion protein PR001488.
  • the results show that the half-life of PR001488 in mice is about 10 days under the Fc terminal detection method; the TGFBRII terminal detection method shows that PR001488 is in mice The half-life is about 5 days.
  • Example 24 Antitumor activity of the fusion protein on the in vivo animal model of colon cancer CT26-human PD-1/PD-L1 transgenic mice
  • This example evaluates the test drug in mouse colon cancer cell CT26-human PDL1(tg)-mouse PDL1(KO) (the endogenous mouse PD-L1 gene of mouse colon cancer cell CT26 is knocked out and introduced into exogenous human PD-L1 transgenic, Jicui Yaokang Biotechnology) transplantation immune checkpoint humanized mouse BALB/c-human PD-1/PD-L1 (introducing BALB/c mice into exogenous human PD-1 transgene and human at the same time PD-L1 genetically modified, Jicui Yao Kang Biotechnology) in the anti-tumor effect.
  • mice Inoculate CT26-human PDL1(tg)-mPDL1(KO) cells in logarithmic growth phase into humanized mouse BALB/c-human PD-1/PD-L1 right axillary subcutaneously, when the average tumor volume reaches 80- At 120 mm 3 , after removing the mice with too large tumor volume, the mice were randomly divided into 3 groups according to the tumor volume, with 6 mice in each group. There are 3 test groups, namely: human IgG1 (10.4 mg/kg, human IgG1 isotype control group), M7824 (12.3 mg/kg, positive control) and PR001902 (12.2 mg/kg, fusion protein of this application).
  • the intraperitoneal administration was started on the day of grouping, and the drug was given once every three days for a total of 6 administrations (q3d ⁇ 6). After starting the administration, weigh the body weight and tumor volume twice a week.
  • the tumor-bearing mice were euthanized and the tumor was stripped and weighed.
  • the experimental results such as the tumor volume and mouse body weight of each group of animals were calculated as mean ⁇ standard error (Mean ⁇ SEM). Multi-group comparisons were performed using one-way ANOVA (one way ANOVA) test method to compare whether there were significant differences between the different treatment groups and the control group. The data was analyzed using SPSS 18.0. P ⁇ 0.05 indicates a significant difference.

Abstract

提供一种PD-L1抗原结合蛋白，其能够以1×10 -8M或更低的KD值结合源自灵长类动物的PD-L1。所述的抗原结合蛋白能够阻断PD-1和CD80与PD-L1的结合，刺激免疫细胞中细胞因子的分泌，能够抑制肿瘤生长和/或肿瘤细胞增殖。还提供一种融合蛋白，其包括人TGFBRII或其片段和所述抗原结合蛋白。还提供所述抗原结合蛋白和/或所述融合蛋白在预防和治疗肿瘤或癌症中的应用。

Description

一种抗PD-L1抗原结合蛋白及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种抗PD-L1抗原结合蛋白及其应用，以及一种抗PD-L1和抗TGFB的融合蛋白及其应用。
背景技术
程序性死亡受体配体1(PD-L1)，也称为分化簇274(CD274)或B7同源蛋白1(B7-H1)，是一个40KDa的I型跨膜蛋白，一般在活化的T细胞、B细胞、单核细胞、树突状细胞、巨噬细胞及许多非造血细胞上表达。
PD-L1能与程序性死亡受体1(PD-1)和B7-1(CD80)结合。其中PD-L1/PD-1信号通路是免疫反应中非常重要的共抑制信号途径，负调节T细胞免疫应答，抑制T细胞活性，减弱细胞因子的分泌。研究发现PD-L1能在许多肿瘤组织中表达，包括胃癌、肺癌、乳腺癌、胰腺癌、卵巢癌、结肠癌、肥大细胞性肿瘤以及恶性黑色素瘤等，以及在浸润肿瘤微环境的骨髓细胞中表达，保护肿瘤细胞逃避免疫攻击。转化生长因子-B(TGFB)是对免疫系统有显著影响的强效细胞因子，参与许多增殖性和非增殖性细胞过程，如细胞增殖和分化、胚胎发育、细胞外基质形成、骨骼发育、伤口愈合、造血作用以及免疫和炎症反应。现有治疗技术手段无论是化学疗法或是肿瘤靶向疗法，影响其疗效的一个重要瓶颈是肿瘤细胞产生免疫耐受。肿瘤细胞在肿瘤微环境中利用多种免疫抑制机制来逃避机体免疫系统识别和攻击，这些免疫抑制机制包括免疫抑制型的细胞因子(如TGFB)、调节性T细胞(Tregs)、共抑制信号通路分子、髓系抑制性细胞等等。
免疫抑制的多种机制可能会阻碍免疫治疗具有有效性。在一些情况下，肿瘤对于单药免疫治疗来说是难治的并且只有一小部分的癌症有完全反应。因此，研发具备阻断PD-L1/PD-1信号通路能力的药物以及在抑制PD-1/PD-L1通路的基础上靶向中和肿瘤微环境的免疫抑制型细胞因子TGFB，能为肿瘤及多种免疫系统相关疾病的治疗带来全新的解决方法。
发明内容
本申请提供了一种PD-L1抗原结合蛋白，其能够以1×10 -8M或更低的KD值结合源自灵长类动物的PD-L1。所述的抗原结合蛋白能够阻断PD-1和CD80与PD-L1的结合，刺激免疫细胞中IFN-γ和/或IL2的分泌，能够抑制肿瘤生长和/或肿瘤细胞增殖。本申请还提供了一种融合蛋白，其包括人TGFBRII或其片段和所述抗原结合蛋白。本申请还提供了所述抗原结 合蛋白和/或所述融合蛋白在预防和治疗肿瘤或癌症中的应用。
一方面，本申请提供了一种结合PD-L1的分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:193所示的氨基酸序列；优选地，所述VH包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:194所示的氨基酸序列；优选地，所述VL包含SEQ ID NOs:108-114、116和118中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体或其抗原结合片段；优选地，所述抗体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；优选地，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv并且/或者dAb。
在某些实施方式中，所述的分离的抗原结合蛋白包含轻链可变区VL和重链可变区VH，轻链可变区VL包含LCDR1、LCDR2和LCDR3，所述LCDR1包含如SEQ ID NO:181所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:182所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:191所示的氨基酸序列；其中重链可变区VH包含HCDR1、HCDR2和HCDR3，所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:179所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:180所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含SEQ ID NOs:181、48-51中任一项所示的氨基酸序列；所述LCDR2包含SEQ ID NOs:182、63-64中任一项所示的氨基酸序列；所述LCDR3包含SEQ ID NOs:191、77-79中任一项所示的氨基酸序列；HCDR1包含如SEQ ID NO:5所示的氨基酸序列；HCDR2包含SEQ ID NOs:179、14、16和17中任一项所示的氨基酸序列；HCDR3包含SEQ ID NOs:180、29-33中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列；所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列；所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列；所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列；所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列；所述HCDR3包含如SEQ ID NO:29所示；或者，
所述LCDR1包含如SEQ ID NO:49所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1 包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:30所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:79所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:31所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:32所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:33所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:30所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:30所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO: 63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:32所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:32所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:33所示的氨基酸序列；或者，
所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述抗体重链可变区VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4；优选地，所述H-FR1的C末端与所述HCDR1的N末端相连，包含SEQ ID NO:1所示的氨基酸序列；并且/或者所述H-FR2位于所述HCDR1与所述HCDR2之间，包含SEQ ID NO:9所示的氨基酸序列；并且/或者所述H-FR3位于所述HCDR2与所述HCDR3之间，包含SEQ ID NO:192所示的氨基酸序列；并且/或者所述H-FR4的N末端与所述HCDR3的C末端相连，包含SEQ ID NO:38所示的氨基酸序列；进一步优选地，所述H-FR3包含SEQ ID NOs:21、22和24中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体轻链可变区VL包括框架区L-FR1，L-FR2，L-FR3，和L- FR4；优选地，所述L-FR1的C末端与所述LCDR1的N末端相连，包含SEQ ID NO:185所示的氨基酸序列；并且/或者所述L-FR2位于所述LCDR1与所述LCDR2之间，包含SEQ ID NO:186所示的氨基酸序列；并且/或者所述L-FR3位于所述LCDR2与所述LCDR3之间，包含SEQ ID NO:187所示的氨基酸序列；并且/或者所述L-FR4的N末端与所述LCDR3的C末端相连，包含SEQ ID NO:188所示的氨基酸序列。
在某些实施方式中，所述L-FR1包含SEQ ID NOs:41-44中任一项所示的氨基酸序列；并且/或者所述L-FR2包含SEQ ID NOs:56-59中任一项所示的氨基酸序列；并且/或者所述L-FR3包含SEQ ID NOs:69-72中任一项所示的氨基酸序列；并且/或者所述L-FR4包含SEQ ID NOs:85-86中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体重链可变区VH包含SEQ ID NO:193所示的氨基酸序列；所述抗体轻链可变区VL包含SEQ ID NO:194所示的氨基酸序列；
优选地，所述抗体重链可变区VH包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列；
优选地，所述抗体轻链可变区VL包含SEQ ID NOs:108-114、116和118中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体重链可变区VH包含SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包括SEQ ID NO:108所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:109所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:91所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:110所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:111所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:92所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:112所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:93所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:113所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:114所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:95所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:116所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:97所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:118所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:98所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:118所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:99所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:113所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:100所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:113所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:101所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:114所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:102所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:114所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:103所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:116所示的氨基酸序列；或者，
所述抗体重链可变区VH包含如SEQ ID NO:104所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:116所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区；优选地，其中所述抗体重链恒定区包含如SEQ ID NOs:172-175中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含抗体重链HC，且所述HC包含如SEQ ID NOs:122-125、127和129-137中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包括抗体轻链恒定区，其中所述抗体轻链恒定区包含如SEQ ID NO:170所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白包含抗体轻链LC，且所述LC包含如SEQ ID NOs:150-156、158和160-163中任一项所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白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-L1；
2)能够阻断PD-1与PD-L1的结合；
3)能够阻断CD80与PD-L1的结合；
4)能够刺激免疫细胞中IFN-γ并且/或者IL2的分泌；
5)能够抑制肿瘤生长并且/或者肿瘤细胞增殖；
6)与对照抗体相比，结合与源自灵长类动物的PD-L1的不完全重叠的表位，
其中所述对照抗体包含SEQ ID NO:52所示的LCDR1、SEQ ID NO:65所示的LCDR2和SEQ ID NO:81所示的LCDR3，且所述对照抗体包含SEQ ID NO:6所示的HCDR1、SEQ ID NO:15所示的HCDR2和SEQ ID NO:34所示的HCDR3；
或者，
其中所述对照抗体包含SEQ ID NO:53所示的LCDR1、SEQ ID NO:66所示的LCDR2和SEQ ID NO:82所示的LCDR3，且所述对照抗体包含SEQ ID NO:5所示的HCDR1、SEQ ID NO:18所示的HCDR2和SEQ ID NO:35所示的HCDR3；
或者，
所述对照抗体包含SEQ ID NO:55所示的LCDR1、SEQ ID NO:68所示的LCDR2和SEQ ID NO:84所示的LCDR3，且所述对照抗体包含SEQ ID NO:8所示的HCDR1、SEQ ID NO:20所示的HCDR2和SEQ ID NO:37所示的HCDR3。
在某些实施方式中，所述灵长类动物包括人并且/或者猴。
另一方面，本申请提供了一种融合蛋白，其包含：a)人TGFBRII或其片段；以及b)所述的分离的抗原结合蛋白。
在某些实施方式中，所述的融合蛋白包含第一多肽和第二多肽，其中所述第一多肽包含所述分离的抗原结合蛋白的重链或其片段以及所述人TGFBRII或其片段；且所述第二多肽包含所述分离的抗原结合蛋白的轻链或其片段；优选地，所述抗体重链或其片段与所述人TGFBRII或其片段框内融合而形成所述第一多肽；进一步优选地，其中所述抗体重链或其片段的C端直接或间接与所述人TGFBRII或其片段的N端连接。
在某些实施方式中，所述第一多肽的所述重链或其片段包括HCDR1、HCDR2和HCDR3，HCDR1包含SEQ ID NO:5所示的氨基酸序列；HCDR2包含SEQ ID NOs:179、14、16和17中任一项所示的氨基酸序列；HCDR3包含SEQ ID NOs:180、29-33中任一项所示的氨基酸序列；并且/或者所述第二多肽包括LCDR1、LCDR2和LCDR3，LCDR1包含SEQ ID NOs:181、48-51中任一项所示的氨基酸序列；LCDR2包含SEQ ID NOs:182、63-64中任一项所示的氨基酸序列；LCDR3包含SEQ ID NOs:191、77-79中任一项所示的氨基酸序列。
在某些实施方式中，LCDR1包含如SEQ ID NO：51所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：78所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：16所示的氨基酸序列，HCDR3包含如SEQ ID NO：29所示的氨基酸序列；或者，
LCDR1包含如SEQ ID NO：50所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示 的氨基酸序列，LCDR3包含如SEQ ID NO：77所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：16所示的氨基酸序列，HCDR3包含如SEQ ID NO：33所示的氨基酸序列；或者，
LCDR1包含如SEQ ID NO：50所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：77所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：17所示的氨基酸序列，HCDR3包含如SEQ ID NO：33所示的氨基酸序列；或者，
LCDR1包含如SEQ ID NO：51所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：78所示的氨基酸序列；HCDR1包含SEQ ID NO：5所示的氨基酸序列，HCDR2包含SEQ ID NO：17所示的氨基酸序列，HCDR3包含如SEQ ID NO：29所示的氨基酸序列；或者，
LCDR1包含如SEQ ID NO：51所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：78所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：14所示的氨基酸序列，HCDR3包含如SEQ ID NO：29所示的氨基酸序列。
在某些实施方式中，所述抗体重链或其片段通过连接子与所述人TGFBRII或其片段相连；优选地，其中所述连接子为肽连接子，且所述肽连接子包含SEQ ID NOs:167-169中任一项所示的氨基酸序列。
在某些实施方式中，所述人TGFBRII或其片段包含人TGFBRII的胞外结构域；优选地，所述人TGFBRII或其片段包含SEQ ID NOs:176-178中任一项所示的氨基酸序列。
在某些实施方式中，所述第一多肽的所述重链或其片段与所述第二多肽的所述轻链或其片段组合后形成特异性结合PD-L1的抗原结合部分。
在某些实施方式中，所述第一多肽包含SEQ ID NOs:138、139、142、143和145-148中任一项所示的氨基酸序列；其中所述第二多肽包含SEQ ID NOs:162-163中任一项所示的氨基酸序列。
在某些实施方式中，所述第一多肽包含如SEQ ID NO:138所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:139所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:142所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:143所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:145所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:163所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:146所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:163所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:147所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:148所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列。
另一方面，本申请提供了分离的一种或多种核酸分子，其编码所述的分离的抗原结合蛋白或所述的融合蛋白。
另一方面，本申请提供了载体，其包含所述的核酸分子。
另一方面，本申请提供了细胞，其包含所述的核酸分子或所述的载体。
另一方面，本申请提供了制备所述的分离的抗原结合蛋白或所述的融合蛋白的方法，所述方法包括在使得所述的分离的抗原结合蛋白所述的融合蛋白表达的条件下，培养所述的细胞。
另一方面，本申请提供了嵌合抗原受体，其包含所述的分离的抗原结合蛋白。
另一方面，本申请提供了基因修饰的细胞，其包含所述的嵌合抗原受体。
另一方面，本申请提供了抗体药物偶联物，其包含细胞毒性剂，以及所述的分离的抗原结合蛋白。
另一方面，本申请提供了药物组合物，其包含所述的分离的抗原结合蛋白、所述的融合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的嵌合抗原受体、所述的基因修饰的细胞和/或所述的抗体药物偶联物，以及任选地药学上可接受的载体；较佳地，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
另一方面，本申请提供了所述的分离的抗原结合蛋白、所述的融合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的嵌合抗原受体、所述的基因修饰的细胞和/或所述的抗体药物偶联物和/或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、缓解并且/或者治疗肿瘤或癌症，抑制肿瘤生长并且/或者抑制肿瘤细胞增殖；优选地，所述肿瘤或癌症为 PD-L1表达异常的肿瘤或癌症；优选地，所述肿瘤或癌症包括结直肠癌。
另一方面，本申请提供了一种预防、缓解或治疗肿瘤，抑制肿瘤生长并且/或者抑制肿瘤细胞增殖的方法，所述方法包括向有需要的受试者施用所述的分离的抗原结合蛋白、所述的融合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的嵌合抗原受体、所述的基因修饰的细胞和/或所述的抗体药物偶联物和/或所述的药物组合物；优选地，其中所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症；优选地，所述肿瘤或癌症包括结直肠癌。
另一方面，本申请提供了一种所述的分离的抗原结合蛋白、所述的融合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的嵌合抗原受体、所述的基因修饰的细胞和/或所述的抗体药物偶联物和/或所述的药物组合物，其用于治疗癌症，抑制肿瘤生长并且/或者抑制肿瘤细胞增殖。
另一方面，本申请提供了一种抑制PD-L1和或CD80与PD-1结合的方法，所述方法包括施用所述的分离的抗原结合蛋白、所述的融合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的嵌合抗原受体、所述的基因修饰的细胞和/或所述的抗体药物偶联物和/或所述的药物组合物。
另一方面，本申请提供了一种试剂盒，其包括所述的分离的抗原结合蛋白、所述的融合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的嵌合抗原受体、所述的基因修饰的细胞和/或所述的抗体药物偶联物和/或所述的药物组合物；优选地，所述试剂盒还包括(i)施用装置；和/或(ii)使用说明。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结合过表达人PD-L1的CHO-K1细胞；
图2显示的是本申请所述抗原结合蛋白结合过表达食蟹猴PD-L1的CHO-K1细胞。
图3显示的是本申请所述抗原结合蛋白结合人PD-L1蛋白；
图4显示的是本申请所述抗原结合蛋白结合食蟹猴PD-L1蛋白；
图5显示的是本申请所述抗原结合蛋白阻断人PD-1与过表达人PD-L1的CHO-K1细胞的结合；
图6显示的是本申请所述抗原结合蛋白阻断人PD-1与过表达人PD-L1的CHO-K1细胞的结合；
图7显示的是本申请所述抗原结合蛋白阻断生物素化的人PD-1蛋白与人PD-L1蛋白的结合；
图8显示的是本申请所述抗原结合蛋白阻断生物素化的人B7-1(CD80)蛋白与人PD-L1蛋白的结合；
图9显示的是本申请所述抗原结合蛋白对PD-1信号通路的抑制作用；
图10显示的是本申请所述抗原结合蛋白对PD-1信号通路的抑制作用；
图11显示的是本申请所述抗原结合蛋白与人PD-L1的亲和力：(A)PR000265的单浓度抗原结合解离曲线，(B)PR000266的单浓度抗原结合解离曲线，(C)PR000265的多浓度抗原结合解离曲线，(D)PR000151的多浓度抗原结合解离曲线，(E)PR001598的多浓度抗原结合解离曲线；
图12显示的是本申请所述抗原结合蛋白在混合淋巴细胞反应(MLR)中刺激细胞因子分泌：在第一组供体配对的MLR实验中，(A)120小时后上清中的IFN-γ水平，(B)72小时后上清中的IL-2水平；在第二组供体配对的MLR实验中，(C)120小时后上清中的IFN-γ水平，(D)72小时后上清中的IL-2水平；
图13显示的是本申请所述抗原结合蛋白的抗体依赖的细胞毒性结果：(A-C)三个不同供体的PBMC对肿瘤细胞MDA-MB-231的杀伤；(D-F)三个不同供体的PBMC对肿瘤细胞NCI-H292的杀伤；
图14显示的是本申请所述抗原结合蛋白的体内抑制肿瘤活性，分别为肿瘤体积变化(A)和小鼠体重变化(B)；
图15显示的是本申请所述融合蛋白结合过表达人PD-L1的CHO-K1细胞：(A)表20-1所列融合蛋白，(B)表20-2所列融合蛋白；
图16显示的是本申请所述融合蛋白阻断人PD-1与过表达人PD-L1的CHO-K1细胞的结合：(A)表21-1所列融合蛋白，(B)表21-2所列融合蛋白；
图17显示的是本申请所述融合蛋白对PD-1信号通路的抑制作用；
图18显示的是本申请所述融合蛋白结合TGFB1：(A)表23-1所列融合蛋白，(B)表23-2所列融合蛋白，(C)表23-3所列融合蛋白；
图19显示的是本申请所述融合蛋白对TGFB1诱导Smad信号通路激活的抑制作用；
图20显示的是本申请所述融合蛋白在混合淋巴细胞反应(MLR)中，在没有或外加TGFB1的条件下，刺激T淋巴细胞分泌细胞因子的水平：(A)72小时后上清中的IL-2水平，(B)120小时后上清中的IFN-γ水平；
图21显示的是本申请所述融合蛋白的抗体依赖的细胞毒性结果：(A-C)三个不同供体的PBMC对肿瘤细胞MDA-MB-231的杀伤；(D-F)三个不同供体的PBMC对肿瘤细胞NCI-H292的杀伤；
图22显示的是本申请所述融合蛋白PR001902的药代动力学结果；
图23显示的是本申请所述融合蛋白PR001488的药代动力学结果；
图24显示的是本申请所述融合蛋白对肿瘤动物体内模型的抗肿瘤活性，分别为肿瘤体积变化(A)和小鼠体重变化(B)。
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。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，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。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。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。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(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、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“融合蛋白”通常是指由两种或多种多肽构成的蛋白质，在天然状态下通常不结合，但是各自的氨基和羧基末端可通过直接或间接结合在一起以形成一个连续多肽。在某些情形中，提及本申请所述的融合蛋白时，术语“融合蛋白”可与“抗体”互换的使用。在某些情形中，提及本申请所述的融合蛋白时，术语“融合蛋白”可与“抗原结合蛋白”互换的使用。
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”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，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)。可变域中更高度保守的部分被称为框架(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，恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞毒性。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat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))和基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)。在本申请中，还使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基。抗体CDR定义方法请参见表1。
表1抗体CDR定义方法
  Kabat Chothia Combined
LCDR1 L24--L34 L24--L34 L24-L34
LCDR2 L50--L56 L50--L56 L50-L56
LCDR3 L89--L97 L89--L97 L89-L97
HCDR1 H31--H35 H26--H32 H26-H35
HCDR2 H50--H65 H52--H56 H50-H65
HCDR3 H95--H102 H95--H102 H95-H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。
在本申请中，术语“分离的”抗原结合蛋白通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。
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，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本发明使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。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“特异性结合”通常是指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。根据该定义，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。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
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“PD-L1”通常是指程序性死亡配体1蛋白、其功能变体和/或其功能片段。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结合其受体，例如程序性死亡1(PD-1)，所述PD-1在活化的T细胞、B细胞和巨噬细胞中表达(Ishida et al.,1992 EMBO J,11:3887-3395；Okazaki et al.,Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice.Science,2001；291:319-22)。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(Freeman et al.,Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation,J.Exp.Med.2000,192:1027-1034；Carter et al.,PD-1:PD-L inhibitory pathway affects both CD4(+)and CD8(+)T cells and is overcome by IL-2.Eur.J.Immunol.2002,32:634–643)。术语“PD-L1”涵盖任何脊椎动物来源的任何天然PD-L1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人)和啮齿类(例如，小鼠和大鼠)。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。又例如可在NCBI Gene ID No.60533下找到关于小鼠PD-L1基因(包括基因组DNA序列)的信息。又例如可在NCBI Gene ID No.102145573下找到关于食蟹猴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。示例性的全长小鼠PD-L1蛋白序列可在NCBI登录号NP_068693或Uniprot登录号Q9EP73下找到。示例性的全长食蟹猴PD-L1蛋白序列可在NCBI登录号XP_005581836或Uniprot登录号G7PSE7下找到。
在本申请中，术语“PD-1”通常是指程序性死亡1受体(也称为CD279)、其功能变体和/或其功能片段。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。在PD-1的定义之内还包括与天然存在的PD-1的氨基酸序列不同但保持特异性结合PD-L1的能力的变体。在PD-1的定义之内进一步包括增强PD-L1的生物活性的变体。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“CD80”通常是指分化簇蛋白80(也称为B7-1)、其功能变体和/或其功能片段。CD80通常在专职抗原递呈细胞(APC)上表达。CD80能与PD-L1结合。在CD80的定义之内还包括与天然存在的CD80的氨基酸序列不同但保持特异性结合PD-L1的能力的变体。如本文使用的术语“CD80”包括人CD80(hCD80)、hCD80的变体、异构体和种属同源体、以及具有至少一种含hCD80的常见表位的类似物。例如，术语“CD80”还涵盖来自其他种属比如其它哺乳动物(例如，大鼠、小鼠、兔、非人灵长类动物、猪或牛)的CD80。完整的人CD80序列可以在Uniprot登录号P33681中找到。
在本申请中，术语“TGFBRII”通常是指一种转化生长因子B(TGFB)的受体(也称为TGFBR2)。细胞表面的TGFBR可以被转化生长因子(TGFB)结合并激活，通过SMAD通路进行信号传递，具有调节生长，抗炎和免疫调节的活性。完整的人TGFBRII序列可以在Uniprot登录号P37173中找到。
在本申请中，术语“特定抗PD-L1抗体”通常是指可以与本申请的抗体竞争结合PD-L1的抗体。本申请的抗原结合蛋白可包括与特定抗PD-L1抗体具有相同氨基酸序列的那些抗体。
在本申请中，术语“对照抗体”通常是指在本申请实施例中以提供相对其来评价实验结果的标准的PD-L1抗体。在某些情形中，对照抗体可以指本申请实施例中的阳性对照，例如PR000151、PR001598和PR002466。
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(诸如，和具体地，食蟹猴(食蟹猴(Macaca fascicularis))和或恒河猴(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(黑猩猩(Pan troglodytes))，并且还包括智人。
在本申请中，术语“表位”通常是指抗原结合蛋白(如抗体)特异性结合的抗原的某个区或区域。表位通常由诸如氨基酸或碳水化合物或糖侧链分子的化学活性表面群组组成，且通常具有特异性三维结构特征以及特异性电荷特征。表位可为“线性表位”或“构型表位”。在线性表位中，蛋白质与相互作用的分子(诸如抗体)之间的所有相互作用点沿蛋白质的一级氨基酸序列线性发生。在构型表位中，相互作用点于彼此分离的蛋白质上的氨基酸上交叉 发生。给定抗原结合蛋白(如抗体)结合何种表位的方法(如，表位作图(Epitope Mapping))是本领域所公知的，包括例如免疫印迹和免疫沉淀测定，例如，测试重叠或邻接的肽(例如，来自PD-L1)与给定抗原结合蛋白(如抗-PD-L1抗体)的反应性。确定表位空间构象的方法包括本领域技术和本申请所述的那些技术，例如，X射线晶体学、二维核磁共振和HDX-MS(可参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology,Vol.66,G.E.Morris,Ed.(1996))。术语“不完全重叠”的表位通常是指在提及两种或多种抗原结合蛋白(如抗体)时，各抗体结合不完全相同或不完全重叠的氨基酸残基群，如通过给定方法所测定的。用于确定本申请所述抗原结合蛋白是否与参比抗体结合相同表位的技术包括例如表位作图方法，诸如，抗原：抗体复合物的晶体的x射线分析，其提供表位的原子分辨率，和氢/氘交换质谱法(HDX-MS)。其他方法监测抗体与抗原片段或抗原的突变变化形式的结合，其中由于抗原序列内氨基酸残基的修饰所致的结合损失经常被视为可指示表位组分(例如，丙氨酸扫描诱变-Cunningham及Wells(1985)Science 244:1081)。另外，亦可使用表位定位的计算组合方法。
在本申请中，术语“受试者”通常是指哺乳动物。哺乳动物包括但不限于驯化动物(例如奶牛、绵羊、猫、狗和马)、灵长类(例如人和非人灵长类，诸如猴)、兔以及啮齿类(例如，小鼠和大鼠)。
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体或其抗原结合片段的个体细胞、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“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
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。所述组合物是无菌的。“无菌”组合物是灭菌的，或不含所有活的微生物及它们的孢子。
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-L1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。部分地根据施用是否为短暂的或长期的，给药可通过任何适合的途径进行，例如通过注射(诸如静脉内或皮下注射)进行。本文涵盖各种给药过程，包括但不限于单次施用或各种时间点内的多次施用、推注施用和脉冲输注。
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，肿瘤可以包括结肠癌。
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。
抗原结合蛋白
在一个方面，本申请提供了一种抗原结合蛋白，其包含抗体轻链可变区VL中的至少一个CDR。
在本申请中，所述抗原结合蛋白可包含LCDR1，且所述LCDR1可包含SEQ ID NO:181所示的氨基酸序列：
RASQSIX 7X 8WLA(SEQ ID NO:181)；其中，X 7＝F，S或Y；X 8＝I或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:48所示抗原结合蛋白的LCDR1相比，所述LCDR1可至少包含在选自以下位置处的氨基酸取代：在X 7和/或X 8处的氨基酸取代。
在某些情形中，与SEQ ID NO:48所示抗原结合蛋白的LCDR1相比，所述LCDR1可至少包含在X 7和/或X 8处的氨基酸取代，其中X 7处的氨基酸可被取代为Y或F；X 8处的氨基酸可被取代为S。
例如，所述LCDR1可包含SEQ ID NOs:48-51中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR2，且所述LCDR2可包含SEQ ID NO:182所示的氨基酸序列：
KASSLEX 7(SEQ ID NO:182)；其中，X 7＝T或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:63所示抗原结合蛋白的LCDR2相比，所述LCDR2可至少包含在选自以下位置处的氨基酸取代：在X 7处的氨基酸取代。
在某些情形中，与SEQ ID NO:63所示抗原结合蛋白的LCDR2相比，所述LCDR2可至少包含在X 7处的氨基酸取代，其中X 7处的氨基酸可被取代为T。
例如，所述LCDR2可包含SEQ ID NOs:63-64中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR3，且所述LCDR3可包含SEQ ID NO:183所示的氨基酸序列：
QQYX 4X 5X 6SRT(SEQ ID NO:183)；其中，X 4＝Y或H；X 5＝G，T或S；X 6＝Y或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:77所示抗原结合蛋白的LCDR3相比，所述LCDR3可至少包含在选自以下位置处的氨基酸取代：在X 4、X 5和/或X 6处的氨基酸取代。
在某些情形中，与SEQ ID NO:77所示抗原结合蛋白的LCDR3相比，所述LCDR3可至少包含在X 4、X 5和/或X 6处的氨基酸取代，其中X 4处的氨基酸可被取代为H；X 5处的氨基酸可被取代为S或T；X 6处的氨基酸可被取代为S。
例如，所述LCDR3可包含SEQ ID NOs:77-80中任一项所示的氨基酸序列。
在另一些情形中，所述LCDR3可包含SEQ ID NO:191所示的氨基酸序列：
QQYYX 5X 6SRT(SEQ ID NO:191)；其中，X 5＝G或S；X 6＝Y或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在一些具体的情形中，与SEQ ID NO:77所示抗原结合蛋白的LCDR3相比，所述LCDR3可至少包含在选自以下位置处的氨基酸取代：在X 5和/或X 6处的氨基酸取代。
在一些具体的情形中，与SEQ ID NO:77所示抗原结合蛋白的LCDR3相比，所述LCDR3可至少包含在X 5和/或X 6处的氨基酸取代，其中X 5处的氨基酸可被取代为S；X 6处的氨基酸可被取代为S。
例如，所述LCDR3可包含SEQ ID NOs:77-79中任一项所示的氨基酸序列。
在本申请中，所述的抗原结合蛋白可包含框架区L-FR1。所述L-FR1的C末端与所述LCDR1的N末端相连，且所述的L-FR1可包含SEQ ID NO:185所示的氨基酸序列：
DIQMTQSPSTLSX 13SVGX 17RVTX 21TC(SEQ ID NO:185)；其中，X 13＝T或A；X 17＝D或H；X 21＝I或V。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:41所示的抗原结合蛋白的L-FR1相比，所述L-FR1可至少包含在选自以下位置处的氨基酸取代：在X 13、X 17和/或X 21处的氨基酸取代。
在某些情形中，与SEQ ID NO:41所示的抗原结合蛋白的L-FR1相比，所述L-FR1可至少包含在X 13、X 17和/或X 21处的氨基酸取代，其中，X 13处的氨基酸可被取代为A；X 17处的氨基酸可被取代为H；X 21处的氨基酸可被取代为V。
例如，所述L-FR1可包含SEQ ID NOs:41-44中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含L-FR2。所述的L-FR2位于所述LCDR1与所述LCDR2之间，且所述的L-FR2可包含SEQ ID NO:186所示的氨基酸序列：
WYQQX 5PGKAPX 11LLIY(SEQ ID NO:186)；其中，X 5＝K或H；X 11＝K，N或D。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:56所示的抗原结合蛋白的L-FR2相比，所述L-FR2至少包含在选自以下位置处的氨基酸取代：在X 5和/或X 11处的氨基酸取代。
在某些情形中，与SEQ ID NO:56所示的抗原结合蛋白的L-FR2相比，所述L-FR2可至少包含在X 5和/或X 11处的氨基酸取代，其中，X 5处的氨基酸可被取代为H；X 11处的氨基酸可被取代为D或N。
例如，所述L-FR2可包含SEQ ID NOs:56-59中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含L-FR3.所述的L-FR3位于所述LCDR2与所述LCDR3之间，且所述的L-FR3包含SEQ ID NO:187所示的氨基酸序列：
GVPSRFSGX 9GSGTEFTLTISSLQPDDFX 28TYX 31C(SEQ ID NO:187)；其中，X 9＝S或N；X 28＝A或T；X 31＝Y或F。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:69所示的L-FR3相比，所述L-FR3可至少包含在选自以下位置处的氨基酸取代：在X 9、X 28和/或X 31处的氨基酸取代。
在某些情形中，与SEQ ID NO:69所示的抗原结合蛋白的L-FR3相比，所述L-FR3可至少包含在X 9、X 28和/或X 31处的氨基酸取代，其中，X 9处的氨基酸可被取代为N；X 28处的氨基酸可被取代为T；X 31处的氨基酸可被取代为F。
例如，所述L-FR3可包含SEQ ID NOs:69-72中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含L-FR4，所述的L-FR4的N末端与所述LCDR3的C末端相连，且所述的L-FR4可包含SEQ ID NO:188所示的氨基酸序列：
FGQGTKVEIX 10(SEQ ID NO:188)；其中，X 10＝K或R。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:85所示的抗原结合蛋白的L-FR4相比，所述L-FR4至少包含在选自以下位置处的氨基酸取代：在X 10处的氨基酸取代。
在某些情形中，与SEQ ID NO:85所示的L-FR4相比，所述L-FR4可至少包含在X 10处的氨基酸取代，其中，X 10处的氨基酸可被取代为R。
例如，所述L-FR4可包含SEQ ID NOs:85-86中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链可变区VL，且所述VL可包含SEQ ID NO:190所示的氨基酸序列：
DIQMTQSPSTLSX 13SVGX 17RVTX 21TCRASQSIX 30X 31WLAWYQQX 39PGKAPX 45LLIYK ASSLEX 56GVPSRFSGX 65GSGTEFTLTISSLQPDDFX 84TYX 87CQQYX 92X 93X 94SRTFGQGTKVE IX 107(SEQ ID NO:190)；其中，X 13＝T或A；X 17＝D或H；X 21＝I或V；X 30＝F，S或Y；X 31＝I或S；X 39＝K或H；X 45＝K，N或D；X 56＝S或T；X 65＝S或N；X 84＝A或T；X 87＝Y或F；X 92＝Y或H；X 93＝G，T或S；X 94＝Y或S；X 107＝K或R。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:108所示的VL相比，所述VL可至少包含在选自以下位置处的氨基酸取代：在X 13、X 17、X 21、X 30、X 31、X 39、X 45、X 56、X 65、X 84、X 87、X 92、X 93、X 94、X 107处的氨基酸取代。
在某些情形中，与SEQ ID NO:108所示的VL相比，所述VL可至少包含在X 13、X 17、X 21、X 30、X 31、X 39、X 45、X 56、X 65、X 84、X 87、X 92、X 93、X 94、X 107处的氨基酸取代，其中，X 13处的氨基酸可被取代为A，X 17处的氨基酸可被取代为H，X 21处的氨基酸可被取代为V，X 30处的氨基酸可被取代为S或Y，X 31处的氨基酸可被取代为S，X 39处的氨基酸可被取代为H，X 45处的氨基酸可被取代为N或D，X 56处的氨基酸可被取代为T，X 65处的氨基酸可被取代为N，X 84处的氨基酸可被取代为T，X 87处的氨基酸可被取代为F，X 92处的氨基酸可被取代为H，X 93处的氨基酸可被取代为T或S，X 94处的氨基酸可被取代为S，X 107处的氨基酸可被取代为R。
例如，所述VL区可包含SEQ ID NOs:108-116和118中任一项所示的氨基酸序列。
在某些情形中，本申请所述VL可包含SEQ ID NO:194所示的氨基酸序列：
DIQMTQSPSTLSX 13SVGX 17RVTX 21TCRASQSIX 30X 31WLAWYQQX 39PGKAPX 45LLIYK ASSLEX 56GVPSRFSGX 65GSGTEFTLTISSLQPDDFX 84TYX 87CQQYYX 93X 94SRTFGQGTKVEI X 107(SEQ ID NO:194)；其中，X 13＝T或A；X 17＝D或H；X 21＝I或V；X 30＝F，S或Y；X 31＝I或S；X 39＝K或H；X 45＝K，N或D；X 56＝S或T；X 65＝S或N；X 84＝A或T；X 87＝Y或F；X 93＝G或S；X 94＝Y或S；X 107＝K或R。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:108所示的VL相比，所述VL可至少包含在选自以下位置处的氨基酸取代：在X 13、X 17、X 21、X 30、X 31、X 39、X 45、X 56、X 65、X 84、X 87、X 93、X 94、X 107处的氨基酸取代。
在某些情形中，与SEQ ID NO:108所示的VL相比，所述VL可至少包含在X 13、X 17、X 21、X 30、X 31、X 39、X 45、X 56、X 65、X 84、X 87、X 93、X 94、X 107处的氨基酸取代，其中，X 13处的氨基酸可被取代为A，X 17处的氨基酸可被取代为H，X 21处的氨基酸可被取代为V，X 30处的氨基酸可被取代为S或Y，X 31处的氨基酸可被取代为S，X 39处的氨基酸可被取代为H，X 45处的氨基酸可被取代为N或D，X 56处的氨基酸可被取代为T，X 65处的氨基酸可被取代为N，X 84处的氨基酸可被取代为T，X 87处的氨基酸可被取代为F，X 93处的氨基酸可被取代为S，X 94处的氨基酸可被取代为S，X 107处的氨基酸可被取代为R。
例如，所述VL区可包含SEQ ID NOs:108-114、116和118中任一项所示的氨基酸序列。
本申请所述抗原结合蛋白可包含轻链恒定区CL，且所述抗体轻链恒定区可包括人Igκ恒 定区。例如，所述CL区可包含下述所示的氨基酸序列：SEQ ID NO:170。
本申请所述抗原结合蛋白可包含抗体轻链LC，所述抗体轻链可包含SEQ ID NOs:150-158和160-163中任一项所示的氨基酸序列。或者，本申请所述的抗原结合蛋白的抗体轻链可包含SEQ ID NO:150-156、158和160-163中任一项所示的氨基酸序列。
本申请所述的抗原结合蛋白可包含抗体重链可变区VH中的至少一个CDR。
在本申请中，所述抗原结合蛋白可包含HCDR1，且所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列：
GFTFSSY(SEQ ID NO:5)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述抗原结合蛋白可包含HCDR2，且所述HCDR2可包含SEQ ID NO:179所示的氨基酸序列：
KQX 3X 4SE(SEQ ID NO:179)；其中，X 3＝D或E；X 4＝G或A。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:14所示抗原结合蛋白的HCDR2相比，所述HCDR2可至少包含在选自以下位置处的氨基酸取代：在X 3和/或X 4处的氨基酸取代。
在某些情形中，与SEQ ID NO:14所示抗原结合蛋白的HCDR2相比，所述HCDR2可至少包含在X 3和/或X 4处的氨基酸取代，其中，X 3处的氨基酸可被取代为E，X 4处的氨基酸可被取代为A。
例如，所述HCDR2可包含SEQ ID NOs:14、16和17中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR3，且所述HCDR3可包含SEQ ID NO:180所示的氨基酸序列：
DRX 3VAGAX 8X 9X 10(SEQ ID NO:180)；其中，X 3＝A或P；X 8＝F或S；X 9＝D或A；X 10＝I或F。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:29所示抗原结合蛋白的HCDR3相比，所述HCDR3可至少包含在选自以下位置处的氨基酸取代：在X 3、X 8、X 9和/或X 10处的氨基酸取代。
在某些情形中，与SEQ ID NO:29所示抗原结合蛋白的HCDR3相比，所述HCDR3可至少包含在X 3、X 8、X 9和/或X 10处的氨基酸取代，其中X 3处的氨基酸可被取代为P；X 8处的氨基酸可被取代为S；X 9处的氨基酸可被取代为A；X 10处的氨基酸可被取代为F。
例如，所述HCDR3可包含SEQ ID NOs:29-33中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含框架区H-FR1。所述H-FR1的C末端与所述HCDR1的N末端相连，且所述的H-FR1可包含SEQ ID NO:1所示的氨基酸序列：
EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:1)。例如，该序列可以是根据Chothia 定义规则确定的序列。
在本申请中，所述抗原结合蛋白可包含H-FR2，所述的H-FR2位于所述HCDR1与所述HCDR2之间，且所述的H-FR2可包含SEQ ID NO:9所示的氨基酸序列：
WMSWVRQAPGKGLEWVANI(SEQ ID NO:9)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述抗原结合蛋白可包含H-FR3，所述的H-FR3位于所述HCDR2与所述HCDR3之间，且所述的H-FR3可包含SEQ ID NO:184所示的氨基酸序列：
KYYX 4DSVKGRFTISRDNAKNSX 22YLQMNSLRAEX 33TAVX 37YCAR(SEQ ID NO:184)；其中，X 4＝V或G；X 22＝L或Q；X 33＝D或E；X 37＝Y或F。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:21所示的H-FR3相比，所述H-FR3可至少包含在选自以下位置处的氨基酸取代：在X 4、X 22、X 33和/或X 37处的氨基酸取代。
在某些情形中，与SEQ ID NO:21所示的抗体的H-FR3相比，所述H-FR3可至少包含在X 4、X 22、X 33和/或X 37处的氨基酸取代，其中，X 4处的氨基酸可被取代为G；X 22处的氨基酸可被取代为Q；X 33处的氨基酸可被取代为E；X 37处的氨基酸可被取代为F。
例如，所述H-FR3可包含SEQ ID NOs:21-24中任一项所示的氨基酸序列。
或者，本申请所述的H-FR3可包含SEQ ID NO:192所示的氨基酸序列：
KYYX 4DSVKGRFTISRDNAKNSLYLQMNSLRAEX 33TAVX 37YCAR(SEQ ID NO:192)；其中，X 4＝V或G；X 33＝D或E；X 37＝Y或F。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:21所示的H-FR3相比，所述H-FR3可至少包含在选自以下位置处的氨基酸取代：在X 4、X 33和/或X 37处的氨基酸取代。
在某些情形中，与SEQ ID NO:21所示的抗体的H-FR3相比，所述H-FR3可至少包含在X 4、X 33和/或X 37处的氨基酸取代，其中，X 4处的氨基酸可被取代为G；X 33处的氨基酸可被取代为E；X 37处的氨基酸可被取代为F。
例如，所述H-FR3可包含SEQ ID NOs:21-22和24中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含H-FR4，所述的H-FR4的N末端与所述HCDR3的C末端相连，且所述的H-FR4可包含SEQ ID NO:38所示的氨基酸序列：
WGQGTMVTVSS(SEQ ID NO:38)。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，所述抗原结合蛋白可包含重链可变区VH，且所述VH可包含SEQ ID NO: 189所示的氨基酸序列：
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQX 54X 5 5SEKYYX 61DSVKGRFTISRDNAKNSX 79YLQMNSLRAEX 90TAVX 94YCARDRX 101VAGAX 106X 107X 108WGQGTMVTVSS(SEQ ID NO:189)；其中，X 54＝D或E；X 55＝G或A；X 61＝V或G；X 79＝L或Q；X 90＝D或E，X 94＝Y或F；X 101＝A或P；X 106＝F或S；X 107＝D或A；X 108＝I或F。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:90所示的VH相比，所述VH可至少包含在选自以下位置处的氨基酸取代：在X 54、X 55、X 61、X 79、X 90、X 94、X 101、X 106、X 107和/或X 108处的氨基酸取代。
在某些情形中，与SEQ ID NO:90所示的VH相比，所述VH可至少包含在X 54、X 55、X 61、X 79、X 90、X 94、X 101、X 106、X 107和/或X 108处的氨基酸取代，其中，X 54处的氨基酸可被取代为E，X 55处的氨基酸可被取代为A，X 61处的氨基酸可被取代为G，X 79处的氨基酸可被取代为Q，X 90处的氨基酸可被取代为E，X 94处的氨基酸可被取代为F，X 101处的氨基酸可被取代为P，X 106处的氨基酸可被取代为S，X 107处的氨基酸可被取代为A，X 108处的氨基酸可被取代为F。
例如，所述VH区可包含SEQ ID NOs:90-95和97-104中任一项所示的氨基酸序列。
在某些情形中，本申请所述VH可包含SEQ ID NO:193所示的氨基酸序列：
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQX 54X 5 5SEKYYX 61DSVKGRFTISRDNAKNSLYLQMNSLRAEX 90TAVX 94YCARDRX 101VAGAX 106X 107X 108WGQGTMVTVSS(SEQ ID NO:193)；其中，X 54＝D或E；X 55＝G或A；X 61＝V或G；X 90＝D或E，X 94＝Y或F；X 101＝A或P；X 106＝F或S；X 107＝D或A；X 108＝I或F。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:90所示的VH相比，所述VH可至少包含在选自以下位置处的氨基酸取代：在X 54、X 55、X 61、X 90、X 94、X 101、X 106、X 107和/或X 108处的氨基酸取代。
在某些情形中，与SEQ ID NO:90所示的VH相比，所述VH可至少包含在X 54、X 55、X 61、X 90、X 94、X 101、X 106、X 107和/或X 108处的氨基酸取代，其中，X 54处的氨基酸可被取代为E，X 55处的氨基酸可被取代为A，X 61处的氨基酸可被取代为G，X 90处的氨基酸可被取代为E，X 94处的氨基酸可被取代为F，X 101处的氨基酸可被取代为P，X 106处的氨基酸可被取代为S，X 107处的氨基酸可被取代为A，X 108处的氨基酸可被取代为F。
例如，所述VH区可包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列。
本申请所述抗原结合蛋白可包含重链恒定区CH，且所述抗体重链恒定区可以包含人IgG恒定区。在某些情形中，所述人IgG恒定区可包含人IgG1恒定区。所述人IgG1恒定区可包括天然和人工合成的IgG1恒定区或其突变体。所述突变可包含以下一个或多个位置的突变：L234，L235，N297或K447。例如，可包含一个、两个或更多位置处的突变。突变可包括氨基酸的缺失、插入或置换。例如，所述人IgG1恒定区可包括的突变如下：1)N297A，2)K447缺失，3)N297A和K447缺失，4)L234A，L235A和K447缺失。例如，所述融合蛋白的人IgG1恒定区可包含SEQ ID NOs:172-175中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1-3。其中，所述LCDR1可包含SEQ ID NO:181所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:182所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:183所示的氨基酸序列。或者，本申请所述的抗原结合蛋白的LCDR1可包含SEQ ID NO:181所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:182所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:191所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:48-51；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:63-64；且所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:77-80。在另一些情形中，本申请所述抗原结合蛋白的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:48-51；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:63-64；且所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:77-79。
例如，本申请所述的抗原结合蛋白可包含与PR000064相同的LCDR1-3。其中LCDR1可包含SEQ ID NO:48所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，且LCDR3可包含SEQ ID NO:77所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000065相同的LCDR1-3，其中LCDR1可包含SEQ ID NO:49所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，LCDR3可包含SEQ ID NO:78所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000066、PR000261、PR000262相同的LCDR1-3。其中LCDR1可包含SEQ ID NO:48所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000067相同的LCDR1-3，其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，LCDR3可包含SEQ ID NO:77所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000068相同的LCDR1-3。其中LCDR1可 包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，且LCDR3可包含SEQ ID NO:79所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000069、PR000263、PR000264相同的LCDR1-3，其中LCDR1可包含SEQ ID NO:51所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，LCDR3可包含SEQ ID NO:77所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000070、PR000265、PR000266相同的LCDR1-3。其中LCDR1可包含SEQ ID NO:51所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000071相同的LCDR1-3，其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，LCDR3可包含SEQ ID NO:80所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000072、PR000267、PR000268相同的LCDR1-3。其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，LCDR3可包含SEQ ID NO:77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L-FR1-4。其中，所述L-FR1可包含SEQ ID NO:185所示的氨基酸序列；所述L-FR2可包含SEQ ID NO:186所示的氨基酸序列；所述L-FR3可包含SEQ ID NO:187所示的氨基酸序列；且所述L-FR4可包含SEQ ID NO:188所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述L-FR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:41-44；所述L-FR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:56-59；所述L-FR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:69-72；且所述L-FR4可包含下述任一项中所示的氨基酸序列：SEQ ID NO:85-86。
例如，本申请所述的抗原结合蛋白可包含与PR000064相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:41所示的氨基酸序列，L-FR2可包含SEQ ID NO:56所示的氨基酸序列，L-FR3可包含SEQ ID NO:69所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000065相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:42所示的氨基酸序列，L-FR2可包含SEQ ID NO:57所示的氨基酸序列，L-FR3可包含SEQ ID NO:69所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000066相同的L-FR1-4。其中L-FR1可包 含SEQ ID NO:43所示的氨基酸序列，L-FR2可包含SEQ ID NO:58所示的氨基酸序列，L-FR3可包含SEQ ID NO:70所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000067相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:42所示的氨基酸序列，L-FR2可包含SEQ ID NO:56所示的氨基酸序列，L-FR3可包含SEQ ID NO:69所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000068相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:42所示的氨基酸序列，L-FR2可包含SEQ ID NO:56所示的氨基酸序列，L-FR3可包含SEQ ID NO:71所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000071相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:42所示的氨基酸序列，L-FR2可包含SEQ ID NO:56所示的氨基酸序列，L-FR3可包含SEQ ID NO:69所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000069、PR000263、PR000264相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:42所示的氨基酸序列，L-FR2可包含SEQ ID NO:59所示的氨基酸序列，L-FR3可包含SEQ ID NO:72所示的氨基酸序列，且L-FR4可包含SEQ ID NO:86所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000070、PR000265、PR000266相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:44所示的氨基酸序列，L-FR2可包含SEQ ID NO:57所示的氨基酸序列，L-FR3可包含SEQ ID NO:69所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000072、PR000267、PR000268相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:42所示的氨基酸序列，L-FR2可包含SEQ ID NO:57所示的氨基酸序列，L-FR3可包含SEQ ID NO:69所示的氨基酸序列，且L-FR4可包含SEQ ID NO:85所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000261、PR000262相同的L-FR1-4。其中L-FR1可包含SEQ ID NO:43所示的氨基酸序列，L-FR2可包含SEQ ID NO:58所示的氨基酸序列，L-FR3可包含SEQ ID NO:69所示的氨基酸序列，且L-FR4可包含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HCDR1-3。其中，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:179所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:180所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述HCDR1可包含下述所示的氨基酸序列：SEQ ID NO:5；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:14、16和17；且所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:29-33。
例如，本申请所述的抗原结合蛋白可包含与PR000064、PR000065、PR000067、PR000070相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000066相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:30所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000068相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:31所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000069相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:32所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000071、PR000072相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000261相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:30所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000262同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:30所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000263相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:32所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000264同的HCDR1-3。其中HCDR1可 包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:32所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000265相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000266相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000267相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000268相同的HCDR1-3。其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:33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H-FR1-4。其中，所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:184所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:38所示的氨基酸序列。或者，所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列；所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列；所述H-FR3可包含SEQ ID NO:192所示的氨基酸序列；且所述H-FR4可包含SEQ ID NO:38所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述H-FR1可包含下述所示的氨基酸序列：SEQ ID NO:1；所述H-FR2可包含下述所示的氨基酸序列：SEQ ID NO:9；所述H-FR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:21-24；且所述H-FR4可包含下述任一项中所示的氨基酸序列：SEQ ID NO:38。在另一些情形中，本申请所述抗原结合蛋白的所述H-FR1可包含下述所示的氨基酸序列：SEQ ID NO:1；所述H-FR2可包含下述所示的氨基酸序列：SEQ ID NO:9；所述H-FR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:21-22和24；且所述H-FR4可包含下述任一项中所示的氨基酸序列：SEQ ID NO:38。
例如，本申请所述的抗原结合蛋白可包含H-FR1-4，其中所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:21所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:38所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含H-FR1-4，其中所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:22所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:38所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含H-FR1-4，其中所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:23所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:38所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含H-FR1-4，其中所述H-FR1可包含SEQ ID NO:1所示的氨基酸序列，所述H-FR2可包含SEQ ID NO:9所示的氨基酸序列，所述H-FR3可包含SEQ ID NO:24所示的氨基酸序列，且所述H-FR4可包含SEQ ID NO:38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LCDR1-3和HCDR1-3。其中，所述LCDR1可包含SEQ ID NO:181所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:182所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:183所示的氨基酸序列；所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:179所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:180所示的氨基酸序列。或者，所述LCDR1可包含SEQ ID NO:181所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:182所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:191所示的氨基酸序列；所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:179所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:180所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:48-51；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:63-64；且所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:77-80；所述HCDR1可包含下述所示的氨基酸序列：SEQ ID NO:5；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:14、16和17；且所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:29-33。在另一些情形中，本申请所述抗原结合蛋白的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:48-51；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:63-64；且所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:77-79；所述HCDR1可包含下述所示的氨基酸序列：SEQ ID NO:5；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:14、16和17；且所述 HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:29-33。
例如，本申请所述的抗原结合蛋白可包含与PR000064相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:48所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，且LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000065相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:49所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，LCDR3可包含SEQ ID NO:78所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000066相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:48所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:30所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000067相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列；LCDR2可包含SEQ ID NO:63所示的氨基酸序列；LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000068相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，且LCDR3可包含SEQ ID NO:79所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:31所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000069相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:51所示的氨基酸序列；LCDR2可包含SEQ ID NO:63所示的氨基酸序列；LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:32所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000070相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:51所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000071相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，且LCDR3可包含SEQ ID NO:80所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000072相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:14所示的氨基酸序列，且HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000261相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:48所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:30所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000262相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:48所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:30所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000263相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:51所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:32所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000264相同的LCDR1-3和HCDR1-3。其 中LCDR1可包含SEQ ID NO:51所示的氨基酸序列，LCDR2可包含SEQ ID NO:63所示的氨基酸序列，LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:32所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000265和PR000416相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:51所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000266相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:51所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，且LCDR3可包含SEQ ID NO:78所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:29所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000267相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:16所示的氨基酸序列，且HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000268相同的LCDR1-3和HCDR1-3。其中LCDR1可包含SEQ ID NO:50所示的氨基酸序列，LCDR2可包含SEQ ID NO:64所示的氨基酸序列，LCDR3可包含SEQ ID NO:77所示的氨基酸序列，其中HCDR1可包含SEQ ID NO:5所示的氨基酸序列，HCDR2可包含SEQ ID NO:17所示的氨基酸序列，且HCDR3可包含SEQ ID NO:33所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含轻链可变区VL和重链可变区VH。所述VL可包含SEQ ID NO:194所示的氨基酸序列，且所述VH可包含SEQ ID NO:193所示的氨基酸序列。或者，所述VL可包含SEQ ID NO:190所示的氨基酸序列，且所述VH可包含SEQ ID NO:189所示的氨基酸序列。
在某些情形中，所述抗原结合蛋白的所述VL可包含SEQ ID NOs:108-116和118中任一项所示的氨基酸序列，且所述VH可包含SEQ ID NOs:90-95、97-104中任一项所示的氨基酸序列。在另一些情形中，所述抗原结合蛋白的所述VL可包含SEQ ID NOs:108-114、116和 118中任一项所示的氨基酸序列，且所述VH可包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000064相同的VL和VH，其中，所述VL可包含SEQ ID NO:108所示的氨基酸序列，且所述VH可包含SEQ ID NO:90所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000065相同的VL和VH，其中，所述VL可包含SEQ ID NO:109所示的氨基酸序列，且所述VH可包含SEQ ID NO:90所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000066相同的VL和VH，其中，所述VL可包含SEQ ID NO:110所示的氨基酸序列，且所述VH可包含SEQ ID NO:91所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000067相同的VL和VH，其中，所述VL可包含SEQ ID NO:111所示的氨基酸序列，且所述VH可包含SEQ ID NO:90所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000068相同的VL和VH，其中，所述VL可包含SEQ ID NO:112所示的氨基酸序列，且所述VH可包含SEQ ID NO:92所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000069相同的VL和VH，其中，所述VL可包含SEQ ID NO:113所示的氨基酸序列，且所述VH可包含SEQ ID NO:93所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000070相同的VL和VH，其中，所述VL可包含SEQ ID NO:114所示的氨基酸序列，且所述VH可包含SEQ ID NO:90所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000071相同的VL和VH，其中，所述VL可包含SEQ ID NO:115所示的氨基酸序列，且所述VH可包含SEQ ID NO:94所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000072相同的VL和VH，其中，所述VL可包含SEQ ID NO:116所示的氨基酸序列，且所述VH可包含SEQ ID NO:95所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000261相同的VL和VH，其中，所述VL可包含SEQ ID NO:118所示的氨基酸序列，且所述VH可包含SEQ ID NO:97所示的氨基酸 序列。
例如，本申请所述的抗原结合蛋白可包含与PR000262相同的VL和VH，其中，所述VL可包含SEQ ID NO:118所示的氨基酸序列，且所述VH可包含SEQ ID NO:98所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000263相同的VL和VH，其中，所述VL可包含SEQ ID NO:113所示的氨基酸序列，且所述VH可包含SEQ ID NO:99所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000264相同的VL和VH，其中，所述VL可包含SEQ ID NO:113所示的氨基酸序列，且所述VH可包含SEQ ID NO:100所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000265或PR000416相同的VL和VH，其中，所述VL可包含SEQ ID NO:114所示的氨基酸序列，且所述VH可包含SEQ ID NO:101所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000266相同的VL和VH，其中，所述VL可包含SEQ ID NO:114所示的氨基酸序列，且所述VH可包含SEQ ID NO:102所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000267相同的VL和VH，其中，所述VL可包含SEQ ID NO:116所示的氨基酸序列，且所述VH可包含SEQ ID NO:103所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000268相同的VL和VH，其中，所述VL可包含SEQ ID NO:116所示的氨基酸序列，且所述VH可包含SEQ ID NO:104所示的氨基酸序列。
在某些情形中，所述抗原结合蛋白可包含轻链恒定区和重链恒定区，其中，所述轻链恒定区可包含SEQ ID NO:170所示的氨基酸序列，且所述重链恒定区可包含SEQ ID NOs:172-175中任一项所示的氨基酸序列。
在某些情形中，所述抗原结合蛋白可包含抗体轻链LC和抗体重链HC，其中，所述抗体轻链LC可包含下述任一项中所示的氨基酸序列：SEQ ID NO:150-158和160-163，且所述抗体重链HC可包含下述任一项中所示的氨基酸序列：SEQ ID NO:122-127和129-137。
在某些情形中，所述抗原结合蛋白可包含抗体轻链LC和抗体重链HC，其中，所述抗体轻链LC可包含下述任一项中所示的氨基酸序列：SEQ ID NO:150-156、158和160-163，且所述抗体重链HC可包含下述任一项中所示的氨基酸序列：SEQ ID NO:122-125、127和129- 137。
例如，本申请所述的抗原结合蛋白可包含与PR000064相同的LC和HC，其中，所述LC可包含SEQ ID NO:150所示的氨基酸序列，且所述HC可包含SEQ ID NO:122所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000065相同的LC和HC，其中，所述LC可包含SEQ ID NO:151所示的氨基酸序列，且所述HC可包含SEQ ID NO:122所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000066相同的LC和HC，其中，所述LC可包含SEQ ID NO:152所示的氨基酸序列，且所述HC可包含SEQ ID NO:123所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000067相同的LC和HC，其中，所述LC可包含SEQ ID NO:153所示的氨基酸序列，且所述HC可包含SEQ ID NO:122所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000068相同的LC和HC，其中，所述LC可包含SEQ ID NO:154所示的氨基酸序列，且所述HC可包含SEQ ID NO:124所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000069相同的LC和HC，其中，所述LC可包含SEQ ID NO:155所示的氨基酸序列，且所述HC可包含SEQ ID NO:125所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000070相同的LC和HC，其中，所述LC可包含SEQ ID NO:156所示的氨基酸序列，且所述HC可包含SEQ ID NO:122所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000071相同的LC和HC，其中，所述LC可包含SEQ ID NO:157所示的氨基酸序列，且所述HC可包含SEQ ID NO:126所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000072相同的LC和HC，其中，所述LC可包含SEQ ID NO:158所示的氨基酸序列，且所述HC可包含SEQ ID NO:127所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000261相同的LC和HC，其中，所述LC可包含SEQ ID NO:160所示的氨基酸序列，且所述HC可包含SEQ ID NO:129所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000262相同的LC和HC，其中，所述LC可包含SEQ ID NO:160所示的氨基酸序列，且所述HC可包含SEQ ID NO:130所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000263相同的LC和HC，其中，所述LC可包含SEQ ID NO:161所示的氨基酸序列，且所述HC可包含SEQ ID NO:131所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000264相同的LC和HC，其中，所述LC可包含SEQ ID NO:161所示的氨基酸序列，且所述HC可包含SEQ ID NO:132所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000265相同的LC和HC，其中，所述LC可包含SEQ ID NO:162所示的氨基酸序列，且所述HC可包含SEQ ID NO:133所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000266相同的LC和HC，其中，所述LC可包含SEQ ID NO:162所示的氨基酸序列，且所述HC可包含SEQ ID NO:134所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000267相同的LC和HC，其中，所述LC可包含SEQ ID NO:163所示的氨基酸序列，且所述HC可包含SEQ ID NO:135所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000268相同的LC和HC，其中，所述LC可包含SEQ ID NO:163所示的氨基酸序列，且所述HC可包含SEQ ID NO:136所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白可包含与PR000416相同的LC和HC，其中，所述LC可包含SEQ ID NO:162所示的氨基酸序列，且所述HC可包含SEQ ID NO:137所示的氨基酸序列。
特定抗PD-L1抗体
本申请所述的分离的抗原结合蛋白，其可与特定抗PD-L1抗体竞争结合PD-L1。在本申请中，所述特定抗PD-L1抗体可包含LCDR1-3。其中，所述LCDR1可包含SEQ ID NO:181所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:182所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:191所示的氨基酸序列。在某些情形中，本申请所述特定抗PD-L1抗体的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:48-51；所述LCDR2可包 含下述任一项中所示的氨基酸序列：SEQ ID NOs:63-64；且所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:77-79。
在本申请中，所述特定抗PD-L1抗体可包含HCDR1-3。其中，所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:179所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:180所示的氨基酸序列。在某些情形中，本申请所述特定抗PD-L1抗体的所述HCDR1可包含下述所示的氨基酸序列：SEQ ID NO:5；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:14、16和17；且所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:29-33。
在本申请中，所述特定抗PD-L1抗体可包含LCDR1-3和HCDR1-3。其中，所述LCDR1可包含SEQ ID NO:181所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:182所示的氨基酸序列；且所述LCDR3可包含SEQ ID NO:191所示的氨基酸序列；所述HCDR1可包含SEQ ID NO:5所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:179所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:180所示的氨基酸序列。在某些情形中，本申请所述特定抗PD-L1抗体的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:48-51；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:63-64；且所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:77-79；所述HCDR1可包含下述所示的氨基酸序列：SEQ ID NO:5；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:14、16和17；且所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NOs:29-33。
在本申请中，所述特定抗PD-L1抗体可包含轻链可变区VL和重链可变区VH。所述VL可包含SEQ ID NO:194所示的氨基酸序列，且所述VH可包含SEQ ID NO:193所示的氨基酸序列。在某些情形中，所述特定抗PD-L1抗体的所述VL可包含SEQ ID NOs:108-114、116和118中任一项所示的氨基酸序列，且所述VH可包含SEQ ID NOs:90-93、95、97-104中任一项所示的氨基酸序列。
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-L1抗原结合蛋白或融合蛋白的物理/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或融合蛋白的抗原结合活性。
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-L1。所述灵长类动物PD-L1抗原结合蛋白对PD-L1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-L1抗原结合蛋白对PD-L1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-L1。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-L1抗原结合蛋白与PD-L1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-L1的宿主细胞(如CHOK1细胞)，所述PD-L1抗原结合蛋白与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0.01nM至约10nM之间，约0.05nM至约5nM之间，约0.05nM至约1nM之间。又例如，在ELISA中，使用人PD-L1抗原蛋白，所述PD-L1抗原结合蛋白与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0.001nM至约1nM之间，约0.01nM至约0.5nM之间，约0.01nM至约0.1nM之间。
在另一个方面，竞争测定可用来鉴别与本申请所述的任一种抗原结合蛋白竞争结合到PD-Ll的抗体。在某些情形中，这种竞争性抗体结合到与由本申请所述的任一种抗原结合蛋白所结合的重叠表位(例如，线性或构象表位)。用于对抗原结合蛋白所结合到的表位进行作图的示例性方法包括但不限于X射线共晶体和低温电子显微镜法(cryo-EM)、基于阵列的寡肽扫描法、定点诱变作图、交联耦合质谱法和生物膜干涉法。
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PD-L1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-1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。又例如，首先将PD-L1抗原蛋白包被在板上，将递减量的未标记的所述抗原结合蛋白和生物素标记的PD-1蛋白混合后，共同孵育。然后，使用ELISA分析细胞，以证实所述抗原结合蛋白可以阻断PD-1和PD-L1的结合。
在另一个方面，本申请所述的抗原结合蛋白能够阻断CD80与PD-L1的结合。在某些情形中，所述的抗原结合蛋白阻断CD80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PD-L1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CD80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CD80与PD-L1结合。又例如，首先将PD-L1抗原蛋白包被在板上，将递减量的未标记的所述抗原结合蛋白和生物素标记的CD80蛋白混合后，共同孵育。然后，使用ELISA分析细胞，以证实所述抗原结合蛋白可以阻断CD80和PD-L1的结合。
本申请所述抗原结合蛋白能够刺激免疫细胞中IFN-γ和/或IL2的分泌。所述免疫细胞可包括淋巴细胞，例如B细胞、T细胞，天然杀伤细胞，髓样细胞，例如单核细胞、巨噬细胞、肥大细胞、嗜碱细胞和粒细胞。可用本领域任何技术人员已知的方法测定免疫细胞中细胞因子的分泌，例如，通过酶联免疫法(ELISA)定量测定免疫细胞(例如T细胞)增殖情况或由免疫细胞产生的细胞因子(例如由T细胞产生的IFN-γ或IL-2)。
对照抗体
本申请所述抗原结合蛋白与对照抗体相比，能够结合与源自灵长类动物的PD-L1的不完全重叠的表位。在某些情形中，所述对照抗体可以是抗PD-L1抗体avelumab(阿维鲁单抗)，即，本申请所述抗体PR001598。PR001598可包含轻链可变区和重链可变区，所述对照抗体的轻链可变区可包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:53所示的氨基酸序列，所述LCDR2包含SEQ ID NO:66所示的氨基酸序列，所述LCDR3包含SEQ ID NO:82所示的氨基酸序列；所述对照抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2包含SEQ ID NO:18所示的氨基酸序列，所述HCDR3包含SEQ ID NO:35所示的氨基酸序列；所述对照抗体的轻链可变区包含SEQ ID NO:119所示的氨基酸序列，所述对照抗体的重链可变区包含SEQ ID NO:105所示的氨基酸序列。
在另一些情形中，所述对照抗体可以是抗PD-L1抗体atezolizumab(阿特珠单抗)，即，本申请所述抗体PR000151。PR000151可包含轻链可变区和重链可变区，所述对照抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:52所示的氨基酸序列，所述LCDR2包含SEQ ID NO:65所示的氨基酸序列，所述LCDR3包含SEQ ID NO:81所示的氨基酸序列；所述对照抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:6所示的氨基酸序列，所述HCDR2包含SEQ ID NO:15所示的氨基酸序列，所述HCDR3包含SEQ ID NO:34所示的氨基酸序列；所述对照抗体的轻链可变 区包含SEQ ID NO:117所示的氨基酸序列，所述对照抗体的重链可变区包含SEQ ID NO:96所示的氨基酸序列。
在另一些情形中，所述对照抗体可以是Hengrui融合蛋白9，即，本申请所述抗体PR002466。PR002466可包含轻链可变区和重链可变区，所述对照抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:55所示的氨基酸序列；所述LCDR2包含SEQ ID NO:68所示的氨基酸序列；所述LCDR3包含SEQ ID NO:84所示的氨基酸序列，所述对照抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:8所示的氨基酸序列；所述HCDR2包含SEQ ID NO:20所示的氨基酸序列；所述HCDR3包含SEQ ID NO:37所示的氨基酸序列；所述对照抗体的轻链可变区包含SEQ ID NO:121所示的氨基酸序列，所述对照抗体的重链可变区包含SEQ ID NO:107所示的氨基酸序列。
本领域测定表位的方法包括但不限于合成肽法，免疫信息学预测法，多肽活性测定，表位肽扫描技术，蛋白质切割法，噬菌体展示技术，X射线衍射与核磁共振分析，以及利用计算机软件进行表位预测。在某些实施方式中，可以使用基于生物膜干涉法(BLI)Octet分子相互作用分析平台进行本申请所述抗原结合蛋白与所述对照抗体对抗原表位的竞争性结合。例如，将第一种抗体与抗原混合，再加入第二种抗体，利用ForteBio Octet测定第二种抗体对第一种抗体的竞争性抑制率，当竞争性抑制率低于85％时(例如，低于84％、低于83％、低于80％或更低)，可以说第一抗体与第二种抗体结合抗原的表位不完全重叠。
融合蛋白
另一方面，本申请提供了一种融合蛋白，其可包含：a)人TGFBRII或其片段；以及b)本申请上文所述的抗原结合蛋白。
本申请所述融合蛋白的所述人TGFBRII或其片段可包含人TGFBRII的胞外结构域。在某些情形中，所述人TGFBRII或其片段可包含SEQ ID NOs:176-178中任一项所示的氨基酸序列。
在本申请中，所述融合蛋白可包含本申请所述的分离的抗原结合蛋白。所述分离的抗原结合蛋白可包含轻链可变区VL和重链可变区VH。在某些情形中，所述VL可包含SEQ ID NO:190所示的氨基酸序列，且所述VH可包含SEQ ID NO:189所示的氨基酸序列。
所述分离的抗原结合蛋白还可包含抗体重链恒定区，且所述重链恒定区可以包含人IgG恒定区。在某些情形中，所述人IgG恒定区可包含人IgG1恒定区。所述人IgG1恒定区可包括天然和人工合成的IgG1恒定区或其突变体。所述突变可包含以下一个或多个位置的突变：L234，L235，N297或K447。例如，可包含一个、两个或更多位置处的突变。突变可包括氨 基酸的缺失、插入或置换。例如，所述人IgG1恒定区可包括的突变如下：1)N297A，2)K447缺失，3)N297A和K447缺失，4)L234A，L235A和K447缺失。例如，所述融合蛋白的人IgG1恒定区可包含SEQ ID NOs:172-175中任一项所示的氨基酸序列。
本申请所述融合蛋白的分离的抗原结合蛋白可包含抗体重链或其片段。在某些情形中，所述抗体重链或其片段可包含下述任一项中所示的氨基酸序列或其一部分：SEQ ID NOs:122-127和129-137。例如，所述抗体重链或其片段可包含下述任一项中所示的氨基酸序列或其一部分：SEQ ID NOs:122和133-137。本申请所述融合蛋白的分离的抗原结合蛋白可包含抗体轻链或其片段。在某些情形中，所述抗体轻链或其片段可包含下述任一项中所示的氨基酸序列或其一部分：SEQ ID NOs:150-158和160-163。例如，所述抗体轻链或其片段可包含下述任一项中所示的氨基酸序列或其一部分：SEQ ID NOs:156和162-163。所述融合蛋白的所述重链或其片段与所述轻链或其片段组合后可形成特异性结合PD-L1的抗原结合部分。
本申请所述融合蛋白包含的所述抗原结合蛋白的所述抗体重链或其片段与所述人TGFBRII或其片段框内融合而形成融合多肽。在某些情形中，所述抗体重链或其片段的N端可以直接或间接与所述人TGFBRII或其片段的C端连接。在某些情形中，所述抗体重链或其片段的C端可以直接或间接与所述人TGFBRII或其片段的N端连接。例如，所述抗体重链或其片段的C端可以直接与所述人TGFBRII或其片段的N端连接。在另一些情形中，所述抗体重链或其片段可以通过连接子与所述人TGFBRII或其片段相连，例如，所述肽连接子可包含SEQ ID NOs:167-169中任一项所示的氨基酸序列。
在本申请中，所述融合蛋白可包含第一多肽和第二多肽。所述第一多肽可包含所述分离的抗原结合蛋白的重链或其片段以及所述人TGFBRII或其片段。在某些情形中，所述第一多肽从N端至C端可以包括所述抗原结合片段的重链或其片段，所述肽连接子，以及所述人TGFBRII或其片段。例如，所述第一多肽可包含SEQ ID NOs:138-139、142-143和145-148中任一项所示的氨基酸序列。所述第二多肽可包含所述分离的抗原结合蛋白的轻链或其片段，例如，所述第二多肽可包含SEQ ID NOs:162-163中任一项所示的氨基酸序列。
例如，所述融合蛋白的所述第一多肽可包含SEQ ID NO:138所示的氨基酸序列，且所述第二多肽可包含SEQ ID NO:162所示的氨基酸序列，例如，所述融合蛋白可包含与PR001487相同的第一多肽和第二多肽；
所述第一多肽可包含SEQ ID NO:139所示的氨基酸序列，且所述第二多肽可包含SEQ ID NO:162所示的氨基酸序列，例如，所述融合蛋白可包含与PR001488相同的第一多肽和第二多肽；
所述第一多肽可包含SEQ ID NO:142所示的氨基酸序列，且所述第二多肽可包含SEQ  ID NO:162所示的氨基酸序列，例如，所述融合蛋白可包含与PR001901相同的第一多肽和第二多肽；
所述第一多肽可包含SEQ ID NO:143所示的氨基酸序列，且所述第二多肽可包含SEQ ID NO:162所示的氨基酸序列，例如，所述融合蛋白可包含与PR001902相同的第一多肽和第二多肽；
所述第一多肽可包含SEQ ID NO:145所示的氨基酸序列，且所述第二多肽可包含SEQ ID NO:163所示的氨基酸序列，例如，所述融合蛋白可包含与PR002247相同的第一多肽和第二多肽
所述第一多肽可包含SEQ ID NO:146所示的氨基酸序列，且所述第二多肽可包含SEQ ID NO:163所示的氨基酸序列，例如，所述融合蛋白可包含与PR002248相同的第一多肽和第二多肽；
所述第一多肽可包含SEQ ID NO:147所示的氨基酸序列，且所述第二多肽可包含SEQ ID NO:162所示的氨基酸序列，例如，所述融合蛋白可包含与PR002249相同的第一多肽和第二多肽；
所述第一多肽可包含SEQ ID NO:148所示的氨基酸序列，且所述第二多肽可包含SEQ ID NO:162所示的氨基酸序列，例如，所述融合蛋白可包含与PR002251相同的第一多肽和第二多肽。
本申请的所述的融合蛋白可包含两条所述第一多肽以及两条所述第二多肽。
在本申请中，所述抗体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。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。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。
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，具体操作可参见Sambrook等人，Molecular Cloning，A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology，Greene Publishing and Wiley-Interscience，New York N.Y.，1993。
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。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录 表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。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。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
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。
在某些情形中，所述方法还可包括分离和/或纯化所述抗体或其抗原结合片段的步骤。例如，可以采用蛋白G-琼脂糖或蛋白A-琼脂糖进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述的抗体或其抗原结合片段。
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和/或所述融合蛋白，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佐剂。
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，并且可包括缓冲剂，诸如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(诸如十八烷基二甲基苄基氯化铵、氯化六烃季铵(hexamethonium chloride)、氯化苯二甲羟铵(benzalkonium chloride)、氯化苄甲乙氧铵(benzethonium chloride)、苯酚、丁醇或苄醇；对羟苯甲酸烷酯，诸如对羟苯甲酸甲酯或丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇和间-甲酚)； 低分子量(小于约10个残基)多肽；蛋白质，诸如血清白蛋白、凝胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯基吡咯烷酮；氨基酸，诸如甘氨酸、谷酰氨酸、天冬酰氨酸、组氨酸、精氨酸或赖氨酸；单糖、双糖以及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，诸如EDTA；糖，诸如蔗糖、甘露醇、海藻糖或山梨醇；成盐反离子，诸如钠离子；金属络合物(例如，Zn-蛋白质络合物)；和/或非离子表面活性剂，诸如TWEEN TM、PLURONICS TM或聚乙二醇(PEG)。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，所述肿瘤或癌症包含结直肠肿瘤或癌症。在某些实施方式中，所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。本申请还提供了以下描述的检测生物样品中PD-L1表达的方法。在某些情形中，所述方法包括使生物样品与本申请所述的抗原结合蛋白和/或融合蛋白在容许所述抗原结合蛋白和/或融合蛋白结合PD-L1的条件下接触，和检测在所述抗原结合蛋白和/或融合蛋白与PD-L1之间是否形成复合物。例如，所述肿瘤或癌症为与非肿瘤或癌症样品相比，PD-L1表达升高的肿瘤或癌症。此类方法可以是体外或体内方法。本申请所述抗原结合蛋白和/或融合蛋白可用于例如免疫测定中，所述免疫测定包括例如免疫组织化学(IHC)、免疫荧光(IF)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FAGS)和酶联免疫吸附测定(ELISA)。在某些情形中，例如当PD-L1为用于选择患者的生物标记时，所述抗原结合蛋白和/或融合蛋白用来选择适于用本申请所述抗原结合蛋白和/或融合蛋白进行的疗法的受试者。本申请还提供了所述抗原结合蛋白和/或融合蛋白在诊断患有病症(例如，癌症或免疫功能失调)的受试者的方法中的用途，所述方法包括：通过使样品与本发明的所述抗原结合蛋白和/或融合蛋白接触并检测结合的所述抗原结合蛋白和/或融合蛋白的存在来确定获自受试者的样品中PD-L1的存在或表达水平。
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。所述方法可以是离体或体外方法。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-L1与CD80结合的方法，包括施用本申请所述的抗原结合蛋白。所述方法可以是离体或体外方法。在某些情形中，所述方法包括使生物样品与本申请所述的抗原结合蛋白和/或CD80在容许所述抗原结合蛋白和/或CD80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CD80与PD-L1之间是否形成复合物。
另一方面，本申请提供了抑制TGFB与TGFBRII结合的方法，包括施用本申请所述的融合蛋白。所述方法可以是离体或体外方法。在某些情形中，所述方法包括使生物样品与本申请所述的融合蛋白和/或表达TGFBRII的报告基因细胞在容许所述融合蛋白和/或表达TGFBRII的报告基因细胞结合TGFB的条件下接触，检测在所述融合蛋白是否抑制了TGFB结合TGFBRII诱导的Smad信号通路。
在一些情况下，生物样品包括组织或细胞样品。例如，生物样品可包括来自正常或癌症患者的细胞或组织。在某些情况下，组织或细胞样品的来源可为如来自新鲜、冷冻和/或防腐器官或组织样品或活检物或吸出物的固体组织；血液或任何血液成分；体液，诸如脑脊髓液、羊水、腹膜液或间质液；来自受试者妊娠或发育的任何时间的细胞。在另一些情形中，所述生物样品获自体外组织或细胞培养物。本申请中的生物样品的实例包括但不限于肿瘤活检、循环肿瘤细胞、血清或血浆、循环血浆蛋白、腹水、来源于肿瘤的或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定石蜡包埋的肿瘤样品或冷冻的肿瘤样品。
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-L1的存在或表达水平。在一些情况下，所述样品可选自由以下组成的组：组织样品、全血样品、血清样品和血浆样品。在一些情况下，所述组织样品可以为肿瘤样品。在一些情况下，所述肿瘤样品可包含肿瘤浸润性免疫细胞、肿瘤细胞、基质细胞及其任何组合。
另一方面，本申请提供了治疗受试者中的癌症、抑制受试者中肿瘤生长和/或抑制肿瘤细胞增殖的方法，包括向有需要的受试者或所述肿瘤细胞施用本申请所述的抗原结合片段和/或所述融合蛋白、所述的分子核酸、所述的载体、所述的宿主细胞和/或所述的药物组合物。可 通过任何合适的方法来施用，所述合适的方法包括例如：以静脉内方式、以肌内方式、以皮下方式、以皮内方式、以经皮方式、以动脉内方式、以腹膜内方式、以损伤内方式、以颅内方式、以关节内方式、以前列腺内方式、以胸膜内方式、以气管内方式、以鞘内方式、以鼻内方式、以阴道内方式、以直肠内方式、以局部方式、以肿瘤内方式、以腹膜方式、以结膜下方式、以囊内方式、以粘膜方式、以心包内方式、以脐内方式、以眼内方式、以眶内方式、以口服方式、以局部方式、以透皮方式、以玻璃体内方式(例如，通过玻璃体内注射)、通过滴眼剂、通过吸入、通过注射、通过植入、通过输注、通过连续输注、通过直接沐浴靶细胞的局部灌注、通过导管、通过灌洗、以乳膏形式或以脂质组合物形式。用于本文描述的方法中的组合物还可以全身方式或以局部方式施用。施用方法可以根据各种因素(例如，所施用的化合物或组合物以及所治疗的病状、疾病或病症的严重性)而变化。在某些实施方案中，以静脉内方式、以肌内方式、以皮下方式、以局部方式、以口服方式、以透皮方式、以腹膜内方式、以眶内方式、通过植入、通过吸入、以鞘内方式、以心室内方式或以鼻内方式施用抗癌疗法(例如，抗PD-L1抗体)。部分地根据施用是否为短暂的或长期的，给药可通过任何适合的途径进行，例如通过注射，诸如静脉内或皮下注射。本文涵盖各种给药排程，包括但不限于单次施用或各种时间点内的多次施用、推注施用和脉冲输注。
本文所述的抗原结合蛋白或药物组合物可以符合良好医疗实践的方式配制、给药和施用。在此情形下的考虑因素包括所治疗的特定病症、所治疗的特定哺乳动物、单个患者的临床病状、病症的病因、药剂递送部位、施用方法、施用排程和医学从业者已知的其他因素。治疗剂(例如，抗PD-L1抗体)无需但任选地与一种或多种当前用来预防或治疗所考虑的病症的药剂一起配制和/或同时施用。此类其他药剂的有效量取决于制剂中存在的治疗剂(例如，抗PD-L1抗体)的量、病症或治疗的类型以及以上论述的其他因素。这些药剂通常可以凭经验/临床上确定为适当的任何剂量且通过凭经验/临床上确定为适当的任何途径加以使用。与单个治疗相比，可减少组合治疗中施用的抗体的剂量。通过常规技术易于监测此疗法的进展。
嵌合抗原受体
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。在某些实施方式中，其可以包含能够结合抗原的胞外域(胞外结合结构域)、铰链结构域、跨膜结构域(跨膜区)和使胞质信号传到结构域的多肽(即胞内信号域)。铰链结构域可以被认为是用于向细胞外抗原结合区提供柔性的一部分。胞内信号域指经由确定的信号传导途径通过产生第二信使而将信息传递到细胞内以调节细胞活性的蛋白质、或通过相应于此类信使而作为效应子发挥作用的蛋白质，产生可以促进CAR的细胞(例如CAR-T细胞))的免疫效应子功能的信号。胞内信号域可以包含信号传导结构域，还 可以包括源自共刺激分子的共刺激胞内结构域。例如，共刺激分子可以选自4-1BB，CD27，ICOS和/或CD28。另一方面，本申请提供了基因修饰的细胞，其可包含所述的嵌合抗原受体。在某些实施方式中，所述基因修饰的细胞可包括真核细胞。在某些实施方式中，所述基因修饰的细胞可包括分离的人细胞。在某些实施方式中，所述基因修饰的细胞可包括免疫细胞如T细胞，或NK细胞。
抗体药物偶联物
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。抗体药物偶联物的靶向性可以来自其中抗体部分(antibody)，毒性大部分可以来自小分子化药毒物部分(payload)，抗体部分也可以自带毒性(ADCC与CDC)。
试剂盒
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。
在某些情形中，所述试剂盒还可以包括施用本申请所述抗原结合蛋白，嵌合抗原受体，基因修饰的细胞，抗体药物偶联物或药物组合物的装置；例如，所述装置取决于内容物的施用方式。在某些情形中，所述试剂盒可包括包装插页，其包括关于试剂盒中的抗原结合蛋白、药物组合物和剂型的信息。通常，此类信息帮助患者和医师有效且安全地使用封装的抗原结合蛋白、药物组合物和剂型。用于此类试剂盒中的容器通常可包含至少一个小瓶、试管、烧瓶、瓶、注射器或其它适合容器，检测和/或治疗组合物中的一种或多种可置于其中，且优选适当等分。在还提供第二治疗剂的情况下，试剂盒也可含有第二不同容器，其中可放置该第二检测和/或治疗组合物。或者，多种化合物可制备成单一药物组合物，且可包装在单一容器装置诸如小瓶、烧瓶、注射器、瓶或其它适合单一容器中。
给药装置
另一方面，本申请提供了一种给药装置(例如塑料或小瓶，例如空心销或注射器圆筒)，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。该设备可以通过肠胃外途径(例如肌肉内部，皮下或静脉内)将一种物质引入患者体内。例如，注射设备可以是注射器(例如预先填充的具有本申请所述抗原结合蛋白或其药物组合物的注射器，例如自动注射器)，其可以包括用于容纳要注射的流体(例如本申请所述抗原结合蛋白或其药物组合物)的针筒 和针(可以用于刺穿皮肤和/或血管)。给药方式可改变。给药途径可以包括口服，肌肉注射，皮下注射，经直肠给药等。
另一方面，本申请提供了以下实施方案：
1.分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:193所示的氨基酸序列。
2.根据实施方案1所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列。
3.根据实施方案1-2中任一项所述的分离的抗原结合蛋白，其包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:194所示的氨基酸序列。
4.根据实施方案3所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NOs:108-114、116和118中任一项所示的氨基酸序列。
5.根据实施方案1-4中任一项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。
6.根据实施方案5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’，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。
7.根据实施方案5-6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。
8.根据实施方案1-7中任一项所述的分离的抗原结合蛋白，其与特定抗PD-L1抗体竞争结合PD-L1，其中所述特定抗PD-L1抗体包含轻链可变区和重链可变区，所述特定抗PD-L1抗体的轻链可变区包含LCDR1、LCDR2和LCDR3，所述LCDR1包含SEQ ID NO:181所示的氨基酸序列，所述LCDR2包含SEQ ID NO:182所示的氨基酸序列，所述LCDR3包含SEQ ID NO:191所示的氨基酸序列；所述特定抗PD-L1抗体的重链可变区包含HCDR1、HCDR2和HCDR3，所述HCDR1包含SEQ ID NO:5所示的氨基酸序列，所述HCDR2包含SEQ ID NO:179所示的氨基酸序列，所述HCDR3包含SEQ ID NO:180所示的氨基酸序列。
9.根据实施方案8所述的分离的抗原结合蛋白，其中所述特定抗PD-L1抗体的轻链可变区包含SEQ ID NO:194所示的氨基酸序列，所述特定抗PD-L1抗体的重链可变区包含SEQ ID NO:193所示的氨基酸序列。
10.根据实施方案1-9中任一项所述的分离的抗原结合蛋白，其中所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:5所示的氨基酸序列。
11.根据实施方案10所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:179所示的氨基酸序列。
12.根据实施方案10-11所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NOs:14、16和17中任一项所示的氨基酸序列。
13.根据实施方案10-12中任一项所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:180所示的氨基酸序列。
14.根据实施方案10-13所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NOs:29-33中任一项所示的氨基酸序列。
15.根据实施方案1-14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-FR1，H-FR2，H-FR3，和H-FR4。
16.根据实施方案15所述的分离的抗原结合蛋白，其中所述H-FR1的C末端与所述HCDR1的N末端相连，且所述H-FR1包含SEQ ID NO:1所示的氨基酸序列。
17.根据实施方案15-16中任一项所述的分离的抗原结合蛋白，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:9所示的氨基酸序列。
18.根据实施方案15-17中任一项所述的分离的抗原结合蛋白，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:192所示的氨基酸序列。
19.根据实施方案15-18中任一项所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NOs:21、22和24中任一项所示的氨基酸序列。
20.根据实施方案15-19中任一项所述的分离的抗原结合蛋白，其中所述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:38所示的氨基酸序列。
21.根据实施方案1-20中任一项所述的分离的抗原结合蛋白，其包含抗体重链可变区VH，其中所述VH包含SEQ ID NO:193所示的氨基酸序列。
22.根据实施方案21所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列。
23.根据实施方案1-22中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
24.根据实施方案23所述的分离的抗原结合蛋白，其中所述抗体重链恒定区包含SEQ ID NOs:172-175中任一项所示的氨基酸序列。
25.根据实施方案1-24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 NOs:122-125、127和129-137中任一项所示的氨基酸序列。
26.根据实施方案3-25中任一项所述的分离的抗原结合蛋白，其中所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR1包含SEQ ID NO:181所示的氨基酸序列。
27.根据实施方案26所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NOs: 48-51中任一项所示的氨基酸序列。
28.根据实施方案26-27中任一项所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:182所示的氨基酸序列。
29.根据实施方案26-28中任一项所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NOs:63-64中任一项所示的氨基酸序列。
30.根据实施方案26-29中任一项所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:191所示的氨基酸序列。
31.根据实施方案26-30中任一项所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NOs:77-79中任一项所示的氨基酸序列。
32.根据实施方案3-31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-FR1，L-FR2，L-FR3，和L-FR4。
33.根据实施方案32所述的分离的抗原结合蛋白，其中所述L-FR1的C末端与所述LCDR1的N末端相连，且所述L-FR1包含SEQ ID NO:185所示的氨基酸序列。
34.根据实施方案32-33中任一项所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NOs:41-44中任一项所示的氨基酸序列。
35.根据实施方案32-34中任一项所述的分离的抗原结合蛋白，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:186所示的氨基酸序列。
36.根据实施方案32-35中任一项所述的分离的抗原结合蛋白，其中所述L-FR2包含SEQ ID NOs:56-59中任一项所示的氨基酸序列。
37.根据实施方案32-36中任一项所述的分离的抗原结合蛋白，其中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:187所示的氨基酸序列。
38.根据实施方案32-37中任一项所述的分离的抗原结合蛋白，其中所述L-FR3包含SEQ ID NOs:69-72中任一项所示的氨基酸序列。
39.根据实施方案32-38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:188所示的氨基酸序列。
40.根据实施方案32-39中任一项所述的分离的抗原结合蛋白，其中所述L-FR4包含SEQ ID NOs:85-86中任一项所示的氨基酸序列。
41.根据实施方案1-40中任一项所述的分离的抗原结合蛋白，其包含抗体轻链可变区VL，其中所述VL包含SEQ ID NO:194所示的氨基酸序列。
42.根据实施方案41所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NOs:108-114、116和118中任一项所示的氨基酸序列。
43.根据实施方案1-42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，其中所述抗体轻链恒定区包含SEQ ID NO:170所示的氨基酸序列。
44.根据实施方案1-43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 NOs:150-156、158和160-163中任一项所示的氨基酸序列。
45.根据实施方案1-44中任一项所述的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-L1；
2)能够阻断PD-1与PD-L1的结合；
3)能够阻断CD80与PD-L1的结合；
4)能够刺激免疫细胞中IFN-γ和/或IL2的分泌；
5)能够抑制肿瘤生长和/或肿瘤细胞增殖；
6)与对照抗体相比，结合与源自灵长类动物的PD-L1的不完全重叠的表位，
其中所述对照抗体包含SEQ ID NO:52所示的LCDR1、SEQ ID NO:65所示的LCDR2和SEQ ID NO:81所示的LCDR3，且所述对照抗体包含SEQ ID NO:6所示的HCDR1、SEQ ID NO:15所示的HCDR2和SEQ ID NO:34所示的HCDR3；
或者，
其中所述对照抗体包含SEQ ID NO:53所示的LCDR1、SEQ ID NO:66所示的LCDR2和SEQ ID NO:82所示的LCDR3，且所述对照抗体包含SEQ ID NO:5所示的HCDR1、SEQ ID NO:18所示的HCDR2和SEQ ID NO:35所示的HCDR3；
或者，
所述对照抗体包含SEQ ID NO:55所示的LCDR1、SEQ ID NO:68所示的LCDR2和SEQ ID NO:84所示的LCDR3，且所述对照抗体包含SEQ ID NO:8所示的HCDR1、SEQ ID NO:20所示的HCDR2和SEQ ID NO:37所示的HCDR3。
46.根据实施方案45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。
47.一种融合蛋白，其包含：a)人TGFBRII或其片段；以及b)实施方案1-46中任一项所述的分离的抗原结合蛋白。
48.根据实施方案47所述的融合蛋白，其中所述分离的抗原结合蛋白包含抗体重链或其片段，且所述抗体重链或其片段与所述人TGFBRII或其片段框内融合而形成所述融合多肽。
49.根据实施方案48所述的融合蛋白，其中所述抗体重链或其片段的C端直接或间接与所述人TGFBRII或其片段的N端连接。
50.根据实施方案48-49中任一项所述的融合蛋白，其中所述抗体重链或其片段通过连接子与所述人TGFBRII或其片段相连。
51.根据实施方案50所述的融合蛋白，其中所述连接子为肽连接子，且所述肽连接子包含SEQ ID NOs:167-169中任一项所示的氨基酸序列。
52.根据实施方案47-51中任一项所述的融合蛋白，其中所述人TGFBRII或其片段包含人TGFBRII的胞外结构域。
53.根据实施方案47-52中任一项所述的融合蛋白，其中所述人TGFBRII或其片段包含SEQ ID NOs:176-178中任一项所示的氨基酸序列。
54.根据实施方案47-53中任一项所述的融合蛋白，其包含第一多肽和第二多肽，
其中所述第一多肽包含所述分离的抗原结合蛋白的重链或其片段以及所述人TGFBRII或其片段；且
所述第二多肽包含所述分离的抗原结合蛋白的轻链或其片段。
55.根据实施方案54所述的融合蛋白，其中所述第一多肽的所述重链或其片段与所述第二多肽的所述轻链或其片段组合后形成特异性结合PD-L1的抗原结合部分。
56.根据实施方案54-55中任一项所述的融合蛋白，其中所述第一多肽包含SEQ ID NOs:138、139、142、143和145-148中任一项所示的氨基酸序列。
57.根据实施方案54-56中任一项所述的融合蛋白，其中所述第二多肽包含SEQ ID NOs:162-163中任一项所示的氨基酸序列。
58.根据实施方案54-57中任一项所述的融合蛋白，其中，
所述第一多肽包含SEQ ID NO:138所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:139所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:142所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:143所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:145所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:163所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:146所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:163所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:147所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
所述第一多肽包含SEQ ID NO:148所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列。
59.根据实施方案54-58中任一项所述的融合蛋白，其包含两条所述第一多肽以及两条所述第二多肽。
60.分离的一种或多种核酸分子，其编码实施方案1-46中任一项所述的分离的抗原结合蛋白，和/或实施方案47-59中任一项所述的融合蛋白。
61.载体，其包含根据实施方案60所述的核酸分子。
62.细胞，其包含根据实施方案60所述的核酸分子或根据实施方案61所述的载体。
63.制备实施方案1-46中任一项所述的分离的抗原结合蛋白和/或实施方案47-59中任一项所述的融合蛋白的方法，所述方法包括在使得实施方案1-46中任一项所述的分离的抗原结合蛋白实施方案47-59中任一项所述的融合蛋白表达的条件下，培养根据实施方案49所述的细胞。
64.嵌合抗原受体，其包含实施方案1-46中任一项所述的分离的抗原结合蛋白。
65.基因修饰的细胞，其包含实施方案64所述的嵌合抗原受体。
66.抗体药物偶联物，其包含细胞毒性剂，以及实施方案1-46中任一项所述的分离的抗原结合蛋白。
67.药物组合物，其包含实施方案1-46中任一项所述的分离的抗原结合蛋白、实施方案47-59中任一项所述的融合蛋白、实施方案60所述的核酸分子、实施方案61所述的载体、实施方案62所述的细胞、实施方案64所述的嵌合抗原受体、实施方案65所述的基因修饰的细胞和/或实施方案66所述的抗体药物偶联物，以及任选地药学上可接受的载体。
68.实施方案1-46中任一项所述的分离的抗原结合蛋白、实施方案60所述的核酸分子、实施方案61所述的载体、实施方案62所述的细胞、实施方案64所述的嵌合抗原受体、实施方案65所述的基因修饰的细胞、实施方案66所述的抗体药物偶联物和/或实施方案67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/或治疗肿瘤或癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。
69.实施方案68所述的用途，其中所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。
70.根据实施方案68-69中任一项所述的用途，其中所述肿瘤或癌症包括结直肠癌。
71.预防、缓解或治疗肿瘤，抑制肿瘤生长和/或抑制肿瘤细胞增殖的方法，所述方法包括向有需要的受试者施用实施方案1-46中任一项所述的分离的抗原结合蛋白、实施方案60所 述的核酸分子、实施方案61所述的载体、实施方案62所述的细胞、实施方案64所述的嵌合抗原受体、实施方案65所述的基因修饰的细胞、实施方案66所述的抗体药物偶联物和/或实施方案67所述的药物组合物。
72.实施方案71所述的方法，其中所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症。
73.根据实施方案71-72中任一项所述的方法，其中所述肿瘤或癌症包括结直肠癌。
74.实施方案1-46中任一项所述的分离的抗原结合蛋白、实施方案60所述的核酸分子、实施方案61所述的载体、实施方案62所述的细胞、实施方案64所述的嵌合抗原受体、实施方案65所述的基因修饰的细胞、实施方案66所述的抗体药物偶联物和/或实施方案67所述的药物组合物，其用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。
75.抑制PD-L1与PD-1结合的方法，所述方法包括施用实施方案1-46中任一项所述的分离的抗原结合蛋白、实施方案60所述的核酸分子、实施方案61所述的载体、实施方案62所述的细胞、实施方案64所述的嵌合抗原受体、实施方案65所述的基因修饰的细胞、实施方案66所述的抗体药物偶联物和/或实施方案67所述的药物组合物。
76.抑制CD80与PD-L1结合的方法，所述方法包括施用实施方案1-46中任一项所述的分离的抗原结合蛋白、实施方案60所述的核酸分子、实施方案61所述的载体、实施方案62所述的细胞、实施方案64所述的嵌合抗原受体、实施方案65所述的基因修饰的细胞、实施方案66所述的抗体药物偶联物和/或实施方案67所述的药物组合物。
77.试剂盒，其包括实施方案1-46中任一项所述的分离的抗原结合蛋白、实施方案60所述的核酸分子、实施方案61所述的载体、实施方案62所述的细胞、实施方案64所述的嵌合抗原受体、实施方案65所述的基因修饰的细胞、实施方案66所述的抗体药物偶联物、实施方案67所述的药物组合物，和/或(i)施用装置。
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
下面显示的实施例意在说明本发明的具体实施方案，并且不意在以任何方式限制本说明书或权利要求书的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook，J.，Fritsch，E.F.and Maniais，T.(1989)MolecuLar Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例1抗原结合蛋白或融合蛋白的制备和表征分析
可以利用PD-L1抗原对实验动物进行免疫以获得针对PD-L1特异性结合的抗体分子，该实验动物可以是小鼠、大鼠、兔、羊、骆驼等。通常，其得到的抗体分子是非人源的。在获得非人源抗体后，需要对这些分子利用抗体工程技术进行人源化改造，以降低免疫原性并提高成药性。然而，抗体的人源化过程有其技术复杂性，经过人源化改造的分子往往会降低对抗原的亲和力。另一方面，转基因技术的进步使得可以培育出基因工程化小鼠，其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。这种转基因小鼠产生的抗体具有全人源的序列，因而无需再进一步做人源化改造，大大提高了治疗性抗体开发的效率。Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
1.1用PD-L1抗原免疫小鼠
用可溶的重组人PD-L1-mFc融合蛋白(Novo Protein Inc.货号CM06，批号0330837)对Harbour H2L2小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100微升。在首轮免疫中，每只小鼠接受用50μg抗原蛋白(人PD-L1-mFc)与完全弗氏佐剂(Sigma，货号F5881)以体积比1:1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中，每只小鼠接受用25μg抗原蛋白与Sigma Adjuvant System佐剂(Sigma，货号S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周，通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天；并且在第49、77天，检测小鼠血清抗体滴度。在进行细胞融合前3天，以每只小鼠25μg抗原蛋白的剂量进行最后一次增强免疫。
1.2获得杂交瘤单克隆和抗体序列
当检测小鼠血清中PD-L1特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，分离出数个表达抗-PD-L1单克隆抗体分子的杂交瘤细胞，分离的杂交瘤细胞及其表达的单克隆抗体都使用对应的克隆号来表示，例如：63G11H3G9，91G3H5H3等等。分离的杂交瘤细胞表达具有完整的人可变结构域和大鼠恒定结构域的重链和轻链的抗体分子。对上述单克隆抗体进行进一步的鉴定，根据其对人PD-L1的结合能力、食蟹猴PD-L1的结合能力、抑制PD-L1与PD-1结合能力等参数，选出若干个杂交瘤克隆进行测序。利用常规的杂交瘤测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。在本实施例中，从免疫的Harbour H2L2小鼠得到的抗PD-L1单克隆抗体分子可变结构域的序列是人源抗体序列。抗体可变结 构域的CDR序列可以通过Kabat或者Chothia或者其他CDR定义规则(如Combined定义规则)进行分析(见表1关于CDR定义)。表2列出了杂交瘤细胞及重组抗体的可变区序列以及CDR序列所对应的SEQ ID NO编号，其中CDR使用Chothia定义规则。
1.3制备全人重组抗体
在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列(SEQ ID NO:172)或者其含有氨基酸突变的变体序列(SEQ ID NO:173-175)的哺乳动物细胞表达质粒载体中，以编码产生人IgG抗体的全长重链(或其变体)；抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体κ轻链恒定结构域序列(SEQ ID NO:170)的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长κ轻链；或者将VL通过基因合成并克隆到编码人抗体λ轻链恒定结构域序列(SEQ ID NO:171)的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长λ轻链。表2列出了本实施例中PD-L1抗体的轻、重链可变结构域氨基酸序列，轻链全长氨基酸序列，重链(人IgG1)全长氨基酸序列和根据Chothia定义规则定义的CDR的氨基酸序列。
将编码抗体重链的质粒和编码抗体轻链的质粒同时转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到具有轻重链正确配对组装的纯化的PD-L1重组抗体。具体说来，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo,A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6～8x10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2x10 6细胞/ml。准备30ml培养的细胞。将上述编码抗体重链的质粒和编码抗体轻链的质粒以2:3的比例混合共计30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo,31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)缓冲液平衡含有MabSelect TM(GE Healthcare Life Science,71-5020-91 AE)的重力柱(Bio-Rad,7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,UFC901024)浓缩换液至PBS缓冲液，得到纯化的重组蛋白溶液。最后用NanoDrop(Thermo Scientific TMNanoDrop TMOne)测定浓 度，分装、存储备用。
表2筛选得到的抗PD-L1杂交瘤单克隆及其重组抗体的序列号SEQ ID NO
Figure PCTCN2020118811-appb-000001
1.4 PD-L1抗体的序列分析和表达纯化
抗体的重链可变结构域序列来源于染色体上重链基因群的胚系基因V、D、J基因片段的基因重排和体细胞高频突变等事件；轻链可变结构域序列来源于轻链基因群的胚系基因V、J基因片段的基因重排和体细胞高频突变等事件。基因重排和体细胞高频突变是增加抗体多样性的主要因素。来源于相同胚系V基因片段的抗体也可能产生不同的序列，但总体上相似性较高。利用一些算法，例如IMGT/DomainGapAlign(http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)或者NCBI/IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)可以从抗体的可变结构域序列推测出其发生基因重排时可能的胚系基因片段。将实施例1.3和表2中的抗体序列进行分析，其重链可变结构域(VH)和轻链可变结构域(VL)的胚系基因V基因片段列于表3。
蛋白质或多肽氨基酸链在细胞中翻译合成后有时会引入化学修饰，称为翻译后修饰(PTM)。对于抗体而言，一些PTM的位点是非常保守的，例如，在人的IgG1抗体的恒定结构域的第297位(EU编号)的保守的氨基酸天冬酰胺Asn通常会发生糖基化修饰形成糖 链，而该糖链结构对于抗体结构和相关的效应子功能是至关重要的。但是，如果在抗体的可变结构域尤其是抗原结合区域(如CDR)中存在PTM，那么这些PTM的存在有可能会对抗原的结合有较大的影响，也可能对抗体的物理化学性质带来变化。例如，糖基化、脱酰胺、异构化、氧化等都可能增加抗体分子的不稳定性或异质性，从而增加抗体开发的难度和风险。因而避免一些潜在的PTM对于治疗性抗体的开发是非常重要的。随着经验的积累，人们发现一些PTM是和氨基酸序列的组成尤其是相邻氨基酸组成的“模式”是高度相关的，这样使得可以从蛋白质的一级氨基酸序列预测出潜在的PTM。例如，N-x-S/T(第一位是天冬酰胺，第二位是非脯氨酸以外的任意氨基酸，第三位是丝氨酸或者苏氨酸)的序列模式预测出N-连接糖基化位点。引起PTM的氨基酸序列模式有可能来源于胚系基因序列，例如人胚系基因片段IGHV3-33天然地在FR3区域存在糖基化模式NST；也可能来源于体细胞高频突变。表3列出了实施例1.3的抗体的可变结构域VH和VL的预测的PTM。具体说来，NGS或NLT可能是糖基化位点，DG可能引起天冬氨酸的异构化。
可以通过氨基酸突变来破坏PTM的氨基酸序列模式，从而降低或者去除特定PTM的形成。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。实际操作中，对同一个PTM序列模式可能采用多种突变设计方法。
表4列出了对六个来自实施例1.3的具有潜在PTM位点的抗体的序列进行氨基酸突变得到的新的抗体分子(称为PTM变体)。表5-1列出了本实施例中这些PTM变体的轻、重链可变结构域氨基酸序列，轻链全长氨基酸序列，重链(人IgG1(N297A))全长氨基酸序列，和根据Chothia定义规则定义的CDR的氨基酸序列。所有设计出来的PTM变体按照实施例1.3中描述的方法得到纯化的重组抗体，并在后续的功能实验中进一步验证。
表3 PD-L1抗原结合蛋白序列的胚系基因分析和翻译后修饰位点(PTM)分析
克隆号 抗体 VH胚系V基因 VL胚系V基因 VH PTM VL PTM
63G11H3G9 PR000064 IGHV3-7 IGKV1-5 DG(HCDR2) -
67C1F7G6 PR000065 IGHV3-7 IGKV1-5 DG(HCDR2) -
69B8G8C2 PR000066 IGHV3-7 IGKV1-5 DG(HCDR2) NGS(LFR3)
73G3G9B11 PR000067 IGHV3-7 IGKV1-5 DG(HCDR2) -
82F8H7E1 PR000068 IGHV3-7 IGKV1-5 DG(HCDR2) -
85C5E4G3 PR000069 IGHV3-7 IGKV1-5 DG(HCDR2) -
91G3H5H3 PR000070 IGHV3-7 IGKV1-5 DG(HCDR2) -
95C7C4A9 PR000071 IGHV3-7 IGKV1-5 DG(HCDR2) -
98E11H1C9 PR000072 IGHV3-7 IGKV1-5 DG(HCDR2) -
表4 PD-L1抗原结合蛋白序列的突变位点设计
初始抗体 PTM变体 突变点 变体VH PTMs 变体VL PTMs
PR000066 PR000261 H:D54E；L:N65S / /
PR000066 PR000262 H:G55A；L:N65S / /
PR000069 PR000263 H:D54E / /
PR000069 PR000264 H:G55A / /
PR000070 PR000265 H:D54E / /
PR000070 PR000266 H:G55A / /
PR000072 PR000267 H:D54E / /
PR000072 PR000268 H:G55A / /
表5-1突变PTM后得到的抗体的SEQ ID NO
Figure PCTCN2020118811-appb-000002
Figure PCTCN2020118811-appb-000003
表5-2列出了本申请的实施例中出现的对照抗体PR000151、PR001598和PR000265的Fc类型的转换抗体PR000416的分子的序列号，其中，PR000151是Roche的抗PD-L1抗体atezolizumab(阿特珠单抗)类似物，在本文中，PR000151与atezolizumab可互换地使用；PR001598是Merck KGaA的抗PD-L1抗体avelumab(阿维鲁单抗)类似物，在本文中，PR001598与avelumab可互换地使用。上述抗体通过实施例1.3的方法制备纯化。
表5-2 PR000265的Fc类型转换和对照抗体
抗体说明 抗体编号 轻链 重链 VL VH 恒定区类型
atezolizumab类似物 PR000151 159 128 117 96 hIgG1(N297A)
avelumab类似物 PR001598 164 140 119 105 hIgG1
PR000265的Fc类型转换 PR000416 162 137 114 101 hIgG1
实施例2抗原结合蛋白与过表达人/食蟹猴PD-L1的细胞的结合(FACS)
本实施例为了研究PD-L1抗原结合蛋白体外结合人/食蟹猴PD-L1的活性，采用过表达人PD-L1的CHOK1细胞株(CHO-K1/hPD-L1，南京金斯瑞，M00543)或过表达食蟹猴PD-L1的CHOK1细胞株(CHOK1/cynoPD-L1，南京金斯瑞，M00573)进行细胞水平的结合实验。简言之，消化PD-L1细胞，并用F-12K完全培养基重悬，将细胞密度调整为1x10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,3894)，随后加入100μL/孔，2倍于终浓度的5倍浓度梯度稀释的待测抗原结合蛋白，混合均匀，其中抗原结合蛋白最高终浓度为100nM，共8个浓度，同型抗体hIgG1 iso作为对照。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g，4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(山羊抗人IgG(H+L)第二抗体Alexa Fluor 488 conjugate,Invitrogen,A11013,1:1000稀释)，4℃，避光孵育30分钟。用200μL/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞。使用BD  FACS CANTOII读取荧光发光信号值。使用流式细胞仪BD FACS CANTOII读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
图1和表6的结果显示了本申请所述PD-L1抗原结合蛋白均能结合人PD-L1；图2和表7的结果显示了所述PD-L1抗原结合蛋白均能结合食蟹猴PD-L1；并且呈现浓度依赖效应。
表6结合细胞表面的人PD-L1
抗原结合蛋白 MFI最大值 EC50(nM)
PR000064 7885 0.417
PR000065 7657 0.412
PR000066 7859 0.255
PR000067 7342 0.456
PR000068 7636 0.326
PR000069 7529 0.312
PR000070 7787 0.210
PR000071 6894 0.521
PR000072 7629 0.292
atezolizumab 6765 0.188
hIgG1对照 305 N.A.
表7结合细胞表面的猴PD-L1
抗原结合蛋白 MFI最大值 EC50(nM)
atezolizumab 22447 0.379
hIgG1对照 935 N.A.
PR000265 22556 0.451
PR000266 22362 0.562
PR000267 22401 0.775
PR000268 22244 0.759
实施例3抗原结合蛋白与人/食蟹猴PD-L1蛋白的结合
本实施例利用酶联免疫吸附测定法(ELISA)研究PD-L1抗原结合蛋白体外结合人/食蟹猴PD-L1蛋白的活性。
3.1抗原结合蛋白与人PD-L1蛋白的结合(ELISA)
将人PD-L1(Acrobiosystems,PD1-H5229)用PBS稀释为2μg/ml，加至96孔板(Corning,9018)中，每孔100μl，4℃下孵育过夜。弃去液体后用PBST缓冲液(pH 7.4，含0.05％tween-20)洗板3次，加入250μl 2％BSA封闭液，37℃条件下孵育1小时。弃去封闭液，并用PBST缓冲液(pH7.4，含0.05％吐温-20)洗板3次，将待测抗原结合蛋白，自100nM浓度起，依次进行5倍浓度稀释，共8个浓度梯度，每个孔加入100μl，37℃孵育1小时，同型抗体hIgG1iso作为对照。PBST缓冲液(pH7.4，含0.05％吐温-20)清洗3次后，加入稀释4000倍的羊抗人HRP二抗(Invitrogen,A18805)，37℃条件下，避光孵育1小时。PBST缓冲液(pH7.4，含0.05％tween-20)清洗3次后，添加100μl/孔TMB(Biopanda,TMB-S-003)，室温避光放置约5分钟；每孔加入50μl/孔终止液(BBI life sciences,E661006-0200)终止反应。用酶标仪Enspire(PerkinElemer)检测450nm吸收值(OD450)。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。
图3和表8显示了本申请所述PD-L1抗原结合蛋白均能与人PD-L1蛋白结合，且PTM变体仍保留了亲本抗体的结合活性。
表8结合人PD-L1蛋白
抗原结合蛋白 EC50(nM)
PR000151 0.0259
PR000066 0.0189
PR000261 0.0385
PR000262 0.0338
PR000069 0.0637
PR000263 0.0321
PR000264 0.0343
PR000070 0.0641
PR000265 0.0373
PR000266 0.0415
PR000072 0.0474
PR000267 0.0462
PR000268 0.0412
3.2抗原结合蛋白与食蟹猴PD-L1蛋白的结合(ELISA)
将食蟹猴(Cynomolgus)的PD-L1蛋白(Acrobiosystems,PD1-C52H4)用PBS稀释为2μg/ml，加至96孔板(Corning,9018)中，每孔100μl，4℃下孵育过夜。弃去液体后用PBST洗板3次，加入250μl的2％BSA封闭，室温条件下孵育1小时。弃去封闭液，并用PBST缓冲液(pH7.4，含0.05％tween-20)洗板3次，将待测抗原结合蛋白浓度稀释至5μg/ml，加入100μl/孔，37℃孵育1小时。PBST缓冲液(pH7.4，含0.05％tween-20)清洗3次后，加入稀释4000倍稀释的羊抗人HRP二抗(Invitrogen,A18805)，37℃孵育1小时。清洗后添加100μl/孔TMB(Biopanda,TMB-S-003)，室温避光放置5分钟；每孔加入50μl/孔终止液(BBI life sciences,E661006-0200)终止反应。用酶标仪Enspire(PerkinElemer)检测450nm吸收值(OD450)。
结果如图4所示，本申请所述PD-L1抗原结合蛋白均能结合食蟹猴PD-L1蛋白。
实施例4抗原结合蛋白阻断人PD-1与过表达人PD-L1的CHO-K1细胞的结合
为了研究人PD-L1结合蛋白体外阻断人PD-1与人PD-L1结合的活性，采用过表达人PD-L1的CHOK1细胞株(CHO-K1/hPD-L1，南京金斯瑞，M00543)进行细胞水平的人PD-1/人PD-L1结合阻断实验。简言之，消化CHO-K1/hPD-L1细胞，并用F-12K完全培养基重悬，将细胞密度调整为1x10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗原结合蛋白，混合均匀，其中抗原结合蛋白最高终浓度为100nM，共8个浓度，同型抗体hIgG1 iso作为对照。将细胞放置于4℃，避光孵育1小时。之后，4℃下离心5分钟，弃上清，随后加入50μL/孔，1μg/mL浓度的生物素标记的人PD-1蛋白(Acrobiosystems，PD1-H82F2)，4℃，避光孵育30分钟。 加入100μL/孔预冷PBS漂洗细胞两次，于500g，4℃下离心5分钟，弃上清。加入100μL/孔荧光二抗PE Streptavidin(BD Pharmingen,554061,1:200稀释)，4℃，避光孵育30分钟。用200μL/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞。使用BD FACS CANTOII读取荧光发光信号值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及IC50值等参数。
图5和表9显示本申请所述PTM突变前的抗原结合蛋白均能阻断人PD-1与细胞表面的人PD-L1结合；图6和表10显示本申请所述PTM变体的抗原结合蛋白均能阻断人PD-1与细胞表面的人PD-L1结合；阻断能力与atezolizumab类似物的相当。
表9阻断人PD-1与细胞表面的人PD-L1结合
抗原结合蛋白 最大抑制率(％) IC50(nM)
PR000064 91.46 0.938
PR000066 91.81 0.539
PR000067 91.16 0.894
PR000068 91.37 0.853
PR000069 92.35 0.836
PR000070 90.74 0.449
PR000071 91.58 0.987
PR000072 92.49 0.746
atezolizumab 91.45 0.536
hIgG1对照 N.A. N.A.
表10阻断人PD-1与细胞表面的人PD-L1结合
抗原结合蛋白 最大抑制率(％) IC50(nM)
atezolizumab 92.79 0.476
hIgG1对照 0.68 N.A.
PR000265 93.57 0.604
PR000266 91.54 0.838
PR000267 91.04 1.040
PR000268 91.34 1.160
实施例5抗原结合蛋白阻断人PD-L1蛋白与其配体蛋白的结合
本实施例利用酶联免疫吸附测定法(ELISA)研究PD-L1抗原结合蛋白体外阻断人PD-L1蛋白与其配体蛋白PD-1或CD80的结合的能力。
5.1抗原结合蛋白阻断人PD-L1与人PD-1的结合(ELISA)
将人PD-L1(Acrobiosystems,EP-101-96tests,A001-214)用PBS稀释成2μg/ml加入到96孔板(Corning,9018)中，每孔100μl，4℃包被过夜。弃去液体后用PBST缓冲液(pH7.4，含0.05％tween-20)洗板3次，加入250μl 2％BSA封闭液，37℃条件下孵育1小时。弃去封闭液，并用PBST缓冲液(pH7.4，含0.05％tween-20)洗板3次，将待测抗原结合蛋白，用PBST with 0.5％(w/v)BSA稀释成不同浓度梯度，与0.6μg/ml人PD-1-Biotin蛋白(Acrobiosystems,EP-101-96tests,A002-214)混合后，加入孔板，同型抗体hIgG1 iso作为对照，37℃条件下孵育1小时。洗涤3次后每孔加入100μl Streptavidin-HRP(Acrobiosystems,EP-101-96tests,A003-214)，37℃条件下，避光反应1小时。洗涤3次后加入100μl TMB显色液，室温下避光显色5分钟。加入终止液终止反应。用酶标仪Enspire(PerkinElemer)检测450nm吸收值(OD450)。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及IC50值等参数。
图7和表11显示了本申请所述抗原结合蛋白能阻断人PD-1与人PD-L1蛋白结合；阻断能力与atezolizumab类似物的相当。
表11阻断人PD-1与人PD-L1蛋白结合
抗原结合蛋白 最大抑制率(％) IC50(nM)
PR000064 91.06 0.313
PR000065 99.03 0.432
PR000066 93.05 0.350
PR000067 92.91 0.333
PR000068 91.59 0.288
PR000069 90.71 0.313
PR000070 90.62 0.200
PR000071 88.98 0.448
PR000072 89.85 0.237
atezolizumab 90.48 0.240
hIgG1对照 N.A. N.A.
5.2抗原结合蛋白阻断人PD-L1与人B7-1(CD80)的结合(ELISA)
将人PD-L1-hFc蛋白(Acrobiosystems,PD1-H5258)用PBS稀释成1μg/ml加入到96孔板(Corning,9018)中，每孔100μl，4℃包被过夜。弃去液体后用PBST缓冲液(pH7.4，含0.05％tween-20)洗板3次，加入250μl 2％BSA封闭液，37℃条件下孵育1.5小时。弃去封闭液，并用PBST缓冲液(pH7.4，含0.05％tween-20)洗板3次，将待测抗原结合蛋白，用PBST with 0.5％(w/v)BSA稀释成不同浓度梯度，与10μg/ml人B7-1-Biotin蛋白(Acrobiosystems,B71-H82F2)混合后，加入孔板，同型抗体hIgG1 iso作为对照，37℃条件下孵育1.5小时。洗涤3次后每孔加入100μl Streptavidin-HRP(SIGMA,S2438)，37℃条件下，避光反应1小时。洗涤3次后加入100μl TMB显色液，室温下避光显色5分钟。加入终止液终止反应。用酶标仪Enspire(PerkinElemer)检测450nm吸收值(OD450)。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及IC50值等参数。
图8和表12显示了本申请所述抗原结合蛋白均能阻断人B7-1(CD80)与人PD-L1蛋白结合。
表12阻断人B7-1(CD80)与人PD-L1结合
抗原结合蛋白 最大抑制率(％) IC50(nM)
PR000064 35.93 0.284
PR000065 43.77 0.312
PR000066 47.24 0.198
PR000067 36.88 0.153
PR000068 34.95 0.199
PR000069 32.95 0.280
PR000070 39.93 0.305
PR000071 37.48 0.351
PR000072 47.63 0.253
atezolizumab 52.11 1.622
hIgG1对照 N.A. N.A.
实施例6利用报告基因细胞系检测抗原结合蛋白对PD-1信号通路的抑制作用
将共表达人PD-L1和OS8(CD3单链抗体跨膜蛋白)的Hep3B(睿智化学(上海)构建)细胞接种到96孔板上，细胞量为1.25×10 4/孔，100μL/孔。37℃在5％CO 2环境下孵育过夜。去除上清液，加入50μL/孔的待测抗原结合蛋白稀释液，起始浓度为100nM，5倍浓度稀释，同型抗体hlgG1 iso为对照组。加入5×10 4/孔的可持续表达PD-1和NFAT-荧光素酶报告基因的Jurkat报告细胞(睿智化学(上海)构建)50μL/孔。37℃在5％CO 2环境下培养6小时。加入ONE-Glo TM荧光素酶试剂(Promega,货号E6110)，室温孵育5分钟，酶标仪检测发光值。
图9和表13以及图10和表14显示了本申请所述抗原结合蛋白对PD-1信号通路有抑制作用；除PR000071以外，其余分子对PD-1信号通路的抑制作用与atezolizumab类似物的相当。
表13对PD-1信号通路的抑制作用
抗原结合蛋白 最大荧光值(RLU) EC50(nM)
PR000064 81597 0.533
PR000066 85890 0.292
PR000067 79845 0.682
PR000068 83254 0.603
PR000069 83317 0.271
PR000070 82697 0.269
PR000071 79738 2.707
PR000072 78043 0.293
atezolizumab 79621 0.190
hIgG1对照 N.A. N.A.
表14对PD-1信号通路的抑制作用
抗原结合蛋白 最大荧光值(RLU) EC50(nM)
atezolizumab 43162 0.416
hIgG1对照 N.A. N.A.
PR000265 50732 0.533
PR000266 48436 0.680
PR000267 42982 0.796
PR000268 45501 0.759
实施例7.利用BLI方法测定抗原结合蛋白与人PD-L1的亲和力
7.1测定抗原结合蛋白与人PD-L1的解离常数，单浓度抗原解离速率的比较
带有组氨酸标签的人PD-L1蛋白购自厂家NovoProtein(货号C315)；测试缓冲液为1x动力学缓冲液(从10x动力学缓冲液(ForteBio，货号18-1105)稀释)，用于亲和力测试以及抗原、抗体的稀释；通过生物膜干涉(BLI)技术，使用Octet分子相互作用分析仪(ForteBio，型号Octet Red96e)，来进行抗原抗体之间的结合动力学分析。
测定抗原和抗体的亲和力时，传感器转动速度为1000转/分钟。先将置于一列的2个AHC传感器在测试缓冲液中平衡10分钟，然后用AHC传感器捕获待测抗原结合蛋白，捕获高度0.9-1.1nm，然后将AHC传感器在测试缓冲液中平衡2分钟，然后将AHC传感器与抗原(60nM以及0nM)结合3分钟，最后解离15分钟。将AHC传感器浸入10mM甘氨酸(pH1.5)溶液进行再生，以洗脱结合在传感器上的蛋白。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，选择单扣除模式(reference well)扣除参照信号，选择“1:1 Local/full fitting”方法进行数据拟合，计算出抗原 与抗体结合的动力学参数，得到k on(1/Ms)值、k dis(1/s)值和K D(M)值。
结果如图11(A-B)和表15-1所示，PR000265和PR000266与PD-L1均具有较高的亲和力。
表15-1抗原结合蛋白与人PD-L1的亲和力
抗原结合蛋白 K on(1/Ms) k dis(1/s) K D(M)
PR000265 2.60E+05 1.06E-04 4.09E-10
PR000266 2.76E+05 9.62E-05 3.49E-10
7.2测定抗原结合蛋白与人PD-L1的结合常数和亲和力(多浓度抗原)
按照实施例7.1的方法，测定抗原(多浓度)和抗体的亲和力。测定抗原(多浓度)和抗体的亲和力时，传感器转动速度为1000转/分钟。先将两列AHC传感器(每列放置8个传感器；第一列称为参照AHC传感器，第二列称为测试AHC传感器)在测试缓冲液中平衡10分钟。然后，用8个测试AHC传感器捕获待测抗原结合蛋白(浓度40nM)，捕获时间30秒，捕获高度约0.7nm；8个测试AHC传感器在测试缓冲液中平衡2分钟后与梯度稀释的抗原蛋白(例如，抗原浓度可以为50-1.56nM的两倍梯度稀释以及0nM)结合，8个传感器可以分别浸入到最多8个不同浓度的抗原中；与抗原结合5分钟，然后解离15分钟；最后将AHC传感器浸入10mM甘氨酸(pH 1.5)溶液进行再生，以洗脱结合在传感器上的蛋白。参照AHC传感器的实验步骤与测试AHC传感器相同，唯一的区别是在第一步将8个参照AHC传感器浸入不含待测分子的测试缓冲液中30秒。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，选择双扣除模式(double reference)扣除参照信号，选择“1:1Global fitting”方法进行数据拟合，计算出抗原与抗原结合蛋白结合的动力学参数，得到k on(1/Ms)值、k dis(1/s)值和K D(M)值。
结果如图11(C-E)和表15-2所示，PR000265与PD-L1的亲和力比PR000151(atezolizumab类似物)和PR001598(avelumab类似物)更高。
表15-2抗原结合蛋白与人PD-L1的亲和力
Figure PCTCN2020118811-appb-000004
Figure PCTCN2020118811-appb-000005
实施例8利用BLI方法测定抗原结合蛋白结合PD-L1的表位竞争
首先将人PD-L1蛋白(NovoProtein,C315)先利用生物素化试剂盒(EZ-Link Sulfo-NHS-LC-Biotin,ThermoFisher,A39257)按照说明书要求进行生物素化。然后使用ForteBio Octet平台对得到的抗原结合蛋白和PR000151(atezolizumab类似物)以及PR001598(avelumab类似物)进行表位竞争实验。第一步，获取抗体的100％信号：用SA传感器捕获生物素化的PD-L1蛋白，捕获高度为0.25nm。将传感器浸入抗体中(50nM)，时间500秒，将该抗体与PD-L1结合的最终信号记录为该抗体的100％信号。第二步，表位竞争实验：用SA传感器捕获生物素化的PD-L1蛋白，捕获高度为0.25nm。将传感器浸入第一抗体中(50nM)，时间500秒，再将SA传感器浸入第一抗体和第二抗体的混合物中(两种抗体的终浓度均为50nM)，时间500秒，将最终信号记录为该第二抗体的信号。抑制率通过下式计算，
抑制率(％)＝(A–B)/A*100
A：某抗体的100％信号(从第一步中获得)，B：该抗体作为第二抗体的信号(从第二步获得)。
若得到的抑制率大于85(％)，则意味着两种抗体的表位完全重叠；若抑制率小于85(％)，则意味着两种抗体结合的表位不完全重叠。
其中，抗体PR000416是将本申请所述抗原结合蛋白PR000265的重链恒定区更换为无突变的人IgG1恒定区得到的抗体。
结果显示PR000265(或PR000416)与对照抗体PR000151或PR001598的表位不完全重叠，见表16-1和表16-2。
表16-1抗原结合蛋白的表位竞争
Figure PCTCN2020118811-appb-000006
表16-2抗原结合蛋白的表位竞争
Figure PCTCN2020118811-appb-000007
Figure PCTCN2020118811-appb-000008
实施例9抗原结合蛋白在混合淋巴细胞反应(MLR)中刺激细胞因子分泌
用Ficoll-Paque Plus(GE Healthcare,17144002)从第一供体全血中分离PBMC细胞，加入重组人源IL-4(R&D Systems,204-GMP)和重组人源GM-CSF(R&D Systems,215-GM/CF)诱导6天后，获得未成熟的人CD14 +树突状细胞(iDC细胞)；继续加入1μg/ml的脂多糖(Lipopolysaccharide,LPS；Sigma,L2630)，诱导24小时，获得成熟的树突状细胞(mDC细胞)。采用T细胞分离试剂盒(StemCell,17951)从第二供体的PBMC细胞中分离得到T淋巴细胞。将1×10 5/孔的T淋巴细胞和1×10 4/孔的mDC细胞按10:1比例接种至96孔板，加入10μg/ml各抗原结合蛋白和及对照抗体，并做10倍或者对应倍数的稀释。于37℃，5％CO 2培养箱孵育5天。分别收集72小时后和120小时后的上清液。采用IL2ELISA试剂盒(Thermo,88-7025-88)检测72小时后上清液中的IL-2的水平；采用IFN-γELISA试剂盒(Thermo,88-7316-88)检测120小时后上清液中的IFN-γ的水平；具体操作参考试剂说明书。
本实施例使用了四个供体的PBMC分成了两组供体配对进行混合淋巴细胞反应(MLR)。结果如图12(A-D)所示，在两次独立的MLR实验中，抗原结合蛋白都能增强激活的T淋巴细胞分泌细胞因子IL-2和IFN-γ。
实施例10抗体依赖的细胞毒性实验
本实施例测定抗体依赖的细胞毒性(ADCC)。本实施例使用PBMC作为效应细胞，使用多种PD-L1高表达的肿瘤细胞系作为靶细胞(如MDA-MB-231细胞(ATCC，HTB-26)，NCI-H292细胞(ATCC，CRL-1848))。具体说来，将新鲜的PBMC离心弃去上清，重悬到10％FBS-RPMI1640培养基中，37°培养箱培养过夜。然后收集PBMC细胞，离心弃上清，重悬到2％FBS-RPMI1640培养基中，计数，调整细胞密度至1×10 7/ml，将PBMC加入到U型底96孔板中，50μl/孔。收集靶细胞，离心弃去上清，重悬到2％FBS-RPMI1640培养基中，计数，调整细胞密度至4×10 5/ml，将靶细胞加入到U型底96孔板中，25μl/孔。效应细胞与靶细胞比例为50:1。稀释抗体至中间浓度，起始浓度为1.6nM，5倍梯度稀释，共3个浓度。将样品加入到U型底96孔板中，25μl/孔，样品最终起始浓度为0.4nM。将细胞板放入培养箱中孵育3.5小时，向靶细胞最大释放孔和体积参照孔加入10μl裂解液，继续孵育30分钟，细胞和样品共同孵育总时间为4小时。取出50μl/孔上清至透明平底96孔板中，加入Promega  LDH试剂盒(Promega LDH-Glo TM,J2380)检测试剂50μl/孔。室温孵育20分钟，加入50μl反应终止液，终止反应。酶标仪读取OD450nm吸光值。根据下面计算公式计算细胞毒性。
细胞毒性(％)＝((ER–CMB)-(ESR–CMB)-(TSR–CMB))/(TMR-VCC)×100％
其中：
ER＝实验孔，样品+效应细胞+靶细胞
ESR＝效应细胞自然释放孔，效应细胞+培养基
TSR＝靶细胞自然释放孔，靶细胞+培养基
TMR＝靶细胞最大释放空，靶细胞+培养基+裂解液
VCC＝体积参照孔，培养基+裂解液
CMB＝培养基参照孔，培养基
本实施例使用了三个供体的PBMC和两个肿瘤细胞系，共六个独立ADCC实验。结果如图13(A-F)所示，PR000416(PR000416是将PR000265更换为无突变的人IgG1恒定区得到的抗体)比PR001598(avelumab类似物)有更强的ADCC作用。
实施例11抗原结合蛋白的体内抑制肿瘤活性
将MC38-hPD-L1(过表达人PD-L1的MC38细胞)结肠癌细胞5×10 5个/0.1mL接种于雌性B-hPD-1人源化小鼠(百奥赛图)右侧皮下，待肿瘤生长到约118mm 3时按肿瘤体积随机分组，每组10只，共5组，分别为：Human IgG1(3mg/kg，人IgG1同型对照组)、PR000151(3mg/kg，Atezolizumab低剂量组)、PR000151(10mg/kg，Atezolizumab高剂量组)、PR000265(3mg/kg，本申请抗体低剂量组)和PR000265(10mg/kg，本申请抗体高剂量组)。给药途径为腹腔注射，每2天给药1次，总计给药13次(q2dx13)。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。
在分组给药第20天时，hIgG1对照组肿瘤体积为1359±171mm 3。抗PD-L1抗体PR000151在3mg/kg和10mg/kg剂量下肿瘤体积分别为898±233mm 3和741±203mm 3，TGI分别为33.9％和45.5％。抗PD-L1抗体PR000265在3mg/kg和10mg/kg剂量下肿瘤体积分别为881±169mm 3和1035±472mm 3，TGI分别为35.1％和23.8％。在分组给药第24天时，human IgG1对照组肿瘤体积为1876±203mm 3。抗PD-L1抗体PR000151在3mg/kg和10mg/kg剂量下肿瘤体积分别为1349±306mm 3和949±266mm 3，TGI分别为28.1％和49.4％。抗PD-L1抗体PR000265在3mg/kg和10mg/kg剂量下肿瘤体积分别为1228±187mm 3和937±225mm 3，TGI分别为34.5％和50.1％。PR000151和PR000265在3mg/kg，10mg/kg剂量下都有 明显的肿瘤抑制效果，且与hIgG1相比，有统计学差异(P<0.05)。PR000151和PR000265药效相似，组间比较未见统计学差异(P>0.05)。
结果如图14所示，PR000151和PR000265有明显肿瘤抑制效果，在实验过程中，各组动物体重均出现增长，表明动物对受试品耐受良好。未对动物产生明显毒性作用，安全性好。
实施例12抗PD-L1和抗TGFB的双功能融合蛋白的制备和表征分析
12.1构建融合蛋白分子
在实施例1得到的抗原结合蛋白的C末端通过长度合适的柔性肽段(Linker)融合人TGFB受体(TGFBRII)胞外区的序列构建双功能融合蛋白分子。将编码多肽序列的质粒转染哺乳动物宿主细胞(如HEK293或者ExpiCHO)，利用实施例1.3所述方法进行融合蛋白的重组表达和纯化，得到本申请所述融合蛋白PR001487、PR001488、PR001901、PR001902、PR002247、PR002248、PR002249和PR002251，各部分结构见表17，抗体轻链、重链和各结构域的序列号见表18。另外，制备蛋白分子M7824(也称为PR001599，其为bintrafusp alfa类似物)，蛋白分子Hengrui融合蛋白9(也称为PR002466，其序列来源于专利WO2018205985A1)，以及TGFBRII Trap(也称为PR002040，其利用抗鸡溶菌酶的抗体克隆TEL16的序列与TGFBRII胞外区截短序列融合得到)作为实施例中的对照，其各部分结构和序列号见表17和表18。
表17本申请所述融合蛋白各部分结构
Figure PCTCN2020118811-appb-000009
Figure PCTCN2020118811-appb-000010
表18本申请所述融合蛋白的序列号SEQ ID NO
Figure PCTCN2020118811-appb-000011
12.2利用HPLC-SEC分析蛋白纯度和多聚体
使用分析型分子尺寸排阻层析色谱法(SEC)来分析蛋白样品的纯度和聚体形式。将分析型色谱柱TSKgel G3000SWxl(Tosoh Bioscience,08541,5μm,7.8mm x 30cm)连接到高压液相色谱仪(HPLC)(型号Agilent Technologies,Agilent 1260Infinity II)，用PBS缓冲液室温下平衡至少1小时。适量蛋白样品(至少10μg，样品浓度调整到1mg/ml)用0.22μm滤膜过滤后注射入系统，并设定HPLC程序：用PBS(pH 7.4)缓冲液将样品以1.0ml/min的流速流过色谱柱，最长时间为20分钟；检测波长280nm。采集后用ChemStation软件对色谱图 进行积分并计算相关数据，生成分析报告，报告出样品内不同分子尺寸组份的滞留时间。
12.3利用HPLC-HIC分析蛋白纯度和疏水性
使用分析型疏水相互作用层析色谱法(HIC)来分析蛋白样品的纯度和疏水性。将分析型色谱柱TSKge1Buty1-NPR(Tosoh Bioscience,14947,4.6mm x 3.5cm)连接到高压液相色谱仪(HPLC)(型号Agilent Technologies,Agilent 1260 Infinity II)，用PBS缓冲液室温下平衡至少1小时。设定方法由16分钟内从100％流动相A(20mM组氨酸，1.8M硫酸铵，pH6.0)至100％流动相B(20mM组氨酸，pH 6.0)的线性梯度，流速设定为0.7ml/min,蛋白样品浓度1mg/ml,进样体积20μl,检测波长280nm。采集后用ChemStation软件对色谱图进行积分并计算相关数据，生成分析报告，报告出样品内不同分子尺寸组份的滞留时间。
12.4利用DSF测定蛋白分子的热稳定性
差示扫描荧光法(Differential Scanning Fluorimetry,DSF)是一种常用的高通量的用来测定蛋白质热稳定性的方法。它使用实时荧光定量PCR仪器通过监测与去折叠的蛋白分子结合的染料的荧光强度的变化，来反映蛋白质的变性的过程，从而反映出蛋白分子的热稳定性。本实施例利用DSF方法来测定蛋白分子热变性温度(Tm)。10μg蛋白加入96-孔PCR板(Thermo,AB-0700/W)，接着加入2μl 100X稀释的染料SYPROTM(Invitrogen,2008138)，然后加入缓冲液使得终体积为40μl每孔。将PCR板密封，放置于实时荧光定量PCR仪器(Bio-Rad CFX96 PCR System)，先于25℃孵育5分钟，然后以0.2℃/0.2分钟的梯度逐渐从25℃升温至95℃，在测试结束时将温度降至25℃。使用FRET扫描模式并使用Bio-Rad CFX Maestro软件进行数据分析并计算出样品的Tm。
12.5融合蛋白的表达和表征分析
实施例12.1所得的融合蛋白的表达和物理化学性质见表19。结果显示，PR001488和PR001902比对照蛋白PR002466、PR001599有更高的产量，PR001488和PR001902比对照蛋白PR001599有更好的亲水性(体现为在HPLC-HIC上更短的滞留时间)。总体上，PR001488和PR001902表现出比PR001599更稳定的物理化学物质。
表19融合蛋白的表达和物理化学性质
Figure PCTCN2020118811-appb-000012
Figure PCTCN2020118811-appb-000013
实施例13融合蛋白结合过表达人PD-L1的CHO-K1细胞
按照实施例2的方法检测本申请所述融合蛋白在体外结合人PD-L1的活性。
图15(A)和表20-1以及图15(B)和表20-2显示了本申请所述融合蛋白均能结合过表达人PD-L1的CHO-K1细胞。
表20-1结合细胞表面的人PD-L1
融合蛋白 MFI最大值 EC50(nM)
atezolizumab 50075 0.3785
PR001487 52996 0.3137
PR001488 51536 0.5311
PR001598 42395 0.5069
PR001599 46184 0.7181
PR001901 46836 0.4884
PR001902 45373 0.5147
PR000416 40359 0.2585
表20-2结合细胞表面的人PD-L1
融合蛋白 MFI最大值 EC50(nM)
PR002247 43412 0.8518
PR002248 43291 0.9836
PR002249 41931 0.5844
PR002251 41242 0.5012
PR000416 37937 0.2313
实施例14融合蛋白阻断人PD-1与过表达人PD-L1的CHO-K1细胞的结合
按照实施例4的方法检测本申请所述融合蛋白体外阻断人PD-1与人PD-L1结合的活性。
图16(A)和表21-1以及图16(B)和表21-2显示了本申请所述融合蛋白均能阻断人PD-1与过表达人PD-L1的CHO-K1细胞的结合。
表21-1阻断人PD-1与细胞表面的人PD-L1结合
融合蛋白 IC50(nM)
PR001487 0.2157
PR001488 0.2661
PR001598 0.4416
PR001599 0.3225
PR001901 0.2598
PR001902 0.3107
PR000416 0.1271
表21-2阻断人PD-1与细胞表面的人PD-L1结合
融合蛋白 IC50(nM)
PR002247 0.3990
PR002248 0.4178
PR002249 0.3999
PR002251 0.3166
PR000416 0.1904
实施例15利用报告基因细胞系检测融合蛋白对PD-1信号通路的抑制作用
按照实施例6的方法利用报告基因细胞系检测本申请所述的融合蛋白对PD-1信号通路的抑制作用。图17和表22显示了本申请所述融合蛋白对PD-1信号通路有抑制作用。
表22对PD-1信号通路的抑制作用
融合蛋白 最大荧光值(RLU) EC50(nM)
PR000416 3656 0.077
PR001598 3786 0.068
PR001599 3626 0.091
PR001487 3600 0.056
PR001488 3549 0.049
实施例16酶联免疫吸附实验法检测融合蛋白体外结合TGFB1的作用
以1μg/ml浓度的人源TGFB1(Novoprotein,CA59)为抗原，按100μl/孔包被96孔板，4℃过夜。200μl/孔1×PBST洗涤3次，加入200μl/孔2％BSA，37℃封闭2小时。加入200μl/孔1xPBST洗涤3次，加入5倍浓度梯度稀释的抗体，其中抗体的最高终浓度为100nM，37℃孵育1小时。200μl/孔1×PBST洗涤3次，每孔加入100μl/孔抗人FC-HRP二抗(Sigma-Aldrich,A0170,1:4000稀释)，37℃孵育1小时。200μl/孔1×PBST洗涤3次，每孔再加入100μl/孔TMB，室温孵育10分钟后加入50μl/孔1M ELISA终止液停止反应。酶标仪上检测450nm及570nm测吸收值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC50值等参数。其中，TGFBRII-Fc是TGFBRII胞外区融合蛋白(Novoprotein,CC10)，PR001599，PR002040为前述对照融合蛋白分子。
图18(A)和表23-1以及图18(B)和表23-2以及图18(C)和表23-3显示了本申请所述融合蛋白均能够有效地与TGFB1结合，并且有药物浓度剂量依赖效应。
表23-1体外结合TGFB1
融合蛋白 OD最大值 EC50(nM)
TGFBRII-Fc 1.798 0.783
PR001487 1.742 1.081
PR001488 1.776 1.254
PR001599 1.815 1.546
PR001901 1.802 1.294
PR001902 1.839 1.492
表23-2体外结合TGFB1
融合蛋白 OD最大值 EC50(nM)
TGFBRII-Fc 1.626 1.132
PR002040 1.627 1.369
PR002247 1.560 1.330
表23-3体外结合TGFB1
融合蛋白 OD最大值 EC50(nM)
TGFBRII-Fc 1.585 1.115
PR002040 1.638 1.281
PR002248 1.515 1.287
PR002249 1.520 1.426
PR002251 1.526 1.195
实施例17融合蛋白对TGFB1诱导Smad信号通路激活的抑制作用
该实验使用高表达人源TGFBRI及融合SEAP报告基因的Smad3/4基因的HEK-Blue TM TGFB细胞(Invivogen，hkb-tgfb,)，在细胞水平上研究了融合蛋白对TGFB1诱导的Smad信号通路的激活的抑制作用。加入10μl/孔，5倍浓度梯度稀释的待测融合蛋白于96孔板中，最高终浓度为50nM。随后，加入10μl/孔，终浓度为1ng/ml的人TGFB1蛋白(R&D Systems，240-B/F)于96孔板中。以TGFB1抗体(Biointron，B5484)和TGFBRII-Fc融合蛋白(Novoprotein，CC10)为对照分子。将180μl，2.5×10 4细胞/孔接种于96孔板中，在二氧化碳培养箱中继续孵育过夜。然后每孔移取20μl细胞上清入另一块96孔板中，然后每孔加入180μl Quanti-Blue工作液(Invivogen，rep-qb1)，37℃下孵育1小时。酶标仪上检测655nm测吸收值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及IC50值等参数。
结果如图19和表24所示，融合蛋白以剂量依赖性形式抑制TGFB1诱导的pSMAD3报 告物活性。PR001902与阳性对照PR001599(M7824)具有相当的功效和IC50；其抑制活性和IC50较阳性对照PR002466(Hengrui融合蛋白9)及TGFB抗体强8倍。
表24对TGFB1诱导Smad信号通路激活的抑制作用
融合蛋白 最大抑制率(％) IC50(nM)
PR002040 111.2 0.0093
PR001599 105.2 0.0090
PR002466 105.2 0.0769
PR001902 112.5 0.0103
TGFBRII-Fc 103.3 0.1402
TGFB1抗体 111.5 0.0876
实施例18利用BLI测定融合蛋白与人PD-L1的亲和力的结果
使用实施例7.2所述方法测试融合蛋白与人PD-L1蛋白的亲和力。
结果如表25显示，本申请的融合蛋白与人PD-L1的亲和力K D值都小于1×10 -8M。
表25融合蛋白与人PD-L1的亲和力
融合蛋白 K D(M) K D误差 k on(1/Ms) k on误差 k dis(1/s) k dis误差 抗原浓度(nM)
PR001488 1.50E-10 1.21E-12 8.07E+05 2.44E+03 1.21E-04 9.06E-07 25-1.563
PR001599 2.82E-10 2.47E-12 5.42E+05 2.16E+03 1.53E-04 1.19E-06 25-1.563
PR001902 6.05E-11 2.40E-12 7.44E+05 5.06E+03 4.50E-05 1.76E-06 12.5-0.78
PR002249 5.95E-11 1.13E-12 1.07E+06 4.36E+03 6.37E-05 1.18E-06 12.5-0.78
PR002251 9.74E-11 1.17E-12 1.17E+06 4.95E+03 1.14E-04 1.28E-06 12.5-0.78
PR002466 7.16E-11 1.18E-12 1.28E+06 7.43E+03 9.16E-05 1.41E-06 6.25-0.39
实施例19利用BLI方法测定融合蛋白与人TGFB1的亲和力
首先将人TGFB1蛋白(NovoProtein，CA59)先利用生物素化试剂盒(EZ-Link Sulfo-NHS-LC-Biotin，ThermoFisher，A39257)按照说明书要求进行生物素化。然后按照类似实施例7.2 的所述方法，测定融合蛋白与人TGFB1的亲和力。特别地，先将两列SA传感器(每列放置8个传感器；第一列称为参照SA传感器，第二列称为测试SA传感器)在含有0.02％吐温的1×PBS的测试缓冲液中平衡10分钟。然后，测试SA传感器捕获生物素化的人TGFB1，设置捕获高度为0.3nm，而参照SA传感器浸入缓冲液中30秒。两列传感器再与梯度稀释的待测融合蛋白(例如，待测融合蛋白浓度可以为6.25-0.39nM的两倍梯度稀释以及0nM)结合；结合3分钟，然后解离10分钟。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，选择双扣除模式(double reference)扣除参照信号，选择“1:1Global fitting”方法进行数据拟合，计算出抗原与抗原结合蛋白结合的动力学参数，得到k on(1/Ms)值、k dis(1/s)值和K D(M)值。
结果如表26显示，本申请融合蛋白与人TGFB1的K D值小于1×10 9M，且比对照分子PR001599和PR002466显示出更高的亲和力。
表26与人TGFB1的亲和力
融合蛋白 K D(M) k on(1/Ms) k dis(1/s)
PR001599 1.04E-10 4.10E+06 4.24E-04
PR001902 7.81E-11 3.19E+06 2.50E-04
PR002466 1.28E-10 4.48E+06 5.73E-04
实施例20利用BLI方法测定融合蛋白结合PD-L1的表位竞争
按照实施例8的方法对融合蛋白和对照分子进行表位竞争实验。结果如表27-1和表27-2所示，PR001488和PR001902与对照分子PR001599或PR002466的表位不完全重叠。
表27-1融合蛋白的表位竞争
Figure PCTCN2020118811-appb-000014
表27-2融合蛋白的表位竞争
Figure PCTCN2020118811-appb-000015
实施例21MLR法体外检测融合蛋白对T细胞的激活作用
为了研究PD-L1/TGFB融合蛋白对T细胞的协同激活作用，收集和纯化人外周血单核细胞(PBMC)，分离单核细胞(Meltenyi，130-050-201)，采用完全培养基(RPI1640含50ng/mL IL-4,100ng/mL GM-CSF)培养6天，再加入1μg/ml LPS诱导24h，生成成熟的DC细胞。收集上述培养的细胞离心，用含2％FBS的PBS洗4次，重悬至新鲜的培养基中，调整密度为2x10 5细胞/mL，100μl/孔接种至96孔细胞培养板。同时，自异体新鲜PBMC中分离Pan T细胞(Meltenyi，130-096-535)新鲜培养基重悬，调整细胞密度为1×10 6细胞/ml，100μl/孔接种至96孔细胞培养板。将不同浓度的抗体50μl/孔，及终浓度为0.3ng/ml的人TGFB1蛋白(R&D Systems，240-B/F)以50μl/孔分别加入上述96孔细胞培养板的对应孔中，并将细胞培养板于37℃，5％CO 2培养箱孵育5天。分别收集72小时后和120小时后的上清液。采用IL2ELISA试剂盒(Thermo,88-7025-88)检测72小时后上清液中的IL-2的水平；采用IFN-γELISA试剂盒(Thermo,88-7316-88)检测120小时后上清液中的IFN-γ的水平；具体操作参考试剂说明书。
结果如图20所示，在外加TGFB1的条件下，融合蛋白能够增强激活的T淋巴细胞分泌细胞因子IL-2(图20(A))和IFN-γ(图20(B))，并且具有比PD-L1单抗PR000416和阳性对照PR001599和PR002446更强的激活作用。
实施例22肿瘤细胞介导的抗体依赖的细胞毒性实验的结果
按照实施例10的方法检测本申请所述融合蛋白的肿瘤细胞介导的抗体依赖的细胞毒性。本实施例使用了三个供体的PBMC和两个肿瘤细胞系，共六个独立ADCC实验。结果如图21(A-F)所示，PR001488比阳性对照PR001599有更强的ADCC作用。
实施例23融合蛋白的药代动力学研究
本实施例测试融合蛋白的药代动力学性能。其方法如下，选取体重18～22克的雌性C57BL/6小鼠3只，按9.53mg/kg PR001902和10mg/kg PR001488的剂量通过静脉注射给与融合蛋白药物；于给药前以及给药后0.5小时、24小时(1天)、第2天、第4天、第7天、第10天和第14天采集全血，将全血静置30分钟使其凝固，随后在4℃下以2,000rpm离心5分钟并将分离的血清样品在-80℃下冻存直至分析。本实施例采用两种ELISA方法来定量测定小鼠血清中的药物浓度。ELISA方法一，即Fc端检测方法，通过包被于96孔板的山羊抗人Fc多克隆抗体来捕获小鼠血清中的含有人Fc的融合蛋白，然后加入HRP标记的山羊抗人Fc第二抗体来检测；ELISA方法二，即TGFBRII端检测方法，通过包被于96孔板的人TGFB1 蛋白来捕获小鼠血清中的含有人TGFBRII结构域的融合蛋白，然后加入HRP标记的的山羊抗人Fc第二抗体来检测。使用Phoenix WinNonlin软件6.4版，选用非房室模型(NCA)对血药浓度数据进行分析以评价其药代动力学。
图22和表28显示的是融合蛋白PR001902的药代动力学数据，结果表明，Fc端检测方法下，PR001902在小鼠体内的半衰期约为11天；TGFBRII端检测方法显示，PR001902在小鼠体内的半衰期约为8.5天。
表28 PR001902的药代动力学
Figure PCTCN2020118811-appb-000016
图23和表29显示的是融合蛋白PR001488的药代动力学数据，结果表明，Fc端检测方法下，PR001488在小鼠体内的半衰期约为10天；TGFBRII端检测方法显示，PR001488在小鼠体内的半衰期约为5天。
表29 PR001488的药代动力学
Figure PCTCN2020118811-appb-000017
实施例24融合蛋白对结肠癌CT26-人PD-1/PD-L1转基因小鼠的动物体内模型的抗肿 瘤活性
本实施例评价测试药物在小鼠结肠癌细胞CT26-人PDL1(tg)-小鼠PDL1(KO)(将小鼠结肠癌细胞CT26的内源性鼠PD-L1基因敲除并引入外源人PD-L1转基因，集萃药康生物科技)移植免疫检查点人源化小鼠BALB/c-人PD-1/PD-L1(将BALB/c小鼠同时引入外源人PD-1转基因和人PD-L1转基因，集萃药康生物科技)中的抗肿瘤作用。接种对数生长期的CT26-人PDL1(tg)-mPDL1(KO)细胞于人源化小鼠BALB/c-人PD-1/PD-L1腋右侧背部皮下，当平均肿瘤体积达到80-120mm 3时，去除肿瘤体积过大的小鼠个体后，小鼠根据肿瘤体积随机分成3组，每组6只。共3个测试组，分别为：人IgG1(10.4mg/kg，人IgG1同型对照组)、M7824(12.3mg/kg，阳性对照)和PR001902(12.2mg/kg，本申请融合蛋白)。分组当天开始腹腔给药，每三天给一次药，共给药6次(q3d×6)。开始给药后，每周称量体重及瘤体积两次，瘤体积计算方式为：肿瘤体积(mm 3)＝0.5×肿瘤长径×肿瘤短径 2。实验结束后将荷瘤小鼠安乐死并剥瘤称重。计算各组动物的肿瘤体积、小鼠体重等实验结果以平均值±标准误差(Mean±SEM)表示。多组比较采用单因素方差分析(one way ANOVA)检验方法比较不同治疗组与对照组相比有无显著性差异。数据使用SPSS 18.0进行分析。P<0.05为具有显著性差异。
结果如图24显示，融合蛋白PR001902与阳性对照M7824均能显著抑制CT26-人PD-1/PD-L1小鼠皮下移植瘤的生长；且在实验过程中，各组动物体重均出现增长，表明动物对受试品耐受良好，未对动物产生明显毒性作用，安全性好。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (39)

  1. 结合PD-L1的分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:193所示的氨基酸序列；优选地，所述VH包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含抗体轻链可变区VL中的至少一个CDR，所述VL包含SEQ ID NO:194所示的氨基酸序列；优选地，所述VL包含SEQ ID NOs:108-114、116和118中任一项所示的氨基酸序列。
  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；优选地，所述抗体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；优选地，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv并且/或者dAb。
  4. 根据权利要求1-3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和重链可变区VH，其中所述轻链可变区VL包含LCDR1、LCDR2和LCDR3，所述LCDR1包含如SEQ ID NO:181所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:182所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:191所示的氨基酸序列；其中重链可变区VH包含HCDR1、HCDR2和HCDR3，所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:179所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:180所示的氨基酸序列。
  5. 根据权利要求4所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NOs:181、48-51中任一项所示的氨基酸序列；所述LCDR2包含SEQ ID NOs:182、63-64中任一项所示的氨基酸序列；所述LCDR3包含SEQ ID NOs:191、77-79中任一项所示的氨基酸序列；HCDR1包含如SEQ ID NO:5所示的氨基酸序列；HCDR2包含SEQ ID NOs:179、14、16和17中任一项所示的氨基酸序列；HCDR3包含SEQ ID NOs:180、29-33中任一项所示的氨基酸序列。
  6. 根据权利要求5所述的分离的抗原结合蛋白，其中所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列；所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列；所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列；所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列；所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列；所述HCDR3包含如SEQ ID NO:29所示；或者，
    所述LCDR1包含如SEQ ID NO:49所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:30所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:79所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:31所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:32所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:14所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:33所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:30所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:48所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序 列且所述HCDR3包含如SEQ ID NO:30所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:32所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:63所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:32所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:51所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:78所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:29所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:16所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:33所示的氨基酸序列；或者，
    所述LCDR1包含如SEQ ID NO:50所示的氨基酸序列、所述LCDR2包含如SEQ ID NO:64所示的氨基酸序列、所述LCDR3包含如SEQ ID NO:77所示的氨基酸序列、所述HCDR1包含如SEQ ID NO:5所示的氨基酸序列、所述HCDR2包含如SEQ ID NO:17所示的氨基酸序列且所述HCDR3包含如SEQ ID NO:33所示的氨基酸序列。
  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，其中所述抗体重链可变区VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4；优选地，所述H-FR1的C末端与所述HCDR1的N末端相连，包含SEQ ID NO:1所示的氨基酸序列；并且/或者所述H-FR2位于所述HCDR1与所述HCDR2之间，包含SEQ ID NO:9所示的氨基酸序列；并且/或者所述H-FR3位于所述HCDR2与所述HCDR3之间，包含SEQ ID NO:192所示的氨基酸序列；并且/或者所述H-FR4的N末端与所述HCDR3的C末端相连，包含SEQ ID NO:38所示的氨基酸 序列；进一步优选地，所述H-FR3包含SEQ ID NOs:21、22和24中任一项所示的氨基酸序列。
  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，其中所述抗体轻链可变区VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4；优选地，所述L-FR1的C末端与所述LCDR1的N末端相连，包含SEQ ID NO:185所示的氨基酸序列；并且/或者所述L-FR2位于所述LCDR1与所述LCDR2之间，包含SEQ ID NO:186所示的氨基酸序列；并且/或者所述L-FR3位于所述LCDR2与所述LCDR3之间，包含SEQ ID NO:187所示的氨基酸序列；并且/或者所述L-FR4的N末端与所述LCDR3的C末端相连，包含SEQ ID NO:188所示的氨基酸序列。
  9. 根据权利要求8所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NOs:41-44中任一项所示的氨基酸序列；并且/或者所述L-FR2包含SEQ ID NOs:56-59中任一项所示的氨基酸序列；并且/或者所述L-FR3包含SEQ ID NOs:69-72中任一项所示的氨基酸序列；并且/或者所述L-FR4包含SEQ ID NOs:85-86中任一项所示的氨基酸序列。
  10. 根据权利要求1-9任一项所述的分离的抗原结合蛋白，所述抗体重链可变区VH包含SEQ ID NO:193所示的氨基酸序列；所述抗体轻链可变区VL包含SEQ ID NO:194所示的氨基酸序列；
    优选地，所述抗体重链可变区VH包含SEQ ID NOs:90-93、95和97-104中任一项所示的氨基酸序列；
    优选地，所述抗体轻链可变区VL包含SEQ ID NOs:108-114、116和118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，所述抗体重链可变区VH包含SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包括SEQ ID NO:108所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:109所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:91所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:110所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:111所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:92所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:112所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:93所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:113所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:90所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:114所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:95所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:116所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:97所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:118所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:98所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:118所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:99所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:113所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:100所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:113所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:101所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:114所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:102所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:114所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:103所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:116所示的氨基酸序列；或者，
    所述抗体重链可变区VH包含如SEQ ID NO:104所示的氨基酸序列，所述抗体轻链可变区VL包含如SEQ ID NO:116所示的氨基酸序列。
  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，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区；优选地，其中所述抗体重链恒定区包含如SEQ ID NOs:172-175中任一项所示的氨基酸序列。
  13. 根据权利要求1-12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包含如SEQ ID NOs:122-125、127和129-137中任一项所示的氨基酸序列。
  14. 根据权利要求1-13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，其中所述抗体轻链恒定区包含如SEQ ID NO:170所示的氨基酸序列。
  15. 根据权利要求1-14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包含如SEQ ID NOs:150-156、158和160-163中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的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-L1；
    2)能够阻断PD-1与PD-L1的结合；
    3)能够阻断CD80与PD-L1的结合；
    4)能够刺激免疫细胞中IFN-γ并且/或者IL2的分泌；
    5)能够抑制肿瘤生长并且/或者肿瘤细胞增殖；
    6)与对照抗体相比，结合与源自灵长类动物的PD-L1的不完全重叠的表位，
    其中所述对照抗体包含SEQ ID NO:52所示的LCDR1、SEQ ID NO:65所示的LCDR2和SEQ ID NO:81所示的LCDR3，且所述对照抗体包含SEQ ID NO:6所示的HCDR1、SEQ ID NO:15所示的HCDR2和SEQ ID NO:34所示的HCDR3；
    或者，
    其中所述对照抗体包含SEQ ID NO:53所示的LCDR1、SEQ ID NO:66所示的LCDR2和SEQ ID NO:82所示的LCDR3，且所述对照抗体包含SEQ ID NO:5所示的HCDR1、SEQ ID NO:18所示的HCDR2和SEQ ID NO:35所示的HCDR3；
    或者，
    所述对照抗体包含SEQ ID NO:55所示的LCDR1、SEQ ID NO:68所示的LCDR2和SEQ ID NO:84所示的LCDR3，且所述对照抗体包含SEQ ID NO:8所示的HCDR1、SEQ ID NO:20所示的HCDR2和SEQ ID NO:37所示的HCDR3。
  17. 根据权利要求16所述的分离的抗原结合蛋白，其中所述灵长类动物包括人并且/或者猴。
  18. 一种融合蛋白，其包含：a)人TGFBRII或其片段；以及b)权利要求1-17中任一项所述的分离的抗原结合蛋白。
  19. 根据权利要求18所述的融合蛋白，其包含第一多肽和第二多肽，其中所述第一多肽包含所述分离的抗原结合蛋白的重链或其片段以及所述人TGFBRII或其片段；且所述第二多肽包含所述分离的抗原结合蛋白的轻链或其片段；优选地，所述抗体重链或其片段与所述人TGFBRII或其片段框内融合而形成所述第一多肽；进一步优选地，其中所述抗体重链或其片段的C端直接或间接与所述人TGFBRII或其片段的N端连接。
  20. 根据权利要求18或19所述的融合蛋白，其中第一多肽的所述重链或其片段包括HCDR1、HCDR2和HCDR3，HCDR1包含SEQ ID NO:5所示的氨基酸序列；HCDR2包含SEQ ID NOs:179、14、16和17中任一项所示的氨基酸序列；HCDR3包含SEQ ID NOs:180、29-33中任一项所示的氨基酸序列；并且/或者所述第二多肽包括LCDR1、LCDR2和LCDR3， LCDR1包含SEQ ID NOs:181、48-51中任一项所示的氨基酸序列；LCDR2包含SEQ ID NOs:182、63-64中任一项所示的氨基酸序列；LCDR3包含SEQ ID NOs:191、77-79中任一项所示的氨基酸序列。
  21. 根据权利要求20所述的融合蛋白，其中，
    LCDR1包含如SEQ ID NO：51所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：78所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：16所示的氨基酸序列，HCDR3包含如SEQ ID NO：29所示的氨基酸序列；或者，
    LCDR1包含如SEQ ID NO：50所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：77所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：16所示的氨基酸序列，HCDR3包含如SEQ ID NO：33所示的氨基酸序列；或者，
    LCDR1包含如SEQ ID NO：50所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：77所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：17所示的氨基酸序列，HCDR3包含如SEQ ID NO：33所示的氨基酸序列；或者，
    LCDR1包含如SEQ ID NO：51所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：78所示的氨基酸序列；HCDR1包含SEQ ID NO：5所示的氨基酸序列，HCDR2包含SEQ ID NO：17所示的氨基酸序列，HCDR3包含如SEQ ID NO：29所示的氨基酸序列；或者，
    LCDR1包含如SEQ ID NO：51所示的氨基酸序列，LCDR2包含如SEQ ID NO：64所示的氨基酸序列，LCDR3包含如SEQ ID NO：78所示的氨基酸序列；HCDR1包含如SEQ ID NO：5所示的氨基酸序列，HCDR2包含如SEQ ID NO：14所示的氨基酸序列，HCDR3包含如SEQ ID NO：29所示的氨基酸序列。
  22. 根据权利要求18-21任一项所述的融合蛋白，其中所述抗体重链或其片段通过连接子与所述人TGFBRII或其片段相连；优选地，其中所述连接子为肽连接子，且所述肽连接子包含SEQ ID NOs:167-169中任一项所示的氨基酸序列。
  23. 根据权利要求18-22任一项所述的融合蛋白，其中所述人TGFBRII或其片段包含人TGFBRII的胞外结构域；优选地，所述人TGFBRII或其片段包含SEQ ID NOs:176-178中任一项所示的氨基酸序列。
  24. 根据权利要求18-23任一项所述的融合蛋白，其中所述第一多肽的所述重链或其片段与所 述第二多肽的所述轻链或其片段组合后形成特异性结合PD-L1的抗原结合部分。
  25. 根据权利要求18-24任一项所述的融合蛋白，其中所述第一多肽包含SEQ ID NOs:138、139、142、143和145-148中任一项所示的氨基酸序列；其中所述第二多肽包含SEQ ID NOs:162-163中任一项所示的氨基酸序列。
  26. 根据权利要求18-25任一项所述的融合蛋白，其中，
    所述第一多肽包含如SEQ ID NO:138所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
    所述第一多肽包含SEQ ID NO:139所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
    所述第一多肽包含SEQ ID NO:142所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
    所述第一多肽包含SEQ ID NO:143所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
    所述第一多肽包含SEQ ID NO:145所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:163所示的氨基酸序列；或者，
    所述第一多肽包含SEQ ID NO:146所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:163所示的氨基酸序列；或者，
    所述第一多肽包含SEQ ID NO:147所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列；或者，
    所述第一多肽包含SEQ ID NO:148所示的氨基酸序列，且所述第二多肽包含SEQ ID NO:162所示的氨基酸序列。
  27. 分离的一种或多种核酸分子，其编码权利要求1-17中任一项所述的分离的抗原结合蛋白或权利要求18-26中任一项所述的融合蛋白。
  28. 载体，其包含根据权利要求27所述的核酸分子。
  29. 细胞，其包含根据权利要求27所述的核酸分子或根据权利要求28所述的载体。
  30. 制备权利要求1-17中任一项所述的分离的抗原结合蛋白或权利要求18-26中任一项所述的融合蛋白的方法，所述方法包括在使得权利要求1-17中任一项所述的分离的抗原结合蛋白权利要求18-26中任一项所述的融合蛋白表达的条件下，培养根据权利要求29所述的细胞。
  31. 嵌合抗原受体，其包含权利要求1-17中任一项所述的分离的抗原结合蛋白。
  32. 基因修饰的细胞，其包含权利要求31所述的嵌合抗原受体。
  33. 抗体药物偶联物，其包含细胞毒性剂，以及权利要求1-17中任一项所述的分离的抗原结合蛋白。
  34. 药物组合物，其包含权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-26中任一项所述的融合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞、权利要求31所述的嵌合抗原受体、权利要求32所述的基因修饰的细胞和/或权利要求33所述的抗体药物偶联物，以及任选地药学上可接受的载体；较佳地，所述药物组合物还含有由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  35. 权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-26中任一项所述的融合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞、权利要求31所述的嵌合抗原受体、权利要求32所述的基因修饰的细胞和/或权利要求33所述的抗体药物偶联物和/或权利要求34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、缓解并且/或者治疗肿瘤或癌症，抑制肿瘤生长并且/或者抑制肿瘤细胞增殖；优选地，所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症；优选地，所述肿瘤或癌症包括结直肠癌。
  36. 预防、缓解或治疗肿瘤，抑制肿瘤生长并且/或者抑制肿瘤细胞增殖的方法，所述方法包括向有需要的受试者施用权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-26中任一项所述的融合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞、权利要求31所述的嵌合抗原受体、权利要求32所述的基因修饰的细胞和/或权利要求33所述的抗体药物偶联物和/或权利要求34所述的药物组合物；优选地，其中所述肿瘤或癌症为PD-L1表达异常的肿瘤或癌症；优选地，所述肿瘤或癌症包括结直肠癌。
  37. 权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-26中任一项所述的融合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞、权利要求31所述的嵌合抗原受体、权利要求32所述的基因修饰的细胞和/或权利要求33所述的抗体药物偶联物和/或权利要求34所述的药物组合物，其用于治疗癌症，抑制肿瘤生长并且/或者抑制肿瘤细胞增殖。
  38. 抑制PD-L1和或CD80与PD-1结合的方法，所述方法包括施用权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-26中任一项所述的融合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞、权利要求31所述的 嵌合抗原受体、权利要求32所述的基因修饰的细胞和/或权利要求33所述的抗体药物偶联物和/或权利要求34所述的药物组合物。
  39. 试剂盒，其包括权利要求1-17中任一项所述的分离的抗原结合蛋白、权利要求18-26中任一项所述的融合蛋白、权利要求27所述的核酸分子、权利要求28所述的载体、权利要求29所述的细胞、权利要求31所述的嵌合抗原受体、权利要求32所述的基因修饰的细胞和/或权利要求33所述的抗体药物偶联物和/或权利要求34所述的药物组合物；优选地，所述试剂盒还包括(i)施用装置；和/或(ii)使用说明。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023109847A1 (zh) * 2021-12-15 2023-06-22 上海华奥泰生物药业股份有限公司 Pd-l1抗原结合蛋白及其用途

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3228311A1 (en) * 2021-08-12 2023-02-16 Ke HE Bispecific recombinant protein and use thereof
CN115925904B (zh) * 2022-08-24 2023-06-16 南京市第二医院 一种新冠病毒单克隆中和抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104961829A (zh) * 2009-11-24 2015-10-07 米迪缪尼有限公司 针对b7-h1的靶向结合剂
WO2016162368A1 (en) * 2015-04-07 2016-10-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Non-invasive imaging of tumor pd-l1 expression
CN106103488A (zh) * 2014-02-10 2016-11-09 默克专利有限公司 靶向TGFβ抑制
CN106939047A (zh) * 2016-01-04 2017-07-11 钜川生物医药 一种pd-l1抗体及其制备方法
WO2018205985A1 (zh) 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途
US20190169621A1 (en) * 2012-04-30 2019-06-06 Biocon Limited Targeted/immunomodulatory fusion proteins in combination with additional therapeutic agents and methods for making same
US20190218296A1 (en) * 2018-01-17 2019-07-18 Apexigen, Inc. Anti-pd-l1 antibodies and methods of use

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2881679C (en) * 2012-08-13 2021-01-26 Regeneron Pharmaceuticals, Inc. Anti-pcsk9 antibodies with ph-dependent binding characteristics
CN107892719B (zh) * 2012-10-04 2022-01-14 达纳－法伯癌症研究所公司 人单克隆抗-pd-l1抗体和使用方法
TWI680138B (zh) * 2014-01-23 2019-12-21 美商再生元醫藥公司 抗ｐｄ-ｌ１之人類抗體
US10435470B2 (en) * 2014-08-05 2019-10-08 Cb Therapeutics, Inc. Anti-PD-L1 antibodies
CN109721657B (zh) * 2017-10-27 2021-11-02 北京比洋生物技术有限公司 阻断pd-1/pd-l1信号传导途径且活化t细胞的融合蛋白及其用途
EP3781265A2 (en) * 2018-04-17 2021-02-24 Celldex Therapeutics, Inc. Anti-cd27 and anti-pd-l1 antibodies and bispecific constructs
CN112574314A (zh) * 2019-09-30 2021-03-30 和铂医药(苏州)有限公司 一种融合蛋白及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104961829A (zh) * 2009-11-24 2015-10-07 米迪缪尼有限公司 针对b7-h1的靶向结合剂
US20190169621A1 (en) * 2012-04-30 2019-06-06 Biocon Limited Targeted/immunomodulatory fusion proteins in combination with additional therapeutic agents and methods for making same
CN106103488A (zh) * 2014-02-10 2016-11-09 默克专利有限公司 靶向TGFβ抑制
WO2016162368A1 (en) * 2015-04-07 2016-10-13 INSERM (Institut National de la Santé et de la Recherche Médicale) Non-invasive imaging of tumor pd-l1 expression
CN106939047A (zh) * 2016-01-04 2017-07-11 钜川生物医药 一种pd-l1抗体及其制备方法
WO2018205985A1 (zh) 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 含有TGF-β受体的融合蛋白及其医药用途
US20190218296A1 (en) * 2018-01-17 2019-07-18 Apexigen, Inc. Anti-pd-l1 antibodies and methods of use

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
"Epitope Mapping Protocols in Methods in Molecular Biology", vol. 66, 1996
"NCBI", Database accession no. NP -001271065
"Uniprot", Database accession no. P37173
AL-LAZIKANI ET AL., J MOL BIOL, vol. 273, 1997, pages 927 - 48
AUSUBE ET AL.: "Current Protocols in Molecular Biology", 1993, GREENE PUBLISHING AND WILEY-INTERSCIENCE
CARTER ET AL.: "PD-1: PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2", EUR. J. IMMUNOL., vol. 32, 2002, pages 634 - 643, XP002980901, DOI: 10.1002/1521-4141(200203)32:3<634::AID-IMMU634>3.0.CO;2-9
CUNNINGHAMWELLS, SCIENCE, vol. 244, 1985, pages 1081
D. J. LIPMANW. R. PEARSON: "Rapid and sensitive protein similarity searches", SCIENCE, vol. 227, 1989, pages 1435 - 1441
FREEMAN ET AL.: "Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation", J. EXP.MED., vol. 192, 2000, pages 1027 - 1034, XP002942545, DOI: 10.1084/jem.192.7.1027
ISHIDA ET AL., EMBO J, vol. 11, 1992, pages 3887 - 3395
KABAT ET AL.: "Protein Sequences of Immunology", 1991, NATIONAL INSTITUTES OF HEALTH
LUAN Y. ET AL.: "A fully human monoclonal antibody targeting PD-L1 with potent anti-tumor activity", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 31, 12 January 2016 (2016-01-12), XP029412996, DOI: 10.1016/j.intimp.2015.12.039 *
OKAZAKI ET AL.: "Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice", SCIENCE, vol. 291, 2001, pages 319 - 22
S. ALTSCHULW. GISHW. MILLERE. W. MYERSD. LIPMAN: "A basic local alignment search tool", J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
SAMBROOK J.FRITSCH, E. F.MANIAIS, T.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
VALECHA G. K.: "Anti–PD-1/PD-L1 antibodies in non-small cell lung cancer: the era of immunotherapy", EXPERT REVIEW OF ANTICANCER THERAPY, vol. 17, no. 1, 23 November 2016 (2016-11-23), pages 47 - 59, XP055798118 *
W. R. PEARSOND. J. LIPMAN: "Improved tools for biological sequence comparison", PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 2444 - 2448

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023109847A1 (zh) * 2021-12-15 2023-06-22 上海华奥泰生物药业股份有限公司 Pd-l1抗原结合蛋白及其用途

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