WO2022012639A1 - Pd-1抗原结合蛋白及其应用 - Google Patents

Pd-1抗原结合蛋白及其应用 Download PDF

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WO2022012639A1
WO2022012639A1 PCT/CN2021/106569 CN2021106569W WO2022012639A1 WO 2022012639 A1 WO2022012639 A1 WO 2022012639A1 CN 2021106569 W CN2021106569 W CN 2021106569W WO 2022012639 A1 WO2022012639 A1 WO 2022012639A1
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seq
hcdr2
hcdr3
hcdr1
amino acid
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PCT/CN2021/106569
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English (en)
French (fr)
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罗海山
单茜茜
王永强
张雪琨
石磊
张云
赵楚楚
周伟
甘馨
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和铂医药(上海)有限责任公司
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Priority to US18/005,055 priority Critical patent/US20240034792A1/en
Priority to CN202180047991.7A priority patent/CN115803346A/zh
Publication of WO2022012639A1 publication Critical patent/WO2022012639A1/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
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61P35/00—Antineoplastic agents
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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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to the field of biomedicine, in particular to an antigen-binding protein that binds to PD-1 and its application.
  • Programmed death-1 is a type I membrane protein with 288 amino acids, mainly expressed on the surface of activated T cells.
  • PD-1 has two ligands, namely programmed death ligand-1 (Programmed Death Ligand-1, PD-L1) and PD-L2.
  • the interaction of PD-1 with PD-L1 and PD-L2 can down-regulate the activity of T cells, weaken the secretion of cytokines, and play an immunosuppressive effect.
  • PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, restore the activity of T cells, and play a role in killing tumor cells, thereby inhibiting tumor growth. Therefore, immunomodulation targeting PD-1/PD-L1 has important implications for tumor suppression.
  • CD73 (also called extracellular-5'-nucleotidase) is an enzyme that can decompose AMP (adenosine monophosphate) into adenosine, and adenosine produced by the decomposition of AMP inhibits immune system cells including T cells function.
  • AMP adenosine monophosphate
  • CD73 has been reported to be expressed on many different tumor cells, and CD73 expression correlates with tumor cell proliferation, migration, neovascularization, invasiveness, metastasis and shorter patient survival. Therefore, CD73 plays an important role in the regulation of immune responses. CD73 can regulate cancer progression in both direct and indirect ways, highlighting its potential as a novel therapeutic target.
  • anti-PD-1/PD-L1 pathway blocking antibody drugs still face many challenges in clinical practice, such as low efficacy, drug resistance and side effects.
  • Preclinical research results show that the combination of PD-1/PD-L1 immune checkpoint inhibitor and CD73 monoclonal antibody to kill tumor cells will have a strong synergistic effect.
  • Several clinical trials (NCT03454451, NCT03835949, and NCT02503774) are also being conducted using a combination of PD-1 or PD-L1 antibodies and CD73 monoclonal antibodies in cancer treatment to improve safety and efficacy.
  • the administration methods and clinical trials of combination drugs are complex and expensive, and two single drugs need to be possessed or developed at the same time, which is difficult and time-consuming.
  • the present application provides an antigen-binding protein capable of binding PD-1, which can block the binding of PD-1 to PD-L1 and PD-L2, stimulate the secretion of IFN- ⁇ and/or IL2 in immune cells, and inhibit the Tumor growth and/or tumor cell proliferation.
  • the present application also provides a heavy chain antibody containing only a "heavy chain", which has the activity of specifically binding to human PD-1 and cynomolgus monkey PD-1. The size of this PD-1 heavy chain antibody is only half of that of traditional IgG antibody. Due to the lack of light chain, this antibody can be used as a bispecific antibody and solves light chain mismatch and heterodimerization. The problem.
  • the present application also provides a fusion protein comprising a PD-1 binding part and a CD73 binding part, which can synergistically increase the secretion of IFN- ⁇ and/or IL2 in immune cells, and further synergistically inhibit tumor growth and/or tumor cell proliferation; At the same time, the fusion protein can recognize CD73 protein, inhibit the activity of CD73 enzymatic activity, and can induce the internalization of CD73 in tumor cells, resulting in further reduction of CD73 activity on the cell surface.
  • the application provides an isolated antigen-binding protein comprising an antibody heavy chain or fragment thereof, the antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises SEQ ID NO:359
  • the amino acid sequence shown the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:360
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:361.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, VHH, heavy chain antibody (HCAb) and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 11-16
  • the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 50-55
  • the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 92-97.
  • the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any of the following group:
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 50
  • HCDR3 SEQ ID NO: 92;
  • HCDR1 SEQ ID NO: 12
  • HCDR2 SEQ ID NO: 50
  • HCDR3 SEQ ID NO: 93;
  • HCDR1 SEQ ID NO: 13
  • HCDR2 SEQ ID NO: 51
  • HCDR3 SEQ ID NO: 94;
  • HCDR1 SEQ ID NO: 14
  • HCDR2 SEQ ID NO: 52
  • HCDR3 SEQ ID NO: 95;
  • HCDR1 SEQ ID NO: 15, HCDR2: SEQ ID NO: 53, and HCDR3: SEQ ID NO: 96;
  • HCDR1 SEQ ID NO: 16
  • HCDR2 SEQ ID NO: 54
  • HCDR3 SEQ ID NO: 97;
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 55
  • HCDR3 SEQ ID NO: 92.
  • the isolated antigen binding protein comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO:368.
  • the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 151-158.
  • the isolated antigen binding protein further comprises an antibody heavy chain constant region.
  • the heavy chain constant region is derived from a human IgG constant region.
  • the heavy chain constant region is derived from a human IgG4 constant region and/or a human IgGl constant region.
  • the heavy chain constant region comprises the amino acid sequence set forth in any one of SEQ ID Nos: 354-355.
  • the isolated antigen binding protein comprises an antibody heavy chain comprising the amino acid sequence set forth in any one of SEQ ID Nos: 238-245.
  • the isolated antigen binding protein comprises an antibody light chain or a fragment thereof, the antibody light chain or fragment thereof comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises SEQ ID NO:365
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:366
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:367.
  • the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 121-125
  • the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 130-135
  • the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 140-145.
  • the LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from any of the following groups:
  • LCDR1 SEQ ID NO: 121
  • LCDR2 SEQ ID NO: 130
  • LCDR3 SEQ ID NO: 140;
  • LCDR1 SEQ ID NO: 122
  • LCDR2 SEQ ID NO: 131
  • LCDR3 SEQ ID NO: 141;
  • LCDR1 SEQ ID NO: 123
  • LCDR2 SEQ ID NO: 132
  • LCDR3 SEQ ID NO: 142;
  • LCDR1 SEQ ID NO: 122
  • LCDR2 SEQ ID NO: 133
  • LCDR3 SEQ ID NO: 143;
  • LCDR1 SEQ ID NO: 124
  • LCDR2 SEQ ID NO: 134
  • LCDR3 SEQ ID NO: 144;
  • LCDR1 SEQ ID NO: 125
  • LCDR2 SEQ ID NO: 135
  • LCDR3 SEQ ID NO: 145.
  • the isolated antigen binding protein comprises an antibody light chain variable region VL, and the VL comprises the amino acid sequence set forth in SEQ ID NO:369.
  • the VL comprises the amino acid sequence set forth in any one of SEQ ID Nos: 231-236.
  • the isolated antigen binding protein further comprises an antibody light chain constant region.
  • the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO:353.
  • the isolated antigen binding protein comprises an antibody light chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 324-329.
  • the isolated antigen binding protein comprises an antibody heavy chain or fragment thereof comprising an antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, the antibody light chain or Fragments thereof comprise LCDR1, LCDR2 and LCDR3, wherein said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from any of the following groups:
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 50
  • HCDR3 SEQ ID NO: 92
  • LCDR1 SEQ ID NO: 121
  • LCDR2 SEQ ID NO: 130
  • LCDR3 SEQ ID NO: 121 :140;
  • HCDR1 SEQ ID NO: 12
  • HCDR2 SEQ ID NO: 50
  • HCDR3 SEQ ID NO: 93
  • LCDR1 SEQ ID NO: 122
  • LCDR2 SEQ ID NO: 131
  • LCDR3 SEQ ID NO: 122 :141;
  • HCDR1 SEQ ID NO: 13
  • HCDR2 SEQ ID NO: 51
  • HCDR3 SEQ ID NO: 94
  • LCDR1 SEQ ID NO: 123
  • LCDR2 SEQ ID NO: 132
  • LCDR3 SEQ ID NO: 123 :142;
  • HCDR1 SEQ ID NO: 14, HCDR2: SEQ ID NO: 52, and HCDR3: SEQ ID NO: 95, LCDR1: SEQ ID NO: 122, LCDR2: SEQ ID NO: 133, and LCDR3: SEQ ID NO: 122 :143;
  • HCDR1 SEQ ID NO: 15, HCDR2: SEQ ID NO: 53, and HCDR3: SEQ ID NO: 96, LCDR1: SEQ ID NO: 124, LCDR2: SEQ ID NO: 134, and LCDR3: SEQ ID NO: 124 :144;
  • HCDR1 SEQ ID NO: 16, HCDR2: SEQ ID NO: 54, and HCDR3: SEQ ID NO: 97, LCDR1: SEQ ID NO: 125, LCDR2: SEQ ID NO: 135, and LCDR3: SEQ ID NO: 125 :145; and,
  • HCDR1 SEQ ID NO: 11
  • HCDR2 SEQ ID NO: 55
  • HCDR3 SEQ ID NO: 92
  • LCDR1 SEQ ID NO: 121
  • LCDR2 SEQ ID NO: 130
  • LCDR3 SEQ ID NO: 130 :140.
  • the isolated antigen binding protein comprises an antibody heavy chain variable region VH and an antibody light chain variable region VL, and the VH and VL comprise amino acid sequences selected from any of the following groups:
  • VH SEQ ID NO: 151 and VL: SEQ ID NO: 231;
  • VH SEQ ID NO: 152 and VL: SEQ ID NO: 232;
  • VH SEQ ID NO: 153 and VL: SEQ ID NO: 233;
  • VH SEQ ID NO: 154 and VL: SEQ ID NO: 234;
  • VH SEQ ID NO: 155 and VL: SEQ ID NO: 235;
  • VH SEQ ID NO: 156 and VL: SEQ ID NO: 232;
  • VH SEQ ID NO: 157 and VL: SEQ ID NO: 236;
  • VH SEQ ID NO: 158 and VL: SEQ ID NO: 231.
  • the isolated antigen binding protein has one or more of the following properties:
  • the application provides an isolated antigen-binding protein comprising an antibody heavy chain or fragment thereof, the antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises SEQ ID NO: The amino acid sequence shown in 362, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:363, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:364.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, VHH, heavy chain antibody (HCAb) and/or dAb.
  • the antigen-binding fragment is a heavy chain antibody (HCAb). In certain embodiments, the antigen-binding fragment is a VHH.
  • the antigen-binding fragment is the antibody selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding fragment is that the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18-33, and the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 57-68 The amino acid sequence shown, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 99-101 and 103-111.
  • the antigen-binding fragment comprises an amino acid sequence selected from any of the following groups for the HCDR1, HCDR2 and HCDR3:
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 57, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 57, and HCDR3: SEQ ID NO: 99;
  • HCDR1 SEQ ID NO: 19
  • HCDR2 SEQ ID NO: 57
  • HCDR3 SEQ ID NO: 99;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 57, and HCDR3: SEQ ID NO: 101;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 58, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 59
  • HCDR3 SEQ ID NO: 103;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 60
  • HCDR3 SEQ ID NO: 103;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 61
  • HCDR3 SEQ ID NO: 103;
  • HCDR1 SEQ ID NO: 21, HCDR2: SEQ ID NO: 60, and HCDR3: SEQ ID NO: 103;
  • HCDR1 SEQ ID NO: 21, HCDR2: SEQ ID NO: 59, and HCDR3: SEQ ID NO: 103;
  • HCDR1 SEQ ID NO: 21, HCDR2: SEQ ID NO: 61, and HCDR3: SEQ ID NO: 103;
  • HCDR1 SEQ ID NO: 20
  • HCDR2 SEQ ID NO: 58
  • HCDR3 SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 58, and HCDR3: SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 58, and HCDR3: SEQ ID NO: 105;
  • HCDR1 SEQ ID NO: 21, HCDR2: SEQ ID NO: 58, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 22
  • HCDR2 SEQ ID NO: 58
  • HCDR3 SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 58, and HCDR3: SEQ ID NO: 106;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 58, and HCDR3: SEQ ID NO: 107;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 58, and HCDR3: SEQ ID NO: 103;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 62, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 61, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 59, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 60, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 63, and HCDR3: SEQ ID NO: 100;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 24, HCDR2: SEQ ID NO: 65, and HCDR3: SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 26
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 25
  • HCDR2 SEQ ID NO: 66
  • HCDR3 SEQ ID NO: 108
  • HCDR1 SEQ ID NO: 25
  • HCDR2 SEQ ID NO: 65
  • HCDR3 SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 26
  • HCDR2 SEQ ID NO: 67
  • HCDR3 SEQ ID NO: 109;
  • HCDR1 SEQ ID NO: 27, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 110;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 28
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 66, and HCDR3: SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 68
  • HCDR3 SEQ ID NO: 108
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 66, and HCDR3: SEQ ID NO: 109;
  • HCDR1 SEQ ID NO: 30, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 29
  • HCDR2 SEQ ID NO: 66
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 68
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 29, HCDR2: SEQ ID NO: 65, and HCDR3: SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 30, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 109;
  • HCDR1 SEQ ID NO: 31, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 66
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 32
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 109;
  • HCDR1 SEQ ID NO: 23
  • HCDR2 SEQ ID NO: 66
  • HCDR3 SEQ ID NO: 109
  • HCDR1 SEQ ID NO: 33
  • HCDR2 SEQ ID NO: 65
  • HCDR3 SEQ ID NO: 111.
  • the isolated antigen binding protein comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO:254.
  • the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 160-166 and 168-221.
  • the isolated antigen binding protein further comprises an antibody heavy chain constant region.
  • the heavy chain constant region is derived from a human IgG constant region.
  • the heavy chain constant region is derived from a human IgG4 constant region and/or a human IgGl constant region.
  • the heavy chain constant region comprises the amino acid sequence set forth in any one of SEQ ID Nos: 354-355.
  • the isolated antigen binding protein comprises an antibody heavy chain comprising the amino acid sequence set forth in any of SEQ ID NOs: 247-253 and 255-309.
  • the isolated antigen binding protein has one or more of the following properties:
  • c) can block the binding of PD-1 and PD-L2;
  • e is capable of inhibiting tumor growth and/or tumor cell proliferation.
  • an isolated antigen-binding protein comprising an antibody heavy chain or a fragment thereof, the antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises SEQ ID NO: The amino acid sequence shown in 10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 81, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 102.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', Fv fragment, F(ab') 2 , scFv, di-scFv, VHH, heavy chain antibody (HCAb) and/or dAb.
  • the antigen-binding fragment is a heavy chain antibody (HCAb).
  • HCAb heavy chain antibody
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 18, 25, 28, and 34-38
  • the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 64-66
  • the amino acid sequence shown and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 108, 111 and 112.
  • the HCDR1, HCDR2 and HCDR3 comprise amino acid sequences selected from any of the following group:
  • HCDR1 SEQ ID NO: 25
  • HCDR2 SEQ ID NO: 66
  • HCDR3 SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 25
  • HCDR2 SEQ ID NO: 65
  • HCDR3 SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 25
  • HCDR2 SEQ ID NO: 65
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 34
  • HCDR2 SEQ ID NO: 65
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 108;
  • HCDR1 SEQ ID NO: 18, HCDR2: SEQ ID NO: 64, and HCDR3: SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 35
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 36
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 36
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 112;
  • HCDR1 SEQ ID NO: 37
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 28
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 111
  • HCDR1 SEQ ID NO: 38
  • HCDR2 SEQ ID NO: 64
  • HCDR3 SEQ ID NO: 111.
  • the isolated antigen binding protein comprises an antibody heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO:167.
  • the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 196, 197, 200, 201, 202, and 222-230.
  • the isolated antigen binding protein further comprises an antibody heavy chain constant region.
  • the heavy chain constant region is derived from a human IgG constant region.
  • the heavy chain constant region is derived from a human IgG4 constant region and/or a human IgGl constant region.
  • the heavy chain constant region comprises the amino acid sequence set forth in any one of SEQ ID Nos: 354-355.
  • the isolated antigen binding protein comprises an antibody heavy chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 310-323.
  • the isolated antigen binding protein has one or more of the following properties:
  • c) can block the binding of PD-1 and PD-L2;
  • e is capable of inhibiting tumor growth and/or tumor cell proliferation.
  • the application provides a fusion protein comprising a first targeting moiety and a second targeting moiety, the first targeting moiety comprising a PD-1 binding moiety, wherein the PD-1
  • the binding moieties comprise the isolated antigen binding proteins described herein.
  • the second targeting moiety comprises a CD73 binding moiety.
  • the CD73-binding portion comprises an antibody or antigen-binding fragment thereof that specifically binds CD73.
  • the CD73 binding moiety comprises an antibody heavy chain or fragment thereof comprising HCDR1, HCDR2 and HCDR3, respectively comprising SEQ ID NO: 17.
  • the CD73 binding portion comprises an antibody heavy chain variable region VH comprising the amino acid sequence set forth in SEQ ID NO:159.
  • the CD73 binding portion comprises an antibody light chain or fragment thereof comprising LCDR1, LCDR2 and LCDR3, and wherein LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO: 123, the amino acid sequences shown in SEQ ID NO: 130 and SEQ ID NO: 146.
  • the CD73 binding portion comprises an antibody light chain variable region VL comprising the amino acid sequence set forth in SEQ ID NO:237.
  • the CD73 binding portion comprises a Fab.
  • the CD73 binding portion comprises an antibody heavy chain
  • the antibody heavy chain comprises the amino acid sequence set forth in SEQ ID NO:246.
  • the CD73 binding portion comprises an antibody light chain
  • the antibody light chain comprises the amino acid sequence set forth in SEQ ID NO:330.
  • the PD-1 binding moiety is N-terminal or C-terminal to the CD73 binding moiety.
  • the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the VH of the PD-1 binding moiety and the VH of the CD73 binding moiety , the second polypeptide chain includes the VL of the CD73 binding portion.
  • the VH of the PD-1 binding moiety is N-terminal to the VH of the CD73 binding moiety.
  • the VH of the PD-1 binding moiety is C-terminal to the VH of the CD73 binding moiety.
  • the first polypeptide chain further comprises an antibody heavy chain constant region.
  • the first polypeptide chain comprises the VH of the PD-1 binding moiety, the VH of the CD73 binding moiety, and the antibody heavy chain constant region, in order from the N-terminus to the C-terminus.
  • the VH of the PD-1 binding moiety is indirectly linked to the VH of the CD73 binding moiety through a linker peptide.
  • the linker peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 334-352.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:333.
  • the second polypeptide chain further comprises an antibody light chain constant region.
  • the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:330.
  • the fusion protein includes two of the first polypeptide chains and two of the second polypeptide chains.
  • the application provides an immunoconjugate comprising the isolated antigen binding protein or the fusion protein.
  • the application provides isolated one or more nucleic acid molecules encoding said isolated antigen binding protein and/or said fusion protein.
  • the application provides a vector comprising the nucleic acid molecule.
  • the application provides cells comprising the nucleic acid molecule or the vector.
  • the present application provides a method for preparing the isolated antigen-binding protein and/or the fusion protein, the method comprising making the isolated antigen-binding protein and/or the fusion protein
  • the cells are cultured under conditions of expression.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen-binding protein, the fusion protein, the conjugate, the nucleic acid molecule, and the carrier and/or the cells, and optionally a pharmaceutically acceptable carrier.
  • the present application provides the isolated antigen binding protein, the fusion protein, the conjugate, the nucleic acid molecule, the vector, the cell and/or the Use of the pharmaceutical composition in the preparation of a medicament for treating a disease or condition mediated by PD-1.
  • the PD-1 mediated disease or disorder comprises a tumor, autoimmune disease or inflammation.
  • the tumor comprises a solid tumor or a non-solid tumor.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises lung cancer, liver cancer, melanoma, urothelial cancer, head and neck squamous cell carcinoma, lymphoma, gastric cancer, and/or esophageal cancer.
  • a method of increasing T cell activity in a subject comprising administering to a subject in need an effective amount of said antigen binding protein, said fusion protein, said conjugate, said nucleic acid
  • administering comprising administering to a subject in need an effective amount of said antigen binding protein, said fusion protein, said conjugate, said nucleic acid
  • said antigen binding protein comprising administering to a subject in need an effective amount of said antigen binding protein, said fusion protein, said conjugate, said nucleic acid
  • the molecule, the carrier, the cell and/or the pharmaceutical composition comprising administering to a subject in need an effective amount of said antigen binding protein, said fusion protein, said conjugate, said nucleic acid.
  • the present application provides a method for preventing, alleviating or treating a PD-1-mediated disease or disorder, comprising administering an effective amount of the antigen-binding protein, the fusion protein to a subject in need thereof , the conjugate, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition; optionally, in combination with one or more other tumor treatment methods.
  • the PD-1 mediated disease or disorder comprises a tumor, autoimmune disease or inflammation.
  • the tumor comprises a solid tumor or a non-solid tumor.
  • the tumor comprises lung cancer, liver cancer, melanoma, urothelial cancer, head and neck squamous cell carcinoma, lymphoma, gastric cancer, and/or esophageal cancer.
  • the present application provides the antigen binding protein, the fusion protein, the conjugate, the nucleic acid molecule, the carrier, the cell and/or the drug A composition for preventing, ameliorating or treating a PD-1 mediated disease or disorder.
  • the PD-1 mediated disease or disorder comprises a tumor, autoimmune disease or inflammation.
  • the tumor comprises a solid tumor or a non-solid tumor.
  • the tumor comprises lung cancer, liver cancer, melanoma, urothelial cancer, head and neck squamous cell carcinoma, lymphoma, gastric cancer, and/or esophageal cancer.
  • the present application provides a kit or a drug delivery device, the kit or drug delivery device comprising the antigen binding protein, the fusion protein, the conjugate, the nucleic acid molecule, The carrier, the cell and/or the pharmaceutical composition.
  • the present application provides a method for inhibiting the binding of PD-L1 protein and PD-1 protein, the method comprising using the antigen binding protein, the fusion protein, the conjugate, the The nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the method is an in vitro method.
  • the method is a method for non-diagnostic and/or therapeutic purposes.
  • the present application provides a method for inhibiting the binding of PD-L2 protein and PD-1 protein, the method comprising using the antigen binding protein, the fusion protein, the conjugate, the The nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the method is an in vitro method.
  • the method is a method for non-diagnostic and/or therapeutic purposes.
  • the present application provides a method for detecting the presence and/or content of PD-1 protein, the method comprising using the antigen binding protein, the fusion protein, the conjugate, the The nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the method is an in vitro method.
  • the method is a method for non-diagnostic and/or therapeutic purposes.
  • the one or more other tumor treatment methods include chemotherapy and/or radiation therapy.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises lung cancer, liver cancer, melanoma, urothelial cancer, head and neck squamous cell carcinoma, lymphoma, gastric cancer, and/or esophageal cancer.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises lung cancer, liver cancer, melanoma, urothelial cancer, head and neck squamous cell carcinoma, lymphoma, gastric cancer, and/or esophageal cancer.
  • Figure 1A shows the binding activities of the antigen binding proteins PR000673, PR000674, PR000675, PR000676, PR000677, PR000678 and PR000679 described in the present application to human PD-1 protein.
  • Figure 1B shows the binding activity of the antigen-binding protein PR000680 described in the present application to human PD-1 protein.
  • Figure 1C shows the binding activities of the antigen-binding proteins PR002474, PR002476, PR002479 and PR002481 described in the present application to human PD-1 protein.
  • Figure 2 shows the binding activity of the antigen binding proteins R002474, PR002476, PR002479 and PR002481 described in the present application to the cynomolgus monkey PD-1 protein.
  • Figure 3A shows the binding activity of the antigen binding proteins PR000673, PR000674, PR000675, PR000676, PR000677, PR000678 and PR000680 described in the present application to human PD-1 protein expressed on CHO-K1 cells.
  • Figure 3B shows the binding activities of the antigen binding proteins R002473, R002474, PR002475, PR002476, R002477, PR002478, PR002479 and PR002481 described in the present application to human PD-1 protein expressed on CHO-K1 cells.
  • Figure 3C shows the binding activity of the antigen binding proteins PR005090, PR005093, PR005096, PR005097, PR005098, PR005099, PR005100, PR005101, PR005102, PR005103 and PR005104 of the present application to human PD-1 proteins expressed on CHO-K1 cells.
  • Figure 3D shows the binding activity of the antigen binding proteins PR005090, PR005145, PR005149, PR005150, PR005141, PR005142, PR005144, PR005147 and PR005148 of the present application to human PD-1 protein expressed on CHO-K1 cells.
  • Figure 3E shows the binding activities of the antigen binding proteins PR002481, PR005585, PR005583, PR005578, PR005577, PR005575, PR005572 and PR005569 described in the present application to human PD-1 protein expressed on CHO-K1 cells.
  • Figure 3F shows the binding activity of the antigen binding proteins PR002481, PR005584, PR005582, PR005581, PR005574, PR005573, PR005571 and PR005570 described in the present application to human PD-1 protein expressed on CHO-K1 cells.
  • Figure 4 shows the binding activity of the antigen binding proteins PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680 described in the present application to the cynomolgus monkey PD-1 protein expressed on CHO-K1 cells.
  • Figure 5A shows the activity of the antigen binding proteins PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680 described in the present application to block the binding of human PD-1 protein and PD-L1 protein expressed on CHO-K1 cells .
  • Figure 5B shows the activity of the antigen binding proteins PR002473, PR002474, PR002475, PR002476, PR002477, PR002478, PR002479 and PR002481 described in the present application to block the binding of human PD-1 protein and PD-L1 protein expressed on CHO-K1 cells .
  • Figure 5C shows the activity of the antigen binding proteins PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680 described in this application to block the binding of human PD-1 protein and PD-L2 protein expressed on CHO-K1 cells .
  • Figure 6A shows the inhibitory effect of the antigen binding proteins PR000673, PR000674, PR000678, PR000679 and PR000680 described in the present application on PD-1 signaling pathway (detected using reporter gene cell lines).
  • Figure 6B shows the inhibition of PD-1 signaling pathway by the antigen binding proteins PR005090, PR005093, PR005096, PR005097, PR005098, PR005099, PR005100, PR005101, PR005102, PR005103 and PR005104 of the present application (detected by reporter gene cell line) .
  • Figure 6C shows the inhibitory effect of the antigen binding proteins PR005090, PR005145, PR005149, PR005150, PR005141, PR005142, PR005144, PR005147 and PR005148 of the present application on PD-1 signaling pathway (detected using reporter gene cell lines).
  • Figure 6D shows the inhibitory effect of the antigen binding proteins PR002481, PR005570, PR005571, PR005573 and PR005574 described in the present application on PD-1 signaling pathway (detected by reporter gene cell line).
  • Figure 6E shows the inhibitory effect of the antigen binding proteins PR002481, PR005577, PR005581, PR005582 and PR005584 described in the present application on PD-1 signaling pathway (detected using reporter gene cell lines).
  • Figure 6F shows the inhibitory effects of the antigen binding proteins PR002481, PR005569, PR005572, PR005575, PR005583 and PR005585 described in the present application on PD-1 signaling pathway (detected using reporter gene cell lines).
  • Figures 7A, 7B and 7C show the ability of the antigen binding proteins PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680 described in the present application to activate T cells (mixed lymphocyte reaction assay).
  • FIGS 8A and 8B show the ability of the antigen binding proteins PR000674, PR000678 and PR000679 described in the present application to activate T cells (mixed lymphocyte reaction assay).
  • Figure 8C shows the ability of the antigen binding protein PR002481 described in the present application to activate T cells (mixed lymphocyte reaction assay).
  • Figure 9 shows the ability of the antigen binding proteins PR005569, PR005572, PR005575, PR005583, and PR005585 and PR002481 described herein to activate T cells (mixed lymphocyte reaction assay).
  • Figure 10 shows an exemplary molecular structure of the fusion protein described in this application.
  • Figure 11 shows the binding activity of the fusion protein PR003568 described in the present application to human CD73 cells expressed on CHO-K1 cells.
  • Figure 12 shows the binding activity of the fusion protein PR003568 described in the present application to human PD-1 cells expressed on CHO-K1 cells.
  • Figure 13 shows that the fusion protein PR003568 described in this application inhibits the enzymatic activity of CD73.
  • Figure 14 shows the inhibitory effect of the fusion protein PR003568 described in this application on PD-1 signaling pathway (detected by reporter gene cell line).
  • Figures 15A and 15B show the ability of the fusion proteins described in the present application to activate T cells to secrete cytokines (mixed lymphocyte reaction assay).
  • PD-1 generally refers to programmed cell death 1, also referred to as “programmed cell death 1", “CD279”, “cluster of differentiation 279", “PD1”, “PDCD1”.
  • PD-1 is normally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs) and is involved in apoptosis.
  • PD-1 generally contains an extracellular IgV domain, a transmembrane domain and an intracellular domain.
  • PD-1 binds two ligands, PD-L1 and PD-L2.
  • the "PD-1” includes any native PD-1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rat).
  • the term encompasses "full length", unprocessed PD-1 and any form of PD-1 produced by cellular processing.
  • PD-1 can exist as a transmembrane protein or as a soluble protein.
  • PD-1 includes complete PD-1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-1, and functional variants, isoforms, derivatives, and analogs of PD-1, Epitope analogs.
  • the PD-1 may include human PD-1 and/or monkey (eg, cynomolgus monkey) PD-1.
  • human PD-1 amino acid sequence can be found in NCBI Accession No. NP_005009.2.
  • exemplary cynomolgus monkey PD-1 amino acid sequence can be found in NCBI Accession No. NP_001271065.1.
  • the term "PD-L1" generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1, cluster of differentiation 274, (3)274 or CD274, which is associated with PD-1 binding downregulates T cell activation and cytokine secretion.
  • P-L1 includes any native PD-L1 from any vertebrate source, including mammals, such as primates (eg, humans and cynomolgus monkeys) and rodents (eg, mice and rats) ).
  • the term encompasses "full length", unprocessed PD-L1 as well as any form of PD-L1 produced by cellular processing.
  • PD-L1 can exist as a transmembrane protein or as a soluble protein.
  • "PD-L1" includes complete PD-L1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of PD-L1, and functional variants, isoforms, derivatives, and analogs of PD-L1, as well as those having at least one in common with PD-L1 Epitope analogs.
  • the basic structure of PD-L1 includes four domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain.
  • Exemplary human PD-L1 amino acid sequences can be found under NCBI Accession No. NP_001254653 or UniProt Accession No. Q9NZQ7.
  • An exemplary cynomolgus monkey PD-L1 amino acid sequence can be found under NCBI accession number XP_005581836.1.
  • CD73 is also referred to as a 5' exonucleotidase, generally referring to the ability to convert extracellular nucleosides 5' monophosphates to nucleosides, ie, adenosine monophosphate (AMP) to adenosine glycoside enzyme (nucleotidase).
  • the term “CD73” includes any variant or isoform of CD73 that is naturally expressed by a cell. CD73, or any variants and isoforms thereof, can be isolated from the cells or tissues in which they are naturally expressed, or can be recombinantly produced using techniques well known in the art and/or those described herein.
  • the amino acid sequence of human CD73 can be found in GenBank under Accession No. AAH65937.1 (5'-nucleotidase, ecto), or under NP_002517 (isoform 1 preprotein) and NP_001191742 (isoform 2 preprotein).
  • antigen binding protein generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone moiety that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding protein to the antigen.
  • antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv and/or dAbs), immunoconjugates, multispecific antibodies (eg bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain
  • Fab' generally refers to a fragment that differs from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain
  • F(ab"') 2 generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.
  • Fv generally refers to the smallest antibody fragment containing the entire antigen recognition and binding site.
  • the fragment may consist of a heavy chain variable region and a light chain variable region in a tightly non-covalently bound dimer;
  • dsFv generally refers to disulfide-stabilized Fv fragments, The bond between its single light chain variable region and single heavy chain variable region is a disulfide bond.
  • dAb fragment generally refers to antibody fragments consisting of VH domains.
  • scFv generally refers to a monovalent molecule formed by covalently linking and pairing one heavy chain variable domain and one light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have a general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • antibody is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies , multispecific antibodies (eg, bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, heavy chain antibodies, and camelized single domain antibodies (eg, heavy chain variable domain antibodies).
  • Antibodies generally have the structure of immunoglobulins, and may comprise proteins of at least two heavy (HC) and two light (LC) chains interconnected by disulfide bonds, or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region.
  • VH heavy chain variable region
  • immunoglobulins can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ , ⁇ , ⁇ chains, respectively , ⁇ chain, ⁇ chain.
  • immunoglobulins can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • each light chain comprises a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region contains one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four framework regions (FRs), arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) , mostly adopt a ⁇ -sheet configuration, connected by three CDRs, forming loop connections, and in some cases forming part of the ⁇ -sheet structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions and together with the CDRs from the other chain form the antigen-binding site of the antibody.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • variable generally refers to the fact that some portion of the sequence of the variable domains of an antibody varies strongly which contributes to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs). The more highly conserved portion of the variable domain is called the framework (FR).
  • CDRs complementarity determining regions
  • HVRs hypervariable regions
  • the CDRs of antibodies can be defined by a variety of methods, such as the Kabat definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Besse Starr, Maryland (1991)), Chothia definition rules based on the location of structural loop regions (see, J Mol Biol 273:927-48, 1997) and IMGT-ONTOLOGY-based concepts and IMGT Scientific chart rules The IMGT defines the rules.
  • the CDRs of the antigen binding proteins are classified according to the Chothia definition rule. The details are shown in Table 1.
  • LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 Chothia L24--L34 L50--L56 L89--L97 H26--H32 H52--H56 H95--H102
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, isolated and/or recovered from components of the environment in which it is produced (eg, native or recombinant). Contaminant components of its producing environment are often substances that interfere with its research, diagnostic or therapeutic use, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • An isolated antigen binding protein or antibody will generally be prepared by at least one purification step.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies in the population are identical except for possible minor natural mutations.
  • Monoclonal antibodies are usually highly specific for a single antigenic site.
  • 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 contamination by other immunoglobulins.
  • the modifier "monoclonal” denotes a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring the production of the antibody by any particular method.
  • the monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve antibody properties, such as binding affinity.
  • Fully human antibody generally refers to the antibody expressed by the human antibody gene-encoding gene transferred into a genetically engineered antibody gene-deficient animal. All parts of an 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 to the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.
  • VHH generally refers to heavy chain variable domain antibodies.
  • the VHH typically lacks the antibody light and/or heavy chain constant regions and contains only part of the heavy chain variable domain.
  • heavy chain antibody may also be referred to as HCAb, and generally refers to an antibody that lacks the light chain of an antibody and only contains a heavy chain relative to a diabody (immunoglobulin).
  • the heavy chain antibody may comprise two antibody heavy chains.
  • binding generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can be determined in the presence of a molecule
  • a target in the context of a heterogeneous population (including biological molecules).
  • an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody that specifically binds a target is an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets.
  • An antibody is said to "specifically bind" to an antigen when it binds to an epitope more readily through its antigen-binding domain than it would bind to a random, unrelated epitope.
  • the term "between” generally means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and its N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment.
  • indirect connection In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR3.
  • first amino acid fragment" and "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • KD K D
  • K D K D
  • KD dissociation rate constant
  • kon association rate (kon)
  • ka association rate
  • association and dissociation rate constants are well known in the art and include, but are not limited to, Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , co-immunoprecipitation (Co-IP) and protein chip technology.
  • BBI Biofilm Interferometry
  • RIA Radioimmunoassay
  • SPR Surface Plasmon Resonance
  • FRET Fluorescence Resonance Energy Transfer
  • Co-IP co-immunoprecipitation
  • the measured affinity for a particular protein-protein interaction can vary if measured under different conditions (eg, salt concentration, pH).
  • isolated nucleic acid molecule or isolated polynucleotide
  • isolated nucleic acid molecule generally refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, which is not related to the polynucleus found in nature All or a portion of the nucleotides are associated, or linked, to polynucleotides to which they are not linked in nature.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • the term "cell” generally refers to an individual cell, cell line or cell that can or already contains a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antibody or antigen-binding fragment thereof described herein. cell culture.
  • the cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein.
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be mammalian cells.
  • the mammalian cells can be CHO-K1 cells.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector has been introduced.
  • the recombinant host cells include not only certain specific cells, but also progeny of these cells.
  • the term "pharmaceutically acceptable carrier” generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed of.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers may include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, Sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.
  • treatment generally refers to the desire to alter the natural course of the disease in the individual being treated, and may be a clinical intervention to achieve prevention or during the course of a clinical disease.
  • Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or ameliorating the disease state, and alleviating or improving prognosis.
  • antibodies eg, anti-PD-1 antibodies
  • administration generally refers to the administration of a dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition comprising an anticancer therapeutic agent) to a subject (eg, a patient).
  • a pharmaceutical composition eg, a pharmaceutical composition comprising an anticancer therapeutic agent
  • Administration can be by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired for topical treatment, intralesional administration.
  • Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • tumor generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • the tumor can be a tumor with high expression of PD-1 or PD-L1 in cells and tissues.
  • Tumors can include solid tumors and/or non-solid tumors (eg, hematological tumors, lymphomas).
  • immunoconjugate generally refers to a substance formed by linking an antigen-binding protein with other active agents, which can be small molecule active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes or spectral probes.
  • kit generally refers to a packaged product comprising components for administering the antigen binding proteins of the present application for the treatment of PD-1 mediated related disorders.
  • the components of the kit may be contained in separate vials (ie, a kit with separate parts), or provided in a single vial.
  • Kits can include reagents such as buffers, protein stabilizing reagents, signal generating systems (eg, fluorescent signal generating systems), antibodies, control proteins, and test containers.
  • the kit may also contain instructions for carrying out the method.
  • the term "delivery device” includes: (i) an infusion module for administering to a subject a pharmaceutical composition comprising an active ingredient; (ii) a drug for infusion A pharmaceutical composition comprising an active ingredient selected from the group consisting of antigen-binding proteins, multispecific antibodies, immune cells, antibody-drug conjugates or combinations thereof; and (iii) any The selected drug efficacy monitoring module.
  • the term "combination" generally means that two or more therapeutic agents may be co-administered to a subject in admixture, simultaneously as a single agent, or sequentially as a single agent in any order.
  • the protein, polypeptide and/or amino acid sequence involved in this application should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
  • the variant may be one in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence of the protein and/or the polypeptide (eg, the antigen-binding protein described herein).
  • protein or peptide may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to alteration.
  • the substitutions can be conservative substitutions.
  • a part of the amino acid sequence of the antigen binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class.
  • both the variable and constant portions of an antibody can be derived from the variable and constant regions of an antibody of an animal species (eg, human).
  • the homologue may be at least about 85% (eg, having at least about 85%) the amino acid sequence of the protein and/or the polypeptide (eg, the antigen binding protein described herein). %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology protein or polypeptide.
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences.
  • Perfect sequence homology can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (eg, A, T, C, G) in the two sequences or position of the same amino acid residue (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To obtain the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to yield the percent sequence homology.
  • Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in WRPearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an antigen-binding protein
  • the antigen-binding protein may comprise at least one CDR in the variable region VH of an antibody heavy chain
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:368.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR1.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR1 of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 11-16.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR2.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR2 of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 50-55.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR3.
  • the HCDR3 of the antigen binding protein can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 92-97.
  • the antigen binding protein of the present application may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:359, and the HCDR2 may comprise the amino acid shown in SEQ ID NO:360 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:361.
  • the antigen binding protein of the present application may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 11-16, and the HCDR2 may comprise SEQ ID NO : the amino acid sequence shown in any one of 50-55, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 92-97.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 50, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:92.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 12, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 50, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:93.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 51, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:94.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 14, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 52, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:95.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 15, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 53, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:96.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 54, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:97.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 55, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:92.
  • the antigen binding protein may comprise a framework region HFWR1, the C-terminus of HFWR1 is directly or indirectly linked to the N-terminus of HCDR1.
  • the HFWR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 1-7.
  • the antigen binding protein may comprise a framework region HFWR2 located between the HCDR1 and the HCDR2.
  • the HFWR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 39-44.
  • the antigen binding protein may comprise a framework region HFWR3 located between the HCDR2 and the HCDR3.
  • the HFWR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 69-75.
  • the antigen binding protein may comprise a framework region HFWR4, the N-terminus of the HFWR4 is linked to the C-terminus of the HCDR3.
  • the HFWR4 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 113-115.
  • the antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:368.
  • the antigen binding protein of the present application can comprise at least one CDR in the VL of the variable region of the antibody light chain, and the VL can comprise the amino acid sequence shown in SEQ ID NO:369.
  • the antigen binding proteins described herein may comprise a light chain complementarity determining region LCDR1.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the LCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 121-125.
  • the antigen binding proteins described herein may comprise a light chain complementarity determining region LCDR2.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the LCDR2 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 130-135.
  • the antigen binding proteins described herein may comprise a light chain complementarity determining region LCDR3.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the LCDR3 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 140-145.
  • the antigen-binding protein of the present application may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 365, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 366, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:367.
  • the antigen-binding protein of the present application may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 121-125, and the LCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 130-135 The amino acid sequence shown in any one, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 140-145.
  • the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 121, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 130, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 140.
  • the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 122, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 131, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 141.
  • the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 123, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 132, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 142.
  • the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 122, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 133, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 143.
  • the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 124, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 134, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 144.
  • the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 125, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 135, And the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 145.
  • the antigen binding protein may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 121, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 130, And the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 140.
  • the antigen binding protein may comprise a framework region LFWR1, and the C-terminus of LFWR1 is directly or indirectly linked to the N-terminus of LCDR1.
  • the LFWR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 116-119.
  • the antigen binding protein may comprise a framework region LFWR2 located between the LCDR1 and the LCDR2.
  • the LFWR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 126-129.
  • the antigen binding protein may comprise a framework region LFWR3 located between the LCDR2 and the LCDR3.
  • the LFWR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 136-139.
  • the antigen binding protein may comprise a framework region LFWR4, the N-terminus of the LFWR4 is linked to the C-terminus of the LCDR3.
  • the LFWR4 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 147-150.
  • the antigen binding protein may comprise the light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:369.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, and the HCDR2 may comprise SEQ ID NO: 50
  • the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 92
  • the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 121
  • the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 130
  • the amino acid sequence of , and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 140.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 12, and the HCDR2 may comprise SEQ ID NO: 50
  • the amino acid sequence shown, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 93
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 122
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 131
  • the amino acid sequence of , and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 141.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the HCDR2 may comprise SEQ ID NO: 51
  • the amino acid sequence shown, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 94
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 123
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 132
  • the amino acid sequence of , and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 142.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 14, and the HCDR2 may comprise SEQ ID NO: 52
  • the amino acid sequence shown, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 95
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 122
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 133
  • the amino acid sequence of , and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 143.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 15, and the HCDR2 may comprise SEQ ID NO: 53
  • the amino acid sequence shown, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 96
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 124
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 134
  • the amino acid sequence of , and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 144.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 16, and the HCDR2 may comprise SEQ ID NO: 54
  • the amino acid sequence shown, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 97
  • the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 125
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 135
  • the amino acid sequence of , and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 145.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 11, and the HCDR2 may comprise SEQ ID NO: 55
  • the amino acid sequence shown, the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 92
  • the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 121
  • the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 130
  • the amino acid sequence of , and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 140.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:368
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:369.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 151-158
  • the VL may comprise SEQ ID NO: 231- The amino acid sequence shown in any one of 236.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 151
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 231.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 152
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 232.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 153
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 233.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 154
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 234.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 155
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 235.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 156
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 232.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 157
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 236.
  • the antigen binding protein may comprise VH and VL
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 158
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 231.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 238, and the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 324.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 239
  • the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 325.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 240
  • the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 326.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 241
  • the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 327.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 242
  • the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 328.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 243
  • the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 325.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 244
  • the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 329.
  • the antigen binding protein may comprise an antibody heavy chain and an antibody light chain
  • the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 245
  • the antibody light chain may comprise SEQ ID NO: The amino acid sequence shown at 324.
  • the application provides an antigen-binding protein
  • the antigen-binding protein may comprise at least one CDR in the VH of the variable region of the antibody heavy chain, and the VH may comprise the amino acid sequence shown in SEQ ID NO:254.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR1.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 18-33.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR2.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 57-68.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR3.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 99-101 and 103-111.
  • the antigen binding protein of the present application may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:362, and the HCDR2 may comprise the amino acid shown in SEQ ID NO:363 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:364.
  • the antigen binding protein of the present application may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 18-33, and the HCDR2 may comprise SEQ ID NO : the amino acid sequence shown in any one of 57-68, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 99-101 and 103-111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 57, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 57, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:99.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 19, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 57, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO:99.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 57, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 101.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 60, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 59, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 61, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 60, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 59, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 61, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 104.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 105.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 22, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 106.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 107.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 58, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 103.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 62, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 61, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 59, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 60, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 63, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 24, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 65, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 66, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 65, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 26, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 67, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 109.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 110.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 66, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 68, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 66, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 109.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 66, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 68, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 29, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 65, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 109.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 31, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 66, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 32, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 109.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 66, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 109.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 33, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 65, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise a framework region HFWR1, the C-terminus of HFWR1 is directly or indirectly linked to the N-terminus of HCDR1.
  • the HFWR1 may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the antigen binding protein may comprise a framework region HFWR2 located between the HCDR1 and the HCDR2.
  • the HFWR2 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 46-49.
  • the antigen binding protein may comprise a framework region HFWR3 located between the HCDR2 and the HCDR3.
  • the HFWR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 77-80 and 82-91.
  • the antigen binding protein may comprise a framework region HFWR4, the N-terminus of the HFWR4 is linked to the C-terminus of the HCDR3.
  • the HFWR4 may comprise the amino acid sequence shown in any one of SEQ ID NO: 113.
  • the antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 160-166 and 168-221.
  • the antigen binding protein may comprise an antibody heavy chain which may comprise the amino acid sequence shown in any one of SEQ ID NOs: 247-253 and 255-309.
  • the present application provides an antigen-binding protein
  • the antigen-binding protein may comprise at least one CDR in the VH of an antibody heavy chain variable region
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 167.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR1.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 18, 25, 28, and 34-38.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR2.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 64-66.
  • the antigen binding proteins described herein may comprise the heavy chain complementarity determining region HCDR3.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 108, 111 and 112.
  • the antigen binding protein described in the present application may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the HCDR2 may comprise the amino acid shown in SEQ ID NO: 81 sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 102.
  • the antigen binding protein of the present application may comprise heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 18, 25, 28 and 34-38, the The HCDR2 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 64-66, and the HCDR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 108, 111 and 112.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 66, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 65, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 25, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 65, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 65, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 36, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 36, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 112.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 37, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 28, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 38, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 64, And the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 111.
  • the antigen binding protein may comprise a framework region HFWR1, the C-terminus of HFWR1 is directly or indirectly linked to the N-terminus of HCDR1.
  • the HFWR1 may comprise the amino acid sequence shown in SEQ ID NO:9.
  • the antigen binding protein may comprise a framework region HFWR2 located between the HCDR1 and the HCDR2.
  • the HFWR2 may comprise the amino acid sequence shown in SEQ ID NO:47.
  • the antigen binding protein may comprise a framework region HFWR3 located between the HCDR2 and the HCDR3.
  • the HFWR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 82, 84, 85, 87, 88 and 89.
  • the antigen binding protein may comprise a framework region HFWR4, the N-terminus of the HFWR4 is linked to the C-terminus of the HCDR3.
  • the HFWR4 may comprise the amino acid sequence shown in SEQ ID NO: 113.
  • the antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in any one of SEQ ID NOs: 196, 197, 201, 202 and 222-230
  • the antigen binding protein may comprise an antibody heavy chain which may comprise the amino acid sequence shown in any one of SEQ ID NOs: 310-323.
  • the physical/chemical properties and/or biological activities of the PD-1 antigen binding proteins or fusion proteins described herein can be identified, screened or characterized by various assays known in the art.
  • the present application can be tested, for example, by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (eg, Western blot), flow cytometry (eg, FACS), immunohistochemistry, immunofluorescence, and the like Antigen-binding activity of an antigen-binding protein or fusion protein.
  • Antigen binding proteins eg, PD-1 antibodies
  • Antigen binding proteins eg, PD-1 antibodies
  • Antigen-binding proteins eg, PD-1 antibodies
  • Antigen-binding proteins that "specifically bind" the PD-1 antigen can generally bind PD-1, but not other proteins that lack the PD-1 sequence.
  • Antigen-binding proteins eg, PD-1 antibodies
  • PD-1 antibodies are capable of specifically binding PD-1 antigen or a labeled form thereof (eg, fluorescently labeled PD-1 antigen), but not binding epitopes lacking PD-1 of other proteins.
  • an antigen binding protein eg, an antibody
  • binds to a PD-1 antigen can be determined using any assay known in the art. Examples of assays known in the art to determine binding affinity include surface plasmon resonance (SPR) or biofilm interferometry (BLI).
  • the antigen binding proteins described in this application can bind to human PD-1 protein.
  • the antigen binding proteins described herein can also cross-react with PD-1 of monkeys (eg, cynomolgus monkeys). For example, detected by flow cytometry and ELISA.
  • cross-reactivity refers to the ability of an antibody to react with homologous proteins from other species.
  • the binding activity of the antigen binding proteins described herein to PD-1 can be detected using flow cytometry or enzyme-linked immunosorbent assay.
  • the PD-1 antigen-binding protein and PD-1 have an EC50 value of between about 0.0001 nM to about 100 nM
  • the PD-1 antigen-binding protein and PD-1 have an EC50 value of between about 0.0001 nM to about 100 nM
  • the PD-1 antigen-binding protein and PD-1 have an EC50 value of between about 0.0001 nM to about 100 nM
  • the PD-1 antigen-binding protein and PD-1 have an EC50 value of between about 0.0001 nM to about 100 nM
  • the PD-1 antigen-binding protein and PD-1 have an EC50 value of between about 0.0001 nM to about 100 nM
  • the PD-1 antigen-binding protein and PD-1 have an EC50 value of between about 0.0001 nM to about 100
  • the PD-1 antigen binding protein and PD-1 have an EC50 value of between about 0.0001 nM to about 100 nM, eg, between about 0.001 nM to about 10 nM , between about 0.001 nM and about 5 nM, between about 0.001 nM and about 1 nM, or between about 0.01 nM and about 1 nM.
  • the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L1.
  • the antigen binding protein blocking the binding of PD-1 to PD-L1 can be determined by flow cytometry FACS, enzyme-linked immunosorbent assay ELISA.
  • host cells stably expressing PD-L1 eg, CHO-K1 cells
  • PD-L1 eg, CHO-K1 cells
  • biotin-labeled PD-1 protein e.g, CHO-K1 cells
  • the PD-L1 antigen protein is first coated on the plate, and the unlabeled antigen-binding protein and the biotin-labeled PD-1 protein are mixed in decreasing amounts, and then incubated together. Cells were then analyzed using ELISA to confirm that the antigen binding protein could block the binding of PD-1 and PD-L1.
  • the antigen binding proteins described herein are capable of blocking the binding of PD-1 to PD-L2.
  • the blocking of the binding of PD-1 to PD-L2 by the antigen binding protein can be determined by flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA).
  • FACS flow cytometry
  • ELISA enzyme-linked immunosorbent assay
  • host cells stably expressing PD-1 eg, CHO-K1 cells
  • ELISA enzyme-linked immunosorbent assay
  • the PD-1 antigen protein is first coated on the plate, and the unlabeled antigen-binding protein and the biotin-labeled PD-L2 protein are mixed with decreasing amounts, and then incubated together. Cells were then analyzed using ELISA to confirm that the antigen binding protein could block the binding of PD-1 to PD-L2.
  • the antigen binding proteins described in the present 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.
  • the secretion of cytokines from immune cells can be measured by methods known to any person skilled in the art, for example, by enzyme-linked immunosorbent assay (ELISA) to quantify the proliferation of immune cells (eg, T cells) or cytokines produced by immune cells (eg, by enzyme-linked immunosorbent assay). IFN- ⁇ or IL-2 produced by T cells).
  • ELISA enzyme-linked immunosorbent assay
  • the application provides a fusion protein comprising a first targeting moiety and a second targeting moiety, the first targeting moiety comprising a PD-1 binding moiety, wherein the PD-1
  • the binding moieties comprise the isolated antigen binding proteins described herein.
  • the second targeting moiety of the fusion protein may comprise a CD73 binding moiety.
  • the CD73-binding portion may comprise an antibody or antigen-binding fragment thereof that specifically binds CD73.
  • the CD73 binding portion of the fusion protein may comprise an antibody heavy chain or fragment thereof, the antibody heavy chain or fragment thereof may comprise HCDR1, and the HCDR1 may comprise the amino acid shown in SEQ ID NO: 17 sequence.
  • the CD73-binding portion of the fusion protein may comprise an antibody heavy chain or fragment thereof, the antibody heavy chain or fragment thereof may comprise HCDR2, and the HCDR2 may comprise the amino acid shown in SEQ ID NO: 56 sequence.
  • the CD73 binding portion of the fusion protein may comprise an antibody heavy chain or fragment thereof, the antibody heavy chain or fragment thereof may comprise HCDR3, and the HCDR3 may comprise the amino acid shown in SEQ ID NO: 98 sequence.
  • the CD73 binding portion of the fusion protein may comprise an antibody heavy chain or fragment thereof, the antibody heavy chain or fragment thereof may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may be respectively
  • the amino acid sequences shown in SEQ ID NO: 17, SEQ ID NO: 56 and SEQ ID NO: 98 are included in sequence.
  • the CD73-binding portion of the fusion protein may include an antibody heavy chain variable region VH, and the VH may include the amino acid sequence shown in SEQ ID NO: 159.
  • the CD73-binding portion of the fusion protein may comprise an antibody light chain or a fragment thereof, the antibody light chain or a fragment thereof may comprise LCDR1, and the LCDR1 may comprise the amino acid shown in SEQ ID NO: 123 sequence.
  • the CD73 binding portion of the fusion protein may comprise an antibody light chain or a fragment thereof, the antibody light chain or a fragment thereof may comprise LCDR2, and the LCDR2 may comprise the amino acid shown in SEQ ID NO: 130 sequence.
  • the CD73-binding portion of the fusion protein may comprise an antibody light chain or a fragment thereof, the antibody light chain or a fragment thereof may comprise LCDR3, and the LCDR3 may comprise the amino acid shown in SEQ ID NO: 146 sequence.
  • the CD73 binding portion of the fusion protein may comprise an antibody light chain or fragment thereof
  • the antibody light chain or fragment thereof may comprise LCDR1, LCDR2 and LCDR3
  • the LCDR1, LCDR2 and LCDR3 may be respectively
  • SEQ ID NO: 123, SEQ ID NO: 130 and SEQ ID NO: 146 are included in sequence.
  • the CD73-binding portion of the fusion protein may include an antibody light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:237.
  • the CD73 binding portion of the fusion protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 may comprise SEQ ID NO: 17, SEQ ID NO: 17, SEQ ID NO: 17, respectively.
  • the amino acid sequences shown in NO: 56 and SEQ ID NO: 98, and the LCDR1, LCDR2 and LCDR3 may respectively comprise the amino acid sequences shown in SEQ ID NO: 123, SEQ ID NO: 130 and SEQ ID NO: 146, respectively.
  • the CD73 binding portion of the fusion protein may comprise VH and VL, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 159, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 237 amino acid sequence.
  • the CD73 binding portion of the fusion protein may comprise Fab.
  • the CD73-binding portion of the fusion protein may comprise an antibody heavy chain, and the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:246.
  • the CD73-binding portion of the fusion protein may comprise an antibody light chain, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:330.
  • the PD-1 binding moiety in the fusion protein, may be located at the N-terminus of the CD73 binding moiety. In the present application, in the fusion protein, the PD-1 binding moiety may be located at the C-terminus of the CD73 binding moiety.
  • the fusion protein can include a first polypeptide chain and a second polypeptide chain.
  • the first polypeptide chain of the fusion protein may comprise the VH of the PD-1 binding portion and the VH of the CD73 binding portion.
  • the second polypeptide chain of the fusion protein may comprise the VL of the CD73 binding moiety.
  • the first polypeptide chain of the fusion protein may sequentially comprise the VH of the PD-1 binding portion and the VH of the CD73 binding portion.
  • the first polypeptide chain of the fusion protein may comprise an antibody heavy chain constant region.
  • the first polypeptide chain of the fusion protein may sequentially comprise the VH of the PD-1 binding part, the VH of the CD73 binding part and the antibody heavy chain constant region .
  • the first polypeptide chain of the fusion protein may comprise the VH of the PD-1 binding portion and the antibody heavy chain of the CD73 binding portion.
  • the first polypeptide chain of the fusion protein may sequentially comprise the VH of the PD-1 binding part and the antibody heavy chain of the CD73 binding part.
  • the PD-1 binding portion and the CD73 binding portion can be directly linked.
  • the PD-1 binding portion and the CD73 binding portion can be linked by a linking peptide.
  • the linker peptide may comprise the amino acid sequence shown in any one of SEQ ID NOs: 334-352.
  • the first polypeptide chain can include the amino acid sequence set forth in SEQ ID NO:333.
  • the second polypeptide chain of the fusion protein may also comprise an antibody light chain constant region.
  • the first polypeptide chain can include the amino acid sequence set forth in SEQ ID NO:330.
  • the fusion protein may include two of the first polypeptide chains and two of the second polypeptide chains.
  • the fusion protein may be a homodimer.
  • the fusion protein is a symmetrical structure.
  • FIG. 10 An exemplary structure of the fusion protein described herein can be shown in Figure 10, which comprises two polypeptide chains: a first polypeptide chain, also called a short chain, from the amino terminus to the carboxy terminus, which comprises VL_A-CL; the second Polypeptide chain, also called long chain, from amino terminus to carboxyl terminus, which comprises VH_B-L-VH_A-CH1-h-CH2-CH3.
  • VH_A and VL_A are the heavy chain variable region and light chain variable region of any CD73 antibody, respectively.
  • VH_B is the heavy chain variable region of the PD-1 heavy chain antibody described in this application.
  • CL is an arbitrary light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains, respectively, of any heavy chain constant region.
  • L is any linker peptide (eg, those mentioned herein) and h is any hinge region or derived sequence of an IgG antibody.
  • the fusion protein has the biological activity of the antigen binding protein, eg, binding to PD-1 antibody, blocking the binding of PD-1 to its ligands (eg, PD-L1 and/or PD-L2) , stimulate T cells to secrete cytokines, etc.
  • the fusion protein described in the present application can also bind to the CD73 protein to inhibit the enzymatic activity of CD73.
  • the application provides one or more nucleic acid molecules that can encode the isolated antigen binding proteins described herein and/or the multispecific antibodies described herein.
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the present application provides a vector, which can comprise the nucleic acid molecule described herein.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host. Such control elements are well known to those of skill in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector may include, for example, a plasmid, cosmid, virus, phage or other vectors commonly used, for example, in genetic engineering.
  • the vector is an expression vector.
  • the present application provides a cell, which may comprise the nucleic acid molecule described herein or the vector described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into such host cells, eg, eukaryotic cells, such as cells from plants, fungi or yeast cells, and the like.
  • the vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the present application provides a pharmaceutical composition, which can comprise the antigen-binding protein and/or the multispecific antibody described in the present application, the nucleic acid molecule, the carrier, and the host. cells, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed, and may include buffers, antioxidants, preservatives, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic Sexual polymers, amino acids, carbohydrates, salt-forming counterions, metal complexes, and/or nonionic surfactants.
  • the pharmaceutical compositions herein may also contain more than one active compound, typically those active compounds with complementary activities that do not adversely affect each other. The type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.
  • compositions described in the present application may comprise prophylactically and/or therapeutically effective amounts of the antigen-binding proteins, multispecific antibodies.
  • the prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) a disease or disorder and/or any complications thereof in a subject having or at risk of developing it.
  • an immunoconjugate which may comprise a cytotoxic agent, and an antigen binding protein as described herein.
  • Immunoconjugate usually refers to using a specific linker to connect antigen-binding protein and small molecule cytotoxic drug, and its main components can include antigen-binding protein, linker and small molecule cytotoxic drug.
  • the present application provides a kit, which may comprise the antigen binding protein, carrier, nucleic acid molecule, cell, immunoconjugate, and/or pharmaceutical composition described herein. It may comprise the antigen binding proteins, carriers, nucleic acid molecules, cells, immunoconjugates, and/or pharmaceutical compositions described herein in a single conventional container, optionally together with one or more A combination of therapeutic agents, optionally formulated together in a kit.
  • the present application provides a drug delivery device that can be used to administer the antigen binding proteins, carriers, nucleic acid molecules, cells, immunoconjugates, and/or pharmaceutical compositions described herein .
  • the present application provides methods for preparing the antigen binding proteins.
  • the method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • Any method suitable for producing monoclonal antibodies can be used to produce the antigen binding proteins of the present application.
  • an animal can be immunized with a linked or naturally occurring PD-1 protein or fragment thereof.
  • Appropriate methods of immunization can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • PD-1 can be used as an immunogen (antigen) for generating non-human antibodies specific for PD-1 and screening the antibodies for biological activity.
  • the priming immunogen can be full-length mature human PD-1, including native homodimers, or peptides containing single/multiple epitopes.
  • the immunogens can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE.
  • the antibody is an IgG antibody, and the IgG1 or IgG4 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity can be achieved by screening antibodies using the biological assays described in the Examples below.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof are useful in the compounds and methods of the present application.
  • sequences of the DNA molecules of the antigen-binding proteins or fragments thereof of the present application can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences for the light and heavy chains can be fused together to form single chain antibodies.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the nucleic acid molecule can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • the present application also relates to vectors comprising suitable nucleic acid molecules as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • animal cells can include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
  • the steps of transforming host cells with recombinant DNA described in this application can be performed using techniques well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express the polypeptides encoded by the nucleic acid molecules of the present application. Depending on the host cell used, it is cultured with conventional media under appropriate conditions. Typically, the transformed host cells are cultured under conditions suitable for expression of the antigen binding proteins of the present application.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography and other techniques in the art
  • the antigen-binding protein of the present application can be obtained by conventional separation and purification methods well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as flow cytometric sorting (FACS) or enzyme-linked immunosorbent assay (ELISA).
  • FACS flow cytometric sorting
  • ELISA enzyme-linked immunosorbent assay
  • the present application provides a method of inhibiting the binding of PD-1 to PD-L1, comprising administering the antigen binding proteins described herein.
  • the method can be an ex vivo or in vitro method.
  • the method can include contacting a biological sample with an antigen binding protein and/or PD-L1 described herein under conditions that allow the antigen binding protein and/or PD-1 to bind PD-L1 , detecting whether a complex is formed between the antigen binding protein and PD-1, and detecting whether a complex is formed between PD-1 and PD-L1.
  • the present application provides methods of inhibiting the binding of PD-1 to PD-L2, comprising administering the antigen binding proteins described herein.
  • the method can be an ex vivo or in vitro method.
  • the method comprises contacting a biological sample with an antigen binding protein and/or PD-L2 described herein under conditions that allow the antigen binding protein and/or PD-L2 to bind PD-1, It is detected whether a complex is formed between the antigen binding protein and PD-1, and whether a complex is formed between PD-L1 and PD-1.
  • the antigen binding protein and/or pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical compositions of the present application can inhibit or delay the development or progression of a disease, can reduce tumor size (or even substantially eliminate a tumor), and/or can alleviate and/or stabilize a disease state.
  • the present application provides a use of the antigen binding protein and/or the multispecific antibody in the preparation of a medicine.
  • the medicament can be used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the present application provides a method of stimulating immune cells to secrete cytokines, comprising administering the isolated antigen binding protein and/or the polypeptide.
  • the cytokine may be IL-2.
  • the immune cells can be lymphocytes.
  • T lymphocytes can be lymphocytes.
  • the method can be an ex vivo or in vitro method.
  • the method may be of a non-diagnostic and/or therapeutic purpose.
  • the present application provides a method for detecting the presence and/or content of PD-1 protein, comprising administering the isolated antigen binding protein and/or the fusion protein.
  • the method can be an ex vivo or in vitro method.
  • the method may be of a non-diagnostic and/or therapeutic purpose.
  • the present application also provides the use of an antigen binding protein in a method of diagnosing a subject with a tumor or cancer, the method comprising: determining by contacting a sample with the antigen binding protein of the present application and detecting the presence of bound antibody The presence or expression level of PD-1 in a sample obtained from a subject.
  • the PD-1 antigen can usually be used to immunize experimental animals, and the experimental animals can be mice, rats, rabbits, sheep, camels, and the like.
  • the resulting antibody molecule is of non-human origin.
  • these molecules need to be humanized using antibody engineering techniques to reduce immunogenicity and improve druggability.
  • the process of humanizing antibodies has its technical complexities, and humanized molecules often have reduced affinity for the antigen.
  • advances in transgenic technology have made it possible to create genetically engineered mice that carry a human immunoglobulin immune repertoire and have their endogenous murine immune repertoire depleted.
  • the antibodies produced by this transgenic mouse have fully human sequences, so there is no need for further humanization transformation, which greatly improves the efficiency of therapeutic antibody development.
  • H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin immune repertoire, which produces antibodies with complete human antibody variable domains and rat constant domains, that is, antibodies in the form of H2L2 Has two antibody heavy chains and two antibody light chains.
  • HCAb mice (Harbour Antibodies BV, WO 2002/085945A3) are transgenic mice carrying a human immunoglobulin immune repertoire, capable of producing novel "heavy chain-only" antibodies that are half the size of traditional IgG antibodies . It produces antibodies with only human antibody “heavy chain” variable domains and mouse Fc constant domains. Due to the lack of light chain, the antibody almost solves the problems of light chain mismatch and heterodimerization, enabling this technology platform to develop products that are difficult to achieve with traditional antibody platforms. That is, an antibody in the form of an HCAb contains two antibody heavy chains and no antibody light chain.
  • each mouse received immunization with an immunogenic reagent formulated with 25 ⁇ g of antigenic protein mixed with Ribi adjuvant (Sigma Adjuvant System, #S6322).
  • the interval between each booster round is at least two weeks, and usually no more than five rounds of booster rounds.
  • the immunization time was 0, 14, 28, 42, 56, and 70 days; and on the 49th and 77th days, the serum antibody titers of mice were detected.
  • each mouse received immunization with an immunogenic reagent formulated with 25 micrograms of antigenic protein mixed with Ribi adjuvant (Sigma Adjuvant System, #S6322).
  • Immunization protocol 2 using HEK293/hPD-1 (Shanghai ChemPartner) stable cell line overexpressing human PD-1 for immunization.
  • Each mouse was injected intraperitoneally with 2 ⁇ 10 6 cell suspension per immunization.
  • the interval between each booster round is at least two weeks, and usually no more than five rounds of booster rounds.
  • the immunization time was 0, 14, 28, 42, 56, and 70 days; and on the 49th and 77th days, the serum antibody titers of mice were detected.
  • a final boost at a dose of 25 micrograms of antigenic protein per mouse was performed 5 days before splenic B cell isolation of HCAb mice.
  • mice The blood of mice was collected, and the blood was diluted 10 times to obtain 5 concentrations (1:100, 1:1000, 1:10000, 1:100000, 1:1000000), and coated with human PD-1-His (Shanghai ChemPartner)
  • the ELISA plate was used for ELISA detection to determine the titer of anti-human PD-1 in mouse blood, and two concentrations of mouse blood (1:100, 1:1000) were used to detect the high expression of PD-1 by flow cytometry Specific reactivity of CHO-K1/hPD-1 cells (Shanghai ChemPartner) and CHO-K1 blast cells.
  • the blank control group (PB) was the serum of the mice before immunization.
  • the spleen cells of the mice were taken out and fused with the myeloma cell line to obtain hybridoma cells; the hybridoma cells were screened and cloned for multiple rounds Thereafter, at least 8 hybridomas expressing anti-PD-1 monoclonal antibody molecules are isolated.
  • the isolated hybridoma cells and their expressed monoclonal antibodies are indicated by the corresponding clone numbers, for example: 4004_10H9A12, 4004_12H9C1, etc.
  • the isolated hybridomas express antibody molecules with intact heavy and light chains of human variable domains and rat constant domains.
  • the above monoclonal antibodies were further identified, and several hybridomas were selected according to their binding ability to human PD-1, cynomolgus monkey PD-1 binding ability, and inhibiting the binding ability of PD-1 and PD-L1.
  • Clones were sequenced.
  • the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence are obtained by conventional hybridoma sequencing methods.
  • the sequences of the variable domains of the anti-PD-1 monoclonal antibody molecules obtained from immunized Harbour H2L2 mice were human antibody sequences.
  • the CDR sequences are divided by the Chothia definition rules.
  • the spleen cells of the mice were taken out to separate B cells, and the CD138-positive plasma cells and human PD-1 antigen were sorted by BD FACS AriaII Cell Sorter. positive B cell population.
  • the RNA of B cells was extracted, cDNA was reverse transcribed (SuperScript IV First-Strand synthesis system, Invitrogen, #18091200), and then the human VH gene was amplified by PCR with specific primers.
  • PCR primers 5'-GGTGTCCAGTGT(G/C)AGGTGCAGCTG-3' SEQ ID NO:357), 5'- AATCCCTGGGCACTGAAGAGACGGTGACC-3' (SEQ ID NO:358).
  • the amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc domain sequence (SEQ ID NO: 355).
  • the constructed plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression to obtain HCAb antibodies. Binding of HCAb-expressing supernatants to a stable cell line of CHO-K1/hPD-1 (GenScript, #M00529) overexpressing human PD-1 was detected, while using a positive antibody as a positive control, by Miroball fluorescence flow instrument (Sptlabtech) filter. The specific steps were as follows: CHO-K1/hPD-1 cells were washed with serum-free F12K medium (Thermofisher, #21127022), and resuspended to 1 ⁇ 10 6 /ml with serum-free medium.
  • Draq5 fluorescent probe (CTS, #4048L) (1 ⁇ L Draq5 to 1 ml CHO-K1/hPD-1 cells, 1:1000 dilution) and incubate for 30 minutes in the dark. After centrifuging the cells, the cells were washed with culture medium, and the cell density was adjusted to 1.0 ⁇ 10 5 cells/ml. Then add Alexa diluted 1:1000 488, AffiniPure Goat Anti-Human IgG, Fc ⁇ Fragment Specific secondary antibody (Jackson, #109-545-098), 30ul of the mixture per well was added to a 384-well plate (Greiner, #781091).
  • the heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic hypermutation of the germline gene V, D, and J gene segments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement and somatic hypermutation of germline genes V and J gene segments of the light chain gene group. Gene rearrangements and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene segment may also yield different sequences, but overall the similarity is high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • the anti-PD-1 positive control antibody Pembrolizumab analog of the present application the corresponding antibody number is PR000150. Its corresponding amino acid sequence is derived from the IMGT database, the antibody heavy chain sequence is SEQ ID NO: 332, and the antibody light chain sequence is SEQ ID NO: 331.
  • the antibody light chain variable domain sequence (VL) obtained from Harbour H2L2 mice was synthesized and cloned into a mammalian cell expression plasmid vector encoding a human antibody kappa light chain constant domain sequence to encode the production of The full-length light chain of the antibody.
  • the antibody heavy chain variable domain sequence (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG4 antibody heavy chain constant domain sequence (SEQ ID NO: 354) to encode a complete IgG4 antibody-producing plasmid vector. long heavy chain.
  • the plasmid encoding the heavy chain of the Harbour H2L2 antibody and the plasmid encoding the light chain of the antibody are simultaneously transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and conventional recombinant protein expression and purification techniques can be used. of purified recombinant antibodies.
  • the plasmid encoding the heavy chain of Harbour HCAb antibody is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and conventional recombinant protein expression and purification techniques can be used to obtain recombinant antibodies with HCAb heavy chain.
  • HEK293 cells were expanded in FreeStyleTM F17 Expression Medium (Thermo, #A1383504). Before the transient transfection started, the cell concentration was adjusted to 6-8 ⁇ 10 5 cells/ml, and cultured at 37° C. in an 8% CO 2 shaker for 24 hours at a cell concentration of 1.2 ⁇ 10 6 cells/ml. Prepare 30 ml of cultured cells. The heavy chain plasmid encoding H2L2 antibody and the light chain plasmid were mixed at a ratio of 2:3 or the heavy chain plasmid encoding HCAb was dissolved in 1.5 ml Opti-MEM reduced serum medium (Thermo, #31985088), and filtered with a 0.22 ⁇ m filter.
  • Opti-MEM reduced serum medium Thermo, #31985088
  • a gravity column (Bio-Rad, #7311550) containing MabSelectTM (GE Healthcare Life Science, #71-5020-91 AE) was equilibrated with PBS (pH 7.4) and washed for 2-5 column volumes. The supernatant sample was passed through the column. Rinse the column with 5-10 column volumes of PBS. The target protein was eluted with 0.1M glycine at pH 3.5, adjusted to neutrality with Tris-HCl at pH 8.0, and finally concentrated and exchanged to PBS buffer with an ultrafiltration tube (Millipore, #UFC901024) to obtain the purified antibody solution. Then use NanoDrop (Thermo Scientific TM NanoDrop TM One) to measure the concentration, aliquot and store for later use.
  • PBS pH 7.4
  • the protein samples were analyzed for purity and aggregate form using analytical size exclusion chromatography (SEC).
  • An analytical column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 ⁇ m, 7.8 mm x 30 cm) was connected to a high pressure liquid chromatograph (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), equilibrated with PBS buffer at room temperature At least 1 hour.
  • An appropriate amount of protein sample (at least 10 ⁇ g, the sample concentration is adjusted to 1mg/ml) is filtered with a 0.22 ⁇ m filter and injected into the system, and the HPLC program is set: use PBS (pH 7.4) buffer to flow the sample at a flow rate of 1.0ml/min The longest time is 25 minutes; the detection wavelength is 280nm.
  • ChemStation software is used to integrate the chromatogram and calculate the relevant data, and generate an analysis report, reporting the retention time of components of different molecular sizes in the sample.
  • the affinity of the anti-PD-1 HCAb antibody PR002481 was modified by the method of site-directed saturation mutation. This affinity maturation method is divided into 2 rounds.
  • the 28 amino acids of the three CDRs of the molecule PR002481 were scanned point by point.
  • a small library of yeast with single point saturation mutations at 28 amino acid positions was created. Pools of the 3 CDRs were each mixed to form a yeast mutant pool of 3 CDRs.
  • the three CDR mutation libraries were sorted on a flow cytometer, the sorted yeast cells were sequenced, and these positive molecules were further identified. According to their binding ability to human PD-1, several mutations were selected. hot spot.
  • the CDR sequences of antibody variable domains were analyzed by Chothia CDR definition rules.
  • the hot spots found by the saturation mutation in the first round were randomly combined to build a yeast library containing all mutation combinations.
  • the combinatorial library was then sorted on a flow cytometer.
  • the sorted yeast cells were sequenced and identified for their binding ability to human PD-1, and several mutants were selected.
  • the antibody heavy chain variable domain sequence (VH) obtained by affinity maturation was synthesized and cloned into a mammalian cell expression plasmid vector encoding Flag and 6xHis tags to encode the PD1 single domain antibody (VH). )molecular.
  • the VH gene fragment of the antibody obtained by affinity maturation was constructed into a mammalian cell expression plasmid vector encoding the heavy chain Fc domain sequence of a human IgG1 antibody (SEQ ID NO: 355) to encode the full-length heavy chain of the HCAb.
  • the present invention obtains monovalent PR002481 variants (both are single-domain antibodies, with Flag-6His tag) through the above-mentioned affinity maturation method and their sequences are shown in Table 6 below (involving HCDR1, SEQ ID NOs: 18, 20-33; HCDR2 , SEQ ID NOs: 58-68; HCDR3, SEQ ID NOs: 100, 103-111).
  • PR005090 is the single domain antibody (with Flag-6His tag) corresponding to the parent sequence of PR002481.
  • the single-domain antibodies in Table 6 were evaluated.
  • the single-domain antibodies were selected to express HCAb by comprehensive analysis or directly or to recombine the existing CDR-expressed HCAb in Table 6.
  • Some hot spot mutations are introduced into the CDR region to design and generate new CDRs for the expression of HCAb antibodies.
  • the HCAb antibodies obtained through mutation and screening of the present invention are shown in Table 7 (related to HCDR1, SEQ ID NOs: 18, 25, 28, 34-38 ; HCDR2, SEQ ID NOs: 64-65; HCDR3, SEQ ID NOs: 108, 111-112).
  • variable domain sequence encoding the PR002481 affinity variant in the above Example 1.5 After obtaining the variable domain sequence encoding the PR002481 affinity variant in the above Example 1.5, conventional recombinant DNA technology can be used to fuse and express the heavy chain variable domain sequence and the corresponding purification tag to obtain a recombinant HCAb single-domain antibody molecule. .
  • the plasmid encoding the recombinant HCAb single-domain antibody is transfected into mammalian host cells (such as Chinese hamster ovary cell CHO), and the corresponding purified recombinant antibody can be obtained by using conventional recombinant protein expression and purification techniques.
  • mammalian host cells such as Chinese hamster ovary cell CHO
  • ExpiCHO-S TM cells (Gibco, #A29127) are expanded in ExpiCHO TM Expression Medium (Gibco, #A2910001). Before transient transfection begins, adjust the cell concentration to 3-4 ⁇ 10 6 cells/ml, incubate at 37°C in 8% CO 2 shaker for 24 hours, and then dilute to 7-10 ⁇ 10 6 cells/ml. 6 x 10 6 cells/ml, prepare 10 ml of cultured cells.
  • ExpiFectamine TM CHO reagent solution slowly added to the plasmid solution, invert and mix, slowly drop the mixed solution of plasmid and transfection reagent while shaking the culture flask, and incubate at 37°C 8% CO 2 shaker for 8-9 days. Cell viability was observed after 8 days. The culture was collected, centrifuged at 3300 g for 10 minutes, and the supernatant was taken, and then the supernatant was filtered through a 0.22 ⁇ m filter to remove impurities.
  • HCAb antibody The specific production and purification method of HCAb antibody is as follows: HEK293 cells are expanded and cultured in FreeStyleTM F17 Expression Medium (Thermo, #A1383504). Before the transient transfection started, the cell concentration was adjusted to 6-8 ⁇ 10 5 cells/ml, and cultured at 37° C. in an 8% CO 2 shaker for 24 hours at a cell concentration of 1.2 ⁇ 10 6 cells/ml. Prepare 30 ml of cultured cells. The heavy chain plasmid encoding the HCAb was dissolved in 1.5 ml Opti-MEM reduced serum medium (Thermo, #31985088) and filter sterilized through a 0.22 ⁇ m filter.
  • Opti-MEM reduced serum medium Thermo, #31985088
  • Opti-MEM 1.5 ml of Opti-MEM was dissolved in 120 ⁇ L of 1 mg/ml PEI (Polysciences Inc, #23966-2), and it was allowed to stand for 5 minutes. Slowly add PEI to the plasmid, incubate at room temperature for 10 minutes, slowly drop the plasmid PEI mixed solution while shaking the culture flask, and culture at 37°C in a 8% CO 2 shaker for 5 days. Cell viability was observed after 5 days. The culture was collected, centrifuged at 3300g for 10 minutes, and the supernatant was taken; then the supernatant was centrifuged at high speed to remove impurities.
  • PEI Polysciences Inc, #23966-2
  • the protein samples were analyzed for purity and aggregate form using analytical size exclusion chromatography (SEC).
  • the analytical column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 ⁇ m, 7.8 mm x 30 cm) was connected to a high pressure liquid chromatograph (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 Hour.
  • An appropriate amount of protein sample (at least 10 ⁇ g, the sample concentration is adjusted to 1mg/ml) is filtered with a 0.22 ⁇ m filter and injected into the system, and the HPLC program is set: use PBS (pH 7.4) buffer to flow the sample at a flow rate of 1.0ml/min The longest time is 20 minutes; the detection wavelength is 280nm.
  • ChemStation software is used to integrate the chromatogram and calculate the relevant data, and generate an analysis report, reporting the retention time of components of different molecular sizes in the sample.
  • Protein samples were analyzed for purity and hydrophobicity using analytical hydrophobic interaction chromatography (HIC).
  • the analytical column TSKge1 Buty1-NPR (Tosoh Bioscience, 14947, 4.6mm x 3.5cm) was connected to a high pressure liquid chromatograph (HPLC) (model Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour.
  • the set-up method consisted of a linear gradient from 100% mobile phase A (20 mM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0) in 16 minutes, with flow rate set It was 0.7ml/min, the protein sample concentration was 1mg/ml, the injection volume was 20 ⁇ l, and the detection wavelength was 280nm.
  • ChemStation software is used to integrate the chromatogram and calculate the relevant data, and generate an analysis report, reporting the retention time of components of different molecular sizes in the sample.
  • DSF Differential Scanning Fluorimetry
  • the PCR plate was sealed and placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad CFX96 PCR System), incubated at 25°C for 5 minutes, and then gradually heated from 25°C to 95°C with a gradient of 0.2°C/0.2 minutes, at the end of the test. Reduce the temperature to 25°C. Data analysis was performed using FRET scan mode and using Bio-Rad CFX Maestro software and the Tm of the samples was calculated.
  • Human PD-1-His protein (ACROBiosystems, #PD1-H5221) was diluted with PBS to 2 ⁇ g/ml, added to a 96-well plate (Corning, cat #9018), 100 ⁇ l per well, and incubated overnight at 4°C. After discarding the liquid, wash the plate three times with PBST buffer (pH 7.4, containing 0.05% tween-20), add 250 ⁇ l of 2% BSA blocking solution, and incubate at 37°C for 1 hour.
  • PBST buffer pH 7.4, containing 0.05% tween-20
  • the blocking solution was discarded, and the plate was washed three times with PBST buffer (pH 7.4, containing 0.05% Tween-20), and the antigen-binding protein to be tested was diluted 5 times from the concentration of 100 nM in sequence, with a total of 8 to 10
  • PBST buffer pH 7.4, containing 0.05% Tween-20
  • the antigen-binding protein to be tested was diluted 5 times from the concentration of 100 nM in sequence, with a total of 8 to 10
  • For each concentration gradient add 100 ⁇ l to each well, incubate at 37°C for 1 hour, and use the isotype antibody as a control.
  • 4000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added, and incubated at 37°C for 1 hour in the dark.
  • Figures 1A-1C show that the anti-PD-1 H2L2 antibodies PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680 of the present application can all bind to human PD-1 antigen, and the binding activity is the same as that of the positive control antibody pembrolizumab is comparable.
  • FIG. 1C show that the anti-PD-1 HCAb antibodies PR002474, PR002476, PR002479 and PR002481 described in this application can all bind to human PD-1 antigen, and the binding activity is comparable to that of the positive control antibody pembrolizumab.
  • Cynomolgus monkey (Cynomolgus) PD-1-His protein (AcroBiosystems, #PD1-C5223) was diluted with PBS to 2 ⁇ g/ml and added to a 96-well plate (Corning, #9018), 100 ⁇ l per well, at 4°C Incubate overnight. After discarding the liquid, the plate was washed three times with PBST, 250 ⁇ l of 2% BSA was added to block, and the plate was incubated at room temperature for 1 hour.
  • PBST buffer pH 7.4, containing 0.05% tween-20
  • dilute the concentration of the antigen-binding protein to be tested to 5 ⁇ g/ml
  • add 100 ⁇ l/well and incubate at 37°C for 1 hour.
  • 4000-fold diluted goat anti-human HRP secondary antibody (Invitrogen, #A18805) was added and incubated at 37°C for 1 hour.
  • TMB Biopanda, #TMB-S-003
  • stop solution BBI life sciences, #E661006-0200
  • the results are shown in Figure 2.
  • the anti-PD-1 HCAb antibodies PR002474, PR002476, PR002479 and PR002481 described in the present application can all bind to the cynomolgus monkey PD-1 protein, and the binding activity is comparable to that of the positive control antibody pembrolizumab.
  • Binding experiments at the cellular level were performed using CHO-K1 cell lines (CHO-K1/hPD-1, CHO-K1/cyno PD-1, derived from GenScript) overexpressing human or cynomolgus PD-1. Briefly, cellular CHO-K1-hPD-1 cells were digested and resuspended in F-12K complete medium to adjust the cell density to 1 ⁇ 10 6 cells/mL.
  • 100 ⁇ L cells/well were seeded on a 96-well V-bottom plate (Corning, Cat#3894), and then 100 ⁇ L/well, 2 times the final concentration of the 5-fold dilution of the antigen-binding protein to be tested was added, and mixed well, wherein the antigen-binding protein was The highest final concentration was 100 nM or 300 nM, ranging from 8 to 11 concentrations, with hIgG as a control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour. After that, 100 ⁇ L/well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g at 4° C. for 5 minutes, and the supernatant was discarded.
  • Figure 3A shows that the anti-PD-1 H2L2 antibodies PR000673, PR000674, PR000675, PR000676, PR000677, PR000678 and PR000680 of the present application can all bind to CHO-K1 cells overexpressing human PD-1, and some of the antibodies have binding activities Comparable to the positive control antibody pembrolizumab.
  • Figure 3B shows that the anti-PD-1 HCAb antibodies PR002473, PR002474, PR002475, PR002476, PR002477, PR002478, PR002479 and PR002481 of the present application can all bind to CHO-K1 cells overexpressing human PD-1.
  • Figure 3C shows that the HCAb antibodies PR005093, PR005096, PR005097, PR005098, PR005099, PR005100, PR005101, PR005102, PR005103 and PR005104 of the PR002481 mutants can all bind to CHO-K1 cells overexpressing human PD-1 with better affinity than PR005090.
  • the kinetics of the PR002481 affinity matured mutant was determined using the Octet Red96e instrument. Histidine- and avi-tagged human PD-1 protein was purchased from manufacturer ACRO Biosystems (Cat. No. PD1-H82E4) in 1x Kinetic Buffer (from 10x Kinetic Buffer (ForteBio, Cat. No. 18-1105)) dilution) for kinetic testing and dilution of antigens and antibodies. Binding kinetics analysis between antigen and antibody was performed by Biofilm Interferometry (BLI) technique using Octet Molecular Interaction Analyzer (ForteBio, model Octet Red96e).
  • BKI Biofilm Interferometry
  • the sensor When determining the binding kinetics of antigen and antibody, the sensor was rotated at 1000 rpm. First, 8 SA sensors placed in a column were equilibrated in the test buffer for 10 minutes, and then PD-1 was captured by the SA sensor with a capture height of 0.5 nm, and then the SA sensor was equilibrated in the test buffer for 100s, and then diluted to a single concentration with Each antibody and a row of blank buffer wells bind for 180s and dissociate for 600s. Replace with a new column of SA sensors each cycle.
  • Antigen-binding proteins block the binding of human PD-L1 or PD-L2 to CHO-K1 cells overexpressing human PD-1
  • CHO-K1 cell line (CHO-K1-hPD-1) overexpressing human PD-1 was used.
  • Cell-level human PD-1/human PD-L1 and cell-level human PD-1/human PD-L2 binding blocking experiments were performed. Briefly, CHO-K1-hPD-1 cells were digested and resuspended in F-12K complete medium to adjust the cell density to 1 x 106 cells/mL.
  • 100 ⁇ L cells/well were seeded in 96-well V-bottom plate (Corning, Cat#3894), and then 100 ⁇ L/well, 2 times the final concentration, 3 times or 5 times the concentration of the antigen-binding protein to be tested, which was serially diluted, were mixed evenly.
  • the highest final concentration of antigen-binding protein was 100nM or 300nM, with a total of 8 concentrations, and hIgG was used as a control.
  • the cells were placed at 4°C and incubated in the dark for 1 hour.
  • biotin-labeled human PD-L1 protein (AcroBiosystems, PD1-H82F2) at a concentration of 1 ⁇ g/mL or biotin-labeled protein at a concentration of 1 ⁇ g/mL.
  • Human PD-L2 protein (AcroBiosytems, PD2-H82F6) was incubated at 4°C for 30 minutes in the dark. 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.
  • FIGS. 5A and 5B shows the application of the H2L2 antibody PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680, and HCAb antibody PR002473, PR002474, PR002475, PR002476, PR002477, PR002478, PR002479 and PR002481 can hindered
  • the binding of human PD-L1 to human PD-1 on the cell surface was blocked, and the inhibitory ability was comparable to that of the positive control pembrolizumab.
  • Figure 5C shows that the H2L2 antibodies PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680 described in this application can block the binding of human PD-L2 to human PD-1 on the cell surface.
  • Hep3B (constructed by Shanghai ChemPartner) cells overexpressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein), or HEK293T cells overexpressing PD-L1 and OS8, were plated on 96-well plates with a cell volume of 1.25 ⁇ 10 4 /well, 100 ⁇ L/well. 37 [deg.] C under 5% CO 2 environment incubated overnight. Remove the supernatant, add 50 ⁇ L/well of the antigen-binding protein dilution to be tested, the initial concentration is 50 nM, 4-fold dilution (or the initial concentration is 500 nM, 5-fold dilution), hlgG1 is the control group.
  • Figure 6A shows that H2L2 antibodies PR000673, PR000674, PR000678, PR000679 and PR000680 described in this application have inhibitory effects on PD-1 signaling pathway.
  • Figure 6B shows that the PR002481 mutant HCAb antibodies PR005090, PR005093, PR005096, PR005097, PR005098, PR005099, PR005100, PR005101, PR005102, PR005103, and PR005104 inhibited the PD-1 signaling pathway.
  • Figure 6C shows that HCAb antibodies PR005090, PR005145, PR005149, PR005150, PR005141, PR005142, PR005144, PR005147 and PR005148 of the PR002481 mutants inhibit the PD-1 signaling pathway.
  • Figure 6D and Figure 6E show that HCAb antibody of PR002481 mutant has inhibitory effect on PD-1 signaling pathway.
  • Figure 6F shows that HCAb antibodies PR005569, PR005572, PR005575, PR005583 and PR005585 of the PR002481 mutant all inhibited the PD-1 signaling pathway.
  • Example 7 Antigen-binding proteins stimulate cytokine secretion in mixed lymphocyte reaction (MLR)
  • PBMC cells Purchase AllCells PBMC cells, isolate monocytes, add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) after induction for 6 days, get no Mature human CD14+ dendritic cells (iDC cells).
  • IL-4 human interleukin 4
  • R&D recombinant human IL-4
  • GM-CSF R&D, #215-GM/CF
  • iDC cells Mature human CD14+ dendritic cells
  • lipopolysaccharide Lipopolysaccharide, LPS; Sigma, #L2630
  • T lymphocytes were isolated from the second donor PBMC cells using a T cell isolation kit (StemCell, #17951).
  • PBMCs from 12 donors were used to divide into six groups of donor pairs for mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • H2L2 antibodies PR000673, PR000674, PR000675, PR000676, PR000677, PR000678, PR000679 and PR000680, and PR002481 all enhanced cytokine secretion by activated T lymphocytes IL-2 and IFN- ⁇ .
  • Figure 9 shows that the HCAb antibodies PR005569, PR005572, PR005575, PR005583 and PR005585 of the PR002481 mutant can enhance the secretion of cytokines IL-2 and IFN- ⁇ by activated T lymphocytes, and the activation ability is comparable to that of the positive control.
  • This example uses the antigen binding domain Fab of the anti-CD73 IgG antibody PR000846 and the antigen binding domain VH of the anti-PD-1 HCAb antibody PR002481 to construct a PD-1 ⁇ CD73 bispecific antibody molecule.
  • the amino acid sequences of PR000846 light and heavy ligation are shown in Table 10.
  • Antibody number light chain heavy chain VL VH PR000846 SEQ ID NO: 330 SEQ ID NO: 246 SEQ ID NO: 237 SEQ ID NO: 159
  • the positive control molecule is the anti-CD73 IgG monoclonal antibody PR000846, which is also the parent monoclonal antibody at the CD73 end of the CD73 ⁇ PD-1 double antibody molecule.
  • the positive control molecule is the anti-PD-1 IgG monoclonal antibody PR002481, which is also the parent monoclonal antibody at the PD-1 end of the CD73 ⁇ PD-1 double antibody molecule.
  • the Fab end is derived from the conventional antibody PR000846, and VH_A and VL_A are the variable region of the heavy chain and the variable region of the light chain of the antibody PR000846, respectively.
  • the VH end is derived from the heavy chain antibody PR002481, and VH_B is the heavy chain variable region of the heavy chain antibody PR002481.
  • CL is the light chain constant region domain.
  • CH1, CH2 and CH3 are the first, second and third domains, respectively, of the heavy chain constant region.
  • L is the linking peptide GS_15 (SEQ ID NO: 338) and h is the hinge region or derived sequence of an IgG antibody.
  • VH_B is at the N end of VH_A.
  • IgG1 is L234A, L235A mutated.
  • the prepared bispecific antibody with tetravalent symmetric structure is PR003568, comprising two polypeptide chains, wherein the amino acid sequence of the long chain is shown in SEQ ID NO:333, and the amino acid sequence of the short chain is shown in SEQ ID NO:330.
  • the prepared diabody molecule samples and their physicochemical properties are summarized in Table 11.
  • CHO-K1 cell lines (CHO-K1-hCD73, CHO-K1-hPD-1) overexpressing human CD73 or PD-1 were used. 1) Carry out binding experiments at the cellular level. Briefly, CHO-K1-hCD73 and CHO-K1-hPD-1 cells were digested and resuspended in F-12K complete medium to adjust the cell density to 1 ⁇ 10 6 cells/mL.
  • 100 ⁇ L of cells/well were seeded in a 96-well V-bottom plate (Corning, #3894), then 100 ⁇ L/well was added, centrifuged at 500 g for 5 minutes at 4°C, and the supernatant was discarded. Dilute the antibody to the highest final concentration of 50 nM, 3-fold dilution, for a total of 8 concentrations. 100 ⁇ L of the diluted antibody was added to the cells, and the cells were placed at 4°C and incubated in the dark for 1 hour. After that, 100 ⁇ L/well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g at 4° C. for 5 minutes, and the supernatant was discarded.
  • PR003568 has a good binding activity to CD73 and PD-1.
  • the activity of the PD-1 end of the PR003568 bispecific antibody was consistent with that of the parental monoclonal antibody PR002481, and the activity of the CD73 end was slightly lower than that of the parental monoclonal antibody PR000846.
  • Example 10 CD73 ⁇ PD-1 bispecific antibody junction inhibits the enzymatic activity of CD73
  • the activity of soluble recombinant CD73 was determined by the malachite green method by first adding 12.5 ⁇ L of 1 nM recombinant CD73 and 12.5 ⁇ L of 1 nM antibody to a 384-well plate (Corning, #3799) (assay buffer was 25 mM Tris pH 7.5, 5 mM MgCl 2 , 0.005 % Tween-20) for 1 hour at room temperature. Add 25 ⁇ L of AMP (maximum concentration 200 ⁇ M, diluted 2-fold with assay buffer to 8 concentrations) and incubate at room temperature for 15 minutes.
  • the concentration of inorganic phosphate in each well was measured, and the concentration of inorganic phosphate was determined by the malachite green method according to the manufacturer's instructions. After the measurement, the absorbance at 620 nm was recorded with a Molecular Devices (SPECTRAMax plus 384) plate reader. The experimental results were analyzed and graphed using GraphPad Prism 8.0.
  • PR003568 has a good inhibitory activity of CD73, which is comparable to that of the parental monoclonal antibody PR000846.
  • HEK293T eBioscience cells expressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) were plated on a 96-well plate, and the cell amount was 1.25 ⁇ 10 4 /well, 100 ⁇ L/well. Incubate overnight at 37°C under 5% CO2. The supernatant was removed, and 50 ⁇ L/well of the dilution of the antigen-binding protein to be tested was added, with an initial concentration of 100 nM and a 5-fold dilution. hlgG1 was the control group.
  • PR003568 has a good inhibitory effect on the PD-1 signaling pathway, which is comparable to the activity of the parental monoclonal antibody PR002481.
  • Example 12 Activation of T cells by bispecific antibodies in a mixed lymphocyte reaction assay
  • PBMC cells Purchase AllCells PBMC cells, isolate monocytes, add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) after induction for 6 days, get no Mature human CD14+ dendritic cells (iDC cells).
  • IL-4 human interleukin 4
  • R&D recombinant human IL-4
  • GM-CSF R&D, #215-GM/CF
  • iDC cells Mature human CD14+ dendritic cells
  • lipopolysaccharide Lipopolysaccharide, LPS; Sigma, #L2630
  • T lymphocytes were isolated from the second donor PBMC cells using a T cell isolation kit (StemCell, #17951).
  • the MLR activity of the CD73xPD-1 bispecific antibody PR003568 was better than that of the PD-1 monoclonal antibody PR002481.

Abstract

提供一种能够结合PD-1的抗原结合蛋白及其融合蛋白，及相应的制备方法和应用。该融合蛋白能够靶向PD-1和CD73。

Description

PD-1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，具体的涉及一种结合PD-1的抗原结合蛋白及其应用。
背景技术
程序性死亡受体1(Programmed Death-1,PD-1)是具有288个氨基酸的I型膜蛋白，主要表达在激活的T细胞表面。PD-1有两个配体，即程序性死亡配体-1(Programmed Death Ligand-1，PD-L1)和PD-L2。PD-1与PD-L1及PD-L2相互作用会下调T细胞的活性，减弱细胞因子的分泌，起到免疫抑制作用。PD-1/PD-L1通路抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，发挥杀伤肿瘤细胞的作用，进而抑制肿瘤生长。因此，以PD-1/PD-L1为靶点的免疫调节对肿瘤抑制有重要的意义。
CD73(也叫胞外-5’-核苷酸酶)是能够将AMP(单磷酸腺苷)分解成腺苷的酶，通过AMP的分解产生的腺苷抑制以T细胞为代表的免疫系统细胞的功能。已经报导CD73在许多不同肿瘤细胞上表达，CD73表达与肿瘤细胞的增殖、迁移、新血管形成、侵袭力、转移及较短患者存活期相关。因此，CD73在免疫应答的调节中发挥着重要的作用。CD73可以直接及间接方式调控癌症进展，凸显其作为新颖治疗靶的潜能。
目前，PD-1/PD-L1通路的阻断型抗体药物在临床上仍面临诸多挑战，例如有效性低、耐药性和副作用等。临床前的研究结果表明，PD-1/PD-L1免疫检查点抑制剂和CD73单克隆抗体联用杀伤肿瘤细胞，会有强有力的协同效应。一些临床试验(NCT03454451、NCT03835949和NCT02503774)也正在联合使用PD-1或者PD-L1抗体和CD73单克隆抗体治疗癌症，以提高安全性和有效性。但是联合用药的给药方式和临床试验均比较复杂且价格昂贵，且需同时具备或开发两个单药，周期和难度均较大。为解决现有的挑战，亟需研发同时针对PD-1/PD-L1免疫检查点和CD73靶点，且抗肿瘤效果高、安全性好的新产品。
发明内容
本申请提供了一种能够结合PD-1的抗原结合蛋白，其能够阻断PD-1和PD-L1及PD-L2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仅含“重链”的重链抗体，具有与人PD-1和食蟹猴PD-1特异性结合的活性。 该PD-1重链抗体的大小只有传统IgG抗体的一半，由于不含轻链的这一特点，使得该抗体可以用于双特异性抗体，并解决了轻链错配和异源二聚化的问题。本申请还提供了包含PD-1结合部分和CD73结合部分的融合蛋白，其能够以协同增加刺激免疫细胞中IFN-γ和/或IL2的分泌，进一步协同抑制肿瘤生长和/或肿瘤细胞增殖；同时，所述融合蛋白能够识别CD73蛋白，抑制CD73酶活的活性，并可诱导CD73的肿瘤细胞内化，导致细胞表面上的CD73活性进一步降低。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，其中，所述HCDR1包含SEQ ID NO:359所示的氨基酸序列，所述HCDR2包含SEQ ID NO:360所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:361所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv，VHH，重链抗体(HCAb)和/或dAb。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述HCDR1包含SEQ ID NO:11-16中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:50-55中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:92-97中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR1、HCDR2和HCDR3包含选自以下任意一组的氨基酸序列：
(1)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:92；
(2)HCDR1：SEQ ID NO:12，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:93；
(3)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:51，和HCDR3：SEQ ID NO:94；
(4)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:95；
(5)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:53，和HCDR3：SEQ ID NO:96；
(6)HCDR1：SEQ ID NO:16，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:97；以及，
(7)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:55，和HCDR3：SEQ ID NO:92。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链可变区VH，且所述VH包含SEQ ID NO:368所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:151-158中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白还包括抗体重链恒定区。
在某些实施方式中，所述重链恒定区源自人IgG恒定区。
在某些实施方式中，所述重链恒定区源自人IgG4恒定区和/或人IgG1恒定区。
在某些实施方式中，所述重链恒定区包含SEQ ID NO:354-355中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链，所述抗体重链包含SEQ ID NO:238-245中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1、LCDR2和LCDR3，其中，所述LCDR1包含SEQ ID NO:365所示的氨基酸序列，所述LCDR2包含SEQ ID NO:366所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:367所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含SEQ ID NO:121-125中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:130-135中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:140-145中任一项所示的氨基酸序列。
在某些实施方式中，所述LCDR1、LCDR2和LCDR3包含选自以下任意一组的氨基酸序列：
(1)LCDR1：SEQ ID NO:121，LCDR2：SEQ ID NO:130，和LCDR3：SEQ ID NO:140；
(2)LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:131，和LCDR3：SEQ ID NO:141；
(3)LCDR1：SEQ ID NO:123，LCDR2：SEQ ID NO:132，和LCDR3：SEQ ID NO:142；
(4)LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:133，和LCDR3：SEQ ID NO:143；
(5)LCDR1：SEQ ID NO:124，LCDR2：SEQ ID NO:134，和LCDR3：SEQ ID NO:144；以及
(6)LCDR1：SEQ ID NO:125，LCDR2：SEQ ID NO:135，和LCDR3：SEQ ID NO:145。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体轻链可变区VL，且所述VL包含 SEQ ID NO:369所示的氨基酸序列。
在某些实施方式中，所述VL包含SEQ ID NO:231-236中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白还包括抗体轻链恒定区。
在某些实施方式中，所述轻链恒定区包含SEQ ID NO:353所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体轻链，所述抗体轻链包含SEQ ID NO:324-329中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链或其片段和抗体轻链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，所述抗体轻链或其片段包含LCDR1、LCDR2和LCDR3，其中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3包含选自以下任意一组的氨基酸序列：
(1)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:92，LCDR1：SEQ ID NO:121，LCDR2：SEQ ID NO:130，和LCDR3：SEQ ID NO:140；
(2)HCDR1：SEQ ID NO:12，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:93，LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:131，和LCDR3：SEQ ID NO:141；
(3)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:51，和HCDR3：SEQ ID NO:94，LCDR1：SEQ ID NO:123，LCDR2：SEQ ID NO:132，和LCDR3：SEQ ID NO:142；
(4)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:95，LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:133，和LCDR3：SEQ ID NO:143；
(5)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:53，和HCDR3：SEQ ID NO:96，LCDR1：SEQ ID NO:124，LCDR2：SEQ ID NO:134，和LCDR3：SEQ ID NO:144；
(6)HCDR1：SEQ ID NO:16，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:97，LCDR1：SEQ ID NO:125，LCDR2：SEQ ID NO:135，和LCDR3：SEQ ID NO:145；以及，
(7)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:55，和HCDR3：SEQ ID NO:92，LCDR1：SEQ ID NO:121，LCDR2：SEQ ID NO:130，和LCDR3：SEQ ID NO:140。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链可变区VH和抗体轻链可变区VL，且所述VH和VL包含选自以下任意一组的氨基酸序列：
(1)VH：SEQ ID NO:151和VL：SEQ ID NO:231；
(2)VH：SEQ ID NO:152和VL：SEQ ID NO:232；
(3)VH：SEQ ID NO:153和VL：SEQ ID NO:233；
(4)VH：SEQ ID NO:154和VL：SEQ ID NO:234；
(5)VH：SEQ ID NO:155和VL：SEQ ID NO:235；
(6)VH：SEQ ID NO:156和VL：SEQ ID NO:232；
(7)VH：SEQ ID NO:157和VL：SEQ ID NO:236；以及
(8)VH：SEQ ID NO:158和VL：SEQ ID NO:231。
在某些实施方式中，所述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以下的KD值结合人PD-1；
(2)能够阻断PD-1和PD-L1结合；
(3)能够阻断PD-1和PD-L2结合；
(4)能够刺激免疫细胞中IL-2和/或IFN-γ的分泌；以及
(5)能够抑制肿瘤生长和/或肿瘤细胞增殖。
另一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，其中，所述HCDR1包含SEQ ID NO:362所示的氨基酸序列，所述HCDR2包含SEQ ID NO:363所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:364所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv，VHH，重链抗体(HCAb)和/或dAb。
在某些实施方式中，所述抗原结合片段为重链抗体(HCAb)。在某些实施方式中，所述抗原结合片段为VHH。
在某些实施方式中，所述抗原结合片段为所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述抗原结合片段为所述HCDR1包含SEQ ID NO:18-33中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:57-68中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:99-101和103-111中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合片段为所述HCDR1、HCDR2和HCDR3包含选自以下任意一组的氨基酸序列：
(1)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:100；
(2)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:99；
(3)HCDR1：SEQ ID NO:19，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:99；
(4)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:101；
(5)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
(6)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:59，和HCDR3：SEQ ID NO:103；
(7)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:60，和HCDR3：SEQ ID NO:103；
(8)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:61，和HCDR3：SEQ ID NO:103；
(9)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:60，和HCDR3：SEQ ID NO:103；
(10)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:59，和HCDR3：SEQ ID NO:103；
(11)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:61，和HCDR3：SEQ ID NO:103；
(12)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
(13)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:104；
(14)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:105；
(15)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
(16)HCDR1：SEQ ID NO:22，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
(17)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:106；
(18)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:107；
(19)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:103；
(20)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:62，和HCDR3：SEQ ID NO:100；
(21)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:61，和HCDR3：SEQ ID NO:100；
(22)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:59，和HCDR3：SEQ ID NO:100；
(23)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:60，和HCDR3：SEQ ID NO:100；
(24)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:63，和HCDR3：SEQ ID NO:100；
(25)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
(26)HCDR1：SEQ ID NO:24，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:108；
(27)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
(28)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:108；
(29)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:108；
(30)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:67，和HCDR3：SEQ ID NO:109；
(31)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:110；
(32)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
(33)HCDR1：SEQ ID NO:28，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(34)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:111；
(35)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:68，和HCDR3：SEQ ID NO:108；
(36)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(37)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:109；
(38)HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(39)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:111；
(40)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:68，和HCDR3：SEQ ID NO:111；
(41)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111；
(42)HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:109；
(43)HCDR1：SEQ ID NO:31，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(44)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:111；
(45)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(46)HCDR1：SEQ ID NO:32，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:109；
(47)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:109；以及
(48)HCDR1：SEQ ID NO:33，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链可变区VH，且所述VH包含SEQ ID NO:254所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:160-166和168-221中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白还包括抗体重链恒定区。
在某些实施方式中，所述重链恒定区源自人IgG恒定区。
在某些实施方式中，所述重链恒定区源自人IgG4恒定区和/或人IgG1恒定区。
在某些实施方式中，所述重链恒定区包含SEQ ID NO:354-355中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链，所述抗体重链包含SEQ ID NO:247-253和255-309中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白具有下述性质中的一种或多种：
a)能够以1×10 8M以下的KD值结合人PD-1；
b)能够阻断PD-1和PD-L1结合；
c)能够阻断PD-1和PD-L2结合；
d)能够刺激免疫细胞中IL-2和/或IFN-γ的分泌；以及
e)能够抑制肿瘤生长和/或肿瘤细胞增殖。
另一方面，本身请提供了一种分离的抗原结合蛋白，其包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，其中，所述HCDR1包含SEQ ID NO:10所示的氨基酸序列，所述HCDR2包含SEQ ID NO:81所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:102所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv，VHH，重链抗体(HCAb)和/或dAb。
在某些实施方式中，所述抗原结合片段为重链抗体(HCAb)。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述HCDR1包含SEQ ID NO:18、25、28和34-38中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:64-66中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:108、111和112中任一项所示的氨基酸序列。
在某些实施方式中，所述HCDR1、HCDR2和HCDR3包含选自以下任意一组的氨基酸序列：
(1)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:108；
(2)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:108；
(3)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111；
(4)HCDR1：SEQ ID NO:34，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111；
(5)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
(6)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(7)HCDR1：SEQ ID NO:35，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(8)HCDR1：SEQ ID NO:36，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(9)HCDR1：SEQ ID NO:36，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:112；
(10)HCDR1：SEQ ID NO:37，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
(11)HCDR1：SEQ ID NO:28，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；以及
(12)HCDR1：SEQ ID NO:38，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链可变区VH，且所述VH包含SEQ ID NO:167所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:196、197、200、201、202和222-230中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白还包括抗体重链恒定区。
在某些实施方式中，所述重链恒定区源自人IgG恒定区。
在某些实施方式中，所述重链恒定区源自人IgG4恒定区和/或人IgG1恒定区。
在某些实施方式中，所述重链恒定区包含SEQ ID NO:354-355中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体重链，所述抗体重链包含SEQ ID NO:310-323中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白具有下述性质中的一种或多种：
a)能够以1×10 8M以下的KD值结合人PD-1；
b)能够阻断PD-1和PD-L1结合；
c)能够阻断PD-1和PD-L2结合；
d)能够刺激免疫细胞中IL-2和/或IFN-γ的分泌；以及
e)能够抑制肿瘤生长和/或肿瘤细胞增殖。
另一方面，本申请提供了一种融合蛋白，所述融合蛋白包含第一靶向部分和第二靶向部分，所述第一靶向部分包含PD-1结合部分，其中所述PD-1结合部分包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，所述第二靶向部分包含CD73结合部分。
在某些实施方式中，所述CD73结合部分包括特异性结合CD73的抗体或其抗原结合片段。
在某些实施方式中，所述CD73结合部分包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3分别依次包含SEQ ID NO:17、SEQ ID NO:56和SEQ ID NO:98所示的氨基酸序列。
在某些实施方式中，所述CD73结合部分包括抗体重链可变区VH，所述VH包含SEQ ID NO:159所示的氨基酸序列。
在某些实施方式中，所述CD73结合部分包含抗体轻链或其片段，所述抗体轻链或其片 段包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3分别依次包含SEQ ID NO:123、SEQ ID NO:130和SEQ ID NO:146所示的氨基酸序列。
在某些实施方式中，所述CD73结合部分包括抗体轻链可变区VL，所述VL包含SEQ ID NO:237所示的氨基酸序列。
在某些实施方式中，所述CD73结合部分包含Fab。
在某些实施方式中，所述CD73结合部分包含抗体重链，且所述抗体重链包含SEQ ID NO:246所示的氨基酸序列。
在某些实施方式中，所述CD73结合部分包含抗体轻链，且所述抗体轻链包含SEQ ID NO:330所示的氨基酸序列。
在某些实施方式中，所述PD-1结合部分位于所述CD73结合部分的N端或C端。
在某些实施方式中，所述融合蛋白包括第一多肽链和第二多肽链，其中所述第一多肽链包括所述PD-1结合部分的VH和所述CD73结合部分的VH，所述第二多肽链包括所述CD73结合部分的VL。
在某些实施方式中，在所述第一多肽链中，所述PD-1结合部分的VH位于所述CD73结合部分的VH的N端。
在某些实施方式中，在所述第一多肽链中，所述PD-1结合部分的VH位于所述CD73结合部分的VH的C端。
在某些实施方式中，所述第一多肽链还包含抗体重链恒定区。
在某些实施方式中，所述第一多肽链自N端至C端，依次包含所述PD-1结合部分的VH、所述CD73结合部分的VH和所述抗体重链恒定区。
在某些实施方式中，所述PD-1结合部分的VH与所述CD73结合部分的VH通过连接肽间接连接。
在某些实施方式中，所述连接肽包含SEQ ID NO:334-352中任一项所示的氨基酸序列。
在某些实施方式中，所述第一多肽链包含SEQ ID NO:333所示的氨基酸序列。
在某些实施方式中，所述第二多肽链还包含抗体轻链恒定区。
在某些实施方式中，所述第二多肽链包含SEQ ID NO:330所示的氨基酸序列。
在某些实施方式中，所述融合蛋白包括两条所述第一多肽链和两条所述第二多肽链。
另一方面，本申请提供了免疫缀合物，所述免疫缀合物包含所述的分离的抗原结合蛋白或所述的融合蛋白。
另一方面，本申请提供了分离的一种或多种核酸分子，所述分离的一种或多种核酸分子 编码所述的分离的抗原结合蛋白和/或所述的融合蛋白。
另一方面，本申请提供了载体，所述载体包含所述的核酸分子。
另一方面，本申请提供了细胞，所述细胞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和/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和/或所述的融合蛋白表达的条件下，培养所述的细胞。
另一方面，本申请提供了药物组合物，所述药物组合物包含所述的分离的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体和/或所述的细胞，以及任选地药学上可接受的载体。
另一方面，本申请提供了所述的分离的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗PD-1介导的疾病或病症。
在某些实施方式中，所述PD-1介导的疾病或病症包括肿瘤、自体免疫疾病或炎症。在某些实施方式中，所述肿瘤包括实体瘤或非实体瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
一种增加受试者中T细胞活性的方法，其包括向有需要的受试者施用有效量的所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。
另一方面，本申请提供了预防、缓解或治疗PD-1介导的疾病或病症的方法，其包括向有需要的受试者施用有效量的所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物；任选地，与一种或多种其他肿瘤治疗方法联用。
在某些实施方式中，所述PD-1介导的疾病或病症包括肿瘤、自体免疫疾病或炎症。在某些实施方式中，所述肿瘤包括实体瘤或非实体瘤。在某些实施方式中，所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
另一方面，本申请提供了所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，其中用于预防、缓解或治疗PD-1介导的疾病或病症。
在某些实施方式中，所述PD-1介导的疾病或病症包括肿瘤、自体免疫疾病或炎症。在某些实施方式中，所述肿瘤包括实体瘤或非实体瘤。在某些实施方式中，所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
另一方面，本申请提供了试剂盒或给药装置，所述试剂盒或给药装置包括所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。
另一方面，本申请提供了抑制PD-L1蛋白和PD-1蛋白结合的方法，所述方法包括使用所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。
在某些实施方式中，所述方法为体外方法。
在某些实施方式中，所述方法为非诊断和/或治疗目的的方法。
另一方面，本申请提供了抑制PD-L2蛋白和PD-1蛋白结合的方法，所述方法包括使用所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。
在某些实施方式中，所述方法为体外方法。
在某些实施方式中，所述方法为非诊断和/或治疗目的的方法。
另一方面，本申请提供了检测PD-1蛋白的存在和/或含量的方法，所述方法包括使用所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物。
在某些实施方式中，所述方法为体外方法。
在某些实施方式中，所述方法为非诊断和/或治疗目的的方法。
所述的抗原结合蛋白、所述的融合蛋白、所述的缀合物、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物，与一种或多种其他肿瘤治疗方法联用在制备药物中的用途，所述药物用于治疗肿瘤。
在某些实施方式中，所述一种或多种其他肿瘤治疗方法包括化疗和/或放疗。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
所述的分离的抗原结合蛋白，和抗CD73抗体联用在制备药物中的用途，所述药物用于治疗肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
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1A是本申请所述抗原结合蛋白PR000673、PR000674、PR000675、PR000676、PR000677、PR000678和PR000679与人PD-1蛋白的结合活性。
图1B显示的是本申请所述抗原结合蛋白PR000680与人PD-1蛋白的结合活性。
图1C显示的是本申请所述抗原结合蛋白PR002474、PR002476、PR002479和PR002481与人PD-1蛋白的结合活性。
图2显示的是本申请所述抗原结合蛋白R002474、PR002476、PR002479和PR002481与食蟹猴PD-1蛋白的结合活性。
图3A显示的是本申请所述抗原结合蛋白PR000673、PR000674、PR000675、PR000676、PR000677、PR000678和PR000680与CHO-K1细胞上表达的人PD-1蛋白的结合活性。
图3B显示的是本申请所述抗原结合蛋白R002473、R002474、PR002475、PR002476、R002477、PR002478、PR002479和PR002481与CHO-K1细胞上表达的人PD-1蛋白的结合活性。
图3C显示的是本申请所述抗原结合蛋白PR005090、PR005093、PR005096、PR005097、PR005098、PR005099、PR005100、PR005101、PR005102、PR005103和PR005104与CHO-K1细胞上表达的人PD-1蛋白的结合活性。
图3D显示的是本申请所述抗原结合蛋白PR005090、PR005145、PR005149、PR005150、PR005141、PR005142、PR005144、PR005147和PR005148与CHO-K1细胞上表达的人PD-1 蛋白的结合活性。
图3E显示的是本申请所述抗原结合蛋白PR002481、PR005585、PR005583、PR005578、PR005577、PR005575、PR005572和PR005569与CHO-K1细胞上表达的人PD-1蛋白的结合活性。
图3F显示的是本申请所述抗原结合蛋白PR002481、PR005584、PR005582、PR005581、PR005574、PR005573、PR005571和PR005570与CHO-K1细胞上表达的人PD-1蛋白的结合活性。
图4显示的是本申请所述抗原结合蛋白PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680与CHO-K1细胞上表达的食蟹猴PD-1蛋白的结合活性。
图5A显示的是本申请所述抗原结合蛋白PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680阻断CHO-K1细胞上表达的人PD-1蛋白和PD-L1蛋白的结合的活性。
图5B显示的是本申请所述抗原结合蛋白PR002473、PR002474、PR002475、PR002476、PR002477、PR002478、PR002479和PR002481阻断CHO-K1细胞上表达的人PD-1蛋白和PD-L1蛋白的结合的活性。
图5C显示的是本申请所述抗原结合蛋白PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680阻断CHO-K1细胞上表达的人PD-1蛋白和PD-L2蛋白的结合的活性。
图6A显示的是本申请所述抗原结合蛋白PR000673、PR000674、PR000678、PR000679和PR000680对PD-1信号通路的抑制作用(利用报告基因细胞系检测)。
图6B显示的是本申请所述抗原结合蛋白PR005090、PR005093、PR005096、PR005097、PR005098、PR005099、PR005100、PR005101、PR005102、PR005103和PR005104对PD-1信号通路的抑制作用(利用报告基因细胞系检测)。
图6C显示的是本申请所述抗原结合蛋白PR005090、PR005145、PR005149、PR005150、PR005141、PR005142、PR005144、PR005147和PR005148对PD-1信号通路的抑制作用(利用报告基因细胞系检测)。
图6D显示的是本申请所述抗原结合蛋白PR002481、PR005570、PR005571、PR005573和PR005574对PD-1信号通路的抑制作用(利用报告基因细胞系检测)。
图6E显示的是本申请所述抗原结合蛋白PR002481、PR005577、PR005581、PR005582 和PR005584对PD-1信号通路的抑制作用(利用报告基因细胞系检测)。
图6F显示的是本申请所述抗原结合蛋白PR002481、PR005569、PR005572、PR005575、PR005583和PR005585对PD-1信号通路的抑制作用(利用报告基因细胞系检测)。
图7A、图7B和图7C显示的是本申请所述抗原结合蛋白PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680激活T细胞的能力(混合淋巴细胞反应实验)。
图8A和图8B显示的是本申请所述抗原结合蛋白PR000674、PR000678和PR000679激活T细胞的能力(混合淋巴细胞反应实验)。
图8C显示的是本申请所述抗原结合蛋白PR002481激活T细胞的能力(混合淋巴细胞反应实验)。
图9显示的是本申请所述抗原结合蛋白PR005569、PR005572、PR005575、PR005583和PR005585和PR002481激活T细胞的能力(混合淋巴细胞反应实验)。
图10显示的是本申请所述融合蛋白示例性的分子结构。
图11显示的是本申请所述融合蛋白PR003568与CHO-K1细胞上表达的人CD73细胞的结合活性。
图12显示的是本申请所述融合蛋白PR003568与CHO-K1细胞上表达的人PD-1细胞的结合活性。
图13显示的是本申请所述融合蛋白PR003568抑制CD73的酶活性。
图14显示的是本申请所述融合蛋白PR003568对PD-1信号通路的抑制作用(利用报告基因细胞系检测)。
图15A和图15B显示的是本申请所述融合蛋白激活T细胞分泌细胞因子的能力(混合淋巴细胞反应实验)。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“PD-1”通常是指程序性细胞死亡1，也称为“程序性死亡1”、“CD279”、“分化簇279”、“PD1”、“PDCD1”。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，并参与细胞凋亡。PD-1通常包含一个细胞外IgV结构 域，跨膜区和胞内结构域。PD-1可结合两种配体，PD-L1和PD-L2。所述“PD-1”包括任何脊椎动物来源的任何天然PD-1，所述任何脊椎动物来源包括哺乳动物，诸如灵长类(例如，人和食蟹猴)和啮齿类(例如，小鼠和大鼠)。所述术语涵盖“全长”、未加工的PD-1以及由细胞加工所产生的任何形式的PD-1。PD-1可作为跨膜蛋白或作为可溶性蛋白存在。“PD-1”包括完整的PD-1及其片段，还包括PD-1的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与PD-1共同表位的类似物。在本申请中，所述PD-1可以包括人PD-1和/或猴(例如，食蟹猴)PD-1。示例性的人PD-1氨基酸序列可以在NCBI登录号NP_005009.2中找到。示例性的食蟹猴PD-1氨基酸序列可以在NCBI登录号NP_001271065.1中找到。
在本申请中，术语“PD-L1”通常是指程序性细胞死亡1配体1，也可称为B7同源物1、B7-H1、分化簇274、(3)274或CD274，其与PD-1结合后下调T细胞活化和细胞因子分泌。“PD-L1”包括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，人和食蟹猴)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。“PD-L1”包括完整的PD-L1及其片段，还包括PD-L1的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与PD-L1共同表位的类似物。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。示例性的人PD-L1氨基酸序列可在NCBI登录号NP_001254653或UniProt登录号Q9NZQ7下找到。示例性的食蟹猴PD-L1氨基酸序列可在NCBI登录号XP_005581836.1下找到。
在本申请中，术语“CD73”也称为5’外切核苷酸酶，通常是指能够将胞外核苷5'单磷酸转化为核苷，即将单磷酸腺苷(AMP)转化为腺苷的酶(核苷酸酶)。术语“CD73”包括细胞天然表达的CD73的任何变体或同工型。可以从天然表达它们的细胞或组织中分离出CD73或其任何变体和同工型，或者可以使用本领域中众所周知的技术和/或本文所述的那些重组产生。人CD73的氨基酸序列可以参见GenBank中登录号AAH65937.1(5'-核苷酸酶，ecto)，或NP_002517(同工型1前蛋白)和NP_001191742(同工型2前蛋白)下。
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。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’，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“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“抗体”其以最广泛意义使用，且具体涵盖，但不限于，单克隆抗体(包括包含两条轻链和两条重链的全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人类抗体、嵌合抗体、重链抗体和骆驼化单结构域抗体(如，重链可变结构域抗体)。抗体通常具有免疫球蛋白的结构，可以包含通过二硫键互相连接的至少两条重链(HC)和两条轻链(LC)的蛋白，或其抗原结合片段。每条重链包含重链可变区(VH)和重链恒定区。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM，IgD，IgG，IgA和IgE，其相应的重链分别为μ链，δ链，γ链，α链，ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2，IgG3，IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
在某些天然存在的IgG、IgD和IgA抗体中，重链恒定区包含三个结构域，CH1、CH2和CH3。在某些天然存在的抗体中，各轻链包含轻链可变区(VL)和轻链恒定区。轻链恒定区包含一个结构域，CL。VH和VL区可进一步细分为超变性的区域，称为互补决定区(CDR)，其与称为框架区(FR)的较保守的区域交替。各VH和VL包含三个CDR和四个框架区(FR)，从氨基端至羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。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CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs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。抗体的恒定区可介导免疫球蛋白与宿主 组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)结合。
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)。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))、基于结构环区域位置的Chothia定义规则(参见，J Mol Biol 273:927-48,1997)和基于IMGT本体论(IMGT-ONTOLOGY)的概念和IMGT Scientific图表规则的IMGT定义规则。在本申请中，所述抗原结合蛋白的CDR按照Chothia定义规则进行划分。具体如表1所示。
表1 Chothia对CDR的划分
  LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
Chothia L24--L34 L50--L56 L89--L97 H26--H32 H52--H56 H95--H102
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，本申请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“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
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“VHH”通常是指重链可变结构域抗体。对于双链抗体(免疫球蛋白)说，VHH通常缺失抗体轻链和/或重链恒定区，只包含重链可变结构域的部分。
在本申请中，术语“重链抗体”也可称为HCAb，通常是指相对双链抗体(免疫球蛋白)来说，缺失抗体轻链，只包含重链的抗体。所述重链抗体可以包含两条抗体重链。
在本申请中，术语“结合”、“特异性结合”或“对…特异性的”通常是指可测量且可再现的相互作用，诸如抗原和抗体之间的结合，其可以确定在存在分子(包括生物学分子)的异质群体的情况中靶物的存在。例如，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更大的亲和力、亲合力、更容易和/或以更大的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。
在本申请中，术语“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“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“分离的核酸分子”或“分离的多核苷酸”通产是指基因组、mRNA、cDNA或合成来源的DNA或RNA或其一定组合，其不与在自然界中发现的多核苷酸的全部或一部分缔合，或连接至其在自然界中不连接的多核苷酸。
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“药学上可接受的载体”通常包括药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。通常，生理学可接受的载体是pH缓冲水溶液。生理学可接受载体的例子可包括缓冲剂，抗氧化剂，低分子量(少于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其它碳水化合物，螯合剂，糖醇，成盐反荷离子，诸如钠；和/或非离子表面活性剂。
在本申请中，术语“治疗”通常是指期望改变所治疗个体的天然病程，且可为实现防治或在临床病变过程中进行的临床介入。合乎需要的治疗效果包括但不限于防止疾病发生或复发性、减轻症状、减弱疾病的任何直接或间接病理学后果、防止转移、降低疾病进展速率、改善或缓解疾病状态以及缓和或改善预后。在一些情形中，抗体(例如，抗PD-1抗体)可用来延迟疾病发展或减缓疾病进展。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的化合物(例如，抗癌治疗剂)或药物组合物(例如，包含抗癌治疗剂的药物组合物)的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。胃肠外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。
在本申请中，术语“肿瘤”通常是指所有赘生性细胞生长和增殖(无论恶性还是良性)以及所有癌前和癌性细胞和组织。在本申请中，所述肿瘤可以为细胞和组织的PD-1或PD-L1高表达的肿瘤。肿瘤可包括实体瘤和/或非实体瘤(例如，血液瘤、淋巴瘤)。
在本申请中，术语“免疫缀合物”通常是指抗原结合蛋白与其它活性剂连接形成的物质，其他活性剂可以是小分子活性剂，例如化疗剂、毒素、免疫治疗剂、成像探针或光谱探针。
在本申请中，术语“试剂盒”通常是指包含用于给予本申请的抗原结合蛋白治疗PD-1介导的相关病症的组分的包装产品。试剂盒的组分可包含在分开的小瓶中(即具有分开部分的试剂盒)，或在单个小瓶内提供。试剂盒可包含试剂，诸如缓冲剂、蛋白稳定试剂、信号产生体系(例如，荧光信号生成体系)、抗体、对照蛋白、以及测试容器。试剂盒还可以包含实施所述方法的说明书。
在本申请中，术语“给药装置”包括：(i)输注模块，所述输注模块用于对受试者施用包括具有一活性成分的药物组合物；(ii)用于输注的药物组合物，所述药物组合物中含有一 活性成分，所述活性成分选自下组：抗原结合蛋白、多特异性抗体、免疫细胞、抗体药物偶联物或其组合；以及(iii)任选的药效监控模块。
在本申请中，术语“联用”通常是指是指两种或更多种治疗剂可以在混合物中、作为单一药剂同时地或作为单一药剂以任何顺序先后地共同向受检者施用。
在本申请中涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，本申请所述的抗原结合蛋白)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述抗原结合蛋白的氨基酸序列的一部分可以与来自特定物种的抗体中相应的氨基酸序列同源，或者属于特定的类别。例如，抗体的可变区及恒定部分均可以来自一个动物物种(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，本申请所述的抗原结合蛋白)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。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
抗原结合蛋白
一方面，本申请提供了一种抗原结合蛋白，所述抗原结合蛋白可包含抗体重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:368所示的氨基酸序列。
本申请所述抗原结合蛋白可包含重链互补决定区HCDR1。在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:359所示的氨基酸序列：GX 2X 3X 4SX 6X 7X 8X 9，其中，X 2＝D、F、G或L，X 3＝I、S或T，X 4＝F、L或V，X 6＝D、N或S，X 7＝N或Y，X 8＝S或不存在氨基酸，且X 9＝A或不存在氨基酸。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:11-16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互补决定区HCDR2。在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:360所示的氨基酸序列：X 1X 2X 3X 4X 5X 6X 7，其中，X 1＝I、L、S、W或Y，X 2＝G、P、S或Y，X 3＝D、I、R或S，X 4＝F、G或S，X 5＝D、K、S或Y，X 6＝K、N、S、T或W，X 7＝Y或不存在氨基酸。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:50-55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互补决定区HCDR3。在本申请中，所述抗原结合蛋 白的HCDR3可包含SEQ ID NO:361所示的氨基酸序列：X 1X 2X 3X 4X 5X 6X 7X 8X 9X1 0X 11X 12X 13X 14，其中，X 1＝E、L、N或S，X 2＝D、G、P、S或T，X 3＝D、G、H、L或P，X 4＝D或Y，X 5＝S、V、Y或不存在氨基酸，X 6＝G、S或不存在氨基酸，X 7＝N、S或不存在氨基酸，X 8＝G、W或不存在氨基酸，X 9＝S、Y或不存在氨基酸，X 10＝Q、Y或不存在氨基酸，X 11＝L、Y或不存在氨基酸，X 12＝D、F或不存在氨基酸，X 13＝Q、Y或不存在氨基酸，且X 14＝H或不存在氨基酸。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:92-97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互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:359所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:360所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:361所示的氨基酸序列。
本申请所述抗原结合蛋白可包含重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:11-16中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:50-55中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:92-97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HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:11所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:50所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:92所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:50所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:93所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:51所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:94所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:14所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:95所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:53所示的氨基酸序 列，且所述HCDR3可包含SEQ ID NO:96所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:54所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:97所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:11所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:55所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:92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HFWR1，所述HFWR1的C末端与所述HCDR1的N末端直接或间接相连。在本申请中，所述HFWR1可包含SEQ ID NO:1-7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HFWR2，所述HFWR2位于所述HCDR1与所述HCDR2之间。在本申请中，所述HFWR2可包含SEQ ID NO:39-44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框架区HFWR3，所述HFWR3位于所述HCDR2与所述HCDR3之间。在本申请中，所述HFWR3可包含SEQ ID NO:69-75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HFWR4，所述HFWR4的N末端与所述HCDR3的C末端相连。在本申请中，所述HFWR4可包含SEQ ID NO:113-115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重链可变区VH，且所述VH可包含SEQ ID NO:368所示的氨基酸序列。
本申请所述抗原结合蛋白可包含抗体轻链可变区VL中的至少一个CDR，所述VL可包含SEQ ID NO:369所示的氨基酸序列。
本申请所述抗原结合蛋白可包含轻链互补决定区LCDR1。在本申请中，所述抗原结合蛋白的LCDR1可包含SEQ ID NO:365所示的氨基酸序列：RASQX 5X 6SX 8X 9LA，其中，X 5＝S或T，X 6＝I或V，X 8＝I、R或S，X 9＝D、S、W或Y。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的LCDR1可包含SEQ ID NO:121-125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互补决定区LCDR2。在本申请中，所述抗原结合蛋 白的LCDR2可包含SEQ ID NO:366所示的氨基酸序列：X 1X 2X 3X 4X 5X 6X 7，其中，X 1＝D、G或K，X 2＝A或T，X 3＝A或S，X 4＝K、N、S或T，X 5＝L或R，X 6＝A或E，X 7＝S或T。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的LCDR2可包含SEQ ID NO:130-135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互补决定区LCDR3。在本申请中，所述抗原结合蛋白的LCDR3可包含SEQ ID NO:367所示的氨基酸序列：X 1QX 3X 4X 5X 6X 7X 8X 9，其中，X 1＝H或Q，X 3＝F、H、R或Y，X 4＝N或S，X 5＝N、S或Y，X 6＝Y或W，X 7＝I、P或W，X 8＝F、I、L或T，且X 9＝T或不存在氨基酸。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的LCDR3可包含SEQ ID NO:140-145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LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:365所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:366所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:367所示的氨基酸序列。
本申请所述抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:121-125中任一项所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:130-135中任一项所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:140-145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LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:121所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:130所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:140所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:122所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:131所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:141所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:123所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:132所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:142所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:122所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:133所示的氨基酸序 列，且所述LCDR3可包含SEQ ID NO:143所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:124所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:134所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:144所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:125所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:135所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:145所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1、LCDR2和LCDR3，所述LCDR1可包含SEQ ID NO:121所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:130所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:140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LFWR1，所述LFWR1的C末端与所述LCDR1的N末端直接或间接相连。在本申请中，所述LFWR1可包含SEQ ID NO:116-119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LFWR2，所述LFWR2位于所述LCDR1与所述LCDR2之间。在本申请中，所述LFWR2可包含SEQ ID NO:126-129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LFWR3，所述LFWR3位于所述LCDR2与所述LCDR3之间。在本申请中，所述LFWR3可包含SEQ ID NO:136-139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LFWR4，所述LFWR4的N末端与所述LCDR3的C末端相连。在本申请中，所述LFWR4可包含SEQ ID NO:147-150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:369所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:11所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:50所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:92所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:121所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:130所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:140所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和 LCDR3，所述HCDR1可包含SEQ ID NO:12所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:50所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:93所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:122所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:131所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:141所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:13所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:51所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:94所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:123所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:132所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:142所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:14所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:52所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:95所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:122所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:133所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:143所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:15所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:53所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:96所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:124所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:134所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:144所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:16所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:54所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:97所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:125所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:135所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:145所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，所述HCDR1可包含SEQ ID NO:11所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:55所示的氨基酸序列，所述HCDR3可包含SEQ ID NO:92所示的氨基酸序列，所述LCDR1可包含SEQ ID NO:121所示的氨基酸序列，所述LCDR2可包含SEQ ID NO:130所示的氨基酸序列，且所述LCDR3可包含SEQ ID NO:140所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:368所 示的氨基酸序列，且所述VL可包含SEQ ID NO:369所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:151-158中任一项所示的氨基酸序列，且所述VL可包含SEQ ID NO:231-236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VH和VL，所述VH可包含SEQ ID NO:151所示的氨基酸序列，且所述VL可包含SEQ ID NO:231所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:152所示的氨基酸序列，且所述VL可包含SEQ ID NO:232所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:153所示的氨基酸序列，且所述VL可包含SEQ ID NO:233所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:154所示的氨基酸序列，且所述VL可包含SEQ ID NO:234所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:155所示的氨基酸序列，且所述VL可包含SEQ ID NO:235所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:156所示的氨基酸序列，且所述VL可包含SEQ ID NO:232所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:157所示的氨基酸序列，且所述VL可包含SEQ ID NO:236所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含VH和VL，所述VH可包含SEQ ID NO:158所示的氨基酸序列，且所述VL可包含SEQ ID NO:231所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:238所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:324所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:239所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:325所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:240所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:326所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:241所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:327所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:242所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:328所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:243所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:325所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:244所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:329所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含抗体重链和抗体轻链，所述抗体重链可包含SEQ ID NO:245所示的氨基酸序列，且所述抗体轻链可包含SEQ ID NO:324所示的氨基酸序列。
一方面，本申请提供了一种抗原结合蛋白，所述抗原结合蛋白可包含抗体重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:254所示的氨基酸序列。
本申请所述抗原结合蛋白可包含重链互补决定区HCDR1。在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:362所示的氨基酸序列：GFX 3VX 5X 6X 7，其中，X 3＝A、F、N或S，X 5＝D、R、S或V，X 6＝F、G、R、S或Y，F 7＝F、H、N或Y。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:18-33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互补决定区HCDR2。在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:363所示的氨基酸序列：X 1X 2X 3GX 5，其中，X 1＝D或H，X 2＝G、I、K、R或S，X 3＝A、G或S，且X 5＝G或S。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:57-68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互补决定区HCDR3。在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:364所示的氨基酸序列：AX 2X 3VX 5EX 7X 8GYNYPFNY，其中，X 2＝I或T，X 3＝A、E、M、Q或R，X 5＝A、P或R，X 7＝A、E或K，X 8＝G、N或Q。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:99-101和103-111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互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:362所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:363所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:364所示的氨基酸序列。
本申请所述抗原结合蛋白可包含重链互补决定区HCDR1、HCDR2和HCDR3，所述 HCDR1可包含SEQ ID NO:18-33中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:57-68中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:99-101和103-111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HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:57所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:57所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:99所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:19所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:57所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:99所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:57所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:101所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:60所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:59所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:61所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:60所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:59所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:61所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:104所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:105所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:21所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:22所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:106所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:107所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:58所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:103所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:62所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:61所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:59所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:60所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:63所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:100所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:24所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:65所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:26所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:66所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:65所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:26所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:67所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:109所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:27所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:110所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:28所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:66所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:68所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:66所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:109所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:29所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:66所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:68所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:29所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:65所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:30所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:109所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:31所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:66所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:32所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:109所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:23所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:66所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:109所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:33所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:65所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111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HFWR1，所述HFWR1的C末端与所述HCDR1的N末端直接或间接相连。在本申请中，所述HFWR1可包含SEQ ID NO:9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HFWR2，所述HFWR2位于所述HCDR1与所述HCDR2之间。在本申请中，所述HFWR2可包含SEQ ID NO:46-49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HFWR3，所述HFWR3位于所述HCDR2与所述HCDR3之间。在本申请中，所述HFWR3可包含SEQ ID NO:77-80和82-91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HFWR4，所述HFWR4的N末端与所述HCDR3的C末端相连。在本申请中，所述HFWR4可包含SEQ ID NO:113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重链可变区VH，且所述VH可包含SEQ ID NO:160-166和168-221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抗体重链可包含SEQ ID NO:247-253和255-309中任一项所示的氨基酸序列。
一方面，本申请提供了一种抗原结合蛋白，所述抗原结合蛋白可包含抗体重链可变区VH中的至少一个CDR，所述VH可包含SEQ ID NO:167所示的氨基酸序列。
本申请所述抗原结合蛋白可包含重链互补决定区HCDR1。在本申请中，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:10所示的氨基酸序列：GFX 3VX 5X 6X 7，其中，X 3＝N或T，X 5＝A或S，X 6＝F或S，X 7＝F、H或Y。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR1可包含SEQ ID NO:18、25、28和34-38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互补决定区HCDR2。在本申请中，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:81所示的氨基酸序列：DKX 3GS，其中，X 3＝A、G或S。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR2可包含SEQ ID NO:64-66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互补决定区HCDR3。在本申请中，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:102所示的氨基酸序列：ATX 2VREKX 8GYNYPFNY，其中，X 2＝A或E，X 8＝N或Q。例如，该序列可以是根据Chothia定义规则确定的序列。
例如，所述抗原结合蛋白的HCDR3可包含SEQ ID NO:108、111和112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互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:10所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:81所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:102所示的氨基酸序列。
本申请所述抗原结合蛋白可包含重链互补决定区HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18、25、28和34-38中任一项所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64-66中任一项所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108、111和112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HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:66所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:65所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:25所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:65所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:34所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:65所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:108所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:18所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包 含SEQ ID NO:36所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:36所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:112所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:37所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:28所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含SEQ ID NO:38所示的氨基酸序列，所述HCDR2可包含SEQ ID NO:64所示的氨基酸序列，且所述HCDR3可包含SEQ ID NO:111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HFWR1，所述HFWR1的C末端与所述HCDR1的N末端直接或间接相连。在本申请中，所述HFWR1可包含SEQ ID NO:9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HFWR2，所述HFWR2位于所述HCDR1与所述HCDR2之间。在本申请中，所述HFWR2可包含SEQ ID NO:47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HFWR3，所述HFWR3位于所述HCDR2与所述HCDR3之间。在本申请中，所述HFWR3可包含SEQ ID NO:82、84、85、87、88和89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HFWR4，所述HFWR4的N末端与所述HCDR3的C末端相连。在本申请中，所述HFWR4可包含SEQ ID NO:113所示的氨基酸序列。
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:196、197、201、202和222-230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抗体重链可包含SEQ ID NO:310-323中任一项所示的氨基酸序列。
可通过本领域已知的各种测定鉴别、筛选或表征本申请所述的PD-1抗原结合蛋白或融 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。本申请所述的抗原结合蛋白(例如，PD-1抗体)能够特异性结合PD-1抗原。“特异性结合”PD-1抗原的抗原结合蛋白(例如，PD-1抗体)通常可以结合PD-1，但不结合缺乏PD-1序列的其它蛋白。本申请所述的抗原结合蛋白(例如，PD-1抗体)能够特异性结合PD-1抗原或其标记形式(例如，荧光标记的PD-1抗原)，但不会结合缺乏PD-1表位的其它蛋白。抗原结合蛋白(例如，抗体)是否结合PD-1抗原可使用本领域中已知的任何测定法确定。本领域中已知测定结合亲和力的分析的实例包括表面等离子共振(SPR)或生物膜干涉技术(BLI)。
本申请所述的抗原结合蛋白可以结合人PD-1蛋白。在某些情形中，本申请所述的抗原结合蛋白还可以与猴(例如，食蟹猴)的PD-1交叉反应。例如，通过流式分析技术和酶联免疫反应所检测的。如本文所用，“交叉反应性”是指抗体与来自其它物种的同源蛋白反应的能力。
在某些情形中，本申请所述抗原结合蛋白与PD-1的结合活性可使用流式细胞术或酶联免疫法测定进行检测。例如，在FACS检测中，使用稳定表达人或猴PD-1的宿主细胞(如CHO-K1细胞)，所述PD-1抗原结合蛋白与PD-1的EC50值在约0.0001nM至约100nM之间，例如，约0.001nM至约80nM之间，约0.01nM至约60nM之间，约0.05nM至约50nM之间，约0.05nM至约40nM之间，约0.05nM至约30nM之间，约0.05nM至约20nM之间，约0.05nM至约10nM之间或约0.05nM至约5nM之间。又例如，在ELISA中，使用人PD-1抗原蛋白，所述PD-1抗原结合蛋白与PD-1的EC50值在约0.0001nM至约100nM之间，例如，约0.001nM至约10nM之间，约0.001nM至约5nM之间，约0.001nM至约1nM之间或约0.01nM至约1nM之间。
在另一个方面，本申请所述的抗原结合蛋白能够阻断PD-1与PD-L1的结合。在某些情形中，所述的抗原结合蛋白阻断PD-1与PD-L1的结合可通过流式细胞技术FACS、酶联免疫法ELISA测定。例如，首先将稳定表达PD-L1的宿主细胞(如CHO-K1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PD-1与PD-L2的结合。在某些情形中，所述的抗原结合蛋白阻断PD-1与PD-L2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-1的宿主细胞(如CHO-K1细胞)与递减量的未标记的所述抗原结合蛋白孵育，随后用生物素标记的PD-L2蛋白孵育。然后，使用FACS分析细胞，以证实所述抗原结合蛋白阻断PD-1与PD-L2结合。又例如，首先将PD-1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-L2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与PD-L2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)。
融合蛋白
另一方面，本申请提供了一种融合蛋白，所述融合蛋白包含第一靶向部分和第二靶向部分，所述第一靶向部分包含PD-1结合部分，其中所述PD-1结合部分包含本申请所述的分离的抗原结合蛋白。
在本申请中，所述融合蛋白的第二靶向部分可包含CD73结合部分。在本申请中，所述CD73结合部分可包含特异性结合CD73的抗体或其抗原结合片段。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体重链或其片段，所述抗体重链或其片段可包含HCDR1，所述HCDR1可包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体重链或其片段，所述抗体重链或其片段可包含HCDR2，所述HCDR2可包含SEQ ID NO:56所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体重链或其片段，所述抗体重链或其片段可包含HCDR3，所述HCDR3可包含SEQ ID NO:98所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体重链或其片段，所述抗体重链或其片段可包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:17、SEQ ID NO:56和SEQ ID NO:98所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包括抗体重链可变区VH，所述VH可包含SEQ ID NO:159所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体轻链或其片段，所述抗体轻链或其片段可包含LCDR1，所述LCDR1可包含SEQ ID NO:123所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体轻链或其片段，所述抗体轻链或其片段可包含LCDR2，所述LCDR2可包含SEQ ID NO:130所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体轻链或其片段，所述抗体轻链或其片段可包含LCDR3，所述LCDR3可包含SEQ ID NO:146所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体轻链或其片段，所述抗体轻链或其片段可包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:123、SEQ ID NO:130和SEQ ID NO:146所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包括抗体轻链可变区VL，所述VL可包含SEQ ID NO:237所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中，所述HCDR1、HCDR2和HCDR3可分别依次包含SEQ ID NO:17、SEQ ID NO:56和SEQ ID NO:98所示的氨基酸序列，且所述LCDR1、LCDR2和LCDR3可分别依次包含SEQ ID NO:123、SEQ ID NO:130和SEQ ID NO:146所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含VH和VL，且所述VH可包含SEQ ID NO:159所示的氨基酸序列，所述VL可包含SEQ ID NO:237所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含Fab。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体重链，且所述抗体重链可包含SEQ ID NO:246所示的氨基酸序列。
在本申请中，所述融合蛋白的所述CD73结合部分可包含抗体轻链，且所述抗体轻链可包含SEQ ID NO:330所示的氨基酸序列。
在本申请中，所述融合蛋白中，所述PD-1结合部分可以位于所述CD73结合部分的N端。在本申请中，所述融合蛋白中，所述PD-1结合部分可以位于所述CD73结合部分的C端。
在本申请中，所述的融合蛋白可包括第一多肽链和第二多肽链。在本申请中，所述融合蛋白的第一条多肽链可包含所述PD-1结合部分的VH和所述CD73结合部分的VH。在本申请中，所述融合蛋白的第二条多肽链可包含所述CD73结合部分的VL。
在本申请中，自N端至C端，所述融合蛋白的第一条多肽链可依次包含所述PD-1结合 部分的VH和所述CD73结合部分的VH。
在本申请中，所述融合蛋白的第一条多肽链可包含抗体重链恒定区。在本申请中，自N端至C端，所述融合蛋白的第一条多肽链可依次包含所述PD-1结合部分的VH、所述CD73结合部分的VH和所述抗体重链恒定区。
在本申请中，所述融合蛋白的第一条多肽链可包含所述PD-1结合部分的VH和所述CD73结合部分的抗体重链。在本申请中，自N端至C端，所述融合蛋白的第一条多肽链可依次包含所述PD-1结合部分的VH和所述CD73结合部分的抗体重链。
在本申请中，在所述融合蛋白的第一条多肽链中，所述PD-1结合部分和所述CD73结合部分可直接连接。
在本申请中，在所述融合蛋白的第一条多肽链中，所述PD-1结合部分和所述CD73结合部分可通过连接肽连接。在本申请中，所述连接肽可包含SEQ ID NO:334-352中任一项所示的氨基酸序列。
例如，所述第一多肽链可以包括SEQ ID NO:333所示的氨基酸序列。
在本申请中，所述融合蛋白的第二条多肽链还可包含抗体轻链恒定区。例如，所述第一多肽链可以包括SEQ ID NO:330所示的氨基酸序列。
在本申请中，所述融合蛋白可包括两条所述第一多肽链和两条所述第二多肽链。在本申请中，所述融合蛋白可以为同二聚体。在本申请中，所述融合蛋白为对称结构。
本申请所述融合蛋白的实例性结构可以如图10所示，其包含两条多肽链：第一多肽链，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；第二多肽链，也称长链，从氨基末端到羧基末端，其包含VH_B-L-VH_A-CH1-h-CH2-CH3。其中，VH_A和VL_A分别为任意的CD73抗体的重链可变区和轻链可变区。VH_B为本申请所述PD-1重链抗体的重链可变区。CL是任意的轻链恒定区结构域。CH1、CH2和CH3分别是任意的重链恒定区的第一、第二和第三结构域。L是任意的连接肽(例如，本文提到的那些连接肽)，h是任意的IgG抗体的铰链区或衍生序列。
在本申请中，所述融合蛋白具有所述抗原结合蛋白的生物活性，例如，结合PD-1抗体、阻断PD-1与其配体(例如，PD-L1和/或PD-L2)的结合，刺激T细胞分泌细胞因子等。此外，本申请所述融合蛋白还能结合CD73蛋白，抑制CD73的酶活性。
核酸分子、载体和细胞
另一方面，本申请提供了一种或多种核酸分子，其可以编码本申请所述的分离的抗原结合蛋白和/或所述的多特异性抗体。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。
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。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。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。
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。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。
药物组合物
另一方面，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/或所述多特异性抗体，所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的载体(carrier)。所述药学上可接受的载体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，抗氧化剂，防腐剂，低分子量(小于约10个残基)多肽，蛋白质，亲水性聚合物，氨基酸，碳水化合物，成盐反离子，金属络合物，和/或非离子表面活性剂。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。
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原结合蛋白、多特异性抗体。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，本申请提供了一种免疫缀合物，其可以包含细胞毒性剂，以及本申请所述的 抗原结合蛋白。免疫缀合物通常是指采用特定的连接子将抗原结合蛋白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原结合蛋白、连接子和小分子细胞毒药物。
另一方面，本申请提供了一种试剂盒，其可以包含本申请所述的抗原结合蛋白，载体，核酸分子，细胞，免疫缀合物，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抗原结合蛋白，载体，核酸分子，细胞，免疫缀合物，和/或本申请所述的药物组合物，也可任选地与一种或多种治疗剂组合，任选地一起配制于试剂盒中。
另一方面，本申请提供了一种给药装置，它可以用来施用本申请所述抗原结合蛋白，载体，核酸分子，细胞，免疫缀合物，和/或本申请所述的药物组合物。
制备方法
另一方面，本申请提供了制备所述的抗原结合蛋白的方法。所述方法可包括，在使得所述的抗原结合蛋白表达的条件下，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。
任何适于产生单克隆抗体的方法都可用于产生本申请的抗原结合蛋白。例如，可以用连接或天然存在的PD-1蛋白或其片段，免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的PD-1都可以作为免疫原(抗原)，用于产生对PD-1特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是全长的成熟人PD-1，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本申请中，抗体是IgG抗体，使用IgG1或IgG4亚型。可以通过用下文实施例中描述的生物学测定筛选抗体实现必需恒定结构域序列的优化，以产生所需生物学活性。同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本申请的化合物和方法中是可以用的。
本申请的抗原结合蛋白或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小 片段，然后再进行连接可获得序列很长的片段。然后可将该核酸分子引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本申请还涉及包含上述的适当核酸分子以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。例如，动物细胞可以包括(但并不限于)：CHO-S、CHO-K1、HEK-293细胞。
本申请中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本申请的核酸分子所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。通常，在适合本申请抗原结合蛋白表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本申请的抗原结合蛋白。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如流式细胞分选技术(FACS)或酶联免疫吸附测定(ELISA))来测定。
方法和用途
另一方面，本申请提供了抑制PD-1与PD-L1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-L1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-1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-1与PD-L2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PD-L2在容许所述抗原结合蛋白和/或PD-L2结合PD-1的条件下接触，检测在所述抗原结合蛋白与PD-1之间是否形成复合物，和检测PD-L1与PD-1之间是否形成复合物。所述抗原结合蛋白和/或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。
另一方面，本申请提供了一种所述抗原结合蛋白和/或所述多特异性抗体在制备药物中的用途。所述药物可用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。
另一方面，本申请提供了刺激免疫细胞分泌细胞因子的方法，其包括施用所述分离的抗 原结合蛋白和/或所述的多肽。所述细胞因子可以是IL-2。所述免疫细胞可以是淋巴细胞。例如，T淋巴细胞。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非诊断和/或治疗目的的方法。
另一方面，本申请提供了一种用于检测PD-1蛋白的存在和/或含量的方法，其包括施用所述分离的抗原结合蛋白和/或所述的融合蛋白。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非诊断和/或治疗目的的方法。
本申请还提供了抗原结合蛋白在诊断患有肿瘤或癌症的受试者的方法中的用途，所述方法包括：通过使样品与本申请的抗原结合蛋白接触并检测结合的抗体的存在来确定获自受试者的样品中PD-1的存在或表达水平。
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。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1抗原结合蛋白的制备
为获得针对PD-1特异性结合的抗体分子，通常可以利用PD-1抗原对实验动物进行免疫，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，即H2L2形式的抗体 具有两条抗体重链和两条抗体轻链。
Harbour HCAb小鼠(Harbour Antibodies BV，WO 2002/085945A3)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生全新的仅“重链”抗体，该抗体的大小只有传统IgG抗体的一半。其产生的抗体仅具有人的抗体“重链”可变结构域和小鼠Fc恒定结构域。由于不含轻链的这一特点，该抗体几乎解决了轻链错配和异源二聚化的问题，使得这一技术平台能够开发出传统抗体平台难以实现的产品。即HCAb形式的抗体包含两条抗体重链，不包含抗体轻链。
1.1 PD-1免疫小鼠
(1)免疫H2L2小鼠
用可溶的重组人PD-1-hFc融合蛋白(Shanghai ChemPartner)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-1-hFc)与完全弗氏佐剂(Sigma,#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Ribi佐剂(Sigma Adjuvant System,#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。
(2)免疫HCAb小鼠
6～8周龄Harbour人源抗体转基因小鼠采用了2组免疫方案对Harbour HCAb小鼠进行多轮免疫。免疫方案1，用重组的人PD-1-hFc(Shanghai ChemPartner)抗原蛋白进行免疫。每只小鼠每次免疫时通过皮下经腹股沟注射或通过腹腔注射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50微克抗原蛋白与完全弗氏佐剂(Sigma,#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25微克抗原蛋白与Ribi佐剂(Sigma Adjuvant System,#S6322)混合配制的免疫原试剂的免疫。免疫方案2，用过表达人PD-1的HEK293/hPD-1(Shanghai ChemPartner)稳定细胞系进行免疫。每只小鼠每次免疫时腹腔注射2×10 6细胞悬液。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HCAb小鼠脾B细胞分离前5天，以每只小鼠25微克抗原蛋白的剂量进行最后一次增强免疫。
采集小鼠血液，对血液进行10倍稀释取5个浓度(1:100、1:1000、1:10000、1:100000、1:1000000)，在包被有人PD-1-His(Shanghai ChemPartner)的ELISA板进行ELISA检测来确定小鼠血液中抗人PD-1的滴度，并经流式细胞术检测2个浓度的小鼠血液(1:100、1:1000)对PD-1高表达的CHO-K1/hPD-1细胞(Shanghai ChemPartner)和CHO-K1母细胞的特异反应性。空白对照组(PB)为免疫前老鼠的血清。
1.2获得杂交瘤单克隆和抗体序列
(1)获得抗PD-1的H2L2抗体序列
当检测H2L2小鼠血清中PD-1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，分离出至少8个表达抗-PD-1的单克隆抗体分子的杂交瘤。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：4004_10H9A12，4004_12H9C1等。分离的杂交瘤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-1的结合能力、食蟹猴PD-1的结合能力、抑制PD-1与PD-L1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-1单克隆抗体分子可变结构域的序列是人源抗体序列。在本申请中，所述CDR序列通过Chothia定义规则划分。
(2)获得抗PD-1的HCAb抗体序列
当检测小鼠血清中PD-1特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用BD FACS AriaII Cell Sorter分选CD138阳性的浆细胞和人PD-1抗原阳性的B细胞群。提取B细胞的RNA，反转录cDNA(SuperScript IV First-Strand synthesis system,Invitrogen,#18091200),然后用特异性的引物PCR扩增人VH基因。PCR引物5’-GGTGTCCAGTGT(G/C)AGGTGCAGCTG-3’(SEQ ID NO:357),5’-AATCCCTGGGCACTGAAGAGACGGTGACC-3’(SEQ ID NO:358)。将扩增的VH基因片段构建到编码人IgG1抗体重链Fc结构域序列(SEQ ID NO:355)的哺乳动物细胞表达质粒pCAG载体中。
构建好的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达获得HCAb的抗体。检测表达HCAb的上清与过表达人PD-1的CHO-K1/hPD-1(GenScript，#M00529)稳定细胞系的结合，同时用阳性抗体作为阳性对照，进行Miroball荧光流式仪器(Sptlabtech)筛选。具体步骤是：用无血清的F12K培养基(Thermofisher，#21127022)洗涤CHO-K1/hPD- 1细胞，将其用无血清的培养基重悬至1×10 6/ml。加入Draq5荧光探针(CTS，#4048L)(1μL Draq5至1ml CHO-K1/hPD-1细胞中，1:1000稀释)，在避光处孵育30分钟。离心细胞后用培养基洗涤细胞，调整细胞密度至1.0×10 5细胞/ml。再加入1:1000稀释后的Alexa
Figure PCTCN2021106569-appb-000001
488,AffiniPure Goat Anti-Human IgG,Fcγ Fragment Specific二抗(Jackson，#109-545-098)，取每孔30ul的该混合物加入384孔板(Greiner，#781091)。再在384孔板中加入10μL阳性对照或者表达HCAb的上清，孵育2小时。在Miroball荧光流式仪器上读取荧光值。阳性克隆抗体进一步用FACS检测与CHO-K1/hPD-1细胞的结合，以及和食蟹猴PD-1-his蛋白(Novoprotein，#CM98)的ELISA检测来验证结合交叉结合活性。利用常规的测序手段对阳性抗体进行测序，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。
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。
上述获得的抗体的胚系基因分析如下表2所示，抗体的序列号和各片段序列如下表3和表4所示。
同时，本申请的抗PD-1的阳性对照抗体Pembrolizumab类似物，对应的抗体编号为PR000150。其相应的氨基酸序列来源于IMGT数据库，抗体重链序列SEQ ID NO:332，抗体轻链序列SEQ ID NO:331。
表2抗PD-1 H2L2和HCAb抗体的序列胚系基因分析
Figure PCTCN2021106569-appb-000002
Figure PCTCN2021106569-appb-000003
表3抗PD-1 H2L2和HCAb抗体的序列号
Figure PCTCN2021106569-appb-000004
Figure PCTCN2021106569-appb-000005
表4抗PD-1 H2L2和HCAb抗体的氨基酸序列
Figure PCTCN2021106569-appb-000006
Figure PCTCN2021106569-appb-000007
1.3重组全人源抗体的制备
上述实施例1.2和1.3中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，重组抗体分子的轻重链序列如表3中所示。
在本实施例中Harbour H2L2小鼠得到的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的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。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IgG4抗体重链恒定结构域序列(SEQ ID NO：354)的哺乳动物细胞表达质粒载体中，以编码产生IgG4抗体的全长重链。
将编码Harbour H2L2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重组抗体。或者将编码Harbour HCAb抗体重链的质粒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HCAb重链重组抗体。
具体说来，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo，#A1383504)扩培。瞬时转染开始之前，调节细胞浓度至6～8×10 5细胞/ml，于37℃ 8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞。将上述编码H2L2抗体的重链质粒和轻链质粒以2:3的比例混合或者编码HCAb的重链质粒溶解于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 Inc，#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)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pH 8.0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，得到纯化的抗体溶液。然后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。
1.4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 1260 Infinity 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25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。
上述获得的全人源H2L2和HCAb抗体的产量和纯度等如下表5所示。
表5 PD-1抗体的产量和纯度分析
Figure PCTCN2021106569-appb-000008
1.5优化HCAb抗体序列提高结合PD-1的亲和力
本实施例通过定点饱和突变的方法对抗PD-1的HCAb抗体PR002481进行亲和力的改造。此亲和力成熟的方法分为2轮。
第一轮，对PR002481这个分子的三个CDRs的28氨基酸逐点扫描。建立28个氨基酸位置的单点饱和突变的酵母小库。将3个CDR的小库各自混合，形成3个CDR的酵母突变库。对三个CDR的突变库在流式细胞仪上进行分选，分选出来的酵母细胞进行测序，并对这些阳性分子进一步鉴定，根据其对人PD-1的结合能力，选出若干个突变热点。在本实施例中抗体可变结构域的CDR序列通过Chothia CDR定义规则进行分析。
第二轮，将第一轮中饱和突变找到的热点进行随机组合，建立一个包含所有突变组合的酵母库。然后对组合库在流式细胞仪上进行分选。分选下来的酵母细胞通过测序以及对人PD-1的结合能力鉴定，选出若干个突变体。
在本实施例中通过亲和力成熟得到的抗体重链可变结构域序列(VH)通过基因合成并克隆到编码Flag和6xHis标签的哺乳动物细胞表达质粒载体中，以编码产生PD1单域抗体(VH)分子。
或者，将亲和力成熟得到的抗体的VH基因片段构建到编码人IgG1抗体重链Fc结构域序列(SEQ ID NO:355)的哺乳动物细胞表达质粒载体中，以编码HCAb的全长重链。
本发明通过上述亲和力成熟的方法得到单价形式PR002481变体(均为单域抗体，带Flag-6His标签)及其序列如下表6所示(涉及HCDR1，SEQ ID NOs:18，20-33；HCDR2，SEQ ID NOs:58-68；HCDR3,SEQ ID NOs:：100，103-111)。其中，PR005090即为PR002481母本序列对应的单域抗体(带Flag-6His标签)。对表6中的单域抗体进行评价，据于Koff排序，表达量及报告基因活性的结果，通过综合分析或直接挑选单域抗体表达HCAb或重组表6中已有CDR表达HCAb，或在已有CDR区域引入一些热点突变设计产生新CDR用于HCAb抗体的表达，本发明经过突变、筛选获得的HCAb抗体如表7所示(涉及HCDR1，SEQ ID NOs:18，25，28，34-38；HCDR2，SEQ ID NOs:64-65；HCDR3,SEQ ID NOs:：108，111-112)。
表6单价形式PR002481变体(单域)抗体的序列表
Figure PCTCN2021106569-appb-000009
Figure PCTCN2021106569-appb-000010
Figure PCTCN2021106569-appb-000011
Figure PCTCN2021106569-appb-000012
表7 PR002481变体(HCAb)抗体的序列表
Figure PCTCN2021106569-appb-000013
Figure PCTCN2021106569-appb-000014
1.6 PR002481亲和力变体的生产和纯化
上述实施例1.5中得到编码PR002481亲和力变体可变结构域序列以后，可以采用常规的重组DNA技术，将重链可变结构域序列和相应的纯化标签进行融合表达，得到重组HCAb单域抗体分子。
将编码重组HCAb单域抗体的质粒转染哺乳动物宿主细胞(如中国仓鼠卵巢细胞CHO)，利用常规的重组蛋白表达和纯化技术，可以得到相应的纯化重组抗体。
单域抗体的生产纯化方法具体为：将ExpiCHO-S TM细胞(Gibco,#A29127)在ExpiCHO TMExpression Medium培养基(Gibco,#A2910001)扩培。瞬时转染开始之前，调节细胞浓度至3～4×10 6细胞/ml，于37℃ 8％CO 2摇床中培养24小时，细胞浓度至7～10×10 6细胞/ml，再稀释至6×10 6细胞/ml，准备10ml培养的细胞。将上述编码HCAb单域抗体的质粒8μg(质粒与细胞的比例为0.8μg:1ml)溶解于0.4ml OptiPRO TM SFM培养基(Gibco,#12309019)，并用0.22μm滤膜过滤除菌。再取32μl ExpiFectamine TM CHO试剂(Gibco,#A29129)加入0.37ml OptiPRO TM SFM培养基(Gibco,#12309019)。立即把ExpiFectamine TM CHO试剂溶液缓慢加入质粒溶液中，颠倒混匀，边摇晃培养瓶边缓慢滴入质粒和转染试剂混合溶液，于37℃8％CO 2摇床中培养8-9天。8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，然后将上清用0.22μm滤膜过滤去除杂质。用PBS(pH7.4)平衡含有Ni Sepharose excel(GE Healthcare Life Science，#17531802)的重力柱(Bio-Rad，#7311550)，2-5倍柱体积冲洗。将上清样品过柱。先后用5-10倍柱体积的PBS和5-10倍柱体积的20mM咪唑溶液(pH7.4)冲洗柱子。再用500mM咪唑溶液(pH7.4)洗脱目的蛋白，最后用超滤管(Millipore，#UFC901024)浓缩换液至PBS缓冲液，得到纯化的抗体溶液。然后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。
HCAb抗体的生产纯化方法具体为：将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo，#A1383504)扩培。瞬时转染开始之前，调节细胞浓度至6～8×10 5细胞/ml，于37℃ 8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞。将编码HCAb的重链质粒溶解于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 Inc，#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)平衡含有MabSelect TM(GE Healthcare Life Science，#71-5020-91AE)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pH 8.0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，得到纯化的抗体溶液。然后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。
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 1260 Infinity 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。
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TSKge1 Buty1-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。
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 CFX96PCR 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。
PR002481亲和力成熟变体HCAb的表达和物理化学性质见表8，结果显示，测试抗体均有较高的产率，纯化的纯度较高，且物理化学性质稳定。
表8 PR002481亲和力成熟变体(HCAb)的理化性质
Figure PCTCN2021106569-appb-000015
Figure PCTCN2021106569-appb-000016
实施例2抗原结合蛋白人和食蟹猴PD-1蛋白的结合
2.1抗原结合蛋白与人PD-1蛋白的结合(ELISA)
将人PD-1-His蛋白(ACROBiosystems,#PD1-H5221)用PBS稀释为2μg/ml，加至96孔板(Corning，cat#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至10个浓度梯度，每个孔加入100μl，37℃孵育1小时，同型抗体作为对照。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，置酶标仪(PerkinElemer,#Enspire)中测定450nm吸光度(OD450)值。根据测定结果计算半数最大结合效应浓度(EC50)。
结果如图1A-图1C所示。图1A和图1B显示，本申请所述抗PD-1的H2L2抗体PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680均能与人 PD-1抗原结合，且结合活性与阳性对照抗体pembrolizumab相当。图1C结果显示本申请所述抗PD-1的HCAb抗体PR002474、PR002476、PR002479和PR002481均能与人PD-1抗原结合，且结合活性与阳性对照抗体pembrolizumab相当。
2.2抗原结合蛋白与食蟹猴PD-1蛋白的结合(ELISA)
将食蟹猴(Cynomolgus)的PD-1-His蛋白(AcroBiosystems,#PD1-C5223)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，置酶标仪(PerkinElemer,#Enspire)中测定450nm吸光度(OD450)值。
结果如图2所示，本申请所述抗PD-1的HCAb抗体PR002474、PR002476、PR002479和PR002481均能结合食蟹猴PD-1蛋白，且结合活性与阳性对照抗体pembrolizumab相当。
实施例3抗原结合蛋白与过表达人/食蟹猴PD-1的细胞的结合(FACS)
为了研究PD-1抗原结合蛋白体外结合人/食蟹猴PD-1的活性。采用过表达人或食蟹猴PD-1的CHO-K1细胞株(CHO-K1/hPD-1，CHO-K1/cyno PD-1，来源于GenScript)进行细胞水平的结合实验。简言之，消化细胞CHO-K1-hPD-1细胞，并用F-12K完全培养基重悬，将细胞密度调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#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或300nM，共8至11个浓度，hIgG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
Figure PCTCN2021106569-appb-000017
488 conjugate,Invitrogen，Cat#A11013,1:1000)，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。
图3A结果显示，本申请所述抗PD-1的H2L2抗体PR000673、PR000674、PR000675、PR000676、PR000677、PR000678和PR000680均能结合过表达人PD-1的CHO-K1细胞，且部分抗体的结合活性与阳性对照抗体pembrolizumab相当。
图3B结果显示，本申请所述抗PD-1的HCAb抗体PR002473、PR002474、PR002475、PR002476、PR002477、PR002478、PR002479和PR002481均能结合过表达人PD-1的CHO-K1细胞。
图3C结果显示，PR002481突变体的HCAb抗体PR005093、PR005096、PR005097、PR005098、PR005099、PR005100、PR005101、PR005102、PR005103和PR005104均能结合过表达人PD-1的CHO-K1细胞，亲和力优于PR005090。
图3D结果显示，PR002481突变体的HCAb抗体PR005145、PR005149、PR005150、PR005141、PR005142、PR005144、PR005147和PR005148均能结合过表达人PD-1的CHO-K1细胞，亲和力优于PR005090。
图3E结果显示，PR002481突变体的HCAb抗体PR005585、PR005583、PR005578、PR005577、PR005575、PR005572和PR005569均能结合过表达人PD-1的CHO-K1细胞，亲和力优于亲本PR002481。
图3F结果显示，PR002481突变体的HCAb抗体PR005584、PR005582、PR005581、PR005574、PR005573、PR005571和PR005570均能结合过表达人PD-1的CHO-K1细胞，亲和力优于PR002481。
图4结果显示，本申请所述H2L2抗体PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680均能结合过表达食蟹猴PD-1的CHO-K1细胞，且部分抗体的结合活性与阳性对照抗体pembrolizumab相当。
实施例4利用BLI方法测定抗原结合蛋白与人PD-1的亲和力
该实施例利用Octet Red96e仪器对PR002481亲和力成熟突变体进行了动力学的测定。带有组氨酸和avi标签的人PD-1蛋白购自厂家ACRO Biosystems(货号PD1-H82E4)，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)，用于动力学测试以及抗原、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。
测定抗原和抗体的结合动力学时，传感器转动速度为1000转/分钟。先将置于一列的8个SA传感器在测试缓冲液中平衡10分钟后用SA传感器捕获PD-1，捕获高度0.5nm，然后将SA传感器在测试缓冲液中平衡100s，再与稀释至单浓度的各抗体及一行空白缓冲液孔结合180s，解离600s。每个循环换用一列新的SA传感器。
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 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kon(1/Ms)值、kdis(1/s)值和KD(M)值。
结果表9所示，PR02481的亲和力成熟变体与PD-1均能结合，大部分KD值在1E9至1E8之间，甚至小于1E12，且多数变体的亲和力较PR005090(基于亲本序列的单域抗体)有明显提高。
表9 PR002481亲和力成熟变体与人PD-1蛋白的亲和力
抗体编号 浓度(nM) KD(M) kon(1/Ms) kdis(1/s) kdis ratio Full R^2
PR005090 60 2.08E-08 5.50E+04 1.14E-03   0.9955
PR005093 80 4.82E-09 1.32E+05 6.36E-04 1.80 0.9988
PR005096 80 1.04E-08 5.79E+04 5.99E-04 1.91 0.9983
PR005097 80 1.39E-08 5.66E+04 7.84E-04 1.46 0.9981
PR005098 80 5.88E-09 6.71E+04 3.94E-04 2.90 0.9985
PR005099 80 4.32E-09 1.05E+05 4.55E-04 2.51 0.9991
PR005100 80 1.16E-08 2.60E+04 3.01E-04 3.80 0.9972
PR005101 80 5.88E-08 2.94E+03 1.73E-04 6.61 0.9946
PR005102 80 5.99E-09 4.48E+04 2.68E-04 4.25 0.9985
PR005103 80 7.96E-08 3.16E+03 2.52E-04 4.54 0.9953
PR005104 80 3.08E-09 7.90E+04 2.43E-04 4.70 0.9992
PR005141 NA 1.49E-09 5.21E+04 7.75E-05 14.73 0.9908
PR005142 80 6.76E-09 8.20E+03 5.55E-05 20.60 0.9992
PR005144 80 4.51E-09 1.00E+04 4.52E-05 25.25 0.9993
PR005145 80 5.31E-09 9.86E+03 5.24E-05 21.81 0.9984
PR005146 900 1.02E-06 5.93E+02 6.05E-04 1.89 0.9492
PR005147 80 1.65E-08 1.66E+03 2.74E-05 41.74 0.9713
PR005148 80 1.21E-09 4.72E+04 5.72E-05 19.98 0.999
PR005149 80 1.40E-08 3.45E+03 4.83E-05 23.66 0.998
PR005150 80 <1.0E-12 5.90E+04 <1.0E-07 NA 0.9388
PR002481 600-150 7.70E-08 2.29E+05 1.62E-03 - 0.9928
PR005569 600-150 4.82E-10 9.97E+03 7.68E-04 - 0.9978
PR005572 600-150 3.32E-09 1.19E+04 5.75E-06 - 0.9986
PR005575 600-150 6.46E-09 1.29E+04 4.28E-05 - 0.9991
PR005583 600-150 1.69E-09 8.28E+03 1.65E-05 - 0.9990
PR005585 600-150 3.08E-09 1.32E+04 2.22E-05 - 0.9986
Pembrolizumab 80-20 7.07E-09 2.29E+05 1.62E-03 - 0.9928
实施例5抗原结合蛋白阻断人PD-L1或PD-L2与过表达人PD-1的CHO-K1细胞的结合
为了研究人PD-1结合蛋白体外阻断人PD-1与人PD-L1以及PD-L2结合的活性，采用过表达人PD-1的CHO-K1细胞株(CHO-K1-hPD-1)进行细胞水平的人PD-1/人PD-L1以及进行细胞水平的人PD-1/人PD-L2结合阻断实验。简言之，消化CHO-K1-hPD-1细胞，并用F-12K完全培养基重悬，将细胞密度调整为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#3894)，随后加入100μL/孔，2倍于终浓度的3倍或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或300nM，共8个浓度，hIgG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-L1蛋白(AcroBiosystems,PD1-H82F2)或者1μg/mL浓度的生物素标记的人PD-L2蛋白(AcroBiosytems,PD2-H82F6)，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,Cat#554061，1:200)，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，计算IC50。
结果如图5A、图5B和图5C所示。图5A和图5B分别显示本申请所述H2L2抗体PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680，以及HCAb抗体PR002473、PR002474、PR002475、PR002476、PR002477、PR002478、PR002479和PR002481均能阻断人PD-L1与细胞表面的人PD-1结合，且抑制能力与阳性对照pembrolizumab相当。图5C显示本申请所述H2L2抗体PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680均能阻断人PD-L2与细胞表面的人PD-1结合。
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
将过表达PD-L1和OS8(CD3单链抗体跨膜蛋白)的Hep3B(Shanghai ChemPartner构建)细胞，或者是过表达PD-L1和OS8的HEK293T细胞，铺到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50nM，4倍浓度稀释(或者起始浓度为500nM，5倍浓度稀释)，hlgG1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(Shanghai ChemPartner构建)50μL/孔。37℃在5％CO 2环境下培养6小时。加入ONE-GloTM荧光素酶试剂(Promega,#E6110)，室温孵育5分钟，酶标仪检测发光值。
结果如图6A-图6F所示，本申请抗体对PD-1信号通路的抑制能力与阳性对照相当。
图6A显示本申请所述H2L2抗体PR000673、PR000674、PR000678、PR000679和PR000680对PD-1信号通路有抑制作用。
图6B显示PR002481突变体的HCAb抗体PR005090、PR005093、PR005096、PR005097、PR005098、PR005099、PR005100、PR005101、PR005102、PR005103和PR005104对PD-1信号通路有抑制作用。
图6C显示PR002481突变体的HCAb抗体PR005090、PR005145、PR005149、PR005150、PR005141、PR005142、PR005144、PR005147和PR005148对PD-1信号通路有抑制作用。
图6D和图6E显示PR002481突变体的HCAb抗体对PD-1信号通路有抑制作用。
图6F显示PR002481突变体的HCAb抗体PR005569、PR005572、PR005575、PR005583和PR005585对PD-1信号通路均有抑制作用。
实施例7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
购买AllCells PBMC细胞，分离monocytes细胞，加入重组人源白介素4(IL-4)(R&D，#204-GMP)和人源GM-CSF(R&D，#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阴阳性对照抗体，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。收集第三天和第五天的上清液，采用ELISA试剂盒，分别检测IL-2(ThermoFisher，#88-7025-88)和IFN-γ(ThermoFisher，#88-7316-88)的分泌量。
首先使用了12个供体的PBMC分成了六组供体配对进行混合淋巴细胞反应(MLR)。结果如图7和图8所示，在六次独立的MLR实验中，H2L2抗体PR000673、PR000674、PR000675、PR000676、PR000677、PR000678、PR000679和PR000680，以及PR002481都能增强激活的T淋巴细胞分泌细胞因子IL-2和IFN-γ。
图9显示PR002481突变体的HCAb抗体PR005569、PR005572、PR005575、PR005583和PR005585均能增强激活的T淋巴细胞分泌细胞因子IL-2和IFN-γ，且激活能力与阳性对照相当。
实施例8 CD73×PD-1双特异性抗体的结构和设计
本实施例利用抗CD73的IgG抗体PR000846的抗原结合结构域Fab，和抗PD-1的HCAb抗体PR002481的抗原结合结构域VH，来构建PD-1×CD73的双特异性抗体分子。PR000846轻重连的氨基酸序列如表10所示。
表10抗CD73的H2L2抗体PR000846的序列编号
抗体编号 轻链 重链 VL VH
PR000846 SEQ ID NO:330 SEQ ID NO:246 SEQ ID NO:237 SEQ ID NO:159
在本实施例及后续实施例中，阳性对照分子为抗CD73的IgG单抗PR000846，亦为CD73×PD-1双抗分子的CD73端的亲本单抗。阳性对照分子为抗PD-1的IgG单抗PR002481，亦为CD73×PD-1双抗分子的PD-1端的亲本单抗。
以图10为例进行说明，Fab端来源于常规抗体PR000846，VH_A和VL_A分别为抗体PR000846的重链可变区和轻链可变区。VH端来源于重链抗体PR002481，VH_B为重链抗体PR002481的重链可变区。CL是轻链恒定区结构域。CH1、CH2和CH3分别是重链恒定区的第一、第二和第三结构域。L是连接肽GS_15(SEQ ID NO:338)，h是IgG抗体的铰链区或衍生序列。VH_B在VH_A的N端。IgG1为L234A,L235A突变。制备得到的四价对称结构的双特异性抗体为PR003568，包含两条多肽链，其中长链的氨基酸序列如SEQ ID NO:333所示，短链的氨基酸序列如SEQ ID NO:330所示。制备的双抗抗体分子样品并理化性质分析，总结于表11。
表11双抗分子蛋白的表达和物理化学性质
Figure PCTCN2021106569-appb-000018
实施例9 CD73×PD-1双特异性抗体与过表达人PD-1或人CD73的细胞的结合(FACS)
为了研究CD73×PD-1双特异性抗体与结合人CD73和PD-1的活性，采用过表达人CD73或PD-1的CHO-K1细胞株(CHO-K1-hCD73，CHO-K1-hPD-1)进行细胞水平的结合实验。简言之，消化CHO-K1-hCD73和CHO-K1-hPD-1细胞，并用F-12K完全培养基重悬，将细胞密度调整为1×10 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，于500g，4℃下离心5分钟，弃上清。稀释抗体至最高终浓度50nM，3倍稀释，共8个浓度。取100μL稀释后的抗体加入到细胞中，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
Figure PCTCN2021106569-appb-000019
488 conjugate,Invitrogen，Cat#A11013,1:1000)，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。
如图11和图12所示PR003568具有很好的结合CD73和PD-1的活性。其中PR003568双特异性抗体PD-1端的活性与亲本单克隆抗体PR002481一致，CD73端的活性比亲本单克隆抗体PR000846有稍微下降。
实施例10 CD73×PD-1双特异性抗体结抑制CD73的酶活性
利用孔雀绿法测定可溶性重组CD73的活性，首先在384孔板(Corning,#3799)中加入12.5μL 1nM重组CD73和12.5μL 1nM的抗体(实验缓冲液为25mM Tris pH 7.5，5mM MgCl 2，0.005％吐温-20)，室温孵育1小时。加入25μL AMP(最高浓度200μM,用实验缓冲液2倍稀释至8个浓度)，室温孵育15分钟。检测每个孔中无机磷酸根的浓度，无机磷酸根的浓度是根据制造商的说明书孔雀绿法测定的。测定结束后用Molecular Devices(SPECTRAMax plus384)读板机记录620nm光吸收值。实验结果用GraphPad Prism 8.0分析作图。
如图13所示PR003568具有很好的抑制CD73的酶活活性，和亲本单克隆抗体PR000846活性相当。
实施例11利用报告基因细胞系检测CD73×PD-1双特异性抗体对PD-1信号通路的抑制作用
将表达PD-L1和OS8(CD3单链抗体跨膜蛋白)的HEK293T(eBioscience)细胞铺到96孔板上，细胞量为1.25×10 4/孔，100μL/孔。37℃在5％CO2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，hlgG1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(UPharm)50μL/孔。37℃在5％CO 2环境下培养6小时。加入ONE-GloTM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。
如图14所示PR003568具有很好的PD-1信号通路抑制作用，和亲本单克隆抗体PR002481 活性相当。
实施例12双特异性抗体在混合淋巴细胞反应试验中激活T细胞
购买AllCells PBMC细胞，分离monocytes细胞，加入重组人源白介素4(IL-4)(R&D，#204-GMP)和人源GM-CSF(R&D，#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阴阳性对照抗体，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。收集第三天和第五天的上清液，采用ELISA试剂盒，分别检测IL-2(ThermoFisher，#88-7025-88)和IFN-γ(ThermoFisher，#88-7316-88)的分泌量。
如图15A和图15B所示，CD73×PD-1双特异性抗体PR003568的MLR活性要优于PD-1单抗PR002481。

Claims (105)

  1. 分离的抗原结合蛋白，其包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，其中，所述HCDR1包含SEQ ID NO:359所示的氨基酸序列，所述HCDR2包含SEQ ID NO:360所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:361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，其包括抗体或其抗原结合片段。
  3. 根据权利要求1-2中任一项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，VHH，重链抗体(HCAb)和/或dAb。
  4. 根据权利要求2-3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。
  5. 根据权利要求1-4中任一项所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:11-16中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:50-55中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:92-97中任一项所示的氨基酸序列。
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其中所述HCDR1、HCDR2和HCDR3包含选自以下任意一组的氨基酸序列：
    (1)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:92；
    (2)HCDR1：SEQ ID NO:12，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:93；
    (3)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:51，和HCDR3：SEQ ID NO:94；
    (4)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:95；
    (5)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:53，和HCDR3：SEQ ID NO:96；
    (6)HCDR1：SEQ ID NO:16，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:97；以及，
    (7)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:55，和HCDR3：SEQ ID NO:92。
  7. 根据权利要求1-6中任一项所述的分离的抗原结合蛋白，其包含抗体重链可变区VH，且所述VH包含SEQ ID NO:368所示的氨基酸序列。
  8. 根据权利要求7所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:151-158中任一项所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的分离的抗原结合蛋白，其还包括抗体重链恒定区。
  10. 根据权利要求9所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG恒定区。
  11. 根据权利要求9-10中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG4恒定区和/或人IgG1恒定区。
  12. 根据权利要求9-11中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区包含SEQ  ID NO:354-355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，其包括抗体重链，所述抗体重链包含SEQ ID NO:238-245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，其包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1、LCDR2和LCDR3，其中，所述LCDR1包含SEQ ID NO:365所示的氨基酸序列，所述LCDR2包含SEQ ID NO:366所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:367所示的氨基酸序列。
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:121-125中任一项所示的氨基酸序列，所述LCDR2包含SEQ ID NO:130-135中任一项所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:140-145中任一项所示的氨基酸序列。
  16. 根据权利要求14-15中任一项所述的分离的抗原结合蛋白，其中所述LCDR1、LCDR2和LCDR3包含选自以下任意一组的氨基酸序列：
    (1)LCDR1：SEQ ID NO:121，LCDR2：SEQ ID NO:130，和LCDR3：SEQ ID NO:140；
    (2)LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:131，和LCDR3：SEQ ID NO:141；
    (3)LCDR1：SEQ ID NO:123，LCDR2：SEQ ID NO:132，和LCDR3：SEQ ID NO:142；
    (4)LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:133，和LCDR3：SEQ ID NO:143；
    (5)LCDR1：SEQ ID NO:124，LCDR2：SEQ ID NO:134，和LCDR3：SEQ ID NO:144；以及
    (6)LCDR1：SEQ ID NO:125，LCDR2：SEQ ID NO:135，和LCDR3：SEQ ID NO:145。
  17. 根据权利要求1-16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，且所述VL包含SEQ ID NO:369所示的氨基酸序列。
  18. 根据权利要求17所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:231-236中任一项所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其还包括抗体轻链恒定区。
  20. 根据权利要求19所述的分离的抗原结合蛋白，其中所述轻链恒定区包含SEQ ID NO:353所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包括抗体轻链，所述抗体轻链包含SEQ ID NO:324-329中任一项所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其包含抗体重链或其片段和抗体轻链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，所述抗体轻链或其片段包含LCDR1、LCDR2和LCDR3，其中，所述HCDR1、HCDR2、HCDR3、 LCDR1、LCDR2和LCDR3包含选自以下任意一组的氨基酸序列：
    (1)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:92，LCDR1：SEQ ID NO:121，LCDR2：SEQ ID NO:130，和LCDR3：SEQ ID NO:140；
    (2)HCDR1：SEQ ID NO:12，HCDR2：SEQ ID NO:50，和HCDR3：SEQ ID NO:93，LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:131，和LCDR3：SEQ ID NO:141；
    (3)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:51，和HCDR3：SEQ ID NO:94，LCDR1：SEQ ID NO:123，LCDR2：SEQ ID NO:132，和LCDR3：SEQ ID NO:142；
    (4)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:52，和HCDR3：SEQ ID NO:95，LCDR1：SEQ ID NO:122，LCDR2：SEQ ID NO:133，和LCDR3：SEQ ID NO:143；
    (5)HCDR1：SEQ ID NO:15，HCDR2：SEQ ID NO:53，和HCDR3：SEQ ID NO:96，LCDR1：SEQ ID NO:124，LCDR2：SEQ ID NO:134，和LCDR3：SEQ ID NO:144；
    (6)HCDR1：SEQ ID NO:16，HCDR2：SEQ ID NO:54，和HCDR3：SEQ ID NO:97，LCDR1：SEQ ID NO:125，LCDR2：SEQ ID NO:135，和LCDR3：SEQ ID NO:145；以及，
    (7)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:55，和HCDR3：SEQ ID NO:92，LCDR1：SEQ ID NO:121，LCDR2：SEQ ID NO:130，和LCDR3：SEQ ID NO:140。
  23. 根据权利要求1-22中任一项所述的分离的抗原结合蛋白，其包含抗体重链可变区VH和抗体轻链可变区VL，且所述VH和VL包含选自以下任意一组的氨基酸序列：
    (1)VH：SEQ ID NO:151和VL：SEQ ID NO:231；
    (2)VH：SEQ ID NO:152和VL：SEQ ID NO:232；
    (3)VH：SEQ ID NO:153和VL：SEQ ID NO:233；
    (4)VH：SEQ ID NO:154和VL：SEQ ID NO:234；
    (5)VH：SEQ ID NO:155和VL：SEQ ID NO:235；
    (6)VH：SEQ ID NO:156和VL：SEQ ID NO:232；
    (7)VH：SEQ ID NO:157和VL：SEQ ID NO:236；以及
    (8)VH：SEQ ID NO:158和VL：SEQ ID NO:231。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    a)能够以1×10 8M以下的KD值结合人PD-1；
    b)能够阻断PD-1和PD-L1结合；
    c)能够阻断PD-1和PD-L2结合；
    d)能够刺激免疫细胞中IL-2和/或IFN-γ的分泌；以及
    e)能够抑制肿瘤生长和/或肿瘤细胞增殖。
  25. 分离的抗原结合蛋白，其包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，其中，所述HCDR1包含SEQ ID NO:362所示的氨基酸序列，所述HCDR2包含SEQ ID NO:363所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:364所示的氨基酸序列。
  26. 根据权利要求25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。
  27. 根据权利要求25-26中任一项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，VHH，重链抗体(HCAb)和/或dAb。
  28. 根据权利要求25-27中任一项所述的分离的抗原结合蛋白，其中所述抗原结合片段为重链抗体(HCAb)或VHH。
  29. 根据权利要求25-28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。
  30. 根据权利要求25-29中任一项所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:18-33中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:57-68中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:99-101和103-111中任一项所示的氨基酸序列。
  31. 根据权利要求25-30中任一项所述的分离的抗原结合蛋白，其中所述HCDR1、HCDR2和HCDR3包含选自以下任意一组的氨基酸序列：
    (1)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:100；
    (2)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:99；
    (3)HCDR1：SEQ ID NO:19，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:99；
    (4)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:57，和HCDR3：SEQ ID NO:101；
    (5)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
    (6)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:60，和HCDR3：SEQ ID NO:103；
    (7)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:59，和HCDR3：SEQ ID NO:103；
    (8)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:61，和HCDR3：SEQ ID NO:103；
    (9)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:60，和HCDR3：SEQ ID NO:103；
    (10)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:59，和HCDR3：SEQ ID NO:103；
    (11)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:61，和HCDR3：SEQ ID NO:103；
    (12)HCDR1：SEQ ID NO:20，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
    (13)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:104；
    (14)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:105；
    (15)HCDR1：SEQ ID NO:21，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
    (16)HCDR1：SEQ ID NO:22，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:100；
    (17)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:106；
    (18)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:107；
    (19)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:58，和HCDR3：SEQ ID NO:103；
    (20)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:62，和HCDR3：SEQ ID NO:100；
    (21)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:61，和HCDR3：SEQ ID NO:100；
    (22)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:59，和HCDR3：SEQ ID NO:100；
    (23)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:60，和HCDR3：SEQ ID NO:100；
    (24)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:63，和HCDR3：SEQ ID NO:100；
    (25)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
    (26)HCDR1：SEQ ID NO:24，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:108；
    (27)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
    (28)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:108；
    (29)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:108；
    (30)HCDR1：SEQ ID NO:26，HCDR2：SEQ ID NO:67，和HCDR3：SEQ ID NO:109；
    (31)HCDR1：SEQ ID NO:27，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:110；
    (32)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
    (33)HCDR1：SEQ ID NO:28，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (34)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:111；
    (35)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:68，和HCDR3：SEQ ID NO:108；
    (36)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (37)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:109；
    (38)HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (39)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:111；
    (40)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:68，和HCDR3：SEQ ID NO:111；
    (41)HCDR1：SEQ ID NO:29，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111；
    (42)HCDR1：SEQ ID NO:30，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:109；
    (43)HCDR1：SEQ ID NO:31，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (44)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:111；
    (45)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (46)HCDR1：SEQ ID NO:32，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:109；
    (47)HCDR1：SEQ ID NO:23，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:109；以及
    (48)HCDR1：SEQ ID NO:33，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111。
  32. 根据权利要求25-31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，且所述VH包含SEQ ID NO:254所示的氨基酸序列。
  33. 根据权利要求32所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:160-166和168-221中任一项所示的氨基酸序列。
  34. 根据权利要求25-33中任一项所述的分离的抗原结合蛋白，其还包括抗体重链恒定区。
  35. 根据权利要求34所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG恒定区。
  36. 根据权利要求34-35中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG4恒定区和/或人IgG1恒定区。
  37. 根据权利要求34-36中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区包含SEQ ID NO:354-355中任一项所示的氨基酸序列。
  38. 根据权利要求25-37中任一项所述的分离的抗原结合蛋白，其包括抗体重链，所述抗体重链包含SEQ ID NO:247-253和255-309中任一项所示的氨基酸序列。
  39. 根据权利要求25-38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：
    a)能够以1×10 8M以下的KD值结合人PD-1；
    b)能够阻断PD-1和PD-L1结合；
    c)能够阻断PD-1和PD-L2结合；
    d)能够刺激免疫细胞中IL-2和/或IFN-γ的分泌；以及
    e)能够抑制肿瘤生长和/或肿瘤细胞增殖。
  40. 分离的抗原结合蛋白，其包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，其中，所述HCDR1包含SEQ ID NO:10所示的氨基酸序列，所述HCDR2包含SEQ ID NO:81所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:102所示的氨基酸序列。
  41. 根据权利要求40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。
  42. 根据权利要求40-41中任一项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，VHH，重链抗体(HCAb)和/或dAb。
  43. 根据权利要求40-42中任一项所述的分离的抗原结合蛋白，其中所述抗原结合片段为重链抗体(HCAb)。
  44. 根据权利要求41-43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。
  45. 根据权利要求40-44中任一项所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:18、25、28和34-38中任一项所示的氨基酸序列，所述HCDR2包含SEQ ID NO:64-66中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:108、111和112中任一项所示的氨基酸序列。
  46. 根据权利要求40-45中任一项所述的分离的抗原结合蛋白，其中所述HCDR1、HCDR2和HCDR3包含选自以下任意一组的氨基酸序列：
    (1)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:66，和HCDR3：SEQ ID NO:108；
    (2)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:108；
    (3)HCDR1：SEQ ID NO:25，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111；
    (4)HCDR1：SEQ ID NO:34，HCDR2：SEQ ID NO:65，和HCDR3：SEQ ID NO:111；
    (5)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:108；
    (6)HCDR1：SEQ ID NO:18，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (7)HCDR1：SEQ ID NO:35，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (8)HCDR1：SEQ ID NO:36，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (9)HCDR1：SEQ ID NO:36，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:112；
    (10)HCDR1：SEQ ID NO:28，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；
    (11)HCDR1：SEQ ID NO:37，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111；以及
    (12)HCDR1：SEQ ID NO:38，HCDR2：SEQ ID NO:64，和HCDR3：SEQ ID NO:111。
  47. 根据权利要求40-46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，且所述VH包含SEQ ID NO:167所示的氨基酸序列。
  48. 根据权利要求47所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:196、197、200、201、202和222-230中任一项所示的氨基酸序列。
  49. 根据权利要求40-48中任一项所述的分离的抗原结合蛋白，其还包括抗体重链恒定区。
  50. 根据权利要求49所述的分离的抗原结合蛋白，其中所述重链恒定区源自人IgG恒定区。
  51. 根据权利要求49-50中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区源自人 IgG4恒定区和/或人IgG1恒定区。
  52. 根据权利要求49-51中任一项所述的分离的抗原结合蛋白，其中所述重链恒定区包含SEQ ID NO:354-355中任一项所示的氨基酸序列。
  53. 根据权利要求40-52中任一项所述的分离的抗原结合蛋白，其包括抗体重链，所述抗体重链包含SEQ ID NO:310-323中任一项所示的氨基酸序列。
  54. 根据权利要求40-53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：
    a)能够以1×10 8M以下的KD值结合人PD-1；
    b)能够阻断PD-1和PD-L1结合；
    c)能够阻断PD-1和PD-L2结合；
    d)能够刺激免疫细胞中IL-2和/或IFN-γ的分泌；以及
    e)能够抑制肿瘤生长和/或肿瘤细胞增殖。
  55. 融合蛋白，其包含第一靶向部分和第二靶向部分，所述第一靶向部分包含PD-1结合部分，其中所述PD-1结合部分包含权利要求1-54中任一项所述的分离的抗原结合蛋白。
  56. 根据权利要求55所述的融合蛋白，其中所述第二靶向部分包含CD73结合部分。
  57. 根据权利要求55-56中任一项所述的融合蛋白，其中所述CD73结合部分包括特异性结合CD73的抗体或其抗原结合片段。
  58. 根据权利要求55-57中任一项所述的融合蛋白，其中所述CD73结合部分包含抗体重链或其片段，所述抗体重链或其片段包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3分别依次包含SEQ ID NO:17、SEQ ID NO:56和SEQ ID NO:98所示的氨基酸序列。
  59. 根据权利要求55-58中任一项所述的融合蛋白，其中所述CD73结合部分包括抗体重链可变区VH，所述VH包含SEQ ID NO:159所示的氨基酸序列。
  60. 根据权利要求55-59中任一项所述的融合蛋白，其中所述CD73结合部分包含抗体轻链或其片段，所述抗体轻链或其片段包含LCDR1、LCDR2和LCDR3，且所述LCDR1、LCDR2和LCDR3分别依次包含SEQ ID NO:123、SEQ ID NO:130和SEQ ID NO:146所示的氨基酸序列。
  61. 根据权利要求55-60中任一项所述的融合蛋白，其中所述CD73结合部分包括抗体轻链可变区VL，所述VL包含SEQ ID NO:237所示的氨基酸序列。
  62. 根据权利要求56-61中任一项所述的融合蛋白，其中所述CD73结合部分包含Fab。
  63. 根据权利要求56-62中任一项所述的融合蛋白，其中所述CD73结合部分包含抗体重链， 且所述抗体重链包含SEQ ID NO:246所示的氨基酸序列。
  64. 根据权利要求56-63中任一项所述的融合蛋白，其中所述CD73结合部分包含抗体轻链，且所述抗体轻链包含SEQ ID NO:330所示的氨基酸序列。
  65. 根据权利要求56-64中任一项所述的融合蛋白，其中所述PD-1结合部分位于所述CD73结合部分的N端或C端。
  66. 根据权利要求56-65中任一项所述的融合蛋白，其包括第一多肽链和第二多肽链，其中所述第一多肽链包括所述PD-1结合部分的VH和所述CD73结合部分的VH，所述第二多肽链包括所述CD73结合部分的VL。
  67. 根据权利要求66所述的融合蛋白，其中在所述第一多肽链中，所述PD-1结合部分的VH位于所述CD73结合部分的VH的N端。
  68. 根据权利要求66所述的融合蛋白，其中在所述第一多肽链中，所述PD-1结合部分的VH位于所述CD73结合部分的VH的C端。
  69. 根据权利要求66-68中任一项所述的融合蛋白，其中所述第一多肽链还包含抗体重链恒定区。
  70. 根据权利要求66-69中任一项所述的融合蛋白，其中所述第一多肽链自N端至C端，依次包含所述PD-1结合部分的VH、所述CD73结合部分的VH和所述抗体重链恒定区。
  71. 根据权利要求66-70中任一项所述的融合蛋白，其中所述PD-1结合部分的VH与所述CD73结合部分的VH通过连接肽间接连接。
  72. 根据权利要求71所述的融合蛋白，其中所述连接肽包含SEQ ID NO:334-352中任一项所示的氨基酸序列。
  73. 根据权利要求66-72中任一项所述的融合蛋白，其中所述第一多肽链包含SEQ ID NO:333所示的氨基酸序列。
  74. 根据权利要求66-73中任一项所述的融合蛋白，其中所述第二多肽链还包含抗体轻链恒定区。
  75. 根据权利要求66-74中任一项所述的融合蛋白，其中所述第二多肽链包含SEQ ID NO:330所示的氨基酸序列。
  76. 根据权利要求66-75中任一项所述的融合蛋白，其包括两条所述第一多肽链和两条所述第二多肽链。
  77. 免疫缀合物，其包含权利要求1-54中任一项所述的分离的抗原结合蛋白或权利要求55-76中任一项所述的融合蛋白。
  78. 分离的一种或多种核酸分子，其编码权利要求1-54中任一项所述的分离的抗原结合蛋白 和/或权利要求55-76中任一项所述的融合蛋白。
  79. 载体，其包含根据权利要求78所述的核酸分子。
  80. 细胞，其包含根据权利要求78所述的核酸分子或根据权利要求79所述的载体。
  81. 制备权利要求1-54中任一项所述的分离的抗原结合蛋白和/或权利要求55-76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1-54中任一项所述的分离的抗原结合蛋白和/或权利要求55-76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80所述的细胞。
  82. 药物组合物，其包含权利要求1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体和/或权利要求80所述的细胞，以及任选地药学上可接受的载体。
  83. 权利要求1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物在制备药物中的用途，所述药物用于治疗PD-1介导的疾病或病症。
  84. 根据权利要求83所述的用途，其中所述PD-1介导的疾病或病症包括肿瘤、自体免疫疾病或炎症。
  85. 根据权利要求84所述的用途，其中所述肿瘤包括实体瘤和/或非实体瘤。
  86. 根据权利要求84-85中任一项所述的用途，其中所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
  87. 预防、缓解或治疗PD-1介导的疾病或病症的方法，所述方法包括向有需要的受试者施用权利要求1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物；任选地，与一种或多种其他肿瘤治疗方法联用。
  88. 根据权利要求87所述的方法，其中所述PD-1介导的疾病或病症包括肿瘤、自体免疫疾病或炎症。
  89. 根据权利要求88所述的方法，其中所述肿瘤包括实体瘤和/或非实体瘤。
  90. 根据权利要求88-89中任一项所述的方法，其中所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和食管癌。
  91. 一种增加受试者中T细胞活性的方法，其包括向有需要的受试者施用有效量的权利要求 1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物。
  92. 试剂盒或给药装置，其包含权利要求1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物。
  93. 抑制PD-L1蛋白或PD-L2蛋白与PD-1蛋白结合的方法，其包含使用权利要求1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物。
  94. 根据权利要求93所述的方法，其为体外方法。
  95. 根据权利要求93所述的方法，其为非诊断目的的方法。
  96. 检测PD-1蛋白的存在和/或含量的方法，其包含使用权利要求1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物。
  97. 根据权利要求96所述的方法，其为体外方法。
  98. 根据权利要求96所述的方法，其为非诊断目的的方法。
  99. 权利要求1-54中任一项所述的分离的抗原结合蛋白、权利要求55-76中任一项所述的融合蛋白、权利要求77所述的免疫缀合物、权利要求78所述的核酸分子、权利要求79所述的载体、权利要求80所述的细胞和/或权利要求82所述的药物组合物，与一种或多种其他肿瘤治疗方法联用在制备药物中的用途，所述药物用于治疗肿瘤。
  100. 根据权利要求99所述的用途，其中所述一种或多种其他肿瘤治疗方法包括化疗和/或放疗。
  101. 根据权利要求99-100中任一项所述的用途，其中所述肿瘤包括实体瘤和/或非实体
    瘤。
  102. 根据权利要求99-101中任一项所述的用途，其中所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
  103. 权利要求1-54中任一项所述的分离的抗原结合蛋白，和抗CD73抗体联用在制备药物中的用途，所述药物用于治疗肿瘤。
  104. 根据权利要求103所述的用途，其中所述肿瘤包括实体瘤和/或非实体瘤。
  105. 根据权利要求103-104中任一项所述的用途，其中所述肿瘤包括肺癌、肝癌、黑色素瘤、尿路上皮癌、头颈鳞癌、淋巴瘤、胃癌和/或食管癌。
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