WO2022247826A1 - 靶向pd-l1和cd73的特异性结合蛋白 - Google Patents

靶向pd-l1和cd73的特异性结合蛋白 Download PDF

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WO2022247826A1
WO2022247826A1 PCT/CN2022/094703 CN2022094703W WO2022247826A1 WO 2022247826 A1 WO2022247826 A1 WO 2022247826A1 CN 2022094703 W CN2022094703 W CN 2022094703W WO 2022247826 A1 WO2022247826 A1 WO 2022247826A1
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seq
binding protein
amino acid
antibody
cancer
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French (fr)
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单茜茜
张雪琨
甘馨
罗海山
石磊
王永强
贾星星
王荣超
戎一平
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Harbour Biomed Shanghai Co Ltd
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Harbour Biomed Shanghai Co Ltd
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Priority to EP22810551.6A priority Critical patent/EP4349867A4/en
Priority to CN202280035753.9A priority patent/CN117337306A/zh
Priority to US18/564,598 priority patent/US20240269276A1/en
Publication of WO2022247826A1 publication Critical patent/WO2022247826A1/zh
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of biomedicine, more specifically to the field of antibody therapy.
  • Programmed death ligand 1 (PD-L1), one of the two cell surface glycoprotein ligands of programmed death receptor 1 (PD-1) (the other being PD-L2), binds PD- 1 downregulates T cell activation and cytokine secretion.
  • PD-L1 also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40kDa type I transmembrane protein
  • PD-L1 contains IgV (immunoglobulin variable region)-like region, IgC (immunoglobulin constant region)-like region, transmembrane region and cytoplasmic tail region, wherein the cytoplasmic tail region is related to intracellular signal transduction; IgV region and IgC region are involved in intercellular signal transduction.
  • PD-L1 When PD-L1 binds to PD-1, it recruits Src homology domain 2 protein tyrosine phosphatase (SHP-2), thereby attenuating lymphocyte-specific protein tyrosine kinase (LCK)-induced zeta chain-related protein ( ZAP70) phosphorylates and inhibits the RAS-MEK-ERK/PI3K-Akt-mTOR pathway, thereby effectively inhibiting the transcription of T cells, and finally inhibiting the immune function of T cells, playing an important role in the negative regulation of immune responses. Therefore, it is generally believed that blocking the PD-1/PD-L1 signaling pathway can up-regulate the activation of T cells and activate the endogenous anti-tumor immune response, thereby exerting a therapeutic effect on tumors.
  • SHP-2 Src homology domain 2 protein tyrosine phosphatase
  • LCK lymphocyte-specific protein tyrosine kinase
  • ZAP70 ze
  • the first-generation immune checkpoint inhibitors represented by PD-1/PD-L1 and CTLA-4 monoclonal antibodies have gradually become the representative of tumor immunotherapy, especially in adoptive cell transfer therapy and immune checkpoint blockade (ICB) play an important role in.
  • Tumor immunotherapy targeting PD-1/PD-L1 has been approved for the treatment of many different solid tumors, and won the Nobel Prize in Physiology or Medicine in 2018.
  • clinical data on anti-PD-1/PD-L1 therapy show limited response rates. Some patients develop primary resistance and do not respond to PD-1/PD-L1 therapy, and some responders also develop acquired resistance after the initial response.
  • CD73 Leukocyte differentiation antigen 73
  • 5'-nucleotidase is an enzyme encoded by the NT5E gene in humans.
  • CD73 is a dimer composed of two identical 70-KD subunits linked to the outer surface of the plasma membrane by a glycosylphosphatidylinositol.
  • CD73 is known to catalyze the dephosphorylation of extracellular monophosphate nucleosides to nucleosides, such as adenosine.
  • the accumulation of extracellular adenosine in cancer tissues constitutes an important mechanism of tumor immune evasion.
  • tumor-derived adenosine largely inhibits infiltrating effector T cells via adenylate cyclase-activated A2A receptors (Ohta et al., (2006) Proc Natl Acad Sci USA 103:13132-13137) .
  • CD73 has been reported to be expressed in a variety of tumor cells, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer (Jin et al., Cancer Res 2010;70:2245-55 and Stagg et al., PNAS 2010;107:1547-52).
  • TME tumor micro-environment
  • Adenosine can inhibit the immune killing effect of T cells through the adenosine receptor (A2AR), enabling tumors to achieve immune escape, and CD73 is the key enzyme that catalyzes the production of adenosine.
  • Immune checkpoint blockade of PD-1/PD-L1 has become a milestone in immunotherapy.
  • durable responses occur in only a small proportion of patients, and some patients who respond initially to treatment eventually develop Therefore, there is an urgent need for PD-1/PD-L1 inhibitors with better efficacy and tumor immune targets that can produce synergistic effects with PD-1/PD-L1.
  • the present invention provides an antigen-binding protein capable of specifically binding to PD-L1 or a fragment thereof, which can block the combination of PD-1 and PD-L1.
  • the present invention also provides a multispecific binding protein capable of binding to one or more antigens with high affinity and high specificity, the antigen being PD-L1 or a fragment thereof, and/or CD73 or a fragment thereof.
  • the present invention also provides nucleic acid molecules encoding the antigen binding protein and the multispecific binding protein, expression vectors for producing the antigen binding protein and the multispecific binding protein, host cells and Methods of making said antigen binding proteins, and said specific binding proteins.
  • the present invention also relates to the antigen-binding protein, and the use of the multispecific binding protein in the treatment, prevention and/or diagnosis of diseases, such as immune diseases, acute and chronic inflammatory diseases, and tumor diseases.
  • the present invention provides an isolated antigen-binding protein that specifically binds to PD-L1 or a fragment thereof.
  • the isolated antigen-binding protein specifically binding to PD-L1 or a fragment thereof has one or more of the following properties: (a) can bind to human PD-L1 and/or cynomolgus monkey PD-L1 with high affinity; (b) ) has an inhibitory effect on the PD-1 signaling pathway; (c) is able to enhance the activation of T lymphocytes; and (d) exhibits tumor suppressor activity in vivo.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH comprising the following complementarity determining regions or mutants thereof:
  • HCDR1 shown in the amino acid sequence of SEQ ID NO:9;
  • HCDR2 shown in the amino acid sequence of SEQ ID NO: 23;
  • HCDR3 shown in the amino acid sequence of SEQ ID NO:36.
  • the mutants have 1, 2, 3 or 4 amino acid insertions, deletions or substitutions respectively based on the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH.
  • Those skilled in the art know that one, two or three CDRs in HCDR1, HCDR2 and HCDR3 can be mutated by insertion, deletion or substitution of 1, 2 or 3 amino acids, respectively.
  • one amino acid mutation can be performed on HCDR1, and no amino acid mutation can be performed on HCDR2 and HCDR3, or one amino acid mutation can be performed on each of HCDR1 and HCDR2, and no amino acid mutation can be performed on HCDR3.
  • the amino acid sequence of the HCDR1 is GFX 1 FSX 2 Y
  • the amino acid sequence of the HCDR2 is X 3 YX 4 GX 5 X 6
  • the amino acid sequence of the HCDR3 is NRAX 7 FGVX 8 PDX 9 SDI
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 or X 9 are selected from N, T, D, S, W, R, K, E, I, A , any of V and L.
  • X is T, D, or N ; X is N or S ; X is W or R; X is D or T; X is T or S; E; X7 is I or L; X8 is V or I; and/or, X9 is A or D.
  • the amino acid sequence of HCDR1 is GF(N/T/D)FS(N/S)Y.
  • the amino acid sequence of the HCDR2 is (W/R)Y(D/T)G(T/S)(K/R/E).
  • the amino acid sequence of the HCDR3 is NRA(I/L)FGV(V/I)PD(A/D)SDI.
  • the mutant of HCDR1 has the amino acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14.
  • the mutant of HCDR2 has the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 27.
  • the mutant of HCDR3 has the amino acid sequence shown in any one of SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.
  • the isolated antigen-binding protein specifically binding to PD-L1 and/or its fragments comprises an antibody heavy chain variable region VH, said VH comprising the following complementarity determining regions or mutants thereof:
  • HCDR1 shown in the amino acid sequence of any one of SEQ ID NO: 9-11 and SEQ ID NO: 13-14;
  • HCDR2 shown in the amino acid sequence of any one of SEQ ID NO: 23-24 and SEQ ID NO: 27; and/or
  • HCDR3 shown in the amino acid sequence of any one of SEQ ID NO: 36 and SEQ ID NO: 39-41.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 9, shown in SEQ ID NO: 23 and SEQ ID NO: 36.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 23 and SEQ ID NO: 36.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 11, shown in SEQ ID NO: 24 and SEQ ID NO: 36.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 11, shown in SEQ ID NO: 23 and SEQ ID NO: 36.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 13, shown in SEQ ID NO: 23 and SEQ ID NO: 36.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 23 and SEQ ID NO: 39.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 23 and SEQ ID NO: 40.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 23 and SEQ ID NO: 41.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 27 and SEQ ID NO: 36.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 27 and SEQ ID NO: 39.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 27 and SEQ ID NO: 40.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, shown in SEQ ID NO: 27 and SEQ ID NO: 41.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, shown in SEQ ID NO: 27 and SEQ ID NO: 36.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, shown in SEQ ID NO: 27 and SEQ ID NO: 39.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, shown in SEQ ID NO: 27 and SEQ ID NO: 40.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, shown in SEQ ID NO: 27 and SEQ ID NO: 41.
  • the VH further comprises a heavy chain variable region framework region (VH FWR), and the VH FWR may comprise VH FWR1, VH FWR2, VH FWR3 and VH FWR4.
  • VH FWR heavy chain variable region framework region
  • the VHFWR1 has the amino acid sequence shown in any one of SEQ ID NO: 3-5.
  • the VHFWR2 has the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20.
  • the VHFWR3 has the amino acid sequence shown in SEQ ID NO: 30 or SEQ ID NO: 31.
  • the VHFWR4 has the amino acid sequence shown in SEQ ID NO:43.
  • the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 74-77, SEQ ID NO: 79-80 and SEQ ID NO: 82-92, or has at least 80%, 85%, or Amino acid sequences that are 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical.
  • the isolated antigen-binding protein that specifically binds to PD-L1 and/or its fragments further comprises an Fc region, or a region equivalent to the Fc region of an immunoglobulin, preferably, the Fc region is human Fc. More preferably, the human Fc is human IgG1 Fc.
  • the Fc comprises L234A, L235A and P329G mutations, or L234A and L235A mutations, or comprises one, two or three mutations of S298A, E333A and K334A, more preferably simultaneously comprises three of S298A, E333A and K334A mutation.
  • the isolated antigen binding protein that specifically binds to PD-L1 or a fragment thereof comprises an antigen binding protein with SEQ ID NO: 99, 100, 101, 102, 104, 105, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116 or 117 have at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence weight chain.
  • the isolated antigen-binding protein that specifically binds to PD-L1 or a fragment thereof comprises any of SEQ ID NOs: 99-102, SEQ ID NOs: 104-105 and SEQ ID NOs: 107-117 The amino acid sequence of the heavy chain shown in item.
  • the antigen-binding protein can also be an antigen-binding fragment, eg, in the form of Fab, Fab', F(ab') 2 , Fv, scFv, VH, or HCAb.
  • the number of said Fab, Fab', F(ab') 2 , Fv, scFv or VH is preferably one or more.
  • the present invention provides an isolated nucleic acid encoding the isolated antigen binding protein of the first aspect of the present invention.
  • the present invention provides an expression vector comprising the isolated nucleic acid of the second aspect of the present invention, capable of expressing it as the isolated antigen-binding protein of the first aspect.
  • the expression vector may be a eukaryotic cell expression vector and/or a prokaryotic cell expression vector, such as a plasmid.
  • the present invention provides a host cell comprising the isolated nucleic acid of the second aspect, or the expression vector of the third aspect.
  • the present invention provides an antibody drug conjugate, comprising: the isolated antigen-binding protein of the first aspect of the present invention; and, a drug covalently linked to the antigen-binding protein.
  • the drug is selected from chemotherapeutics, radiotherapeutics, immunosuppressants, and cytotoxic drugs.
  • the present invention provides a chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the first aspect of the present invention
  • the isolated antigen binding protein comprises the first aspect of the present invention The isolated antigen binding protein.
  • the present invention provides modified immune cells comprising the chimeric antigen receptor described in the sixth aspect of the present invention.
  • the present invention provides a multispecific antibody comprising two or more antigen-binding domains, one of which comprises the isolated antigen-binding protein described in the first aspect of the present invention.
  • the present invention provides a method for preparing the isolated antigen-binding protein of the first aspect.
  • the isolated antigen-binding protein of the first aspect is prepared using hybridoma technology or other conventional techniques in the art, such as humanization techniques.
  • the method of preparation includes the step of culturing the host cell of the fourth aspect.
  • HCAb transgenic mice (hereinafter referred to as HCAb transgenic mice) are used to prepare the isolated antigen-binding protein of the first aspect.
  • the HCAb transgenic mouse is a transgenic mouse carrying the immune repertoire of human immunoglobulins, 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.
  • the method for preparing the isolated antigen-binding protein of the first aspect using HCAb transgenic mice comprises the following steps:
  • human PD-L1 antigen is recombinant human PD-L1-mFc, specifically, the antigen is a recombinant human PD-L1 linked to Fc fusion protein;
  • step (d) Purifying the fully human PD-L1 monoclonal antibody obtained in step (c).
  • the IgG antibody is an IgG1 antibody or an IgG4 antibody.
  • the method further includes the step of affinity engineering the fully human PD-L1 monoclonal antibody by mutating the CDR region.
  • the fully human PD-L1 mAb is optimized by germline back mutation and/or post-translational modification (PTM) removal.
  • PTM post-translational modification
  • the present invention provides a pharmaceutical composition, which comprises: the isolated antigen-binding protein described in the first aspect, the antibody drug conjugate described in the fifth aspect, and the antibody drug conjugate described in the sixth aspect.
  • the pharmaceutical composition may also contain other therapeutic agents, including but not limited to, chemotherapeutic agents, radiotherapeutic agents, immunosuppressants, cytotoxic drugs, and the like.
  • the chemotherapeutic agent, radiotherapeutic agent, immunosuppressant or cytotoxic drug in the pharmaceutical composition is the same as or different from the drug in the antibody-drug conjugate described in the fifth aspect.
  • the present invention provides the isolated antigen-binding protein described in the first aspect, the isolated nucleic acid described in the second aspect, the antibody-drug conjugate described in the fifth aspect, and the chimera described in the sixth aspect.
  • Combined antigen receptor, the modified immune cell of the seventh aspect or the multispecific antibody of the eighth aspect or the pharmaceutical composition of the tenth aspect are used in the preparation of prevention, treatment and/or diagnosis of immune diseases, acute And chronic inflammatory diseases, as well as the application of drugs in tumor diseases.
  • the tumor can be breast cancer, renal cell carcinoma, melanoma, colon cancer, and B cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urothelial cancer, liver cancer, esophageal cancer , gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin's lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignancy, etc.
  • the inflammatory disease may be atopic dermatitis, ulcerative colitis and the like.
  • the immune disease may be graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma and the like.
  • the present invention provides a method for detecting PD-L1 in a sample, the method comprising the step of detecting PD-L1 in the sample with the isolated antigen-binding protein of the first aspect.
  • the sample can be a biological sample, for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.
  • a biological sample for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.
  • the method of detecting PD-L1 in a sample may be for non-diagnostic purposes.
  • the present invention provides a multispecific binding protein, the specific binding protein comprising at least two structural domains capable of binding PD-L1 or a fragment thereof, and/or CD73 or a fragment thereof.
  • the specific binding protein comprises a first domain and a second domain, the first domain binds CD73 or a fragment thereof, and the second domain binds PD-L1 or a fragment thereof.
  • the first domain is linked to the second domain to form a bispecific binding protein.
  • the first domain is a CD73 antibody or antigen-binding fragment thereof.
  • the second domain is a PD-L1 antibody or antigen-binding fragment thereof.
  • the first domain and/or the second domain is in the form of an IgG, Fab, Fab', F(ab') 2 , Fv, scFv, VH, or HCAb.
  • the number of said Fab, Fab', F(ab') 2 , Fv, scFv, VH is one or more.
  • the first domain is in the form of an IgG.
  • the IgG heavy chain constant region is a human heavy chain constant region, more preferably a human IgG1, human IgG2, human IgG3 or human IgG4 heavy chain constant region; wherein the human IgG preferably comprises one or both of L234A, L235A and P329G One or three mutations, more preferably L234A and L235A mutations, or one, two or three S298A, E333A and K334A mutations, more preferably S298A, E333A and K334A mutations.
  • the Fc of IgG in the first structural domain is the Fc of human IgG1, more preferably, the Fc comprises L234A, L235A and P329G mutations, or L234A and L235A mutations, or comprises one of S298A, E333A and K334A, Two or three mutations, more preferably comprising the S298A, E333A and K334A mutations.
  • the first domain is in the form of a Fab, more preferably, the first domain comprises 2 Fabs.
  • the second domain comprises one or more VHs.
  • said VH is human VH. More preferably, the second domain has two VHs.
  • the second domain further comprises Fc, a region equivalent to the Fc region of an immunoglobulin, preferably, the Fc is the Fc of human IgG1, more preferably, the Fc comprises L234A, L235A and P329G mutation, or L234A and L235A mutation, or comprise one, two or three of S298A, E333A and K334A mutations, more preferably comprise S298A, E333A and K334A mutations.
  • the first domain and the second domain are linked directly or via a linker peptide L to form a bispecific binding protein.
  • the second domain is linked C-terminally or N-terminally to the first domain.
  • the bispecific binding protein comprises a short chain and a long chain.
  • the short chain has the structure shown by N'-VL 1 -CL 1 -C';
  • the long chain has the structure shown by N'-VH 1 -CH1-h-CH2-CH3-L-VH 2 -C' the structure shown;
  • the short chain has a structure represented by N'-VL 1 -CL 1 -C';
  • the long chain has a structure represented by N'-VH 2 -L-VH 1 -CH1-h-CH2-CH3-C' the structure shown;
  • the short chain has a structure shown by N'-VH 1 -CH-C';
  • the long chain has a structure shown by N'-VL 1 -CL 1 -L-VH 2 -CH2-CH3-C' structure,
  • said VL 1 and VH 1 are VL and VH of the first structural domain respectively
  • said VH 2 is VH of the second structural domain
  • said h is a hinge region
  • said L is a connecting peptide
  • said CL 1 is the CL of the first domain.
  • the hinge region is a common hinge region in the immunoglobulin field, usually contains a large amount of proline, is flexible, and forms 2-5 disulfide bonds.
  • said L is a peptide with a length of 0-30 amino acids.
  • the amino acid sequence of said L is as shown in any one of SEQ ID NO: 136-157.
  • CH3 of the long chain is directly connected to VH2 , that is, the length of L is zero.
  • the CH3 of the long chain is linked to VH2 via linker peptide L.
  • the connecting peptide L is a peptide of 0-30 amino acids in length.
  • the connecting peptide L can be the amino acid sequence shown in any one of SEQ ID NO.136-157.
  • VH 1 and VH 2 of the long chain are directly connected, that is, the length of L is zero.
  • the long-chain VH1 is linked to VH2 via a linker peptide L.
  • the connecting peptide L is a peptide of 0-30 amino acids in length.
  • the connecting peptide L can be the amino acid sequence shown in any one of SEQ ID NO.136-157.
  • VH 2 of the long chain is directly linked to CL 1 , ie the length of L is zero.
  • the long chain VH 2 is linked to CL 1 via a linker peptide L.
  • the connecting peptide L is a peptide of 0-30 amino acids in length.
  • the connecting peptide L can be the amino acid sequence shown in any one of SEQ ID NO.136-157.
  • the first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), the VL comprising a complementarity determining region selected from:
  • LCDR1 shown in the amino acid sequence of any one of SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53;
  • LCDR2 shown in the amino acid sequence of any one of SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59; and/or
  • LCDR3 shown in the amino acid sequence of any one of SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67;
  • the VH comprises a complementarity determining region selected from the group consisting of:
  • HCDR1 shown in the amino acid sequence of any one of SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 10;
  • HCDR2 shown in the amino acid sequence of any one of SEQ ID NO: 22, SEQ ID NO: 25 and SEQ ID NO: 26; and/or
  • HCDR3 shown in the amino acid sequence of any one of SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 38.
  • said first domain comprises a light chain variable region (VL) and a heavy chain variable region (VH), said VL and VH comprising complementarity determining regions selected from:
  • LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 52, SEQ ID NO: 58 and SEQ ID NO: 66, respectively; and, as shown in SEQ ID NO: 12, SEQ ID NO: 25 and SEQ ID NO: 37, respectively HCDR1, HCDR2 and HCDR3 indicated;
  • the first domain comprises VL and VH, wherein the amino acid sequence of the VL is at least 80%, 85% identical to SEQ ID NO:94, SEQ ID NO:95 or SEQ ID NO:96 , 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity; the amino acid sequence of the VH is identical to SEQ ID NO: 73, SEQ ID NO: 78 or SEQ ID NO: 81 has at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity.
  • the first domain comprises VL and VH, wherein the VL has the amino acid sequence shown in any one of SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96; the VH has The amino acid sequence shown in any one of SEQ ID NO:73, SEQ ID NO:78 and SEQ ID NO:81.
  • the first structural domain comprises VL and VH, and the amino acid sequences of the VL and VH are shown in SEQ ID NO:94 and SEQ ID NO:73 respectively; or as shown in SEQ ID NO:95 and SEQ ID NO respectively :78; or as shown in SEQ ID NO:96 and SEQ ID NO:81 respectively.
  • the first domain comprises an amino acid sequence at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical light chain; %, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical heavy chains.
  • the first domain comprises a light chain shown in any one of SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:121; and SEQ ID NO:98, SEQ ID NO:103 and The heavy chain shown in any one of SEQ ID NO: 106.
  • said first domain comprises the light chain shown in SEQ ID NO: 119 and the heavy chain shown in SEQ ID NO: 98.
  • the first domain comprises the light chain shown in SEQ ID NO:120 and the heavy chain shown in SEQ ID NO:103.
  • the first domain comprises the light chain shown in SEQ ID NO:121 and the heavy chain shown in SEQ ID NO:106.
  • the second domain comprises an antibody heavy chain variable region VH comprising the following complementarity determining regions or mutants thereof:
  • HCDR1 shown in the amino acid sequence of SEQ ID NO:9;
  • HCDR2 shown in the amino acid sequence of SEQ ID NO: 23;
  • HCDR3 shown in the amino acid sequence of SEQ ID NO:36.
  • the mutants have 1, 2 or 3 amino acid insertions, deletions or substitutions respectively based on the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the VH.
  • Those skilled in the art know that one, two or three CDRs in HCDR1, HCDR2 and HCDR3 can be mutated by insertion, deletion or substitution of 1, 2 or 3 amino acids, respectively.
  • one amino acid mutation can be performed on HCDR1, and no amino acid mutation can be performed on HCDR2 and HCDR3, or one amino acid mutation can be performed on each of HCDR1 and HCDR2, and no amino acid mutation can be performed on HCDR3.
  • the amino acid sequence of the HCDR1 is GFX 1 FSX 2 Y
  • the amino acid sequence of the HCDR2 is X 3 YX 4 GX 5 X 6
  • the amino acid sequence of the HCDR3 is NRAX 7 FGVX 8 PDX 9 SDI
  • X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 or X 9 are selected from N, T, D, S, W, R, K, E, I, A , any of V and L.
  • X is T, D, or N ; X is N or S ; X is W or R; X is D or T; X is T or S; E; X7 is I or L; X8 is V or I; and/or, X9 is A or D.
  • the amino acid sequence of HCDR1 is GF(N/T/D)FS(N/S)Y.
  • the amino acid sequence of the HCDR2 is (W/R)Y(D/T)G(T/S)(K/R/E).
  • the amino acid sequence of the HCDR3 is NRA(I/L)FGV(V/I)PD(A/D)SDI.
  • the mutant of HCDR1 has the amino acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14.
  • the mutant of HCDR2 has the amino acid sequence shown in SEQ ID NO: 24 or SEQ ID NO: 27.
  • the mutant of HCDR3 has the amino acid sequence shown in any one of SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.
  • the second domain comprises an antibody heavy chain variable region VH comprising the following complementarity determining regions or mutants thereof:
  • HCDR1 shown in the amino acid sequence of any one of SEQ ID NO: 9-11 and SEQ ID NO: 13-14;
  • HCDR2 shown in the amino acid sequence of any one of SEQ ID NO: 23-24 and SEQ ID NO: 27; and/or
  • HCDR3 shown in the amino acid sequence of any one of SEQ ID NO: 36 and SEQ ID NO: 39-41.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 36 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 36 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO: 36 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 11, SEQ ID NO: 23 and SEQ ID NO: 36 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 13, SEQ ID NO: 23 and SEQ ID NO: 36 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 39 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 40 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 23 and SEQ ID NO: 41 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 36 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 39 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 40 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 10, SEQ ID NO: 27 and SEQ ID NO: 41 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 36 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 39 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 40 shown.
  • the second domain comprises an antibody heavy chain variable region VH, the VH comprising HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are SEQ ID NO: 14, SEQ ID NO: 27 and SEQ ID NO: 41 shown.
  • the VH further comprises a heavy chain variable region framework region (VH FWR), and the VH FWR may comprise VH FWR1, VH FWR2, VH FWR3 and VH FWR4.
  • VH FWR heavy chain variable region framework region
  • the VHFWR1 has the amino acid sequence shown in any one of SEQ ID NO: 3-5.
  • the VHFWR2 has the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20.
  • the VHFWR3 has the amino acid sequence shown in SEQ ID NO: 30 or SEQ ID NO: 31.
  • the VHFWR4 has the amino acid sequence shown in SEQ ID NO:43.
  • the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 74-77, SEQ ID NO: 79-80 and SEQ ID NO: 82-92, or has at least 80%, 85%, or Amino acid sequences that are 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical.
  • the second domain further comprises an Fc region, or a region equivalent to the Fc region of an immunoglobulin, preferably, the Fc region is human Fc. More preferably, the human Fc is human IgG1 Fc.
  • the Fc comprises L234A, L235A and P329G mutations, or L234A and L235A mutations, or one, two or three of S298A, E333A and K334A mutations, more preferably S298A, E333A and K334A mutations.
  • the second domain comprises a sequence associated with SEQ ID NO: 99, 100, 101, 102, 104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 or 117 Heavy chains having amino acid sequences that are at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical.
  • the second domain comprises the amino acid sequence of the heavy chain set forth in any one of SEQ ID NOs: 99-102, SEQ ID NOs: 104-105, and SEQ ID NOs: 107-117.
  • the long chain of the specific binding protein comprises at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical amino acid sequence; Or 131 has an amino acid sequence that is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical.
  • the long chain of the specific binding protein has an amino acid sequence selected from any one of SEQ ID NO: 122-130 and SEQ ID NO: 132-133; and the specific binding protein The short chain has an amino acid sequence selected from any one of SEQ ID NO: 119-121 and SEQ ID NO: 131.
  • the specific binding protein has the long chain shown in SEQ ID NO: 122 and the short chain shown in SEQ ID NO: 119.
  • the specific binding protein has the long chain shown in SEQ ID NO: 123 and the short chain shown in SEQ ID NO: 120.
  • the specific binding protein has the long chain shown in SEQ ID NO: 124 and the short chain shown in SEQ ID NO: 121.
  • the specific binding protein has a long chain set forth in SEQ ID NO: 125 and a short chain set forth in SEQ ID NO: 119.
  • the specific binding protein has a long chain set forth in SEQ ID NO: 126 and a short chain set forth in SEQ ID NO: 119.
  • the specific binding protein has a long chain set forth in SEQ ID NO: 127 and a short chain set forth in SEQ ID NO: 119.
  • the specific binding protein has a long chain set forth in SEQ ID NO: 128 and a short chain set forth in SEQ ID NO: 119.
  • the specific binding protein has a long chain set forth in SEQ ID NO: 129 and a short chain set forth in SEQ ID NO: 119.
  • the specific binding protein has the long chain shown in SEQ ID NO: 132 and the short chain shown in SEQ ID NO: 131.
  • the specific binding protein has a long chain set forth in SEQ ID NO: 130 and a short chain set forth in SEQ ID NO: 119.
  • the specific binding protein has the long chain shown in SEQ ID NO: 133 and the short chain shown in SEQ ID NO: 131.
  • the bispecific binding protein is a tetravalent symmetrical structure formed by two first polypeptide chains and two second polypeptide chains.
  • the present invention provides an isolated nucleic acid encoding the specific binding protein or a fragment thereof according to the thirteenth aspect of the present invention.
  • the present invention provides an expression vector capable of expressing the isolated nucleic acid in the fourteenth aspect as the multispecific binding protein or a fragment thereof in the thirteenth aspect.
  • the expression vector may be a eukaryotic cell expression vector and/or a prokaryotic cell expression vector, such as a plasmid.
  • the present invention provides a host cell comprising the isolated nucleic acid of the fourteenth aspect, or the expression vector of the fifteenth aspect.
  • the host cell is a conventional host cell in the art, as long as the expression vector of the fifteenth aspect can stably express the carried nucleic acid as the specific binding protein of the thirteenth aspect or a fragment thereof.
  • the host cells are prokaryotic cells and/or eukaryotic cells, the prokaryotic cells are preferably E.coli cells such as TG1, BL21 (expressing single-chain antibodies or Fab antibodies), and the eukaryotic cells are preferably HEK293 cells or CHO cells cells (expressing full-length IgG antibodies).
  • the host cell of the present invention can be obtained by transforming the expression vector of the fifteenth aspect into a host cell.
  • the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.
  • the present invention provides an antibody drug conjugate comprising the specific binding protein or a fragment thereof in the thirteenth aspect.
  • the antibody drug conjugate also comprises a drug covalently linked to the binding protein or fragment thereof.
  • the drug is selected from chemotherapeutics, radiotherapeutics, immunosuppressants, and cytotoxic drugs.
  • the present invention provides the preparation method of the specific binding protein or the fragment thereof in the thirteenth aspect.
  • the specific binding protein or fragment thereof of the thirteenth aspect is prepared using hybridoma technology or other conventional technologies in the art, such as humanization technology, etc.
  • the preparation method comprises the step of culturing the host cell of the sixteenth aspect.
  • Harbor HCAb transgenic mice (hereinafter referred to as HCAb transgenic mice) are used to prepare the second domain of the specific binding protein of the thirteenth aspect.
  • the HCAb transgenic mouse is a transgenic mouse carrying the immune repertoire of human immunoglobulins, 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.
  • the method for preparing the second domain of the specific binding protein of the thirteenth aspect using HCAb transgenic mice comprises the following steps:
  • the human PD-L1 antigen is recombinant human PD-L1-Fc, specifically, the antigen is a recombinant fusion composed of PD-L1 linked to Fc protein;
  • step (d) Purifying the fully human PD-L1 monoclonal antibody obtained in step (c).
  • the method further includes the step of affinity engineering the fully human PD-L1 monoclonal antibody by mutating the CDR region.
  • the fully human PD-L1 monoclonal antibody can also be optimized by germline back mutation and/or post-translational modification (PTM) removal.
  • PTM post-translational modification
  • H2L2 transgenic mice (hereinafter referred to as H2L2 transgenic mice) are used to prepare the first domain of the specific binding protein of the thirteenth aspect.
  • the H2L2 transgenic mouse is a transgenic mouse carrying a human immunoglobulin immune library, and the antibody produced by it has a complete human antibody variable domain and a rat constant domain.
  • the method for preparing the first domain of the specific binding protein of the thirteenth aspect using H2L2 transgenic mice comprises the following steps:
  • the human CD73 antigen is soluble recombinant human CD73-hFc, specifically, the antigen is a recombinant fusion protein composed of CD73 linked to Fc;
  • Hybridoma cells were obtained by fusion of splenocytes from immunized H2L2 transgenic mice with myeloma cell lines, and the isolated hybridomas expressed heavy and light chain antibodies with complete human variable domains and rat constant domains molecular;
  • the antibody light chain variable domain sequence (VL) obtained in step (b) is genetically synthesized and cloned into a mammalian cell expression vector encoding the human antibody kappa light chain constant domain sequence to encode the antibody-producing full-length light chain;
  • the antibody heavy chain variable domain sequence (VH) obtained in step (b) is synthesized through genes and cloned into a mammalian cell expression vector encoding a human IgG antibody heavy chain constant domain sequence to encode an IgG antibody full-length heavy chain;
  • step (e) Simultaneously transfect the expression vectors of steps (c) and (d) into mammalian host cells, and use conventional recombinant protein expression and purification techniques to obtain recombinant antibodies with correct pairing and assembly of light and heavy chains.
  • the antibody heavy chain variable domain sequence (VH) obtained in step (b) is gene-synthesized and cloned into a mammalian cell expression vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the IgG1 antibody produced full-length heavy chain.
  • the method further includes the step of affinity engineering the fully human CD73 monoclonal antibody by mutating the CDR region.
  • the fully human CD73 mAb can also be optimized by germline back mutation and/or post-translational modification (PTM) removal.
  • PTM post-translational modification
  • the prepared second domain and the prepared first domain are combined into a bispecific binding protein.
  • the bispecific binding protein can simultaneously bind two targets, wherein the first domain can recognize CD73 specifically expressed on the surface of tumor cells, and the second domain can bind PD-L1 molecules on T cells. After the binding protein binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.
  • the prepared second domain and the prepared first domain are constructed into a bispecific binding protein.
  • the bispecific binding protein can simultaneously bind two targets, wherein the first domain can recognize CD73 specifically expressed on the surface of tumor cells, and the second domain can bind PD-L1 molecules expressed by cells.
  • the bispecific binding protein can effectively bind soluble CD73 and PD-L1 on the cell surface, inhibit the enzymatic activity of CD73, block the combination of PD-L1 and PD-1, and activate T cells.
  • the bispecific binding protein has a tetravalent symmetrical structure.
  • the bispecific binding protein has the structure and sequence described in the thirteenth aspect.
  • the present invention provides a pharmaceutical composition, which comprises the specific binding protein of the thirteenth aspect or the antibody-drug conjugate of the seventeenth aspect, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes other ingredients as active ingredients, such as other small molecule drugs or antibodies or polypeptides as active ingredients.
  • the pharmaceutical composition further comprises a therapeutic agent selected from a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressant, and a cytotoxic drug.
  • a therapeutic agent selected from a chemotherapeutic agent, a radiotherapeutic agent, an immunosuppressant, and a cytotoxic drug.
  • the chemotherapeutic agent, radiotherapeutic agent, immunosuppressant and cytotoxic drug therapeutic agent of the pharmaceutical composition are different from or the same as the drug in the antibody drug conjugate of the seventeenth aspect.
  • the pharmaceutically acceptable carrier can be a conventional carrier in the art, and the carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical adjuvant.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the field, preferably including pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the specific binding protein and/or other small molecule drugs or antibodies or polypeptides, and 0.01-99.99% of the pharmaceutical carrier, the percentage being The mass percent of the pharmaceutical composition.
  • the administration route of the pharmaceutical composition can be parenteral, injection or oral administration.
  • the pharmaceutical composition can be prepared in a form suitable for administration, such as solid, semi-solid or liquid form, which can be aqueous solution, non-aqueous solution or suspension, powder, tablet, capsule, granule, injection or infusion form.
  • Administration can be intravascular, subcutaneous, intraperitoneal, intramuscular, inhalation, intranasal, airway instillation, or intrathoracic instillation.
  • the pharmaceutical composition can also be administered in the form of aerosol or spray, such as nasal administration; or, intrathecal, intramedullary or intraventricular administration, and can also be transdermal, transdermal, topical, enteral, intravaginal , sublingual or rectal administration.
  • the pharmaceutical composition can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dose beneficial to the patient.
  • the first domain and the second domain in the specific binding protein may be administered simultaneously or sequentially.
  • the specific binding protein and other active ingredients in the pharmaceutical composition can be administered simultaneously or sequentially.
  • the specific binding protein is a bispecific binding protein.
  • the present invention provides the specific binding protein of the thirteenth aspect, the isolated nucleic acid of the fourteenth aspect, the antibody drug conjugate of the seventeenth aspect or the drug combination of the nineteenth aspect
  • the tumor can be breast cancer, renal cell carcinoma, melanoma, colon cancer, and B cell lymphoma, melanoma, head and neck cancer, bladder cancer, gastric cancer, ovarian cancer, malignant sarcoma, urothelial cancer, liver cancer, esophageal cancer , gastroesophageal junction cancer, nasopharyngeal cancer, small cell lung cancer, cervical cancer, endometrial cancer, pancreatic cancer, prostate cancer, glioma, non-small cell lung cancer, acute myeloid leukemia, Hodgkin's lymphoma, cutaneous squamous cell carcinoma Cell carcinoma, locally advanced or metastatic malignancy, etc.
  • the inflammatory disease may be atopic dermatitis, ulcerative colitis and the like.
  • the immune disease may be graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, asthma and the like.
  • the present invention provides a method for detecting PD-L1 and CD73 in a sample, the method comprising the step of detecting PD-L1 and CD73 in the sample with the specific binding protein of the thirteenth aspect.
  • the sample can be a biological sample, for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.
  • a biological sample for example, whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, Biological samples such as oral secretions and nasal secretions.
  • the present invention provides a kit, which includes one or more kits, comprising the antigen-binding protein of the first aspect, the antibody-drug conjugate of the fifth aspect, or the antibody-drug conjugate of the fifth aspect.
  • a kit which includes one or more kits, comprising the antigen-binding protein of the first aspect, the antibody-drug conjugate of the fifth aspect, or the antibody-drug conjugate of the fifth aspect.
  • the kit includes a first kit and a second kit
  • the first kit includes the antigen-binding protein of the first aspect, the antibody-drug conjugate of the fifth aspect, or the antibody-drug conjugate of the fifth aspect.
  • the pharmaceutical composition according to the tenth aspect, and the second kit contains other therapeutic agents, including but not limited to chemotherapeutic agents, radiotherapeutic agents, immunosuppressants and cytotoxic drugs.
  • the chemotherapeutic agent, radiotherapeutic agent, immunosuppressant and cytotoxic drug in the kit are the same as or different from the antibody drug conjugate of the fifth aspect.
  • the above-mentioned first medicine box and the second medicine box can be used at the same time, or the above-mentioned first medicine box can be used first and then the above-mentioned second medicine box, or the above-mentioned second medicine box can be used first and then the above-mentioned first medicine box can be used.
  • a medicine box can be determined according to the actual needs of specific applications.
  • the present invention provides a kit, the kit includes one or more kits, comprising the specific binding protein of the thirteenth aspect, the antibody-drug conjugate of the seventeenth aspect or the pharmaceutical composition described in the nineteenth aspect.
  • the set of kits includes a first kit, and the first kit includes the bispecific binding protein composed of the first domain and the second domain described in the thirteenth aspect, the tenth The antibody drug conjugate of the seventh aspect or the pharmaceutical composition of the nineteenth aspect.
  • the kit of parts may further include a second kit comprising other therapeutic agents including, but not limited to, chemotherapeutics, radiotherapeutics, immunosuppressive agents and cytotoxic drugs.
  • chemotherapeutic agent, radiotherapeutic agent, immunosuppressant and cytotoxic drug in the kit are the same as or different from the antibody drug conjugate of the seventeenth aspect.
  • the above-mentioned first medicine box and the second medicine box can be used at the same time, or the above-mentioned first medicine box can be used first and then the above-mentioned second medicine box, or the above-mentioned second medicine box can be used first and then the above-mentioned first medicine box can be used.
  • a medicine box can be determined according to the actual needs of specific applications.
  • the present invention provides a drug delivery device comprising the antigen-binding protein of the first aspect, the antibody-drug conjugate of the fifth aspect, or the pharmaceutical composition of the tenth aspect.
  • the drug delivery device is a prefilled syringe.
  • the present invention provides a drug delivery device comprising the antigen-binding protein of the thirteenth aspect, the antibody-drug conjugate of the seventeenth aspect, or the pharmaceutical composition of the nineteenth aspect.
  • the drug delivery device is a prefilled syringe.
  • the present invention provides a method for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, comprising administering to a subject a therapeutically effective amount of the antigen-binding compound of the first aspect
  • the protein, the antibody drug conjugate of the fifth aspect, or the pharmaceutical composition of the tenth aspect comprising administering to a subject a therapeutically effective amount of the antigen-binding compound of the first aspect
  • the protein, the antibody drug conjugate of the fifth aspect, or the pharmaceutical composition of the tenth aspect comprising administering to a subject a therapeutically effective amount of the antigen-binding compound of the first aspect
  • the protein, the antibody drug conjugate of the fifth aspect, or the pharmaceutical composition of the tenth aspect comprising administering to a subject a therapeutically effective amount of the antigen-binding compound of the first aspect
  • the protein, the antibody drug conjugate of the fifth aspect, or the pharmaceutical composition of the tenth aspect comprising administering to a subject a therapeutically effective amount of the antigen-
  • the present invention provides a method for preventing, treating and/or diagnosing immune diseases, acute and chronic inflammatory diseases, and tumor diseases, which comprises administering a therapeutically effective amount of the antigen of the thirteenth aspect to a subject
  • the binding protein, the antibody drug conjugate of the seventeenth aspect, or the pharmaceutical composition of the nineteenth aspect comprises administering a therapeutically effective amount of the antigen of the thirteenth aspect.
  • the bispecific binding protein of the present invention has one or more of the following properties:
  • the bispecific binding protein can specifically bind human PD-L1 and CD73 with high affinity
  • the bispecific binding protein has an inhibitory effect on the PD-1 signaling pathway
  • the bispecific binding protein can enhance the secretion of cytokines IL-2 and IFN- ⁇ from activated T lymphocytes;
  • ABSOR and “approximately” generally mean an acceptable range of error for the measured value given the nature or precision of the measurement. Typically the margin of error is within 20%, typically within 10%, and even more typically within 5% of a given value or range of values.
  • antigen binding molecule or “specific binding protein” refers broadly to a molecule that specifically binds an antigenic determinant.
  • Antigen binding molecules or specific binding proteins include, for example, antibodies, antibody fragments, and backbone antigen binding proteins.
  • antibody of the present invention encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single chain molecules and antibody fragments as long as they exhibit the desired antigen-binding activity.
  • the term "monoclonal antibody” in the present invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible traces of variant antibodies (e.g., containing naturally occurring mutations or arising during the production of monoclonal antibody preparations). , usually present in small amounts), the individual antibodies comprised by the population of antibodies are identical and/or bind the same epitope. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • multispecific antibody of the present invention is used in its broadest sense to encompass antibodies with polyepitopic specificities.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has polyepitopic specificity; Antibodies with VL and VH domains, each VH-VL unit binds to a different target or a different epitope of the same target; antibodies with two or more single variable domains, each single variable domain binds to Binding to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and triabodies (triabodies), antibody fragments linked together covalently or non-covalently Wait.
  • VH heavy chain variable region
  • VL light chain variable region
  • bispecific binding protein or “bispecific antibody” of the present invention refers to the ability to specifically bind at least two different antigenic determinants, for example each composed of an antibody heavy chain variable domain (VH) and antibody light
  • VH antibody heavy chain variable domain
  • VL chain variable domain
  • Bispecific antibodies can be in a 1+1 format, a 2+1 format (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or a 2+2 format (comprising a second antigen or epitope).
  • bispecific antibodies comprise two antigen-binding sites, each specific for a different antigenic determinant.
  • valence in the present invention means that an antigen-binding molecule has a specified number of binding domains present.
  • bispecific antibody is at least “bivalent”, and may be “trivalent”, “tetravalent” or “more valent”).
  • the antibodies have two or more binding sites and are bispecific. That is, antibodies can be bispecific even where there are more than two binding sites (ie, the antibody is trivalent or multivalent).
  • full-length antibody and “intact antibody” of the present invention are used interchangeably herein to refer to an antibody that is substantially similar in structure to a natural antibody.
  • “Native antibody” refers to a naturally occurring immunoglobulin molecule.
  • antibodies of the native IgG class are heterotetrameric glycoproteins of approximately 150,000 Daltons, consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also called variable heavy domain or heavy chain variable domain) and three constant domains (CH1, CH2 and CH3) (also known as the heavy chain constant region).
  • VH variable region
  • CH1 and CH3 constant domains
  • each light chain has a variable region (VL) (also called variable light domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain domain). chain constant region).
  • the heavy chain of an antibody can be of one of five types, alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG), or mu (IgM), which can be further divided into Subtypes such as ⁇ 1 (IgG1), ⁇ 2 (IgG2), ⁇ 3 (IgG3), ⁇ 4 (IgG4), ⁇ 1 (IgA1 ) and ⁇ 2 (IgA2).
  • the light chains of an antibody based on the amino acid sequence of their constant domains, can be of one of two types, kappa and lambda light chains.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • CL light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • Antibody fragments comprise a portion of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2, and Fv; diabodies, triabodies, tetrabodies, crossed Fab fragments; linear antibodies; single chain antibody molecules (e.g. scFv); Multispecific antibodies formed from antibody fragments and single domain antibodies (sdAbs).
  • an antigen binding domain refers to a portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term “antigen-binding domain” refers to a part of an antibody comprising a region that specifically binds and is complementary to a part or all of an antigen. In cases where the antigen molecule is large, the antigen-binding molecule can bind only a specific part of the antigen, called an epitope.
  • An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions).
  • the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • an antigen binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.
  • antigenic determinant in the present invention is synonymous with “antigen” and “epitope”, and refers to a site on a polypeptide macromolecule (such as a stretch of continuous amino acids or a conformational configuration consisting of different regions of non-contiguous amino acids ), the antigen-binding moiety binds to said site, thereby forming an antigen-binding moiety-antigen complex.
  • the antigenic determinant may be present, for example, on the surface of tumor cells, microbially infected cells, other diseased cells, immune cells, free matter in serum and/or in the extracellular matrix (ECM).
  • the proteins used as antigens in the present invention may be proteins in any native form from any vertebrate source, including, for example, primates (e.g., humans) and rodents (e.g., mice and rats) and other mammals.
  • the antigen can also be a human protein, or the antigen can be "full length", unprocessed protein, any form of protein resulting from intracellular processing, or a naturally occurring variant of a protein, such as a splice variant or an allele Variants.
  • Specifically binds means that binding is selective for an antigen and can be distinguished from unwanted or non-specific binding.
  • the ability of an antigen-binding molecule to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) techniques as well as traditional binding assays.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen, as measured by SPR.
  • the solution of the molecule bound to the antigen is ⁇ 1 ⁇ M, ⁇ 100nM, ⁇ 10nM, ⁇ 1nM, ⁇ 0.1nM, ⁇ 0.01nM or ⁇ 0.001nM (for example, 10 -7 M or lower, such as 10 -7 M to 10 -13 M, eg 10- 9 M to 10 -13 M).
  • Binding affinity refers to the strength of the non-covalent interaction between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Binding affinity can generally be expressed in terms of a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (K off and K on , respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • high affinity in the present invention means that the Kd of the antibody to the target antigen is 10-9 M or lower, even 10-10 M or lower.
  • low affinity in the present invention means that the Kd of the antibody is 10 -8 M or higher.
  • An “affinity matured” antibody is one that has one or more modifications in one or more hypervariable regions (HVRs) that result in greater improved affinity.
  • HVRs hypervariable regions
  • single domain antibody and “nanobody” in the present invention have the same meaning, referring to only having the variable region of the heavy chain of an antibody, and constructing a single domain antibody consisting of only one heavy chain variable region, which is fully functional the smallest antigen-binding fragment.
  • HCAb antibody refers to an antibody that lacks the light chain of the antibody and only contains the heavy chain, compared to the double-chain antibody (immunoglobulin), specifically contains the variable structure of the heavy chain domain and Fc constant domain.
  • the specific binding protein comprises a first domain that binds CD73 or a fragment thereof and a second domain that binds PD-L1 or a fragment thereof.
  • a bispecific antibody comprising a first domain that specifically binds CD73 and a second domain that specifically binds PD-L1 "a bispecific antibody that specifically binds CD73 and PD-L1” "specific to CD73 and Bispecific antigen-binding molecule to PD-L1" or "anti-CD73/anti-PD-L1 antibody” are used interchangeably herein and refer to an antibody capable of binding CD73 and PD-L1 with sufficient affinity such that the antibody can be used Bispecific antibodies as diagnostic and/or therapeutic agents targeting CD73 and PD-L1.
  • T effector cells in the present invention refers to T cells with cytolytic activity (for example, CD4+ and CD8+ T cells) and T helper (Th) cells, T effector cells secrete cytokines, and activate and guide other immune cells, Regulatory T cells (Treg cells) are not included.
  • cytolytic activity for example, CD4+ and CD8+ T cells
  • Th cells T helper cells
  • Treg cell refers to a special type of CD4+ T cell that can block the response of other T cells.
  • Treg cells are characterized by the expression of CD4, the alpha subunit of the IL-2 receptor (CD25) and the transcription factor FOXP3, and play a critical role in the induction and maintenance of peripheral self-tolerance against tumor-expressed antigens.
  • CD73 cluster of differentiation 73, also known as 5'-exonucleotidase, generally refers to the ability to convert extracellular nucleoside 5' monophosphates into nucleosides, adenosine monophosphate Enzyme (nucleotidase) that converts (AMP) to adenosine.
  • CD73 is a cell surface enzyme linked by glycosylphosphatidylinositol (GPI). CD73 is widely expressed on the surface of human endothelial cells and lymphocytes, such as Treg, DC, MDSC, NK and other cells.
  • CD73 is also expressed on cancer cells including colon, lung, pancreas, ovary, bladder, leukemia, glioma, glioblastoma, melanoma, thyroid, esophagus, prostate, and breast cancer Wait. High CD73 expression has been reported to be associated with poor prognosis in various cancer indications (eg, lung, melanoma, breast, squamous head and neck cancer, and colorectal cancer).
  • the main function of CD73 is to convert extracellular nucleotides such as 5'-AMP into the highly immunosuppressive molecule adenosine.
  • the term "CD73" of the present invention 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 cells or tissues that naturally express them, or can be recombinantly produced using techniques well known in the art and/or described herein. Accordingly, the antigen binding proteins described herein may cross-react with species other than human (eg, cynomolgus CD73). Alternatively, an antigen binding protein, antibody or domain directed against CD73 of the present invention may be specific for human CD73 and may not exhibit any cross-reactivity with other species.
  • CD73 antibody or "isolated antigen-binding protein that specifically binds CD73 and/or fragments thereof" of the present invention is capable of binding CD73 with sufficient affinity such that the antibody can be used as a diagnostic and/or CD73-targeted antibody or therapeutic agents.
  • a CD73 antibody binds to an irrelevant CD73 protein less than the antibody binds to CD73 as determined by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance using a biosensor system about 10% of capacity.
  • RIA radioimmunoassay
  • FACS flow cytometry
  • the antibody that binds CD73 has a concentration of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10 ⁇ 8 M or less, such as 10 ⁇ 13 M to 10 - 8 M, eg 10 -13 M to 10 -9 M) dissociation constant (KD).
  • concentration of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM e.g., 10 ⁇ 8 M or less, such as 10 ⁇ 13 M to 10 - 8 M, eg 10 -13 M to 10 -9 M
  • KD dissociation constant
  • PD-L1 Programmed Cell Death 1 Ligand 1
  • B7-H1 Cluster of Differentiation CD274 or B7 Homolog 1
  • PD-L1 is a member of the B7 family that regulates the activation or inhibition of the PD-1 receptor.
  • a member of The open reading frame of PD-L1 encodes a 290-amino acid type I transmembrane protein, which includes an extracellular Ig domain (N-terminal V-like domain and IgC-like domain), a hydrophobic transmembrane domain, and a 30-amino acid cytoplasmic tail.
  • the 30 amino acid intracellular (cytoplasmic) domain does not contain significant signaling, but does have a potential site for protein kinase C phosphorylation.
  • PD-L1 is enhanced under the effect of interferon gamma stimulation.
  • PD-L1 is also expressed on nonimmune cells, including pancreatic islets, Kupffer cells of the liver, vascular endothelium, and selected epithelium, such as tracheal and renal tubular epithelium, where its expression is enhanced during inflammatory episodes.
  • Increased levels of PD-L1 expression have also been found on a variety of tumors including, but not limited to, breast cancer, ovarian cancer, cervical cancer, colon cancer, colorectal cancer, lung cancer including non-small cell lung cancer, including renal cell Cancers of the kidney, stomach, esophagus, bladder, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN) and pancreas, melanoma and uveal melanoma.
  • breast cancer breast cancer
  • ovarian cancer cervical cancer
  • colon cancer colorectal cancer
  • lung cancer including non-small cell lung cancer, including renal cell Cancers of the kidney, stomach, esophagus, bladder, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN) and pancreas, melanoma and uveal melanoma.
  • SCCHN squamous cell carcinoma of the head and neck
  • PD-L1 antibody or "isolated antigen-binding protein that specifically binds to PD-L1 and/or fragments thereof" of the present invention is capable of binding to PD-L1, especially a PD-L1 polypeptide expressed on the surface of a cell, and Having sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting PD-L1.
  • PD-L1 antibodies bind to irrelevant, non-PD-L1 proteins less than the stated About 10% of the binding capacity of the antibody to PD-L1.
  • the KD value of the antigen binding protein that binds to human PD-L1 is ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM or ⁇ 0.001 nM (for example, 10 -8 M or more low, eg 10 -13 M to 10 -8 M, eg 10 -13 M to 10 -9 M).
  • human PD-L1 especially human PD-L1 with a histidine tag, is used to determine the corresponding KD value of the binding affinity to obtain the PD-L1 binding affinity.
  • Therapeutic strategies with PD-1/PD-L1 antibodies are a standard treatment strategy in several metastatic tumors and have shown their role in early disease stages and adjuvant therapy, especially in melanoma and non-small cell lung cancer.
  • H2L2 transgenic mouse or "Harbour H2L2 mouse” in this application is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces a protein consisting of two heavy chains with a fully human variable region.
  • the antibodies produced by the transgenic mice are affinity matured, fully humanized variable regions, and have excellent solubility.
  • Hardbour HCAb mouse (WO2002/085945A2) in this application is a transgenic mouse carrying an immune repertoire of human immunoglobulins capable of producing novel "heavy chain only” antibodies the size of traditional IgG antibodies half of. It produces antibodies with only human antibody “heavy chain” variable domains and mouse Fc constant domains. Due to the feature of not containing light chains, 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.
  • variable region refers to the domain of antibody heavy chain or light chain that participates in the binding of antigen-binding molecule to antigen.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • variable in the present invention means that certain segments of the variable domains generally differ in sequence between antibodies.
  • the V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains.
  • HVRs hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, mostly in a ⁇ -sheet configuration, connected by three HVRs that form loops connecting and in some cases forming part of the ⁇ -sheet structure.
  • the HVRs in each chain are held tightly together by the FR regions and, together with the HVRs of the other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda , MD (1991)).
  • the constant domain is not directly involved in the binding of the antibody to the antigen, but has other effector functions, such as participating in the antibody-dependent cellular cytotoxicity of the antibody.
  • hypervariable region refers to the region in the variable domain region of an antibody that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops").
  • native four-chain antibodies typically contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3).
  • HVRs typically contain amino acid residues from hypervariable loops and/or from "complementarity determining regions (CDRs)" that have the highest sequence variability and/or are involved in antigen recognition.
  • CDRs complementarity determining regions
  • CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia et al., J.Mol.Biol.196:901-917 (1987).
  • Exemplary CDR (CDR -L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 (L1), amino acid residues 50-56 (L2), amino acid residues 89- 97 (L3), amino acid residues 31-35 (H1), amino acid residues 50-65 (H2), and amino acid residues 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, No.
  • the Combined definition rule is to combine the range defined by Kabat and Chothia, and a larger range is taken based on this.
  • CDR complementarity determining region
  • the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g. variable region) are to be understood as encompassing the above-mentioned known Any one of the defined complementarity determining regions in the scheme.Although the scope of protection claimed in the present invention is based on the sequence shown in the Chothia definition rules, the corresponding amino acid sequences according to the definition rules of other CDRs should also fall within the scope of the present invention in the scope of protection.
  • Table 1 The positions of the CDR definitions of the present invention in the antibody light chain or heavy chain under different numbers
  • FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR4.
  • HVR and FR sequences typically occur in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • the "class" of an antibody refers to the type of constant domain or constant region that the heavy chain of the antibody has.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • a “humanized antibody” comprises amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody comprises at least one, usually two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs Corresponds to FRs of human antibodies.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, eg, a non-human antibody refers to an antibody that has undergone humanization.
  • a "human antibody” has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • Fc domain or "Fc region” of the present invention is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the IgG Fc region consists of an IgG CH2 domain and an IgG CH3 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain.
  • the CH2 domain may comprise one or more mutations that reduce or eliminate binding of the CH2 domain to one or more Fcy receptors (eg, FcyRI, FcyRIIa, FcyRIIb, FcyRIII) and/or complement. It is hypothesized that reducing or eliminating binding to Fc receptor gamma will reduce or eliminate ADCC mediated by antibody molecules. Similarly, reducing or eliminating binding to complement is expected to reduce or eliminate CDC mediated by antibody molecules. Mutations that reduce or eliminate binding of the CH2 domain to one or more Fc ⁇ receptors and/or complement are known in the art (Wang et al., 2018).
  • LALA mutation involves the replacement of the leucine residues at positions 1.3 and 1.2 of the IMGT of the CH2 domain with alanine (L1.3A and L1.2A).
  • CH2 Mutation of the asparagine (N) at position 84.4 of the IMGT position in the domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q) to glycosylate the conserved N-chain Site mutations to generate a-glycosyl antibodies to reduce IgG1 effector function are also known (Wang et al., 2018).
  • C1q binding complement activation
  • ADCC can be achieved through IMGT of the CH2 domain.
  • Proline at position 114 is reduced by mutation to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can be combined to produce cells with further reduced or no ADCC or CDC activity. antibody molecules.
  • a region equivalent to the Fc region of an immunoglobulin includes naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as those having substitutions, additions or deletions that do not substantially reduce immunoglobulin-mediated effector functions Modified variants with capabilities such as antibody-dependent cellular cytotoxicity. For example, one or more amino acids can be deleted from the N- or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, eg, Bowie, J.U. et al., Science 247:1306-10 (1990)).
  • effector function of the present invention is attributable to the Fc region of an antibody and is a biological activity that varies with the antibody isotype.
  • antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine Secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (such as B cell receptors), and B cell activation, among others.
  • peptide linker or "connecting peptide” of the present invention refers to a peptide comprising one or more amino acids, usually about 2 to 20 amino acids.
  • Peptide linkers are known in the art or described herein.
  • fused to refers to segments (eg antigen binding domain and FC domain) connected by peptide bonds either directly or via one or more peptide linkers.
  • the invention also relates to amino acid sequence variants of the bispecific antibodies of the invention.
  • Amino acid sequence variants of bispecific antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions and/or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions and substitutions can be made to arrive at a final construct having the desired properties, such as antigen binding activity.
  • Sites for substitution typically include HVRs and frameworks (FRs). See Table 2 below for possible amino acid substitutions.
  • polynucleotide or “nucleic acid” or “nucleotide sequence” refers to an isolated nucleic acid molecule or construct such as messenger RNA (mRNA), virus-derived RNA or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA virus-derived RNA
  • pDNA plasmid DNA
  • a polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (eg, amide bonds, such as found in peptide nucleic acids (PNAs)).
  • PNAs peptide nucleic acids
  • nucleic acid molecule refers to any one or more nucleic acid segments, such as DNA or RNA segments, present in a polynucleotide.
  • isolated nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA or RNA, which has been separated from its natural environment.
  • the recombinant polynucleotide encoding the polypeptide contained in the vector is also isolated.
  • isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution.
  • An isolated polynucleotide includes a polynucleotide molecule normally contained in a cell containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the invention, in plus- and minus-strand forms, and in double-stranded form.
  • Isolated polynucleotides or nucleic acids of the invention further include such molecules produced synthetically.
  • a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, ribosomal binding site, or transcription terminator.
  • expression cassette refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow the transcription of a particular nucleic acid in a target cell.
  • Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses or nucleic acid fragments.
  • the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.
  • an expression cassette of the invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.
  • the term "vector” or "expression vector” and “expression construct” in the present invention can be used interchangeably, and a specific gene operably linked to it is introduced into a target cell and directs the expression of a DNA molecule.
  • the vector includes a vector that is a self-replicating nucleic acid structure as well as a vector that is incorporated into the genome of a host cell into which it has been introduced.
  • the expression vectors of the present invention comprise expression cassettes. Expression vectors allow the transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery.
  • the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants/transformants” and “transformed cells”, including the primary transformed cell and progeny derived therefrom.
  • the nucleic acid of the progeny may not be identical to that of the parental cell and may contain mutations.
  • a host cell is any type of cell that can be used to produce a bispecific antigen binding molecule of the invention.
  • Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 Cells or hybridoma cells, yeast cells, insect cells and plant cells, also cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.
  • cultured mammalian cells such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 Cells or hybridoma cells, yeast cells, insect cells and plant cells, also cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.
  • Germlineization backmutation refers to the process of backmutating supersomatic mutations of antibodies into corresponding germline sequences.
  • the heavy chain variable domain sequence of the antibody is derived from the gene rearrangement and somatic high-frequency mutation of the germline gene V, D, and J gene fragments 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 gene V and J gene fragments of the light chain gene group. Gene rearrangement and somatic hypermutation are the main factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • post-translational modification refers to the process of introducing chemical modifications after the protein or polypeptide amino acid chain is translated and synthesized in cells.
  • PTM positions are very conservative.
  • EU numbering conserved amino acid asparagine Asn at position 297 (EU numbering) of the constant domain of human IgG1 antibodies usually undergoes glycosylation modification to form sugar chains, and this sugar chain structure is crucial for antibody structure and related effector functions.
  • PTMs in the variable domain of the antibody especially in the antigen-binding region (such as CDR)
  • the existence of these PTMs may have a greater impact on the binding of the antigen, and may also have a significant impact on the physicochemical properties of the antibody.
  • the sequence pattern of N-x-S/T (the first position is asparagine, the second position is any amino acid except proline, and the third position is serine or threonine) predicts N-linked glycosylation sites.
  • the amino acid sequence pattern that causes PTM may be derived from the germline gene sequence, for example, the human germline gene fragment IGHV3-33 naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic hypermutation. Amino acid mutations can be used to disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of specific PTMs. Depending on the antibody sequence and PTM sequence pattern, there are different mutation design methods.
  • One method is to replace the "hotspot" amino acid (such as N or S in the NS pattern) with an amino acid with similar physicochemical properties (such as mutating N to Q). If the PTM sequence pattern is derived from somatic hypermutation and does not exist in the germline gene sequence, another method may be to replace the sequence pattern with the corresponding germline gene sequence. In practice, multiple mutation design methods may be used for the same PTM sequence pattern.
  • an ADC refers to a binding protein (such as an antibody or antigen-binding fragment thereof) chemically linked to one or more chemical drugs.
  • an ADC includes a binding protein, a drug, and a linker linking the binding protein to the drug.
  • chimeric antigen receptor refers to a receptor that has the desired antigen specificity and signaling domains to propagate an intracellular signal upon antigen binding.
  • T lymphocytes recognize specific antigens via the interaction of the T cell receptor (TCR) with short peptides presented by class I or class II major histocompatibility complex (MHC) molecules.
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • monocytes and macrophages can be engineered, for example, to express a chimeric antigen receptor (CAR).
  • the modified cells can be recruited to the tumor microenvironment where they serve as potent immune effectors by infiltrating the tumor and killing targeted cancer cells.
  • a CAR can include an antigen-binding domain, a transmembrane domain, and an intracellular domain.
  • the antigen binding domain binds to an antigen on a target cell.
  • Examples of cell surface markers that can be used as antigens that bind to the antigen binding domain of the CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (eg, tumor antigens).
  • modified immune cell refers to an immune cell that has been genetically modified to express a CAR.
  • the immune cells are T cells, or cells derived therefrom.
  • the immune cells are natural killer (NK) cells, or cells derived therefrom.
  • the immune cells are B cells, or cells derived therefrom.
  • the immune cells are B cells, or cells derived therefrom.
  • the immune cells are monocytes or macrophages, or cells derived therefrom.
  • kit of parts refers to a combination of one or more active ingredients present in more than one unit. When there are multiple active ingredients in the kit, each active ingredient can be administered simultaneously or sequentially.
  • administration device refers to any means for administering a drug to a subject.
  • prefilled syringe refers to a syringe that has been loaded with drug before it is accessed or used by the operator of the syringe.
  • Prefilled syringes can be of any material (eg, glass, plastic, or metal).
  • the prefilled syringe is a glass syringe.
  • an “effective amount” of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which it is administered.
  • An “effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dosage of administration and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • the bispecific antibodies according to the invention have a synergistic effect.
  • “Synergistic effect” means that the combined effect of two drugs is greater than the sum of their individual effects and statistically different from the control and single drugs.
  • the additive effect of the present invention means that the combined effect of the two drugs is the sum of their individual effects and is statistically different from the control and/or single drug.
  • a “therapeutically effective amount” of a drug refers to that amount, in dosage and administration interval and time, effective to achieve the desired therapeutic or prophylactic effect.
  • a therapeutically effective amount of a drug eliminates, alleviates/reduces, delays, minimizes or prevents the adverse effects of a disease.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • an individual or subject is a human.
  • pharmaceutical composition means a mixture comprising one or more antibodies or antigen-binding fragments thereof of the present disclosure and other chemical components such as physiological/pharmaceutically acceptable carriers or excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • pharmaceutically acceptable carrier refers to an ingredient of a pharmaceutical composition other than the active ingredient that is nontoxic to the subject.
  • Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers and/or preservatives.
  • cancer is meant to describe disorders in mammals characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, tumors, lymphomas, blastomas, sarcomas, and leukemia or lymphoid malignancies. More specific examples of cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal carcinoma, hepatocellular carcinoma, Gastric cancer (including gastrointestinal and gastrointestinal stromal cancer), bone cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer cancer, endometrial or cervical cancer, salivary gland cancer, kidney or ureter cancer, prostate cancer, vaginal cancer, vulvar cancer, thyroid cancer
  • treatment refers to the administration of a therapeutic agent, internally or externally, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure or a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, to a patient who has a or multiple diseases or symptoms, and the therapeutic agent has a therapeutic effect on these diseases or symptoms.
  • a therapeutic agent is administered in a patient or population to be treated in an amount effective to ameliorate one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent.
  • preventing cancer means delaying, inhibiting or preventing the onset of cancer in a mammal in which the onset of carcinogenesis or tumorigenesis has not been proven, but has been identified, for example, by genetic screening or other methods have a susceptibility to cancer.
  • the term also includes treating a mammal with a premalignant condition to halt the progression of the premalignant condition to a malignancy or to cause its regression.
  • sequence number (SEQ ID NO:) of the antibody of the present application is shown in Table 3 below.
  • sequence number (SEQ ID NO:) of the double antibody of the present application is shown in Table 4 below.
  • Figure 1A and Figure 1B show the results of the PD-L1 antigen binding protein described in the present application binding to CHO-K1 cells overexpressing human PD-L1.
  • Figure 1C shows the results of binding of PR000960 post-translational modification (PTM) variants described in the present application to CHO-K1 cells overexpressing human PD-L1.
  • Figure 1D, Figure 1E, and Figure 1F show the results of binding of PR002082 affinity maturation variants described in this application to CHO-K1 cells overexpressing human PD-L1.
  • PTM post-translational modification
  • Fig. 2A shows the results of the PD-L1 antigen binding protein described in the present application binding to CHO-K1 cells overexpressing cynomolgus monkey PD-L1.
  • Figure 2B shows the results of binding of PR000960 post-translational modification (PTM) variants described in the present application to CHO-K1 cells overexpressing cynomolgus monkey PD-L1.
  • Figure 2C shows the binding of PR002082 affinity matured variants described herein to CHO-K1 cells overexpressing monkey PD-L1.
  • PTM post-translational modification
  • Figure 3A shows the inhibitory effect of the PD-L1 antigen-binding protein described in the present application on the PD-1 signaling pathway.
  • Figure 3B shows the inhibitory effect of the PR000960 post-translational modification (PTM) variants described in the present application on the PD-1 signaling pathway.
  • Figure 3C and Figure 3D show the inhibitory effect of the PR002082 affinity maturation variants described in the present application on the PD-1 signaling pathway.
  • PTM post-translational modification
  • Figure 4 shows the results of the PD-L1 antigen binding protein described in this application enhancing the secretion of cytokines IL-2 (Figure 4A) and IFN- ⁇ ( Figure 4B) by activated T lymphocytes in the MLR experiment of donor pair #1.
  • Figure 5 shows the results of the PD-L1 antigen binding protein described in this application enhancing the secretion of cytokines IL-2 (Figure 5A) and IFN- ⁇ (Figure 5B) by activated T lymphocytes in the MLR experiment of donor pair #2.
  • FIG. 6 shows that PR000960 post-translational modification (PTM) variants of the present application enhanced activation of T lymphocytes to secrete cytokines IL-2 ( Figure 6A) and IFN- ⁇ ( Figure 6B) in the MLR experiment of donor pair #3. result.
  • PTM post-translational modification
  • Figure 7 shows the results of the PR002082 affinity matured variants described herein to enhance activation of T lymphocytes to secrete the cytokines IL-2 ( Figure 7A) and IFN- ⁇ ( Figure 7B) in a MLR experiment with donor pair #4.
  • Figure 8 shows the results of the PR002082 affinity-matured variants described herein for enhanced activation of T lymphocytes secreting the cytokines IL-2 ( Figure 8A) and IFN- ⁇ ( Figure 8B) in an MLR experiment with donor pair #5.
  • Figure 9 shows the results of the PR002082 affinity matured variants described herein to enhance activation of T lymphocytes to secrete cytokines IL-2 ( Figure 9A) and IFN- ⁇ ( Figure 9B) in a MLR experiment with donor pair #6.
  • Figure 10 shows the results of the PR002082 affinity matured variants described herein to enhance the secretion of the cytokines IL-2 (Figure 10A) and IFN- ⁇ (Figure 10B) by activated T lymphocytes in a MLR experiment with donor pair #7.
  • Figure 11 shows the results of the PR002082 affinity matured variants described herein to enhance activation of T lymphocytes to secrete the cytokines IL-2 ( Figure 11A) and IFN- ⁇ ( Figure 11B) in a MLR experiment with donor pair #8.
  • Figure 12 shows the results of the PR002082 affinity matured variants described herein to enhance activation of T lymphocytes to secrete the cytokines IL-2 ( Figure 12A) and IFN- ⁇ ( Figure 12B) in a MLR experiment with donor pair #9.
  • Figure 13A shows the in vivo anti-tumor effect of the A375 tumor model in which the human PBMC immune system was reconstituted in NCG mice.
  • Figure 13B shows the results of body weight changes of animals in each group during the experiment.
  • Figure 14 shows a schematic diagram of the structure of the bispecific molecule of the present invention.
  • Figure 15 shows antibody binding to CHO-K1 cells stably expressing human CD73 and human PD-L1.
  • Figure 16 shows the experimental results of antibodies inhibiting CD73 enzymatic activity.
  • Figure 17 shows the inhibitory effect of antibodies on PD-1/PD-L1 signaling pathway detected by reporter gene cell lines.
  • Fig. 18 shows the activation effect of the fusion protein on T cells detected in vitro by MLR method.
  • experimental animals can usually be immunized with PD-L1 antigens.
  • the experimental animals can be mice, rats, rabbits, sheep, camels, etc., but the obtained antibody molecules are non- human source.
  • these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability.
  • the humanization process of antibodies has its own technical complexity, and molecules after humanization often reduce the affinity for antigens.
  • transgenic technology advances in transgenic technology have made it possible to create genetically engineered mice that carry the human immunoglobulin immune repertoire and have their endogenous murine immune repertoire deleted.
  • the antibody produced by this transgenic mouse has a fully human sequence, so there is no need for further humanization, which greatly improves the efficiency of therapeutic antibody development.
  • the Harbor HCAb mouse (Harbour Antibodies BV, WO 2002/085945 A3) is a transgenic mouse carrying an immune repertoire of human immunoglobulins that produces novel "heavy chain only” antibodies the size of traditional IgG antibodies half. It produces antibodies with only human antibody “heavy chain” variable domains and mouse Fc constant domains. Due to the fact that it does not contain light chains, 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.
  • each mouse was immunized with an immunogenic reagent prepared by mixing 25 ⁇ g of antigenic protein with Ribi adjuvant (Sigma Adjuvant System, #S6322).
  • the interval between each booster round is at least two weeks and usually no more than five booster rounds.
  • the immunization time was the 0th day, the 14th day, the 28th day, the 42nd day, the 56th day, and the 70th day; and on the 49th day and the 77th day, the serum antibody titer of the mice was detected.
  • Five days before the isolation of spleen B cells from HCAb mice, the last booster immunization was carried out with a dose of 25 ⁇ g of antigenic protein per mouse.
  • the splenocytes of the mouse are taken out to separate B cells, and the CD138-positive plasma cells and human PD-L1 antigen are sorted by BD FACS AriaII Cell Sorter Positive B cell population.
  • PCR primers 5'-GGTGTCCAGTGTSAGGTGCAGCTG-3'(SEQ ID NO:134), 5'-AATCCCTGGGCACTGAAGAGACGGTGACC-3'(SEQ ID NO:135).
  • the amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the heavy chain Fc domain sequence of human IgG1 antibody.
  • the constructed plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) to express the obtained HCAb antibody.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • use the positive antibody PR000151 (Atezolizumab analogue, the applicant synthesized and expressed it according to the Atezolizumab sequence ) as a positive control for Miroball fluorescence flow instrument (Sptlabtech) screening.
  • the specific steps are: wash CHO-K1/hPD-L1 cells with serum-free F12K medium (Thermofisher, #21127022), and resuspend them to 1 ⁇ 10 6 /mL with serum-free medium.
  • CTS Draq5 fluorescent probe
  • the positive cloned antibody was further detected by FACS for binding to CHO-K1/hPD-L1 cells, and ELISA detection with cynomolgus monkey PD-L1-his protein (Acrobiosystems, #PD-1-C52H4) to verify the binding and cross-binding activity.
  • the positive antibody is sequenced by conventional sequencing means to obtain the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence.
  • the heavy chain variable domain sequence and the corresponding human antibody heavy chain constant domain sequence can be fused and expressed by using conventional recombinant DNA technology , to obtain recombinant antibody molecules.
  • the recombinant antibody with HCAb heavy chain can be obtained by transfecting the plasmid encoding the antibody heavy chain into mammalian host cells (such as human embryonic kidney cells HEK293), and using conventional recombinant protein expression and purification techniques.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504). Before the start of transient transfection, the cell concentration was adjusted to (6-8) ⁇ 10 5 cells/mL, and cultured at 37°C, 8% CO 2 shaker for 24 hours, and the cell concentration was 1.2 ⁇ 10 6 cells/mL. Prepare 30 mL of cultured cells. The above heavy chain plasmid encoding HCAb was dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo, #31985088), and sterilized by filtration with a 0.22 ⁇ m filter membrane.
  • Opti-MEM reduced serum medium Thermo, #31985088
  • a gravity column (Bio-Rad, #7311550) containing MabSelect TM (GE Healthcare Life Science, #71-5020-91AE) was equilibrated with PBS (pH 7.4), washed with 2-5 column volumes. Pass the supernatant sample through the column. Rinse the column with 5-10 column volumes of PBS. The target protein was then eluted with 0.1M glycine at pH 3.5, adjusted to neutrality with Tris-HCl at pH 8.0, and finally concentrated and exchanged into PBS buffer with an ultrafiltration tube (Millipore, #UFC901024) to obtain purified antibodies solution. Then use NanoDrop (Thermo Scientific TM NanoDrop TM One) to measure the concentration, aliquot and store for future use.
  • PBS pH 7.4
  • Table 5 lists the amino acid sequence of the heavy chain variable domain of the PD-L1 antibody in this example, the full-length amino acid sequence of the heavy chain, and the amino acid sequence of the CDR defined according to the Chothia definition rules.
  • the corresponding amino acid sequence of the anti-PD-L1 positive control antibody Atezolizumab analog of the present application is derived from the IMGT database, the antibody heavy chain sequence is SEQ ID NO:97, and the antibody light chain sequence is SEQ ID NO:118.
  • Protein samples were analyzed for purity and aggregated form using analytical size exclusion chromatography (SEC). Connect the analytical chromatographic column TSKgel G3000SWxl (Tosoh Bioscience, #08541, 5 ⁇ m, 7.8 mm ⁇ 30 cm) to a high-pressure liquid chromatography (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), and equilibrate with PBS buffer at room temperature At least 1 hour.
  • SEC analytical size exclusion chromatography
  • Example 1 The antibody sequence in Example 1 was analyzed, the germline gene V gene fragment of the heavy chain variable domain (VH), and the predicted PTM of the variable domain VH are listed in Table 7.
  • Table 8 lists new antibody molecules (referred to as PTM variants) obtained by amino acid mutations from the sequence of the PR000960 antibody with potential PTM sites in Example 1.
  • Table 9 lists the amino acid sequences of the CDRs, VH and HC of the PTM variants of PR000960, and the amino acid sequences of the CDRs defined according to the Chothia definition rules. Purified recombinant antibodies were obtained from all designed PTM variants according to the method described in Example 1.3, and further verified in subsequent functional experiments. The yield and purity analysis results of the PTM variants of PR000960 are shown in Table 10.
  • the yeast display antibody mutation library was established by randomly mutating the CDR region, and the affinity of the PR002082 molecule was modified with the help of the BD FACS AriaIII sorting platform. This affinity maturation method is divided into three rounds.
  • mutations were randomly introduced into the three CDRs of the parent molecule PR002082 to establish a 3CDRs (CDR1, CDR2, CDR3) yeast display mutation library; then enriched by MACS sorting method; followed by multiple rounds of sorting by flow cytometry FACS, Enrich high affinity mutant molecules.
  • CDR1, CDR2, CDR3 yeast display mutation library CDR1, CDR2, CDR3 yeast display mutation library
  • MACS sorting method MACS sorting method
  • multiple rounds of sorting by flow cytometry FACS Enrich high affinity mutant molecules.
  • the CDR sequences of antibody variable domains were analyzed by Chothia's rules.
  • yeast cells with higher binding capacity were sorted out with 0.1nM biotinylated PD-L1-his; then cultured and induced.
  • VH antibody heavy chain variable domain sequence
  • Table 11 Sequence number list of PR002082 variants (SEQ ID NO:)
  • the plasmid encoding the recombinant HCAb single-domain antibody is transfected into mammalian host cells (such as Chinese hamster ovary cells 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 cells CHO
  • ExpiCHO-S TM cells (Gibco, #A29127) were expanded in ExpiCHO TM Expression Medium (Gibco, #A2910001). Before the start of transient transfection, adjust the cell concentration to (3-4) ⁇ 10 6 cells/mL, culture in a 8% CO 2 shaker at 37°C for 24 hours, and the cell concentration to (7-10) ⁇ 10 6 cells/mL , and then diluted to 6 ⁇ 10 6 cells/mL to prepare 10 mL of cultured cells.
  • a gravity column (Bio-Rad, #7311550) containing Ni Sepharose excel (GE Healthcare Life Science, #17531802) was equilibrated with PBS (pH 7.4), washed with 2-5 column volumes. Pass the supernatant sample through the column. Wash the column successively with 5-10 column volumes of PBS and 5-10 column volumes of 20 mM imidazole solution (pH 7.4). The target protein was then eluted with 500 mM imidazole solution (pH 7.4), and finally concentrated and exchanged into PBS buffer with an ultrafiltration tube (Millipore, #UFC901024) to obtain a purified antibody solution. Then use NanoDrop (Thermo Scientific TM NanoDrop TM One) to measure the concentration, aliquot and store for future use. See Table 12 for the preparation of PR002082 affinity matured variants.
  • a CHO-K1 cell line (CHO-K1/hPD-L1, Jinsi, Nanjing, China) overexpressing human PD-L1 was used. Rui, M00543) or the CHO-K1 cell line overexpressing cynomolgus monkey PD-L1 (CHO-K1/cynoPD-L1, Nanjing GenScript, M00573) for binding experiments at the cellular level. Briefly, PD-L1 cells were digested and resuspended with F-12K complete medium to adjust the cell density to 1 x 106 cells/mL.
  • concentration is 100 nM, 8 concentrations in total, and the isotype antibody hIgG1iso is used as a control. Place the cells at 4°C and incubate for 1 hour in the dark. Afterwards, add 100 ⁇ L/well of pre-cooled PBS to rinse the cells twice, centrifuge at 500 g at 4°C for 5 minutes, and discard the supernatant.
  • Fluorescence signal values were read using flow cytometer BD FACS CANTOII or FACS ACEA, and the data were processed and analyzed with the software FlowJo v10 (FlowJo, LLC).
  • the application software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as binding curve and EC 50 value were obtained through four-parameter nonlinear fitting.
  • Figure 1 show that the PD-L1 antigen-binding proteins described in this application can all bind to CHO-K1 cells overexpressing human PD-L1.
  • Figure 1 (D, E, F) results show that the PR002082 affinity maturation variants described in this application can all bind to CHO-K1 cells overexpressing human PD-L1, and the effect is stronger than that of the parental sequence antibody PR002082.
  • Figure 2(A) shows that all the PD-L1 antigen-binding proteins described in this application can bind to CHO-K1 cells overexpressing cynomolgus monkey PD-L1.
  • FIG. 2(B) shows that all the PR000960 post-translational modification (PTM) variants described in this application can bind to CHO-K1 cells overexpressing cynomolgus monkey PD-L1.
  • PTM post-translational modification
  • Example 4 Using the BLI method to determine the affinity of antigen-binding proteins to human PD-L1
  • an Octet Red96e instrument was used to measure the affinity of the PR002082 affinity matured mutant.
  • the specific method is that the human PD-L1 protein with histidine tag was purchased from the manufacturer ACROBiosystems (#PD-1-H5258, and the test buffer was 1 ⁇ kinetic buffer (from 10 ⁇ kinetic buffer (ForteBio, # 18-1105) dilution) for affinity testing and dilution of antigens and antibodies.
  • biofilm interference (BLI) technology use Octet molecular interaction analyzer (ForteBio, model Octet Red96e) to carry out the binding between antigens and antibodies kinetics analysis.
  • the sensor rotation speed is 1000 rpm.
  • Regenerate the HIS1K sensor by immersing it in a 10mM glycine (pH 1.5) solution to elute the protein bound to the sensor.
  • Example 5 Using a reporter gene cell line to detect the inhibitory effect of antigen-binding proteins on the PD-1 signaling pathway
  • 293T (eBioscience) cells expressing PD-L1 and OS8 (CD3 single-chain antibody transmembrane protein) were plated on a 96-well plate with a cell volume of 1.25 ⁇ 10 4 /well, 100 ⁇ L/well. Incubate overnight at 37°C in a 5% CO environment . The supernatant was removed, and 50 ⁇ L/well of the dilution of the antigen-binding protein to be tested was added. The initial concentration was 100 nM, and the concentration was diluted 5 times. hlgG1 was used as the control group.
  • PBMC cells separate mononuclear cells, add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) to induce for 6 days, and obtain Immature human CD14+ dendritic cells (iDC cells).
  • IL-4 human interleukin 4
  • iDC cells Immature human CD14+ dendritic cells
  • Add 1 ⁇ g/mL lipopolysaccharide Lipopolysaccharide, LPS; Sigma, #L2630
  • mDC cells mature dendritic cells
  • IL-2 ThermoFisher, #88-7025-88
  • IFN- ⁇ ThermoFisher, #88-7316-88
  • the PBMCs of 18 donors were divided into 9 groups of donor pairs for mixed lymphocyte reaction (MLR).
  • MLR mixed lymphocyte reaction
  • Example 7 In vivo tumor suppressive activity of antigen binding proteins
  • the in vivo anti-tumor effect of the A375 tumor model in which the human PBMC immune system was reconstituted by NCG mice is shown in Figure 13A.
  • the average tumor volume of mice in the vehicle control group was 1336 mm 3 on day 23 after administration.
  • Example 1 For obtaining the CD73 antibody, refer to the descriptions in Example 1, Example 2 and Example 3 in the description of the published application WO2021032173A1.
  • CD73 protein was used as an immunogen to generate anti-CD73 antibodies.
  • Each mouse was given a first boost of 50 ⁇ g protein followed by a subsequent boost of 25 ⁇ g protein using adjuvant (Sigma, S6322) by subcutaneous and intraperitoneal injection. This immunization was performed every two weeks for a total of 6 times. Final immunization was performed by intraperitoneal injection of immunogen diluted in PBS. Serum titers against human CD73 were tested using ELISA and FACS. At indicated time points, mouse sera were sampled and titrated for ELISA and FACS analysis to test for binding to CD73 protein or CD73 stable cell lines. Good serum titers were observed in the protein immunization cohort.
  • Mouse splenocytes isolated from immunized mice were fused with the mouse myeloma cell line SP2/0 (ATCC, CRL-1581) by an electric field-based electroporator using a cell fusion generator (BEX-LF301), to obtain hybridomas.
  • Human anti-CD73 antibodies were screened in individual wells 9-14 days after fusion. After the hybridomas were subcloned, the monoclonal 38H6 that showed good binding activity to CD73 was selected for sequencing. The heavy and light chain variable regions were identified and obtained. The heavy and light chain variable regions were then synthesized and cloned into a plasmid encoding the human IgG1 constant region and a human Ig ⁇ region, respectively.
  • Recombinant plasmids encoding the antibodies of interest were transiently co-transfected into HEK293-6E or HEK293-F cell cultures using PEI (Polyscience, 24885). 30 ⁇ g of plasmid was mixed with 120 ⁇ L PEI and incubated for 15 minutes at room temperature. Next, the mixture was added dropwise to 293 cells suspended in Opti-MEM at a concentration of 1 ⁇ 10 6 cells/ml. After transfection, the cells were cultured on a shaker at 120 rpm at 37° C. and 5% CO 2 . Cell culture supernatants collected on days 6-7 were used for purification.
  • the supernatant containing the antibody of interest was harvested 6-7 days after transfection by centrifugation and filtration.
  • Monoclonal antibodies were purified by passing rProtein A (GE, 17-1279-02) prepacked columns (Bio-Rad, 7311550). Prepare a Protein A column by packing 0.2 mL of rProtein A resin into a column, then flushing with 10 column volumes of ddHO and 10 column volumes of PBS. The cell culture supernatant was passed through the column, and then washed with 10 column volumes of PBS.
  • the protein was then eluted with 8 column volumes of elution buffer (Thermo, 21004), and mixed with 640 ⁇ L of neutralization buffer (1M Tris-HCl, pH 9.0; Teknova, T1090) immediately after elution.
  • neutralization buffer 1M Tris-HCl, pH 9.0; Teknova, T1090
  • a concentrator Millipore, UFC903024
  • centrifuge at 3800 rpm (Eppendorf, 5810R) at 4°C, exchange the buffer with DPBS more than 500 times, and finally concentrate to an appropriate volume.
  • the concentration of purified anti-CD73 antibody was determined by UV absorbance at 280 nm (NanoDrop).
  • Antibody purity was determined by SEC-HPLC and SDS-PAGE. Recombinant antibody PR000506 was successfully expressed and purified for characterization.
  • VH and VL sequences of the anti-CD73 antibody were further optimized by germline back mutation and PTM removal:
  • the antibody VH or VL sequence is first aligned to the closest human germline sequence by an algorithm such as NCBI/Ig-BLAST, and then the residues in the framework regions that are different from the germline sequence are reversed to Corresponding residues in the germline sequence. Then, germlined antibodies composed of sequence variants are recombinantly produced by mature molecular biology techniques.
  • PTMs Post-translational modifications
  • other PTM sites that occur within the antibody antigen-binding site may reduce the antigen Binding activity or reduced chemical stability.
  • deamidation or isomerization can render molecules unstable and heterogeneous.
  • sequence instability the PTM motif can be removed by mutation. Look for PTM motifs such as isomerization motifs (eg DG) for VH or VL sequences.
  • “Hotspot” residues eg, D or G in the DG motif
  • Antibodies consisting of sequence variants after PTM removal are then produced recombinantly by well-established molecular biology techniques.
  • Antibody number PTM removal light chain heavy chain VL VH PR000846 remove 119 98 94 73 PR001408 remove 120 103 95 78 PR003836 remove 121 106 96 81
  • This example uses the antigen-binding domain Fab of anti-CD73 IgG antibodies PR000846, PR001408, and PR003836, and the antigen-binding structures of anti-PD-L1 HCAb antibody PR002082 and affinity matured variants PR005263, PR005872, PR005875, PR005878, PR006246, and PR006248 domain VH to construct CD73 ⁇ PD-L1 bispecific antibody molecules.
  • the sequence number of the anti-CD73 H2L2 antibody is shown in Table 15, and the sequence number of the anti-PD-L1 HCAb antibody is shown in Table 16.
  • Antibody number heavy chain VH PR005263 107 82 PR005872 111 86 PR005875 112 87 PR005878 113 88 PR006246 115 90 PR006248 117 92 PR002082 105 80
  • Figure 14 lists the molecular structures of the bispecific binding proteins encompassed by the present application, each of which is further described below.
  • the number of polypeptide chains contained in the molecular structure it usually refers to the number of "different polypeptide chains"; for example, conventional IgG antibodies have two different polypeptide chains, namely Heavy chain and light chain, although the IgG antibody molecule itself is a tetrapeptide chain protein molecule containing two identical heavy chains and two identical light chains, when describing its structural characteristics, it specifically refers to its two different polypeptide chains.
  • Binding valence refers to the number of antigen-binding sites in the molecular structure. For example, a conventional IgG antibody can simultaneously bind two identical antigen molecules, and its binding valence is two.
  • Table 17 lists the linking peptide sequences that may be used in the structural design of the application of the present invention.
  • the anti-CD73 H2L2 antibody and the anti-PD-L1 HCAb antibody were used to construct bispecific antibodies with IgG-VH tetravalent symmetrical structure.
  • the binding protein of IgG_HC-VH tetravalent symmetrical structure contains two polypeptide chains: polypeptide chain 1, also known as short chain, from amino terminal to carboxyl terminal, which contains VL_A-CL; polypeptide chain 2, also called The long chain is called, from amino terminus to carboxyl terminus, which comprises VH_A-CH1-h-CH2-CH3-L-VH_B.
  • h is the hinge region or derived sequence of an IgG antibody.
  • CH3 of polypeptide chain 2 is directly fused with VH_B, that is, the length of L is 0.
  • CH3 of polypeptide chain 2 is linked to VH_B via linker peptide L; L can be the sequence listed in Table 17.
  • the anti-CD73 H2L2 antibody and the anti-PD-L1 HCAb antibody were used to construct bispecific antibodies with IgG-VH tetravalent symmetrical structure.
  • the binding protein of the VH-IgG_HC tetravalent symmetrical structure contains two polypeptide chains: polypeptide chain 1, also known as short chain, from the amino terminal to the carboxyl terminal, which contains VL_A-CL; polypeptide chain 2, also called The long chain, from amino terminus to carboxyl terminus, is called VH_B-L-VH_A-CH1-h-CH2-CH3.
  • h is the hinge region or derived sequence of an IgG antibody.
  • VH_A of polypeptide chain 2 is linked to VH_B via connecting peptide L, which can be the sequence listed in Table 17.
  • VH_A and VH_B of polypeptide chain 2 are fused directly, that is, the length of L is 0.
  • the anti-CD73 H2L2 antibody and the anti-PD-L1 HCAb antibody were used to construct bispecific antibodies with IgG-VH tetravalent symmetrical structure.
  • the binding protein of Fab(CL)-VH-Fc symmetric structure (as shown in Figure 14C) contains two polypeptide chains, polypeptide chain 1, also known as short chain, from amino terminal to carboxyl terminal, which contains VH_A-CH1; polypeptide chain 2, also known as long chain, from amino terminal to carboxyl terminal, it contains VL_A-CL-L1-VH_B-L2-CH2-CH3.
  • Linking peptides L1 and L2 of polypeptide chain 2 can be the sequences listed in Table 17.
  • the CL of polypeptide chain 2 is directly fused with VH_B, that is, the length of L1 is 0.
  • the CD73 ⁇ PD-L1 bispecific antibody is IgG1 with Fc mutations L234A and L235A or L234A and L235A and G237A (numbering according to EU index).
  • the CD73 ⁇ PD-L1 bispecific antibody prepared in the present invention is summarized in Table 18; the sequence number of the amino acid sequence of the polypeptide chain of the obtained biantibody molecule is shown in Table 19. See Table 20 for the sequence numbers of the CDRs of the antigen-binding domain of the obtained biantibody molecule.
  • Table 21 The analysis of the physical and chemical properties of the prepared CD73 ⁇ PD-L1 double antibody molecular samples is summarized in Table 21.
  • Table 18 CD73 ⁇ PD-L1 double antibody molecules designed using anti-CD73 IgG antibody and anti-PD-L1 heavy chain antibody
  • Table 20 Sequence numbers of the CDRs of the antigen-binding domain of the CD73 ⁇ PD-L1 biantibody molecule
  • Example 10 Binding of CD73 ⁇ PD-L1 bispecific antibody to cells overexpressing human PD-L1 or human CD73 (FACS)
  • CHO-K1 cell lines overexpressing human CD73 or PD-L1 (CHO-K1-hCD73, CHO-K1-hPD- L1) Perform binding experiments at the cellular level. Briefly, digest CHO-K1-hCD73 and CHO-K1-hPD-L1 cells and resuspend with F-12K complete medium to adjust the cell density to 1 x 106 cells/mL.
  • Figure 15 show that the anti-CD73 antibodies PR000846 and PR001408 of the present application can specifically bind to CHO-K1 cells overexpressing human CD73 ( Figure 15A); the CD73 ⁇ PD-L1 bispecific antibodies of the present application (PR003569 and PR003739) can Binding to CHO-K1 cells overexpressing human CD73 ( Figure 15A); the anti-CD73 antibody PR003836 of the present application can specifically bind to CHO-K1 cells overexpressing human CD73 ( Figure 15B); the CD73 ⁇ PD-L1 bispecific Antibody PR004295 can bind to CHO-K1 cells overexpressing human CD73 (Fig. 15B);
  • the anti-CD73 antibody PR000846 of the present application can specifically bind to CHO-K1 cells overexpressing human CD73 ( Figure 15C, Figure 15D); , PR006662, PR006663, PR006664, PR006667, PR006668) can bind to CHO-K1 cells overexpressing human CD73 ( Figure 15C, Figure 15D); the anti-PD-L1 antibody PR002082 of the present application can bind to CHO-K1 cells overexpressing human PD-L1 K1 cells ( Figure 15E); the CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 of the present application can bind to CHO-K1 cells overexpressing human PD-L1 ( Figure 15E, Figure 15F).
  • the EC50 values of the anti-CD73 antibody, anti-PD-L1 antibody and CD73 ⁇ PD-L1 bispecific antibody of the present application are in the range of 0.3418nM-2.929nM.
  • the initial concentration is 50 nM, with 3-fold serial dilution, and a total of eight concentrations; in Figure 15D, the initial concentration is 100 nM, with a 3-fold serial dilution, and a total of eight concentrations.
  • CD73 ⁇ PD-L1 bispecific antibody inhibits the enzymatic activity of CD73
  • the malachite green method was used to measure the enzyme activity of antibodies inhibiting soluble recombinant CD73.
  • 12.5 ⁇ L 1 nM recombinant CD73 protein and 12.5 ⁇ L 1 nM antibody were added to a 384-well plate (Corning, #3799) (assay buffer was 25 mM Tris pH 7.5, 5 mM MgCl 2 , 0.005% Tween-20), incubated at room temperature for 1 hour. Add 25 ⁇ L AMP (the highest concentration is 200 ⁇ M, diluted to 8 concentrations by 2 times with the assay buffer), and incubate at room temperature for 15 minutes.
  • the concentration of inorganic phosphate in each well was detected, and the determination of the concentration of inorganic phosphate was performed according to the supplier's instructions. After the measurement, the absorbance value at 620 nm was recorded with a Molecular Devices (SPECTRAMax plus384) plate reader. The experimental results were analyzed and graphed with GraphPad Prism 8.0.
  • Example 12 Using a reporter gene cell line to detect the inhibitory effect of CD73 ⁇ PD-L1 bispecific antibody on PD-1 signaling pathway
  • CD73 ⁇ PD-L1 bispecific antibody activates T cells in a mixed lymphocyte reaction (MLR) assay
  • PBMC cells separate CD14+ monocytes, add recombinant human interleukin 4 (IL-4) (R&D, #204-GMP) and human GM-CSF (R&D, #215-GM/CF) to induce 6 days later, Immature human dendritic cells (iDC cells) were obtained.
  • IL-4 human interleukin 4
  • iDC cells Immature human dendritic cells
  • lipopolysaccharide Lipopolysaccharide, LPS; Sigma, #L2630
  • T lymphocytes were isolated from the second donor PBMC cells using the Pan-T cell isolation kit (Miltenyibiotec, #130-096-535).
  • the results in Figure 18 show that the CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 of the present application have an activation effect on T cells detected in vitro by the MLR method.
  • Anti-PD-L1 mAb PR002082 alone compared to human isotype IgG, and compared to anti-CD73 mAbs PR000846, PR001408 and PR003836 alone, and anti-PD-L1 mAb PR002082 with anti-CD73 CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 at a concentration of 10 nM were more effective in activating T cells secrete IL-2 significantly more efficiently.
  • CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 compared with the combination of anti-PD-L1 monoclonal antibody PR002082 and anti-CD73 monoclonal antibody PR000846, and the combination of anti-PD-L1 monoclonal antibody PR002082 and anti-CD73 monoclonal antibody Compared with the combination of anti-PR003836, the effect is more significant, and it can be considered that the CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 have synergistic effects.
  • CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 at 10nM Concentrations are significantly more effective in activating T cells to secrete IFN- ⁇ .
  • CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 compared with the combination of anti-PD-L1 monoclonal antibody PR002082 and anti-CD73 monoclonal antibody PR000846, and the combination of anti-PD-L1 monoclonal antibody PR002082 and anti-CD73 monoclonal antibody Compared with the combination of anti-PR003836, the effect is more significant, and it can be considered that the CD73 ⁇ PD-L1 bispecific antibodies PR003569, PR003739 and PR004295 have synergistic effects.

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Abstract

一种靶向PD-L1和CD73的特异性结合蛋白，所述特异性结合蛋白能够与PD-L1和/或其片段，以及CD73和/或其片段特异性结合。所述特异性结合蛋白在治疗、预防和/或诊断疾病中的用途，所述疾病例如为免疫性疾病，急性和慢性炎性疾病，以及肿瘤疾病。

Description

靶向PD-L1和CD73的特异性结合蛋白 技术领域
本发明涉及生物医药领域，更具体地涉及抗体治疗领域。
背景介绍
程序性死亡配体1(PD-L1)，是程序性死亡受体1(PD-1)的两种细胞表面糖蛋白配体之一(另一种是PD-L2)，其在结合PD-1时下调T细胞激活和细胞因子的分泌。PD-L1也称为分化簇274(CD274)或B7同系物1(B7-H1)，是一种40kDa的I型跨膜蛋白，PD-L1含IgV(免疫球蛋白可变区)样区、IgC(免疫球蛋白恒定区)样区、跨膜区和细胞质尾区，其中细胞质尾区与细胞内的信号转导相关；IgV区和IgC区则参与细胞间的信号转导。PD-L1与PD-1结合时，募集Src同源区2蛋白酪氨酸磷酸酶(SHP-2)，从而减弱淋巴细胞特异性蛋白酪氨酸激酶(LCK)诱导的ζ链-相关蛋白(ZAP70)磷酸化和抑制RAS-MEK-ERK/PI3K-Akt-mTOR通路，进而有效抑制T细胞的转录，最终抑制T细胞的免疫功能，在免疫应答的负性调控方面发挥着重要作用。因此，通常认为，阻断PD-1/PD-L1信号通路可使T细胞活化上调，激活内源性抗肿瘤免疫反应，从而发挥对肿瘤的治疗作用。
以PD-1/PD-L1、CTLA-4单抗为代表的第一代免疫检查点抑制剂已逐渐成为肿瘤免疫治疗的代表，特别是在过继细胞转移治疗和免疫检查点阻断(ICB)中发挥重要作用。靶向PD-1/PD-L1的肿瘤免疫治疗已被批准用于多种不同的实体肿瘤治疗，并于2018年获得诺贝尔生理学或医学奖。然而，抗PD-1/PD-L1治疗的临床数据显示其应答率有限。一些患者产生原发耐药，对PD-1/PD-L1治疗没有应答，一些应答者则在最初反应后也出现了获得性耐药。
而寻找安全有效，并且能与PD-1/PD-L1产生协同效应的新一代肿瘤免疫靶点，是现阶段肿瘤免疫治疗关注的焦点，也由此涌现出如LAG3，TIGIT，TIM3，CD47，OX40，4-1BB等一系列免疫抑制性或激动性靶点。
白细胞分化抗原73(CD73)，也称为5'-核苷酸酶，是人体中由NT5E基因编码的酶。CD73是由两个相同的70-KD亚基组成的二聚体，由一个糖基磷脂酰肌醇连接到质膜的外表面。已知CD73催化细胞外单磷酸核苷去磷酸化成核苷，例如腺苷。癌组织中细胞外腺苷的积累构成肿瘤免疫逃逸的重要机制。其他作用中，肿瘤来源的腺苷通过腺苷酸环化酶激活的A2A受体很大程度上抑制浸润的效应T细胞(Ohta等人,(2006)Proc Natl Acad Sci USA 103:13132-13137)。已报道CD73在多种肿瘤细胞中表达，包括白血病，膀胱癌，神经胶质瘤，成胶质细胞瘤，卵巢癌，黑色素瘤，前列腺癌，甲状腺癌，食道癌和乳腺癌(Jin等人,Cancer Res 2010；70:2245-55和Stagg等人,PNAS 2010；107:1547-52)。
对于实体瘤治疗而言，要克服耐药性提高疗效，很重要的一个方面就是解除肿瘤微环境(Tumor micro-environment，TME)对免疫效应细胞的抑制作用。TME是非常复杂的系统，由多种细胞、胞间质、酶、细胞因子、代谢产物等构成，有显著的低氧、低pH以及高压的特点，与正常组织差异巨大。其间很重要的一个免疫抑制机制由CD73-腺苷代谢信号途径介导。腺苷可以通过腺苷受体(A2AR)抑制T细胞的免疫杀伤作用，使肿瘤实现免疫逃逸，而CD73则是催化腺苷产生的关键酶。
PD-1/PD-L1的免疫检查点阻断已成为免疫治疗的里程碑。但是，尽管抗PD-1/PD-L1治疗在实体瘤治疗中显示出令人印象深刻的效果，持久的应答却只在一小部分患者中发生，一些最初对治疗有反应的患者最终产生了后天抵抗力，因此，亟待出现疗效更好的PD-1/PD-L1抑制剂以及能与PD-1/PD-L1产生协同效应的肿瘤免疫靶点。
发明内容
本发明提供了能够特异性结合PD-L1或其片段的抗原结合蛋白，其能够阻断PD-1与PD-L1的结合。本发明还提供了多特异性结合蛋白，所述多特异性结合蛋白能够以高亲和力、高特异性与一种或多种抗原结合，所述抗原为PD-L1或其片段，和/或CD73或其片段。本发明还提供了编码所述抗原结合蛋白，以及所述多 特异性结合蛋白的核酸分子，用于产生所述抗原结合蛋白，以及所述多特异性结合蛋白的表达载体，宿主细胞以及用于制备所述抗原结合蛋白，以及所述特异性结合蛋白的方法。本发明还涉及所述抗原结合蛋白，以及多特异性结合蛋白在治疗、预防和/或诊断疾病中的用途，所述疾病例如为免疫性疾病，急性和慢性炎性疾病，以及肿瘤疾病。
第一方面，本发明提供了一种特异性结合PD-L1或其片段的分离的抗原结合蛋白。
所述特异性结合PD-L1或其片段的分离的抗原结合蛋白具有以下一种或者多种特性：(a)能够以高亲和力结合人PD-L1和/或食蟹猴PD-L1；(b)对PD-1信号通路有抑制作用；(c)能够增强激活T淋巴细胞；以及(d)显示体内肿瘤抑制活性。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含以下的互补决定区或其突变体：
SEQ ID NO：9的氨基酸序列所示的HCDR1；
SEQ ID NO：23的氨基酸序列所示的HCDR2；和/或
SEQ ID NO：36的氨基酸序列所示的HCDR3。
所述突变体为在所述VH的HCDR1、HCDR2、HCDR3的氨基酸序列的基础上分别具有1、2、3或4个氨基酸的插入、缺失或取代。本领域技术人员知晓，可以对HCDR1、HCDR2和HCDR3中的1个，2个或3个CDR分别进行1、2或3个氨基酸的插入、缺失或取代突变。例如，可以对HCDR1进行1个氨基酸的突变，对HCDR2和HCDR3不进行氨基酸的突变，或者，对HCDR1和HCDR2分别进行1个氨基酸的突变，对HCDR3不进行氨基酸的突变。
在一些实施方案中，所述HCDR1的氨基酸序列为GFX 1FSX 2Y，所述HCDR2的氨基酸序列为X 3YX 4GX 5X 6，所述HCDR3的氨基酸序列为NRAX 7FGVX 8PDX 9SDI，其中，X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8或X 9为选自N、T、D、S、W、R、K、E、I、A、V和L中的任一种。在一些实施方案中，X 1为T、D或N；X 2为N或S；X 3为W或R；X 4为D或T；X 5为T或S；X 6为K、R或E；X 7为I或L；X 8为V或I；和/或，X 9为A或D。
在一些实施方案中，所述HCDR1的氨基酸序列为GF(N/T/D)FS(N/S)Y。在一些实施方案中，所述HCDR2的氨基酸序列为(W/R)Y(D/T)G(T/S)(K/R/E)。在一些实施方案中，所述HCDR3的氨基酸序列为NRA(I/L)FGV(V/I)PD(A/D)SDI。
在一些实施方案中，HCDR1的突变体具有SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：13和SEQ ID NO：14中任一项所示的氨基酸序列。
在一些实施方案中，HCDR2的突变体具有SEQ ID NO：24或SEQ ID NO：27所示的氨基酸序列。
在一些实施方案中，HCDR3的突变体具有SEQ ID NO：39、SEQ ID NO：40和SEQ ID NO：41中任一项所示的氨基酸序列。
优选地，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含以下的互补决定区或其突变体：
SEQ ID NO：9-11和SEQ ID NO：13-14中任一项的氨基酸序列所示的HCDR1；
SEQ ID NO：23-24和SEQ ID NO：27中任一项的氨基酸序列所示的HCDR2；和/或
SEQ ID NO：36和SEQ ID NO：39-41中任一项的氨基酸序列所示的HCDR3。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：9，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：11，SEQ ID NO：24 和SEQ ID NO：36所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：11，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：13，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：39所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：40所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：41所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：36所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：39所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：40所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：41所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：36所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：39所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：40所示。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：41所示。
优选地，所述VH还包含重链可变区框架区(VH FWR)，所述VH FWR可以包含VH FWR1，VH FWR2，VH FWR3和VH FWR4。
在一些实施方案中，所述VH FWR1具有SEQ ID NO：3-5中任一项所示的氨基酸序列。
在一些实施方案中，所述VH FWR2具有SEQ ID NO：17或SEQ ID NO：20所示的氨基酸序列。
在一些实施方案中，所述VH FWR3具有SEQ ID NO：30或SEQ ID NO：31所示的氨基酸序列。
在一些实施方案中，所述VH FWR4具有SEQ ID NO：43所示的氨基酸序列。
优选地，所述VH包含SEQ ID NO：74-77，SEQ ID NO：79-80和SEQ ID NO：82-92中任一项所示的氨基酸序列，或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。
在一些实施方案中，所述特异性结合PD-L1和/或其片段的分离的抗原结合蛋白还包含Fc区，或者与免疫球蛋白的Fc区等同的区域，优选地，所述Fc区为人Fc。更优选地，所述人Fc为人IgG1Fc。
优选地，所述Fc包含L234A、L235A和P329G突变，或L234A和L235A突变，或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地同时包含S298A，E333A和K334A三个突变。
在一些实施方案中，所述特异性结合PD-L1或其片段的分离的抗原结合蛋白包含与SEQ ID NO：99、100、101、102、104、105、107、108、109、110、111、112、113、114、115、116或117具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列的重链。在一些实施方案中，所述特异性结合PD-L1或其片段的分离的抗原结合蛋白包含SEQ ID NO：99-102，SEQ ID NO：104-105和SEQ ID NO：107-117中任一项所示重链的氨基酸序列。
在一些实施方案中，所述抗原结合蛋白还可以是抗原结合片段，例如可以为Fab、Fab’、F(ab’) 2、Fv、scFv、VH、或者HCAb的形式。所述Fab、Fab’、F(ab’) 2、Fv、scFv或VH的数量优选为一个或多个。
第二方面，本发明提供了分离的核酸，其编码本发明第一方面所述的分离的抗原结合蛋白。
第三方面，本发明提供了表达载体，其包含本发明第二方面所述的分离的核酸，能够将其表达为第一方面的分离的抗原结合蛋白。
所述表达载体可以是真核细胞表达载体和/或原核细胞表达载体，例如为质粒。
第四方面，本发明提供了宿主细胞，所述宿主细胞包含第二方面的分离的核酸，或第三方面的表达载体。
第五方面，本发明提供了抗体药物偶联物，其包含：本发明第一方面所述的分离的抗原结合蛋白；和，与所述抗原结合蛋白共价连接的药物。在一些实施方案中，所述药物选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物。
第六方面，本发明提供了嵌合抗原受体，其包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，其中所述胞外抗原结合结构域包含本发明第一方面所述的分离的抗原结合蛋白。
第七方面，本发明提供了修饰的免疫细胞，其包含本发明第六方面所述的嵌合抗原受体。
第八方面，本发明提供了多特异性抗体，其包含两个或两个以上的抗原结合结构域，其中一个抗原结合结构域包含本发明第一方面所述的分离的抗原结合蛋白。
第九方面，本发明提供了第一方面的分离的抗原结合蛋白的制备方法。
在一些实施方案中，利用杂交瘤技术或者本领域的其他常规技术制备，例如人源化技术等制备第一方面的分离的抗原结合蛋白。
在一些实施方案中，所述制备方法包括培养第四方面的宿主细胞的步骤。
在一些实施方案中，利用Harbour HCAb转基因小鼠(以下简称HCAb转基因小鼠)制备第一方面的分离的抗原结合蛋白。
所述HCAb转基因小鼠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。
优选地，所述利用HCAb转基因小鼠制备第一方面的分离的抗原结合蛋白的方法包括如下步骤：
(a)利用人PD-L1抗原免疫HCAb转基因小鼠，更优选地，所述人PD-L1抗原为重组人PD-L1-mFc，具体地，所述抗原为人PD-L1连接Fc构成的重组融合蛋白；
(b)利用免疫后的HCAb转基因小鼠脾B细胞的RNA反转录后的cDNA，以及特异性引物扩增人VH基因；
(c)将扩增的VH基因构建到编码人IgG抗体重链Fc结构域序列的哺乳动物细胞表达载体中；和
(d)纯化步骤(c)获得的全人源PD-L1单抗。
优选地，所述IgG抗体为IgG1抗体或IgG4抗体。
在一些实施方案中，所述方法还包括通过对CDR区域进行突变对全人源PD-L1单抗进行亲和力改造的步骤。
优选地，通过胚系化回复突变和/或翻译后修饰(PTM)去除对全人源PD-L1单抗进行优化。
第十方面，本发明提供了一种药物组合物，所述药物组合物包含：第一方面所述的分离的抗原结合蛋白，第五方面所述的抗体药物偶联物，第六方面所述的嵌合抗原受体，第七方面所述的修饰的免疫细胞或第八方面所述的多特异性抗体；以及，药学上可接受的载体。
所述药物组合物还可以包含其他治疗剂，包括但不限于，化学治疗剂、放射治疗剂、免疫抑制剂、细胞毒性药物等。在一些实施方案中，所述药物组合物中的化学治疗剂、放射治疗剂、免疫抑制剂或细胞毒性药物，与第五方面所述的抗体药物偶联物中的药物相同或不同。
第十一方面，本发明提供了第一方面所述的分离的抗原结合蛋白、第二方面所述的分离的核酸、第五方面所述的抗体药物偶联物，第六方面所述的嵌合抗原受体，第七方面所述的修饰的免疫细胞或第八方面所述的多特异性抗体或第十方面的药物组合物在制备用于预防、治疗和/或诊断免疫性疾病，急性和慢性炎性疾病，以及肿瘤疾病的药物中的应用。
所述肿瘤可以为乳腺癌、肾细胞癌、黑色素瘤、结肠癌，以及B细胞淋巴瘤，黑色素瘤，头颈癌，膀胱癌，胃癌，卵巢癌，恶性肉瘤，尿路上皮癌，肝癌，食道癌，胃食管交界癌，鼻咽癌，小细胞肺癌，宫颈癌，子宫内膜癌，胰腺癌，前列腺癌，胶质瘤，非小细胞肺癌，急性粒细胞白血病，霍奇金淋巴瘤，皮肤鳞状细胞癌，局部晚期或转移性恶性肿瘤等。
所述炎性疾病可以为特应性皮炎、溃疡性结肠炎等。
所述免疫性疾病可以为移植物抗宿主病、类风湿性关节炎、系统性红斑狼疮、哮喘等。
第十二方面，本发明提供了用于检测样品中的PD-L1的方法，所述方法包括用第一方面的分离的抗原结合蛋白检测样品中的PD-L1的步骤。
所述样品可以是生物学样品，例如，全血，红血细胞浓缩物，血小板浓缩物，白细胞浓缩物，组织，骨髓吸出物，血浆，血清，脑脊液，粪便，尿液，培养的细胞，唾液，口腔分泌物，和鼻腔分泌物等生物学样品。
在一些实施方案中，所述检测样品中的PD-L1的方法可以为非诊断目的的。
第十三方面，本发明提供了多特异性结合蛋白，所述特异性结合蛋白包含至少两个结构域，能够结合PD-L1或其片段，和/或CD73或其片段。
在一些实施方案中，所述特异性结合蛋白包含第一结构域和第二结构域，所述第一结构域结合CD73或其片段，并且所述第二结构域结合PD-L1或其片段。所述第一结构域与所述第二结构域连接形成双特异性结合蛋白。
在一些实施方案中，所述第一结构域为CD73抗体或其抗原结合片段。
在一些实施方案中，所述第二结构域为PD-L1抗体或其抗原结合片段。
在一些实施方案中，所述第一结构域和/或所述第二结构域为IgG、Fab、Fab’、F(ab’) 2、Fv、scFv、VH、或者HCAb的形式。
所述Fab、Fab’、F(ab’) 2、Fv、scFv、VH的数量为一个或多个。
在一些实施方案中，所述第一结构域为IgG的形式。优选地，所述IgG的重链恒定区为人重链恒定区，更优选为人IgG1、人IgG2、人IgG3或人IgG4重链恒定区；其中人IgG优选包含L234A、L235A和P329G中的一个、两个或三个突变，更优选地包含L234A和L235A的突变，或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地包含S298A，E333A和K334A突变。
优选地，所述第一结构域中IgG的Fc为人IgG1的Fc，更优选地，所述Fc包含L234A、L235A和P329G突变，或L234A和L235A突变，或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地包含S298A，E333A和K334A突变。
在一些实施方案中，所述第一结构域为Fab的形式，更优选地，所述第一结构域包括2个Fab。
在一些实施方案中，所述第二结构域包含一个或多个VH。
优选地，所述VH为人VH。更优选地，所述第二结构域具有两个VH。
在一些实施方案中，所述第二结构域还包含Fc，与免疫球蛋白的Fc区等同的区域，优选地，所述Fc为人IgG1的Fc，更优选地，所述Fc包含L234A、L235A和P329G突变，或L234A和L235A突变，或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地包含S298A，E333A和K334A突变。
在一些实施方案中，所述第一结构域与所述第二结构域直接连接或经连接肽L连接形成双特异性结合蛋白。
在一些实施方案中，所述第二结构域连接在所述第一结构域的C末端或N末端。
在一些实施方案中，所述双特异性结合蛋白包含短链和长链。
优选地，所述短链具有N’-VL 1-CL 1-C’所示的结构；所述长链具有N’-VH 1-CH1-h-CH2-CH3-L-VH 2-C’所示的结构；
或者，所述短链具有N’-VL 1-CL 1-C’所示的结构；所述长链具有N’-VH 2-L-VH 1-CH1-h-CH2-CH3-C’所示的结构；
或者，所述短链具有N’-VH 1-CH-C’所示的结构；所述长链具有N’-VL 1-CL 1-L-VH 2-CH2-CH3-C’所示的结构，
其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L为连接肽，所述CL 1是第一结构域的CL。其中，所述的铰链区为免疫球蛋白领域常见的铰链区，通常含大量脯氨酸，具有挠性，形成2-5个二硫键。
优选地，所述L为长度为0-30个氨基酸长度的肽。在一些实施方案中，所述L的氨基酸序列如SEQ ID NO：136-157任一所示。
在一些实施方案中，所述长链的CH3与VH 2直接连接，即L的长度为0。
在一些实施方案中，所述长链的CH3经由连接肽L连接到VH 2。在一些实施方案中，所述连接肽L为长度为0-30个氨基酸长度的肽。在一些实施方案中，所述连接肽L可以是如SEQ ID NO.136-157中任一项所示的氨基酸序列。
在一些实施方案中，所述长链的VH 1与VH 2直接连接，即L的长度为0。
在一些实施方案中，所述长链的VH 1经由连接肽L连接到VH 2。在一些实施方案中，所述连接肽L为长度为0-30个氨基酸长度的肽。在一些实施方案中，所述连接肽L可以是如SEQ ID NO.136-157中任一项所示的氨基酸序列。
在一些实施方案中，所述长链的VH 2与CL 1直接连接，即L的长度为0。
在一些实施方案中，所述长链的VH 2经由连接肽L连接到CL 1。在一些实施方案中，所述连接肽L为长度为0-30个氨基酸长度的肽。在一些实施方案中，所述连接肽L可以是如SEQ ID NO.136-157中任一项所示的氨基酸序列。
在一些实施方案中，所述第一结构域包含轻链可变区(VL)和重链可变区(VH)，所述VL包含选自如下的互补决定区：
SEQ ID NO：51，SEQ ID NO：52和SEQ ID NO：53中任一项的氨基酸序列所示的LCDR1；
SEQ ID NO：57，SEQ ID NO：58和SEQ ID NO：59中任一项的氨基酸序列所示的LCDR2；和/或
SEQ ID NO：65，SEQ ID NO：66和SEQ ID NO：67中任一项的氨基酸序列所示的LCDR3；
所述VH包含选自如下的互补决定区：
SEQ ID NO：8，SEQ ID NO：12和SEQ ID NO：10中任一项的氨基酸序列所示的HCDR1；
SEQ ID NO：22，SEQ ID NO：25和SEQ ID NO：26中任一项的氨基酸序列所示的HCDR2；和/或
SEQ ID NO：35，SEQ ID NO：37和SEQ ID NO：38中任一项的氨基酸序列所示的HCDR3。
优选地，所述第一结构域包含轻链可变区(VL)和重链可变区(VH)，所述VL和VH包含选自如下的互补决定区：
分别如SEQ ID NO：51，SEQ ID NO：57和SEQ ID NO：65所示的LCDR1，LCDR2和LCDR3；和，分别如SEQ ID NO：8，SEQ ID NO：22和SEQ ID NO：35所示的HCDR1，HCDR2和HCDR3；
分别如SEQ ID NO：52，SEQ ID NO：58和SEQ ID NO：66所示的LCDR1，LCDR2和LCDR3；和，分别如SEQ ID NO：12，SEQ ID NO：25和SEQ ID NO：37所示的HCDR1，HCDR2和HCDR3；
分别如SEQ ID NO：53，SEQ ID NO：59和SEQ ID NO：67所示的LCDR1，LCDR2和LCDR3；和，分别如SEQ ID NO：10，SEQ ID NO：26和SEQ ID NO：38所示的HCDR1，HCDR2和HCDR3。
在一些实施方案中，所述第一结构域包含VL和VH，其中，所述VL的氨基酸序列与SEQ ID NO：94，SEQ ID NO：95或SEQ ID NO：96具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性；所述VH的氨基酸序列与SEQ ID NO：73，SEQ ID NO：78或SEQ ID NO：81具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性。
优选地，所述第一结构域包含VL和VH，其中所述VL具有SEQ ID NO：94，SEQ ID NO：95和SEQ ID NO：96中任一项所示的氨基酸序列；所述VH具有SEQ ID NO：73，SEQ ID NO：78和SEQ ID NO：81中任一项所示的氨基酸序列。
优选地，所述第一结构域包含VL和VH，所述VL和VH的氨基酸序列分别如SEQ ID NO:94和SEQ ID NO:73所示；或分别如SEQ ID NO:95和SEQ ID NO:78所示；或分别如SEQ ID NO:96和SEQ ID NO:81所示。
在一些实施方案中，所述第一结构域包含氨基酸序列与SEQ ID NO:119，SEQ ID NO:120或SEQ ID NO:121具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的轻链；以及氨基酸序列与SEQ ID NO:98，SEQ ID NO:103或SEQ ID NO:106具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。
优选地，所述第一结构域包含SEQ ID NO:119，SEQ ID NO:120和SEQ ID NO:121中任一项所示的轻链；以及SEQ ID NO:98，SEQ ID NO:103和SEQ ID NO:106中任一项所示的重链。
优选地，所述第一结构域包含SEQ ID NO:119所示的轻链和SEQ ID NO:98所示的重链。
优选地，所述第一结构域包含SEQ ID NO:120所示的轻链和SEQ ID NO:103所示的重链。
优选地，所述第一结构域包含SEQ ID NO:121所示的轻链和SEQ ID NO:106所示的重链。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含以下的互补决定区或其突变体：
SEQ ID NO：9的氨基酸序列所示的HCDR1；
SEQ ID NO：23的氨基酸序列所示的HCDR2；和/或
SEQ ID NO：36的氨基酸序列所示的HCDR3。
所述突变体为在所述VH的HCDR1、HCDR2、HCDR3的氨基酸序列的基础上分别具有1、2或3个氨基酸的插入、缺失或取代。本领域技术人员知晓，可以对HCDR1、HCDR2和HCDR3中的1个，2个或3个CDR分别进行1、2或3个氨基酸的插入、缺失或取代突变。例如，可以对HCDR1进行1个氨基酸的突变，对HCDR2和HCDR3不进行氨基酸的突变，或者，对HCDR1和HCDR2分别进行1个氨基酸的突变，对HCDR3不进行氨基酸的突变。
在一些实施方案中，所述HCDR1的氨基酸序列为GFX 1FSX 2Y，所述HCDR2的氨基酸序列为X 3YX 4GX 5X 6，所述HCDR3的氨基酸序列为NRAX 7FGVX 8PDX 9SDI，其中，X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8或X 9为选自N、T、D、S、W、R、K、E、I、A、V和L中的任一种。在一些实施方案中，X 1为T、D或N；X 2为N或S；X 3为W或R；X 4为D或T；X 5为T或S；X 6为K、R或E；X 7为I或L；X 8为V或I；和/或，X 9为A或D。
在一些实施方案中，所述HCDR1的氨基酸序列为GF(N/T/D)FS(N/S)Y。在一些实施方案中，所述HCDR2的氨基酸序列为(W/R)Y(D/T)G(T/S)(K/R/E)。在一些实施方案中，所述HCDR3的氨基酸序列为NRA(I/L)FGV(V/I)PD(A/D)SDI。
在一些实施方案中，HCDR1的突变体具有SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：13和SEQ  ID NO：14中任一项所示的氨基酸序列。
在一些实施方案中，HCDR2的突变体具有SEQ ID NO：24或SEQ ID NO：27所示的氨基酸序列。
在一些实施方案中，HCDR3的突变体具有SEQ ID NO：39，SEQ ID NO：40和SEQ ID NO：41中任一项所示的氨基酸序列。
优选地，所述第二结构域包含抗体重链可变区VH，所述VH包含以下的互补决定区或其突变体：
SEQ ID NO：9-11和SEQ ID NO：13-14中任一项的氨基酸序列所示的HCDR1；
SEQ ID NO：23-24和SEQ ID NO：27中任一项的氨基酸序列所示的HCDR2；和/或
SEQ ID NO：36和SEQ ID NO：39-41中任一项的氨基酸序列所示的HCDR3.
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：9，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：11，SEQ ID NO：24和SEQ ID NO：36所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：11，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：13，SEQ ID NO：23和SEQ ID NO：36所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：39所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：40所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：41所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：36所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：39所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：40所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：41所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：36所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：39所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：40所示。
在一些实施方案中，所述第二结构域包含抗体重链可变区VH，所述VH包含HCDR1，HCDR2和HCDR3，其氨基酸序列分别为SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：41所示。
优选地，所述VH还包含重链可变区框架区(VH FWR)，所述VH FWR可以包含VH FWR1，VH FWR2，VH FWR3和VH FWR4。
在一些实施方案中，所述VH FWR1具有SEQ ID NO：3-5中任一项所示的氨基酸序列。
在一些实施方案中，所述VH FWR2具有SEQ ID NO：17或SEQ ID NO：20所示的氨基酸序列。
在一些实施方案中，所述VH FWR3具有SEQ ID NO：30或SEQ ID NO：31所示的氨基酸序列。
在一些实施方案中，所述VH FWR4具有SEQ ID NO：43所示的氨基酸序列。
优选地，所述VH包含SEQ ID NO：74-77，SEQ ID NO：79-80和SEQ ID NO：82-92中任一项所示的氨基酸序列，或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。
在一些实施方案中，所述第二结构域还包含Fc区，或与免疫球蛋白的Fc区等同的区域，优选地，所述Fc区为人Fc。更优选地，所述人Fc为人IgG1Fc。
优选地，所述Fc包含L234A、L235A和P329G突变，或L234A和L235A突变，或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地包含S298A，E333A和K334A突变。
在一些实施方案中，所述第二结构域包含与SEQ ID NO：99、100、101、102、104、105、107、108、109、110、111、112、113、114、115、116或117具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列的重链。在一些实施方案中，所述第二结构域包含SEQ ID NO：99-102，SEQ ID NO：104-105和SEQ ID NO：107-117中任一项所示重链的氨基酸序列。
在一些实施方案中，所述特异性结合蛋白的长链包含与SEQ ID NO：122、123、124、125、126、127、128、129、130、132或133具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；以及所述特异性结合蛋白的短链包含与SEQ ID NO：119、120、121或131具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。在一些实施方案中，所述特异性结合蛋白的长链具有选自SEQ ID NO：122-130和SEQ ID NO：132-133中任一项所示的氨基酸序列；以及所述特异性结合蛋白的短链具有选自SEQ ID NO：119-121和SEQ ID NO：131中任一项所示的氨基酸序列。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：122所示的长链以及SEQ ID NO：119所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：123所示的长链以及SEQ ID NO：120所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：124所示的长链以及SEQ ID NO：121所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：125所示的长链以及SEQ ID NO：119所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：126所示的长链以及SEQ ID NO：119所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：127所示的长链以及SEQ ID NO：119所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：128所示的长链以及SEQ ID NO：119所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：129所示的长链以及SEQ ID NO：119所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：132所示的长链以及SEQ ID NO：131所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：130所示的长链以及SEQ ID NO：119所示的短链。
在一些实施方案中，所述特异性结合蛋白具有SEQ ID NO：133所示的长链以及SEQ ID NO：131所示的短链。
在一些实施方案中，所述双特异性结合蛋白为包含两条第一多肽链以及两条第二多肽链形成的四价对称结构。
第十四方面，本发明提供了分离的核酸，其编码本发明第十三方面所述的特异性结合蛋白或其片段。
第十五方面，本发明提供了表达载体，其能够将第十四方面所述的分离核酸表达为第十三方面的多特异性结合蛋白或其片段。
所述表达载体可以是真核细胞表达载体和/或原核细胞表达载体，例如为质粒。
第十六方面，本发明提供了宿主细胞，所述宿主细胞包含第十四方面的分离的核酸，或第十五方面的表达载体。
所述宿主细胞为本领域常规的宿主细胞，只要能使第十五方面的表达载体稳定地将所携带的核酸表达为第十三方面的特异性结合蛋白或其片段。优选地，所述宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选E.coli细胞如TG1、BL21(表达单链抗体或Fab抗体)，所述真核细胞优选HEK293细胞或CHO细胞(表达全长IgG抗体)。将第十五方面的表达载体转化至宿主细胞中，即可得本发明的宿主细胞。其中所述转化方法为本领域的常规转化方法，较佳地为化学转化法，热激法或电转法。
第十七方面，本发明提供了包含第十三方面的特异性结合蛋白或其片段的抗体药物偶联物。所述抗体药物偶联物还包含与所述结合蛋白或其片段共价连接的药物。在一些实施方案中，所述药物选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物。
第十八方面，本发明提供了第十三方面的特异性结合蛋白或其片段的制备方法。
在一些实施方案中，利用杂交瘤技术或者本领域的其他常规技术制备，例如人源化技术等制备第十三方面的特异性结合蛋白或其片段。
在一些实施方案中，所述制备方法包括培养第十六方面的宿主细胞的步骤。
在一些实施方案中，利用Harbour HCAb转基因小鼠(以下简称HCAb转基因小鼠)制备第十三方面的特异性结合蛋白的第二结构域。
所述HCAb转基因小鼠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。
优选地，所述利用HCAb转基因小鼠制备第十三方面的特异性结合蛋白的第二结构域的方法包括如下步骤：
(a)利用人PD-L1免疫HCAb转基因小鼠，更优选地，所述人PD-L1抗原为重组人PD-L1-Fc，具体地，所述抗原为PD-L1连接Fc构成的重组融合蛋白；
(b)利用免疫后的HCAb转基因小鼠脾B细胞的RNA反转录后的cDNA，以及特异性引物扩增人VH基因；
(c)将扩增的VH基因构建到编码人IgG抗体重链Fc结构域序列的哺乳动物细胞表达载体中；和
(d)纯化步骤(c)获得的全人源PD-L1单抗。
在一些实施方案中，所述方法还包括通过对CDR区域进行突变对全人源PD-L1单抗进行亲和力改造的步骤。
优选地，还可以通过胚系化回复突变和/或翻译后修饰(PTM)去除对全人源PD-L1单抗进行优化。
在一些实施方案中，利用Harbour H2L2转基因小鼠(以下简称H2L2转基因小鼠)制备第十三方面的特异性结合蛋白的第一结构域。
所述H2L2转基因小鼠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。
优选地，所述利用H2L2转基因小鼠制备第十三方面的特异性结合蛋白的第一结构域的方法包括如下步骤：
(a)利用人CD73免疫H2L2转基因小鼠，更优选地，所述人CD73抗原为可溶性的重组人CD73-hFc，具体地，所述抗原为CD73连接Fc构成的重组融合蛋白；
(b)免疫后的H2L2转基因小鼠脾细胞与骨髓瘤细胞系融合得到杂交瘤细胞，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；
(c)将步骤(b)获得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载体中，以编码产生抗体的全长轻链；
(d)将步骤(b)获得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人IgG抗体重链恒定结构域序列的哺乳动物细胞表达载体中，以编码产生IgG抗体的全长重链；
(e)将步骤(c)和(d)的表达载体同时转染哺乳动物宿主细胞，利用常规的重组蛋白表达和纯化技术，可以得到轻重链正确配对组装的重组抗体。
优选地，将步骤(b)获得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人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达载体中，以编码产生IgG1抗体的全长重链。
在一些实施方案中，所述方法还包括通过对CDR区域进行突变对全人源CD73单抗进行亲和力改造的步骤。
优选地，还可以通过胚系化回复突变和/或翻译后修饰(PTM)去除对全人源CD73单抗进行优化。
在一些实施方案中，将所制备的第二结构域和所制备的第一结构域组合成双特异性结合蛋白。所述双特异性结合蛋白可以同时结合两个靶点，其中第一结构域可以识别肿瘤细胞表面特异表达的CD73，而第二结构域可以结合T细胞上的PD-L1分子，所述特异性结合蛋白结合到肿瘤细胞表面后，可以招募并激活肿瘤细胞附近的T细胞，从而杀死肿瘤细胞。
在一些实施方案中，将所制备的第二结构域和所制备的第一结构域构建成双特异性结合蛋白。所述双特异性结合蛋白可以同时结合两个靶点，其中第一结构域可以识别肿瘤细胞表面特异表达的CD73，而第二结构域可以结合细胞表达的PD-L1分子。所述双特异性结合蛋白能够有效结合可溶性以及细胞表面的CD73和PD-L1，能够抑制CD73的酶活，而且还能阻断PD-L1与PD-1的结合，还能够激活T细胞。
优选地，所述双特异性结合蛋白为四价对称结构。
优选地，所述双特异性结合蛋白具有第十三方面中所述的结构和序列。
第十九方面，本发明提供了一种药物组合物，所述药物组合物包含第十三方面所述的特异性结合蛋白或第十七方面的抗体药物偶联物，以及药学上可接受的载体。
在一些实施方案中，所述药物组合物还包括其他的成分作为活性成分，例如其他的小分子药物或者抗体或多肽作为活性成分。
在一些实施方案中，所述药物组合物还包含选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物的治疗剂。在一些实施方案中，所述药物组合物的化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物的治疗剂，与第十七方面的抗体药物偶联物中的药物不同或者相同。
所述的药学上可接受的载体可为本领域常规的载体，所述的载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，优选地包括药学上可接受的赋形剂、填充剂或稀释剂等。更优选地，所述的药物组合物包括0.01～99.99％的所述特异性结合蛋白和/或其他的小分子药物或者抗体或多肽，以及0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
所述的药物组合物的给药途径可以是经肠胃外、注射或口服给药。所述药物组合物可以制备成适于给药的形式，例如固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，粉末、片剂、胶囊、颗粒、注射剂或输注剂的形式。可以经血管内、皮下、腹膜内、肌内、吸入、鼻内、气道滴注或胸腔内滴注施用。所述药物组合物还可以气雾剂或喷雾剂的形式施用，例如经鼻施用；或者，鞘内、髓内或心室内施用，还可以经透皮、经皮、局部、肠内、阴道内、舌下或经直肠施用。所述的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。
在一些实施方案中，所述特异性结合蛋白中的第一结构域和第二结构域可以同时给药或者按顺序给药。
在一些实施方案中，所述药物组合物中的特异性结合蛋白与其他的活性成分可以同时给药或者按顺序给药。
在一些实施方案中，所述特异性结合蛋白是双特异性结合蛋白。
第二十方面，本发明提供了第十三方面所述的特异性结合蛋白、第十四方面所述的分离的核酸、第十七方面的抗体药物偶联物或第十九方面的药物组合物在制备用于预防、治疗和/或诊断免疫性疾病，急性和慢 性炎性疾病，以及肿瘤疾病的药物中的应用。
所述肿瘤可以为乳腺癌、肾细胞癌、黑色素瘤、结肠癌，以及B细胞淋巴瘤，黑色素瘤，头颈癌，膀胱癌，胃癌，卵巢癌，恶性肉瘤，尿路上皮癌，肝癌，食道癌，胃食管交界癌，鼻咽癌，小细胞肺癌，宫颈癌，子宫内膜癌，胰腺癌，前列腺癌，胶质瘤，非小细胞肺癌，急性粒细胞白血病，霍奇金淋巴瘤，皮肤鳞状细胞癌，局部晚期或转移性恶性肿瘤等。
所述炎性疾病可以为特应性皮炎、溃疡性结肠炎等。
所述免疫性疾病可以为移植物抗宿主病、类风湿性关节炎、系统性红斑狼疮、哮喘等。
第二十一方面，本发明提供了用于检测样品中的PD-L1和CD73的方法，所述方法包括用第十三方面的特异性结合蛋白检测样品中的PD-L1和CD73的步骤。
所述样品可以是生物学样品，例如，全血，红血细胞浓缩物，血小板浓缩物，白细胞浓缩物，组织，骨髓吸出物，血浆，血清，脑脊液，粪便，尿液，培养的细胞，唾液，口腔分泌物，和鼻腔分泌物等生物学样品。
第二十二方面，本发明提供了一种套装药盒，该套装药盒包括一个或多个药盒，包含第一方面所述的抗原结合蛋白、第五方面的抗体药物偶联物或第十方面所述的药物组合物。
在一些实施方案中，所述套装药盒包括第一药盒和第二药盒，所述第一药盒包括第一方面所述的抗原结合蛋白、第五方面的抗体药物偶联物或第十方面所述的药物组合物，且所述第二药盒包含其他治疗剂，所述其他治疗剂包括，但不限于化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物。在一些实施方案中，所述套装药盒中的化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物，与第五方面的抗体药物偶联物相同或不同。
在一些实施方案中，上述第一药盒和第二药盒可以同时使用，也可以先使用上述第一药盒再使用上述第二药盒，还可以先使用上述第二药盒再使用上述第一药盒，可以根据具体应用时的实际需求而定。
第二十三方面，本发明提供了一种套装药盒，该套装药盒包括一个或多个药盒，包含第十三方面所述的特异性结合蛋白、第十七方面的抗体药物偶联物或第十九方面所述的药物组合物。
在一些实施方案中，所述套装药盒包括第一药盒，所述第一药盒包括第十三方面所述的第一结构域和第二结构域组成的双特异性结合蛋白、第十七方面的抗体药物偶联物或第十九方面所述的药物组合物。
在一些实施方案中，所述套装药盒可以进一步包括第二药盒，所述第二药盒包括其他治疗剂，所述其他治疗剂包括，但不限于化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物。在一些实施方案中，所述套装药盒中的化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物，与第十七方面的抗体药物偶联物相同或不同。
在一些实施方案中，上述第一药盒和第二药盒可以同时使用，也可以先使用上述第一药盒再使用上述第二药盒，还可以先使用上述第二药盒再使用上述第一药盒，可以根据具体应用时的实际需求而定。
第二十四方面，本发明提供了给药装置，其包含第一方面的抗原结合蛋白，第五方面的抗体药物偶联物，或第十方面的药物组合物。在一些实施方案中，所述给药装置是预填充注射器。
第二十五方面，本发明提供了给药装置，其包含第十三方面的抗原结合蛋白，第十七方面的抗体药物偶联物，或第十九方面的药物组合物。在一些实施方案中，所述给药装置是预填充注射器。
第二十六方面，本发明提供了预防、治疗和/或诊断免疫性疾病、急性和慢性炎性疾病以及肿瘤疾病的方法，其包括向受试者施用治疗有效量的第一方面的抗原结合蛋白，第五方面的抗体药物偶联物，或第十方面的药物组合物。
第二十七方面，本发明提供了预防、治疗和/或诊断免疫性疾病、急性和慢性炎性疾病以及肿瘤疾病的方法，其包括向受试者施用治疗有效量的第十三方面的抗原结合蛋白，第十七方面的抗体药物偶联物，或第十九方面的药物组合物。
本发明的双特异性结合蛋白具有如下的一种或多种特性：
(a)双特异性结合蛋白能够以高亲和力特异性结合人PD-L1和CD73；
(b)能够抑制CD73的酶活性；
(c)双特异性结合蛋白对PD-1信号途径具有抑制作用；
(d)双特异性结合蛋白能够增强激活T淋巴细胞分泌细胞因子IL-2和IFN-γ；以及
(e)显示体内肿瘤抑制活性。
定义
除非另有定义，否则本发明使用的所有技术术语和科技术语都具有如在本发明所属领域中通常使用的相同含义。出于解释本说明书的目的，将应用以下定义，并且在适当时，以单数形式使用的术语也将包括复数形式，反之亦然。
“约”和“大约”通常意指鉴于测量的性质或精度，所测量值的可接受误差范围。通常误差范围在所给出的值或值范围的20％范围内，一般在10％的范围内，甚至更一般在5％的范围内。
术语“抗原结合分子”或“特异性结合蛋白”泛指特异性结合抗原决定簇的分子。抗原结合分子或特异性结合蛋白包括例如抗体、抗体片段和骨架抗原结合蛋白。
本发明的术语“抗体”涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、单特异性和多特异性抗体(例如，双特异性抗体或三特异性抗体)，单链分子以及抗体片段，只要表现出所需的抗原结合活性即可。
本发明的术语“单克隆抗体”是指从基本上同质的抗体群获得的抗体，即，除了可能微量存在的变异抗体(例如含有天然存在的突变或在单克隆抗体制剂的生产过程中产生的，通常以少量存在)之外，所述抗体群所包含的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同抗原决定簇(表位)的不同抗体的多克隆抗体制剂不同，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单个决定簇。
本发明的术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区(VH)和轻链可变区(VL)的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双特异性抗体(diabodies)、和三抗体(triabodies)、共价或非共价连接在一起的抗体片段等。
本发明的术语“双特异性结合蛋白”或“双特异性抗体”是指能够特异性结合至少两种不同的抗原决定簇，例如各自由一对抗体重链可变结构域(VH)和抗体轻链可变结构域(VL)形成的两个结合位点与不同抗原或同一抗原上的不同表位结合。双特异性抗体可以是1+1形式，2+1形式(包含第一抗原或表位的两个结合位点和第二抗原或表位的一个结合位点)或2+2形式(包含第一抗原或表位的两个结合位点和第二抗原或表位的两个结合位点)。通常，双特异性抗体包含两个抗原结合位点，每个抗原结合位点特异性针对不同的抗原决定簇。
本发明的术语“价”表示抗原结合分子存在指定数目的结合结构域。因此，术语“二价”“四价”和“六价”分别表示抗原结合分子中存在两个结合结构域、四个结合结构域和六个结合结构域。所述双特异性抗体至少是“二价的”，并且可以是“三价的”，“四价的”或“更多价的”)。在一些情况下，所述抗体具有两个或更多个结合位点并且是双特异性的。也就是说，即使在存在多于两个结合位点(即抗体是三价的或多价的)的情况下，抗体也可以是双特异性的。
本发明的术语“全长抗体”和“完整抗体”在本文中可互换使用，指代与天然抗体结构基本上相似的抗体。“天然抗体”是指天然存在的免疫球蛋白分子。例如，天然IgG类抗体是约150,000道尔顿的异四聚体糖蛋白，由二硫键键合的两条轻链和两条重链组成。从N末端到C末端，每条重链具有可变区(VH)(也称为可变重链结构域或重链可变结构域)和三个恒定结构域(CH1、CH2和CH3)(也称为重链恒定区)。从N末端到C末端，每条轻链具有可变区(VL)(也称为可变轻链结构域或轻链可变结构域)和轻链恒定结构域(CL)(也称为轻链恒定区)。抗体的重链可以是五种类型中的一种，所述五种类型为α(IgA)、δ(IgD)、ε(IgE)、γ(IgG)或μ(IgM)，还可以进一步分为亚型，例如γ1(IgG1)、γ2(IgG2)、γ3(IgG3)、γ4(IgG4)、α1(IgA1)和α2(IgA2)。抗体的轻链基于其恒定结构域的氨基酸序列，可以是两种类型中的一种，所述两种类型为κ轻链和λ轻链。
在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。 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(C1q)的结合。
“抗体片段”包含完整抗体的一部分。抗体片段的示例包括但不限于Fab、Fab’、F(ab’) 2和Fv；双体抗体、三体抗体、四体抗体、交叉Fab片段；线性抗体；单链抗体分子(例如scFv)；由抗体片段和单结构域抗体(单域抗体)形成的多特异性抗体。
本发明的术语“抗原结合结构域”或“抗原结合位点”是指抗原结合分子中特异性结合抗原决定簇的部分。更具体地，术语“抗原结合结构域”是指抗体的一部分，所述部分包含与抗原的一部分或全部特异性结合并互补的区域。在抗原分子很大的情况下，抗原结合分子可以仅结合抗原的特定部分，该部分称为表位。抗原结合结构域可以由例如一个或多个可变结构域(也称为可变区)提供。优选地，抗原结合结构域包含抗体轻链可变区(VL)和抗体重链可变区(VH)。在一个方面，抗原结合结构域能够结合其抗原并阻断或部分阻断所述抗原的功能。
本发明的术语“抗原决定簇”与“抗原”和“表位”同义，并且是指多肽大分子上的位点(例如一段连续的氨基酸或由非连续氨基酸的不同区域组成的构象构型)，抗原结合部分与所述位点结合，从而形成抗原结合部分-抗原复合物。抗原决定簇可以存在于例如肿瘤细胞的表面、微生物感染细胞的表面、其他患病细胞的表面、免疫细胞的表面、血清中游离物和/或细胞外基质(ECM)中。除非另有说明，本发明中用作抗原的蛋白可以是任何脊椎动物来源的任何天然形式的蛋白质，所述脊椎动物来源包括诸如灵长类动物(例如人类)和啮齿类动物(例如小鼠和大鼠)等哺乳动物。抗原也可以是人蛋白质，或者抗原为“全长”、未加工的蛋白质，以及由细胞内加工而产生的任何形式的蛋白质，或者为天然存在的蛋白质变体，例如剪接变体或等位基因变体。
“特异性结合”是指对于抗原具有结合选择性，并且可以与不需要的或非特异性的结合区分开来。抗原结合分子与特定抗原结合的能力可以通过酶联免疫吸附测定(ELISA)或本领域技术人员熟悉的其他技术(例如表面等离子体共振(SPR)技术以及传统的结合测定进行测量。在一个实施例中，例如通过SPR所测得的，抗原结合分子与不相关蛋白的结合程度小于所述抗原结合分子与抗原的结合程度的约10％。在某些实施例中，与抗原结合的分子的解离常数(Kd)为≤1μM、≤100nM、≤10nM、≤1nM、≤0.1nM、≤0.01nM或≤0.001nM(例如10 -7M或更低，例如10 -7M至10 -13M，例如10- 9M至10 -13M)。
“亲和力”或“结合亲和力”是指分子(例如抗体)的单个结合位点与其结合配体(例如抗原)之间的非共价相互作用的强度。结合亲和力通常可以用解离常数(Kd)表示，解离常数(Kd)是解离速率常数与缔合速率常数(分别为K off和K on)的比率。因此，等效亲和力可以包括不同的速率常数，只要速率常数的比率保持相同即可。亲和力可以通过本领域已知的常规方法测量，例如表面等离子体共振(SPR)。
本发明的术语“高亲和力”是指抗体对靶抗原的Kd为10- 9M或更低，甚至10 -10M或更低。本发明的术语“低亲和力”是指抗体的Kd为10 -8M或更高。
“亲和力成熟的”的抗体是指在一个或多个高变区(HVR)中具有一个或多个修饰的抗体，与不具有此类修饰的亲本抗体相比，此类修饰导致了抗体对抗原的亲和力的改善。
本发明的术语“单域抗体”和“纳米抗体”具有相同的含义，指仅具有抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。
本发明的术语“重链抗体”也称为HCAb抗体，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，具体来说包含重链可变结构域和Fc恒定结构域。
特异性结合蛋白包含第一结构域和第二结构域，所述第一结构域结合CD73或其片段，并且所述第二结构域结合PD-L1或其片段。
术语“包含特异性结合CD73的第一结构域和特异性结合PD-L1的第二结构域的双特异性抗体”“特异性结合CD73和PD-L1的双特异性抗体”“特异于CD73和PD-L1的双特异性抗原结合分子”或“抗CD73/抗PD-L1抗体”在本文中可互换使用，并且是指能够以足够的亲和力结合CD73和PD-L1，使得该抗体可用作靶向CD73和PD-L1的诊断和/或治疗剂的双特异性抗体。
本发明的术语“T效应细胞”指具有细胞溶解活性的T细胞(例如，CD4+及CD8+T细胞)以及T辅助(Th)细胞，T效应细胞分泌细胞因子、且激活并引导其他免疫细胞，但不包括调控性T细胞(Treg细胞)。
本发明的术语“调节性T细胞”或“Treg细胞”是指特殊类型的CD4+T细胞，能阻断其它T细胞的应答。Treg细胞特征在于表达CD4，IL-2受体的α亚基(CD25)和转录因子FOXP3，并且在诱导和维持外周自体耐受中发挥至关重要作用，所述耐受针对肿瘤表达的抗原。
本发明的术语术语“CD73”，即分化簇73，也称为5’-外切核苷酸酶，通常是指能够将细胞外核苷5‘单磷酸转化为核苷，即单磷酸腺苷(AMP)转化为腺苷的酶(核苷酸酶)。CD73是一种由糖基磷脂酰肌醇(GPI)连接的细胞表面酶，CD73广泛表达于人体内皮细胞、淋巴细胞，如Treg，DC，MDSC，NK等细胞的表面。CD73也可在癌细胞上表达，包括结肠癌、肺癌、胰腺癌、卵巢癌、膀胱癌、白血病、胶质瘤、胶质母细胞瘤、黑色素瘤、甲状腺癌、食道癌、前列腺癌和乳腺癌等。据报道，高CD73表达与多种癌症适应症(例如，肺癌、黑色素瘤、乳腺癌、鳞状头颈癌和结直肠癌)的不良预后相关。CD73的主要功能是将细胞外核苷酸(例如5'-AMP)转化为高度免疫抑制分子腺苷。本发明的术语“CD73”包括细胞天然表达的CD73的任何变体或同工型。CD73或其任何变体和同工型可以从天然表达它们的细胞或组织中分离，或者可以使用本领域中熟知的技术和/或本文所述的技术重组产生。相应地，本文所述的抗原结合蛋白可以与人以外的物种(例如食蟹猴CD73)交叉反应。或者，本发明的针对CD73的抗原结合蛋白，抗体或结构域可以对人CD73具有特异性，并且可以不与其他物种表现出任何交叉反应性。
本发明的术语“CD73抗体”或者“特异性结合CD73和/或其片段的分离的抗原结合蛋白”能够结合CD73，并具有足够的亲和力以使得所述抗体可用作靶向CD73的诊断和/或治疗剂。通常情况下，经放射免疫测定(RIA)或流式细胞术(FACS)或使用生物传感器系统通过表面等离子体共振测定，CD73抗体与不相关的CD73蛋白的结合能力小于所述抗体与CD73的结合能力的约10％。在某些实施方案中，结合CD73的抗体具有≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -8M或更低，例如10 -13M到10 - 8M，例如10 -13M到10 -9M)的解离常数(KD)。
本发明的术语程序性细胞死亡1配体1(PD-L1)，也称为分化簇CD274或B7同系物1(B7-H1)，是调节PD-1受体的活化或抑制的B7家族中的一员。PD-L1的开放阅读框编码290个氨基酸的I型跨膜蛋白，其包括胞外Ig结构域(N端V样结构域和IgC样结构域)、疏水性跨膜结构域和30个氨基酸的细胞质尾区。30个氨基酸胞内(细胞质)结构域不含明显的信号传导，但确实具有用于蛋白激酶C磷酸化的潜在位点。研究表明，在干扰素γ刺激的作用下，PD-L1在免疫细胞上的表达增强。PD-L1还在非免疫细胞上表达，包括胰岛、肝脏的库普弗细胞(Kupffer cells)、脉管内皮和选定上皮，例如气管上皮和肾小管上皮，其中其表达在炎症发作期间增强。在多种肿瘤上还发现增大水平的PD-L1表达，所述肿瘤包括但不限于乳癌、卵巢癌、子宫颈癌、结肠癌、结肠直肠癌、包括非小细胞肺癌的肺癌、包括肾细胞癌的肾癌、胃癌、食道癌、膀胱癌、肝细胞癌、头部和颈部鳞状细胞癌(SCCHN)和胰脏癌、黑素瘤和葡萄膜黑素瘤(uveal melanoma)。
本发明的术语“PD-L1抗体”或者“特异性结合PD-L1和/或其片段的分离的抗原结合蛋白”能够结合PD-L1，尤其是在细胞表面上表达的PD-L1多肽，并具有足够的亲和力以使得所述抗体可用作靶向PD-L1的诊断和/或治疗剂。通常情况下，经放射免疫测定(RIA)或流式细胞术(FACS)或使用生物传感器系统通过表面等离子体共振测定，PD-L1抗体与不相关的非PD-L1蛋白的结合能力小于所述抗体与PD-L1的结合能力的约10％。在某些实施方案中，结合人PD-L1的抗原结合蛋白的KD值≤1μM、≤100nM、≤10nM、≤1nM、≤0.1nM、≤0.01nM或≤0.001nM(例如10 -8M或更低，例如10 -13M至10 -8M，例如10 -13M至10 -9M)。在一些实施方案中，在表面等离子体共振测定中，使用人PD-L1，尤其是带有组氨酸标签的人PD-L1测定结合亲和力的相应KD值，以获得PD-L1结合亲和力。PD-1/PD-L1抗体的治疗策略是几种转移性肿瘤中的标准治疗策略，并已显示出它们在早期疾病阶段和辅助治疗中的作用，尤其是黑色素瘤和非小细胞肺癌。
本申请中的术语“H2L2转基因小鼠”或“Harbour H2L2小鼠”是一种携带人免疫球蛋白免疫库的转基因小鼠，所述小鼠产生具有完全人可变区的由两条重链和两条轻链(H2L2)组成的传统四聚体抗体，具有完整的人抗体可变结构域和大鼠恒定结构域。由所述转基因小鼠所产生的抗体亲和力成熟，可变区完全人源化，并且具有优异的溶解性。
本申请中的术语“Harbour HCAb小鼠”(WO2002/085945A2)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的恒定结构域。由于不含轻链这一特点，所述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。
本发明的术语“可变区”或“可变结构域”是指参与抗原结合分子与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域(分别为VH和VL)通常具有相似的结构，其中每个结构域包含四个保守框架区(FR)和三个高变区(HVR)。单个VH或VL结构域可足以赋予抗原结合特异性。
本发明的术语“可变”是指可变域的某些区段在抗体之间在序列上普遍不同。V结构域介导抗原结合并限定特定抗体对于其特定抗原的特异性。然而，可变性在整个可变域并非均匀分布的。相反，集中于轻链与重链可变域内三个称为高变区(HVR)的区段中。可变域的更高度保守部分被称作框架区(FR)。天然重链与轻链的可变域各自包含四个FR区，大部分采用β-折叠构型，由三个HVR连接，其形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的HVR通过FR区紧密保持在一起，并且与其它链的HVR一起促成抗体的抗原结合位点的形成(参见Kabat等,Sequences of Immunological Interest,第5版,National Institute of Health,Bethesda,MD(1991))。恒定域不直接参与抗体与抗原的结合，具有其他效应功能，例如参与抗体的抗体依赖性细胞毒性。
本发明的术语“高变区”或“HVR”是指在抗体可变结构域区域中序列上高变和/或形成结构上限定的环(“高变环”)的区域。通常，天然四链抗体包含六个HVR：三个存在于VH中(H1、H2、H3)，三个存在于VL中(L1、L2、L3)。HVR通常包含来自高变环和/或来自“互补决定区(CDR)”的氨基酸残基，来自“互补决定区(CDR)”的氨基酸残基具有最高的序列可变性和/或参与抗原识别。示例性CDR(CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和CDR-H3)发生在氨基酸残基26-32(L1)、50-52(L2)、91-96(L3)、26-32(H1)、53-55(H2)和96-101(H3)处(Chothia等人,J.Mol.Biol.196:901-917(1987)。示例性CDR(CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2和CDR-H3)发生在氨基酸残基24-34(L1)、氨基酸残基50-56(L2)、氨基酸残基89-97(L3)、氨基酸残基31-35(H1)、氨基酸残基50-65(H2)，以及氨基酸残基95-102(H3)处(Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,MD(1991)。作为比较，在下表1中列出了包含上文引用的参考文献中所定义的CDR的相应氨基酸残基。在本申请中，上述所列CDR的氨基酸序列均是按照Chothia定义规则所示出的(本申请的权利要求中也是按照Chothia定义规则所示出的序列)。但是，本领域人员公知，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定义规则和基于结构环区域位置的Chothia定义规则(参见J Mol Biol273:927-48,1997)。在本申请中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
表1：不同编号下本发明的CDR定义在抗体轻链或重链中的位置
CDR\编号体系 Kabat Chothia Combined
LCDR1 24-34 26-32 24-34
LCDR2 50-56 50-52 50-56
LCDR3 89-97 91-96 89-97
HCDR1 31-35 26-32 26-35
HCDR2 50-65 52-58 50-65
HCR3 95-102 95-102 95-102
“框架”或“FR”是指除高变区(HVR)残基之外的可变结构域残基。可变结构域的FR通常由以下四个FR结构域组成：FR1、FR2、FR3和FR4。因此，HVR和FR序列通常在VH(或VL)中以如下序列出现：FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。
抗体的“类别”是指抗体的重链所具有的恒定结构域或恒定区的类型。存在五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些类别中的若干可以进一步分为亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1 和IgA2。对应于不同类别的免疫球蛋白的重链恒定结构域分别称为α、δ、ε、γ和μ。
“人源化抗体”包含来自非人HVR的氨基酸残基和来自人FR的氨基酸残基。在某些实施例中，人源化抗体包含至少一个，通常两个可变结构域，其中所有或基本上所有HVR(例如CDR)对应于非人抗体的HVR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。“人源化形式”的抗体，例如非人抗体，是指已经经历了人源化的抗体。
“人抗体”具有的氨基酸序列与由人或人细胞产生的或来源于利用人抗体库或其他人抗体编码序列的非人来源的抗体的氨基酸序列相对应。人抗体的该定义特别地排除了包含非人抗原结合残基的人源化抗体。
本发明的术语“Fc结构域”或“Fc区”用于定义含有恒定区的至少一部分的抗体重链C末端区域。该术语包括天然序列Fc区和变体Fc区。IgG Fc区包含IgG CH2结构域和IgG CH3结构域。本文的CH2结构域可以是天然序列CH2结构域或变体CH2结构域。本文的CH3区可以是天然序列CH3结构域或变体CH3结构域。CH2结构域可包含一种或多种减少或消除CH2结构域结合一种或多种Fcγ受体(例如FcγRI、FcγRIIa、FcγRIIb、FcγRIII)和/或补体的突变。推测减少或消除与Fc受体γ的结合将减少或消除抗体分子介导的ADCC。类似地，减少或消除与补体的结合预期将减少或消除抗体分子介导的CDC。减少或消除CH2结构域与一种或多种Fcγ受体和/或补体结合的突变是本领域已知的(Wang等，2018)。这些突变包括所谓的LALA突变”，所述突变涉及将CH2域的IMGT位置的1.3和1.2处的亮氨酸残基替换为丙氨酸(L1.3A和L1.2A)。或者，通过将CH2结构域中IMGT位置的84.4位的天冬酰胺(N)突变为丙氨酸、甘氨酸或谷氨酰胺(N84.4A、N84.4G或N84.4Q)，从而将保守的N-链糖基化位点突变产生a-糖基抗体，以降低IgG1效应子功能也是已知的(Wang等，2018)。作为另一选择，已知补体激活(C1q结合)和ADCC可通过将CH2结构域的IMGT位置114位的脯氨酸突变为丙氨酸或甘氨酸(P114A或P114G)而降低(Idusogie等，2000；Klein等，2016)。这些突变可以被组合，以产生具有进一步降低或没有ADCC或CDC活性的抗体分子。
“与免疫球蛋白的Fc区等同的区域”包括免疫球蛋白Fc区的天然存在等位基因的变体，以及具有产生取代、添加或缺失但基本上不降低免疫球蛋白介导的效应子功能(诸如抗体依赖性细胞毒性)的能力的修饰变体。例如，可以使一个或多个氨基酸从免疫球蛋白的Fc区的N末端或C末端缺失，而基本上不丧失生物学功能。可以根据本领域中已知的一般规则来选择此类变体，以便对活性具有最小的影响(参见例如Bowie,J.U.等人，Science 247:1306-10(1990))。
本发明的术语“效应子功能”可归因于抗体的Fc区，是随着抗体同种型的变化而变化的生物活性。抗体效应子功能的示例包括：C1q结合和补体依赖性细胞毒性(CDC)、Fc受体结合、抗体依赖性细胞介导的细胞毒性(ADCC)、抗体依赖性细胞吞噬作用(ADCP)、细胞因子分泌、免疫复合物介导的抗原呈递细胞的抗原摄取、下调细胞表面受体(例如B细胞受体)，以及B细胞激活等。
本发明的术语“肽接头”或者“连接肽”是指包含一个或多个氨基酸，通常为约2至20个氨基酸的肽。肽接头是本领域中已知的或在本文中描述的。
本发明的术语“融合至”，“融合联结”或“连接至”是指区段(例如抗原结合结构域和FC结构域)或者直接地或经由一个或多个肽接头而通过肽键连接。
本发明还涉及本发明的双特异性抗体的氨基酸序列变体。双特异性抗体的氨基酸序列变体可以通过向编码分子的核苷酸序列中引入适当的修饰或通过肽合成来制备。这类修饰包括例如抗体氨基酸序列中残基的缺失、插入和/或取代。可以进行缺失、插入和取代的任何组合获得最终的构建体，最终构建体具有所需的特性，例如抗原结合活性。用于取代的位点通常包括HVR和框架(FR)。参见下表2中氨基酸的可能取代。
表2：保守氨基酸取代
氨基酸残基 保守取代 优选保守取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp；Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；Nle Leu
Leu(L) Nle；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；Nle Leu
本发明的术语“多核苷酸”或“核酸”或“核苷酸序列”指分离的核酸分子或构建物，例如信使RNA(mRNA)，病毒衍生的RNA或质粒DNA(pDNA)。多核苷酸可包含常规的磷酸二酯键或非常规键(例如酰胺键,诸如在肽核酸(PNA)中找到的)。术语“核酸分子”指多核苷酸中存在的任一个或多个核酸区段，例如DNA或RNA片段。
本发明的术语“分离的”核酸分子或多核苷酸是指已经与其天然环境分隔开的核酸分子，DNA或RNA。在本发明中，载体中包含的编码多肽的重组多核苷酸也是分离的。分离的多核苷酸的其他实例包括在异源宿主细胞中的重组多核苷酸或溶液中纯化的多核苷酸。分离的多核苷酸包括通常是含有该多核苷酸分子的细胞中所含有的多核苷酸分子,但是该多核苷酸分子存在于染色体外或与其天然染色体位置不同的染色体位置。分离的RNA分子包括本发明的体内或体外RNA转录物，为正和负链形式，和双链形式。本发明的分离的多核苷酸或核酸进一步包括合成生成的此类分子。另外，多核苷酸或核酸可以为或可以包括调节元件,诸如启动子,核糖体结合位点,或转录终止子。
本发明的术语“表达盒”指重组或合成生成的多核苷酸，具有允许特定核酸在靶细胞中转录的一系列核酸元件。重组表达盒可导入质粒，染色体，线粒体DNA，质体DNA，病毒或核酸片段。典型地，在其它序列以外，表达载体的重组表达盒部分包括要转录的核酸序列和启动子。在某些实施方案中，本发明的表达盒包含编码本发明的双特异性抗原结合分子或其片段的多核苷酸序列。
本发明的术语“载体”或“表达载体”与“表达构建物”可以互换使用，将与其可操作连接的特定基因导入靶细胞并指导表达的DNA分子。所述载体包括作为自身复制性核酸结构的载体以及并入其已经导入的宿主细胞的基因组的载体。本发明的表达载体包含表达盒。表达载体可以进行大量稳定mRNA的转录。一旦表达载体在靶细胞内，则由细胞转录和/或翻译机制生成由该基因编码的核糖核酸分子或蛋白质。在一个实施方案中，本发明的表达载体包括含有编码本发明的双特异性抗原结合分子或其片段的多核苷酸序列的表达盒。
术语“宿主细胞”，“宿主细胞系”和“宿主细胞培养物”可互换使用，是指其中已经导入外源核酸的细胞，还包括这类细胞的后代。宿主细胞包括“转化体/转化子”和“转化细胞”，包括原代转化细胞以及由其衍生的后代。后代的核酸与亲本细胞可以不完全一致，可含有突变。宿主细胞是可用于生成本发明的双特异性抗原结合分子的任何类型的细胞。宿主细胞包括培养的细胞，例如培养的哺乳动物细胞，诸如CHO细胞，HEK293细胞，BHK细胞，NS0细胞，SP2/0细胞，YO骨髓瘤细胞，P3X63小鼠骨髓瘤细胞，PER细胞，PER.C6细胞或杂交瘤细胞，酵母细胞，昆虫细胞和植物细胞，还包括转基因动物，转基因植物或培养的植物或动物组织内所包含的细胞。
胚系化回复突变是指将抗体的超体细胞突变回复突变为相应胚系序列的过程。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。
术语“翻译后修饰(PTM)”是指蛋白质或多肽氨基酸链在细胞中翻译合成后，引入化学修饰的过程。对于抗体而言，一些PTM的位点是非常保守的，例如，在人的IgG1抗体的恒定结构域的第297位(EU编号)的保守氨基酸天冬酰胺Asn通常会发生糖基化修饰形成糖链，而该糖链结构对于抗体结构和相关的效应子功能是至关重要的。但是，如果在抗体的可变结构域尤其是抗原结合区域(如CDR)中存在PTM，那么这些PTM的存在有可能会对抗原的结合有较大的影响，也可能对抗体的物理化学性质带来变化。例如，糖基化、脱酰胺、异构化、氧化等都可能增加抗体分子的不稳定性或异质性，从而增加抗体开发的难度和风险。因而去除PTM以降低序列不稳定性的过程，从而避免一些潜在的PTM对于治疗性抗体的开发是非常重要的。随着经验的积累，人们发现一些PTM与氨基酸序列的组成，尤其与相邻氨基酸组成的“模式”是高度相关的，这样使得可以从蛋白质的一级氨基酸序列预测出潜在的PTM。例如，N-x-S/T(第一位是天冬酰胺，第二位是非脯氨酸以外的任意氨基酸，第三位是丝氨酸或者苏氨酸)的序列模式预测出N-连接糖基化位点。引起PTM的氨基酸序列模式有可能来源于胚系基因序列，例如人胚系基因片段IGHV3-33天然地在FR3区域存在糖基化模式NST；也可能来源于体细胞高频突变。可以通过氨基酸突变来破坏PTM的氨基酸序列模式，从而降低或者去除特定PTM的形成。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。实际操作中，对同一个PTM序列模式可能采用多种突变设计方法。
术语“抗体药物偶联物”或“ADC”指与一个或多个化学药物化学连接的结合蛋白(如抗体或其抗原结合片段)。在优选的实施方案中，ADC包括结合蛋白，药物，和连接所述结合蛋白与药物的接头。
术语“嵌合抗原受体”或“CAR”指具有期望抗原特异性和信号传导结构域，以在抗原结合时传播细胞内信号的受体。例如，T淋巴细胞经由T细胞受体(TCR)与由I类或II类主要组织相容性复合物(MHC)分子呈递的短肽的相互作用来识别特异性抗原。对于初始活化和克隆扩增来说，初始T细胞依赖于提供额外共刺激信号的抗原呈递细胞(APC)。在一些实施方案中，可将单核细胞和巨噬细胞工程化以例如表达嵌合抗原受体(CAR)。修饰细胞可募集至肿瘤微环境，在此其通过浸润肿瘤并杀伤靶标癌细胞而用作强力免疫效应物。CAR可包括抗原结合结构域、跨膜结构域和细胞内结构域。抗原结合结构域结合至靶细胞上的抗原。可用作结合至CAR的抗原结合结构域的抗原的细胞表面标志物的实例包括与病毒、细菌、寄生虫感染、自身免疫疾病和癌细胞相关者(例如肿瘤抗原)。
术语“修饰的免疫细胞”指免疫细胞已经被基因修饰，以表达CAR。在一些实施方案中，所述免疫细胞是T细胞，或由其衍生的细胞。在一些实施方案中，所述免疫细胞是自然杀伤(NK)细胞，或由其衍生的细胞。在一些实施方案中，所述免疫细胞是B细胞，或由其衍生的细胞。在一些实施方案中，所述免疫细胞是B细胞，或由其衍生的细胞。在一些实施方案中，所述免疫细胞是单核细胞或巨噬细胞，或由其衍生的细胞。
术语“套装药盒”指一种或多种活性成分存在于多以一个单位的组合。套装药盒中存在多种活性成分时，各活性成分可以同时或顺序给药。
术语“给药装置”指用于向受试者给药的任何工具。
术语“预填充注射器(prefilled syringe)”是指在注射器的操作者接近或使用之前已经装载有药物的注射器。预填充注射器可以是任何材料(例如，玻璃、塑料或金属)。在一些实施方案中，预填充注射器是玻璃注射器。
药物的“有效量”指在接受其施用的细胞或组织中导致生理变化所必需的量。“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用的严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
依照本发明的双特异性抗体具有协同效应。“协同效应”是指两种药物的组合效果大于它们单独的效果之 和且在统计学上不同于对照和单一药物。本发明的叠加效应是指两种药物的组合效果是它们单独的效果之和且在统计学上不同于对照和/或单一药物。
药物(例如药用组合物)的“治疗有效量”指在剂量和给药间隔和时间上有效实现想要的治疗或预防效果必需的量。例如，治疗有效量的药物消除、减轻/减少、延迟、最小化或预防疾病的不利影响。
术语“个体”或“受试者”是指哺乳动物。哺乳动物包括但不限于驯养的动物(例如牛，绵羊，猫，犬和马)，灵长类动物(例如人和非人灵长类，诸如猴)，家兔和啮齿类动物(例如小鼠和大鼠)。具体地，个体或受试者是人。
术语“药物组合物”表示含有一种或多种本公开的抗体或其抗原结合片段与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体或赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学可接受的载体”指药用组合物中除了活性组分以外,对受试者无毒的组分。药学可接受赋形剂包括但不限于缓冲剂，稳定剂和/或防腐剂。
术语“癌症”是指描述哺乳动物中以细胞生长不受调节为特征的疾患。癌症的例子包括但不限于肿瘤，淋巴瘤、母细胞瘤、肉瘤和白血病或淋巴样恶性肿瘤。癌症更具体的实例包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、腺癌和肺的鳞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌和胃肠基质癌)、骨癌、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、尿道癌、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫颈癌、唾液腺癌、肾癌或输尿管癌、前列腺癌、阴道癌、外阴癌、甲状腺癌、肛门癌、阴茎癌、黑素瘤、胆管癌、中枢神经系统(CNS)肿瘤、脊椎轴肿瘤、脑干胶质瘤、多形性成胶质细胞瘤、星形细胞瘤、神经鞘瘤、室管膜瘤、成髓细胞瘤、脑膜瘤、鳞状细胞癌、垂体腺瘤和尤文氏肉瘤、浅表扩散性黑素瘤、恶性雀斑样痣黑素瘤、肢端黑素瘤、结节性黑素瘤、多发性骨髓瘤和B细胞淋巴瘤、慢性淋巴细胞白血病(CLL)、急性成淋巴细胞性白血病(ALL)、毛细胞性白血病、慢性成髓细胞性白血病和移植后淋巴增殖性疾病(PTLD)、以及与瘢痣病(phakomatoses)、水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖、脑瘤和脑癌、以及头或颈癌和相关的转移癌。
术语“治疗”是指给予患者内用或外用治疗剂，例如包含本公开的任一种抗体或其抗原结合片段的组合物或编码抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病或症状，所述治疗剂对这些疾病或症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病或症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。
术语“预防癌症”是指在哺乳动物中延迟、抑制或防止癌症发作，所述哺乳动物中癌发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定其具有癌症易感性。该术语还包括治疗具有癌变前病症的哺乳动物以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。
本申请的抗体的序列编号(SEQ ID NO:)见下表3。
表3：
Figure PCTCN2022094703-appb-000001
Figure PCTCN2022094703-appb-000002
本申请的双抗的序列编号(SEQ ID NO:)见下表4。
表4：
蛋白编号 多肽链1 多肽链2
PR003569 122 119
PR003739 123 120
PR004295 124 121
PR006268 125 119
PR006269 126 119
PR006270 127 119
PR006271 128 119
PR006663 129 119
PR006667 132 131
PR006664 130 119
PR006668 133 131
附图说明
图1A、图1B显示本申请所述PD-L1抗原结合蛋白结合过表达人PD-L1的CHO-K1细胞的结果。图1C显示本申请所述PR000960翻译后修饰(PTM)变体结合过表达人PD-L1的CHO-K1细胞的结果。图1D、图1E、图1F显示本申请所述PR002082亲和力成熟变体结合过表达人PD-L1的CHO-K1细胞的结果。
图2A显示本申请所述PD-L1抗原结合蛋白结合过表达食蟹猴PD-L1的CHO-K1细胞的结果。图2B显示本申请所述PR000960翻译后修饰(PTM)变体结合过表达食蟹猴PD-L1的CHO-K1细胞的结果。图2C显示本申请所述PR002082亲和力成熟变体结合过表达猴PD-L1的CHO-K1细胞的结果。
图3A显示本申请所述PD-L1抗原结合蛋白对PD-1信号通路的抑制作用。图3B显示本申请所述PR000960翻译后修饰(PTM)变体对PD-1信号通路的抑制作用。图3C、图3D显示本申请所述PR002082亲和力成熟变体对PD-1信号通路的抑制作用。
图4显示在供体对#1的MLR实验中本申请所述PD-L1抗原结合蛋白增强激活T淋巴细胞分泌细胞因子IL-2(图4A)和IFN-γ(图4B)的结果。
图5显示在供体对#2的MLR实验中本申请所述PD-L1抗原结合蛋白增强激活T淋巴细胞分泌细胞因子IL-2(图5A)和IFN-γ(图5B)的结果。
图6显示在供体对#3的MLR实验中本申请所述PR000960翻译后修饰(PTM)变体增强激活T淋巴细胞分泌细胞因子IL-2(图6A)和IFN-γ(图6B)的结果。
图7显示在供体对#4的MLR实验中本申请所述PR002082亲和力成熟变体增强激活T淋巴细胞分泌细胞因子IL-2(图7A)和IFN-γ(图7B)的结果。
图8显示在供体对#5的MLR实验中本申请所述PR002082亲和力成熟变体增强激活T淋巴细胞分泌细 胞因子IL-2(图8A)和IFN-γ(图8B)的结果。
图9显示在供体对#6的MLR实验中本申请所述PR002082亲和力成熟变体增强激活T淋巴细胞分泌细胞因子IL-2(图9A)和IFN-γ(图9B)的结果。
图10显示在供体对#7的MLR实验中本申请所述PR002082亲和力成熟变体增强激活T淋巴细胞分泌细胞因子IL-2(图10A)和IFN-γ(图10B)的结果。
图11显示在供体对#8的MLR实验中本申请所述PR002082亲和力成熟变体增强激活T淋巴细胞分泌细胞因子IL-2(图11A)和IFN-γ(图11B)的结果。
图12显示在供体对#9的MLR实验中本申请所述PR002082亲和力成熟变体增强激活T淋巴细胞分泌细胞因子IL-2(图12A)和IFN-γ(图12B)的结果。
图13A显示NCG小鼠重建人PBMC免疫系统的A375肿瘤模型的体内抗瘤效果。图13B显示在实验过程中各组动物体重改变的结果。
图14显示本发明的双特异性分子的结构示意图。
图15显示抗体与稳定表达人CD73和人PD-L1的CHO-K1细胞的结合。
图16显示抗体抑制CD73酶活性的实验结果。
图17显示利用报告基因细胞系检测抗体对PD-1/PD-L1信号通路的抑制作用。
图18显示MLR法体外检测融合蛋白对T细胞的激活作用。
具体实施方式
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.和Maniais，T.(1989)MolecuLar Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例
实施例1.PD-L1抗原结合蛋白的制备
为获得针对PD-L1特异性结合的抗体分子，通常可以利用PD-L1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，但是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 HCAb小鼠(Harbour Antibodies BV，WO 2002/085945 A3)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。
1.1 PD-L1免疫HCAb小鼠
用可溶的重组人PD-L1-mFc融合蛋白(Novo Protein Inc.#CM06，批号0330837)对6～8周龄Harbour HCAb人源抗体转基因小鼠进行多轮免疫。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100μL。在首轮免疫中，每只小鼠接受用50μg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天、第14天、第28天、第42天、第56天、第70天；并且在第49天、第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 25μg抗原蛋白的剂量进行最后一次增强免疫。
1.2获得抗PD-L1的HCAb抗体序列
当检测小鼠血清中PD-L1特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用BD FACS AriaII Cell Sorter分选CD138阳性的浆细胞和人PD-L1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’-GGTGTCCA GTGTSAGGTGCAGCTG-3’(SEQ ID NO:134)，5’-AATCCCTGGGCACTGAAGAGACGG TGACC-3’(SEQ ID NO:135)。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)，以表达获得的HCAb抗体。检测表达HCAb的上清与过表达人PD-L1的CHO-K1/hPD-L1(GenScript，#M00543)稳定细胞系的结合，同时用阳性抗体PR000151(Atezolizumab类似物，申请人根据Atezolizumab序列合成表达)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-L1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-L1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 PCTCN2022094703-appb-000003
488 AffiniPure Goat Anti-Human IgG,FcγFragment Specific二抗(Jackson ImmunoResearch，#109-545-098)，取每孔30μL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-L1细胞的结合，以及和食蟹猴PD-L1-his蛋白(Acrobiosystems，#PD-1-C52H4)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。
1.3重组全人源抗体的制备
上述实施例1.2中得到编码抗体分子的重链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人的抗体重链恒定结构域序列进行融合表达，得到重组抗体分子。
将编码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)中扩培。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。将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。
1.4 PD-L1全人重组抗体的序列
表5列出了本实施例中PD-L1抗体的重链可变结构域氨基酸序列，重链全长氨基酸序列和根据Chothia定义规则定义的CDR的氨基酸序列。同时，本申请的抗PD-L1的阳性对照抗体Atezolizumab类似物，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:97，抗体轻链序列SEQ ID NO:118。
表5抗PD-L1 HCAb抗体的序列编号(SEQ ID NO:)
Figure PCTCN2022094703-appb-000004
1.5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×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。
PD-L1抗体的产量和纯度分析结果见表6。
表6：PD-L1抗体的产量和纯度分析
Figure PCTCN2022094703-appb-000005
实施例2抗体序列的优化
2.1 PTM位点的去除
将实施例1中的抗体序列进行分析，其重链可变结构域(VH)的胚系基因V基因片段，以及可变结构域VH预测的PTM，列于表7。
表7抗PD-L1 HCAb抗体的序列胚系基因分析
Figure PCTCN2022094703-appb-000006
表8列出了对自实施例1的具有潜在PTM位点的PR000960抗体的序列，进行氨基酸突变得到的新的抗体分子(称为PTM变体)。表9列出了PR000960的PTM变体的CDR、VH和HC的氨基酸序列，和根据Chothia定义规则定义的CDR的氨基酸序列。所有设计出来的PTM变体按照实施例1.3中描述的方法得到纯化的重组抗体，并在后续的功能实验中进一步验证。PR000960的PTM变体的产量和纯度分析结果见表10。
表8PR000960的突变位点设计
Figure PCTCN2022094703-appb-000007
表9 PR000960的PTM变体的CDR、VH和HC的氨基酸序列
Figure PCTCN2022094703-appb-000008
表10：PR000960翻译后修饰(PTM)变体的产量和纯度分析
Figure PCTCN2022094703-appb-000009
2.2优化HCAb抗体序列提高结合PD-L1的亲和力
通过对CDR区域进行随机突变的方法建立酵母展示抗体突变库，借助于BD FACS AriaIII分选平台对PR002082分子进行亲和力的改造。此亲和力成熟的方法分为三轮。
首先，对母本分子PR002082的三个CDRs随机引入突变，建立3CDRs(CDR1、CDR2、CDR3)酵母展示突变库；然后用MACS分选方法进行富集；随后用流式FACS进行多轮分选，富集高亲和力的突变分子。在本实施例中，抗体可变结构域的CDR序列通过Chothia规则进行分析。
第一轮，用0.1nM生物素化的PD-L1-his分选出结合力更高的酵母细胞；然后继续培养诱导。
第二轮，用0.01nM生物素化的PD-L1-his进一步分选出结合力更高的酵母细胞；然后继续培养诱导。
第三轮，继续降低分选时抗原的浓度，用0.003nM生物素化的PD-L1-his进行最后一轮分选。
最后，将前面三轮分选到的分子测序，找到突变位点进行随机组合，建立组合突变库。然后用FACS继续以更低的抗原浓度分选出更高亲和力突变分子。在本实施例中，经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，以编码产生重组HCAb单域抗体分子。
得到PR002082变体(均为单域抗体)的序列如下表11所示。
表11：PR002082变体的序列编号表(SEQ ID NO:)
Figure PCTCN2022094703-appb-000010
Figure PCTCN2022094703-appb-000011
2.3 PR002082亲和力变体的生产和纯化
上述实施例2.2中得到编码PR002082亲和力变体可变结构域的序列以后，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。
具体说来，将ExpiCHO-S TM细胞(Gibco,#A29127)在ExpiCHO TM Expression 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)。立即将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。PR002082亲和力成熟变体的制备见表12。
表12：PR002082亲和力成熟变体的制备
Figure PCTCN2022094703-appb-000012
Figure PCTCN2022094703-appb-000013
实施例3抗原结合蛋白与过表达人/食蟹猴PD-L1的细胞的结合(FACS)
本实施例为了研究PD-L1抗原结合蛋白在体外结合人/食蟹猴PD-L1的活性，采用过表达人PD-L1的CHO-K1细胞株(CHO-K1/hPD-L1，南京金斯瑞，M00543)或过表达食蟹猴PD-L1的CHO-K1细胞株(CHO-K1/cynoPD-L1，南京金斯瑞，M00573)进行细胞水平的结合实验。简言之，消化PD-L1细胞，并用F-12K完全培养基重悬，将细胞密度调整为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，2倍于终浓度的5倍浓度梯度稀释的待测抗原结合蛋白，混合均匀，其中抗原结合蛋白最高终浓度为100nM，共8个浓度，同型抗体hIgG1iso作为对照。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g 4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(山羊抗人IgG(H+L)第二抗体，Alexa Fluor 488 conjugate,Invitrogen,A11013,1:1000稀释)，4℃，避光孵育30分钟。用200μL/孔预冷PBS洗涤细胞两次，于500g 4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞。使用BD FACS CANTOII读取荧光发光信号值。使用流式细胞仪BD FACS CANTOII或FACS ACEA读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到结合曲线及EC 50值等参数。
图1(A、B)结果显示，本申请所述PD-L1抗原结合蛋白均能结合过表达人PD-L1的CHO-K1细胞。
图1(C)结果显示，本申请所述PR000960翻译后修饰(PTM)变体均能结合过表达人PD-L1的CHO-K1细胞。
图1(D、E、F)结果显示，本申请所述PR002082亲和力成熟变体均能结合过表达人PD-L1的CHO-K1细胞，且作用均强于亲本序列抗体PR002082。
图2(A)结果显示，本申请所述PD-L1抗原结合蛋白均能结合过表达食蟹猴PD-L1的CHO-K1细胞。
图2(B)结果显示，本申请所述PR000960翻译后修饰(PTM)变体均能结合过表达食蟹猴PD-L1的CHO-K1细胞。
图2(C)结果显示，本申请所述PR002082亲和力成熟变体均能结合过表达猴PD-L1的CHO-K1细胞，且作用均强于亲本序列抗体PR002082。
实施例4.利用BLI方法测定抗原结合蛋白与人PD-L1的亲和力
该实施例利用Octet Red96e仪器对PR002082亲和力成熟突变体进行了亲和力的测定。具体方法是，带有组氨酸标签的人PD-L1蛋白购自厂家ACROBiosystems(#PD-1-H5258，测试缓冲液为1×动力学缓冲液(从10×动力学缓冲液(ForteBio，#18-1105)稀释)，用于亲和力测试以及抗原、抗体的稀释。通过生物膜干涉(BLI)技术，使用Octet分子相互作用分析仪(ForteBio，型号Octet Red96e)，来进行抗原抗体之间的结合动力学分析。
测定抗原和抗体的亲和力时，传感器转动速度为1000转/分钟。先将置于一列的2个HIS1K传感器在测试缓冲液中平衡10分钟，然后用HIS1K传感器捕获His PD-L1，捕获高度0.9-1.1nm，然后将HIS1K传感器在测试缓冲液中平衡2分钟，然后将HIS1K传感器与抗原结合蛋白(60nM以及0nM)结合3分钟，最后解离15分钟。将HIS1K传感器浸入10mM甘氨酸(pH 1.5)溶液进行再生，以洗脱结合在传感器上的蛋白。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，选择单扣除模式(reference well)扣除参照信号，选择“1:1 Local/full fitting”方法进行数据拟合，计算出抗原与抗体结合的动力学参数，得到K on(1/Ms)值、K dis(1/s)值和KD(M)值。
结果如表13所示，PR02082的亲和力成熟变体与PD-L1均能结合，且多数变体的亲和力较PR002082有明显提高。
表13：PR002082亲和力成熟变体与人PD-L1蛋白的亲和力
Figure PCTCN2022094703-appb-000014
实施例5.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
将表达PD-L1和OS8(CD3单链抗体跨膜蛋白)的293T(eBioscience)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,#E6110)，室温孵育5分钟，酶标仪检测发光值。结果如图3所示。结果显示本申请所述HCAb抗体对PD-1信号通路有抑制作用。
图3(A)结果显示，本申请所述PD-L1抗原结合蛋白均对PD-1信号通路有抑制作用。
图3(B)结果显示，本申请所述PR000960翻译后修饰(PTM)变体均对PD-1信号通路有抑制作用。
图3(C、D)结果显示，本申请所述PR002082亲和力成熟变体均对PD-1信号通路有抑制作用，且多数变体的抑制作用较PR002082有明显提高。
实施例6.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，分离单核细胞，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)。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10倍或者对应倍数的稀释的各抗原结合蛋白及阴阳性对照抗体。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。
本实施例使用了18个供体的PBMC分成了9组供体配对进行混合淋巴细胞反应(MLR)。
图4和图5结果显示，在两次独立的MLR实验中，本申请所述PD-L1抗原结合蛋白均能增强激活的T淋巴细胞分泌细胞因子IL-2和IFN-γ。
图6结果显示，本申请所述PR000960翻译后修饰(PTM)变体均能增强激活的T淋巴细胞分泌细胞因 子IL-2和IFN-γ。
图7、图8、图9、图10、图11、图12结果显示，在6次独立的MLR实验中，本申请所述PR002082亲和力成熟变体均能增强激活的T淋巴细胞分泌细胞因子IL-2和IFN-γ。
实施例7.抗原结合蛋白的体内抑制肿瘤活性
NCG小鼠重建人PBMC免疫系统的A375肿瘤模型的体内抗瘤效果参见图13A。具体地，溶媒对照组小鼠在给药后第23天平均肿瘤体积为1336mm 3。测试药Atezolizumab(10mg/kg)治疗组在接种后第23天平均肿瘤体积为343mm 3，相较溶媒对照组有显著性差异(p值＝0.002)，肿瘤抑制率TGI(％)为74.33％。测试药PR002079(5mg/kg)治疗组在接种后第23天平均肿瘤体积为728mm 3，相较溶媒对照组有显著性差异(p值＝0.021)，肿瘤抑制率TGI(％)为45.51％。测试药PR002082(5mg/kg)治疗组在接种后第23天平均肿瘤体积为891mm 3，相较溶媒对照组没有显著性差异(p值＝0.139)，肿瘤抑制率TGI(％)为33.31％。
图13B结果显示，在实验过程中，各组动物体重均出现增长，表明动物对受试品耐受良好。未对动物产生明显毒性作用，安全性较好。
实施例8.CD73抗体的获得
CD73抗体的获得参见公开申请WO2021032173A1的说明书中实施例1，实施例2和实施例3的记载。
具体而言，使用Harbour H2L2小鼠，将CD73蛋白用作免疫原以产生抗CD73抗体。通过皮下注射和腹膜内注射，与佐剂一起使用(Sigma，S6322)给每只小鼠第一次加强用50μg蛋白质，随后加强用25μg蛋白质。每两周进行1次这种免疫，共6次。最终免疫是通过经腹膜内注射用PBS稀释的免疫原进行。使用ELISA和FACS测试针对人CD73的血清效价。在指定的时间点，对小鼠血清进行采样并滴定以进行ELISA和FACS分析，以测试与CD73蛋白或CD73稳定细胞系的结合。在蛋白质免疫阵列(protein immunization cohort)中观察到良好的血清效价。
使用细胞融合发生器(BEX-LF301)，通过基于电场的电穿孔器，将从免疫小鼠中分离的小鼠脾细胞与小鼠骨髓瘤细胞系SP2/0(ATCC，CRL-1581)融合，以获得杂交瘤。在融合后9-14天，在各个孔中筛选人抗CD73抗体。杂交瘤亚克隆后，选择对CD73表现出良好结合活性的单克隆38H6进行测序。鉴定并获得重链可变区和轻链可变区。然后合成重链可变区和轻链可变区，并分别克隆到编码人IgG1恒定区的质粒和编码人Igκ区的质粒中。
使用PEI(Polyscience，24885)将编码目标抗体的重组质粒瞬时共转染到HEK293-6E或HEK293-F细胞培养物中。30μg质粒与120μL PEI在室温下混合并孵育15分钟。接下来，将混合物逐滴加入以1×106细胞/毫升的浓度悬浮于Opti-MEM的293细胞中。转染后，将细胞在37℃、5％CO2的条件下，以120rpm摇床培养。在第6-7天收集的细胞培养上清液用于纯化。
在转染后6-7天通过离心和过滤收获含有目标抗体的上清液。使单克隆抗体通过rProtein A(GE，17-1279-02)预装柱(Bio-Rad，7311550)进行纯化。通过将0.2mL rProtein A树脂装填到一个柱中，然后用10柱体积的ddH2O和10柱体积的PBS冲洗来制备Protein A柱。将细胞培养上清液过柱，然后用10倍柱体积的PBS洗涤。然后用8倍柱体积的洗脱缓冲液(Thermo，21004)洗脱蛋白质，并在洗脱后立即与640μL的中和缓冲液(1M Tris-HCl，pH 9.0；Teknova，T1090)进行混合。在浓缩器(Millipore，UFC903024)中，通过3800rpm(Eppendorf，5810R)4℃离心，将缓冲液与DPBS进行500倍以上的交换，最后浓缩至合适的体积。纯化的抗CD73抗体的浓度通过在280nm(NanoDrop)处的UV吸光度确定。通过SEC-HPLC和SDS-PAGE测定抗体纯度。重组抗体PR000506被成功表达并纯化用于表征。
抗CD73抗体的VH和VL序列通过胚系化回复突变和PTM去除操作进行了进一步优化：
在胚系化程序中，抗体VH或VL序列首先通过例如NCBI/Ig-BLAST的算法比对到最接近的人类胚系序列，然后将框架区的具有不同于胚系序列的残基反转为胚系序列中的对应残基。然后通过成熟的分子生物学技术重组产生胚系化后的由序列变体组成的抗体。
在哺乳动物细胞中表达的蛋白质中广泛观察到翻译后修饰(PTM)。除了抗体中保守的PTM位点(例如IgG1抗体CH2结构域上的N-糖基化保守位点)外，在抗体抗原结合位点(即CDR区)内发生的其他PTM位点可能会降低抗原结合活性或降低化学稳定性。例如，脱酰胺或异构化可以使分子变得不稳定和异 质化。为了减少序列不稳定性，可以通过突变去除PTM基序。查找VH或VL序列的PTM基序，例如异构化基序(例如DG)。然后将“热点”残基(例如，DG基序中的D或G)突变为胚系序列中的对应残基或具有类似生物物理特性的其他残基。然后，通过成熟的分子生物学技术重组产生由PTM去除后的序列变体组成的抗体。
获得了以下表14的抗CD73抗体：
表14：
抗体编号 PTM去除 轻链 重链 VL VH
PR000846 去除 119 98 94 73
PR001408 去除 120 103 95 78
PR003836 去除 121 106 96 81
实施例9 CD73/PD-L1双特异性抗体的制备
本实施例利用抗CD73的IgG抗体PR000846、PR001408、PR003836的抗原结合结构域Fab，与抗PD-L1的HCAb抗体PR002082以及亲和力成熟变体PR005263、PR005872、PR005875、PR005878、PR006246、PR006248的抗原结合结构域VH，来构建CD73×PD-L1的双特异性抗体分子。
抗CD73的H2L2抗体的序列编号见表15，抗PD-L1的HCAb抗体的序列编号见表16。
表15：抗CD73的H2L2抗体的序列编号(SEQ ID NO:)
抗体编号 轻链 重链 VL VH
PR000846 119 98 94 73
PR001408 120 103 95 78
PR003836 121 106 96 81
表16：抗PD-L1的HCAb抗体的序列编号(SEQ ID NO:)
抗体编号 重链 VH
PR005263 107 82
PR005872 111 86
PR005875 112 87
PR005878 113 88
PR006246 115 90
PR006248 117 92
PR002082 105 80
图14列出了本申请所包含的双特异性结合蛋白的分子结构，每一个结构会在下文进一步描述。在本实施例以及本发明申请的其他部分，当提及分子结构所含多肽链数目的时候，通常是指“不同的多肽链”的数目； 例如常规IgG抗体有两条不同的多肽链，即重链和轻链，尽管IgG抗体分子本身是含有两条相同的重链和两条相同的轻链的四肽链蛋白分子，但是描述其结构特征时特指其两条不同的多肽链。结合价数是指该分子结构中的抗原结合位点的数目，例如常规IgG抗体能同时结合两个相同的抗原分子，其结合价数为二。
表17列出了本发明申请的结构设计中可能用到的连接肽序列。
表17：连接肽序列表
Figure PCTCN2022094703-appb-000015
Figure PCTCN2022094703-appb-000016
9.1构建IgG_HC-VH四价对称结构的双特异性抗体
利用抗CD73的H2L2抗体和抗PD-L1的HCAb抗体构建IgG-VH四价对称结构的双特异性抗体。IgG_HC-VH四价对称结构(如图14A所示)的结合蛋白包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，也称长链，从氨基末端到羧基末端，其包含VH_A-CH1-h-CH2-CH3-L-VH_B。h是IgG抗体的铰链区或衍生序列。在一个实施方案中，多肽链2的CH3与VH_B直接融合联结，即L的长度为0。在另一个实施方案中，多肽链2的CH3经由连接肽L联结到VH_B；L可以是表17中所列的序列。
9.2构建VH-IgG_HC四价对称结构的双特异性抗体
利用抗CD73的H2L2抗体和抗PD-L1的HCAb抗体构建IgG-VH四价对称结构的双特异性抗体。VH-IgG_HC四价对称结构(如图14B所示)的结合蛋白包含两条多肽链：多肽链1，也称短链，从氨基末端到羧基末端，其包含VL_A-CL；多肽链2，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。h是IgG抗体的铰链区或衍生序列。多肽链2的VH_A经由连接肽L联结到VH_B，连接肽L可以是表17中所列序列。在一个实施方案中，多肽链2的VH_A与VH_B直接融合联结，即L的长度为0。
9.3构建Fab(CL)-VH-Fc结构的双特异性抗体分子
利用抗CD73的H2L2抗体和抗PD-L1的HCAb抗体构建IgG-VH四价对称结构的双特异性抗体。Fab(CL)-VH-Fc对称结构(如图14C所示)的结合蛋白包含两条多肽链，多肽链1，也称短链，从氨基末端到羧基末端，其包含VH_A-CH1；多肽链2，也称长链，从氨基末端到羧基末端，其包含VL_A-CL-L1-VH_B-L2-CH2-CH3。多肽链2的连接肽L1和L2可以是表17中所列序列。在一个实施方案中，多肽链2的CL与VH_B直接融合联结，即L1的长度为0。
CD73×PD-L1双特异性抗体为IgG1，具有Fc突变L234A和L235A或者L234A和L235A和G237A(根据EU索引编号)。
本发明制备得到的CD73×PD-L1双特异性抗体总结在表18；所得双抗分子的多肽链的氨基酸序列的序列编号见表19。所得双抗分子的抗原结合结构域的CDR的序列编号见表20。制备的CD73×PD-L1双抗抗体分子样品理化性质分析总结于表21。
表18：利用抗CD73的IgG抗体和抗PD-L1的重链抗体设计的CD73×PD-L1双抗分子
Figure PCTCN2022094703-appb-000017
Figure PCTCN2022094703-appb-000018
表19：本申请的CD73×PD-L1双抗分子的序列编号
结构编号 抗体编号 多肽链1 多肽链2
2 PR006268 125 119
2 PR006269 126 119
2 PR006270 127 119
2 PR006271 128 119
1 PR006663 129 119
3 PR006667 132 131
1 PR006664 130 119
3 PR006668 133 131
2 PR003569 122 119
2 PR003739 123 120
2 PR004295 124 121
表20：CD73×PD-L1双抗分子的抗原结合结构域的CDR的序列编号
Figure PCTCN2022094703-appb-000019
Figure PCTCN2022094703-appb-000020
表21：CD73×PD-L1双抗分子蛋白的表达和物理化学性质
Figure PCTCN2022094703-appb-000021
实施例10.CD73×PD-L1双特异性抗体与过表达人PD-L1或人CD73的细胞的结合(FACS)
为了研究CD73×PD-L1双特异性抗体与结合人CD73和PD-L1的活性，采用过表达人CD73或PD-L1的CHO-K1细胞株(CHO-K1-hCD73，CHO-K1-hPD-L1)进行细胞水平的结合实验。简言之，消化CHO-K1-hCD73和CHO-K1-hPD-L1细胞，并用F-12K完全培养基重悬，将细胞密度调整为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℃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℃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 PCTCN2022094703-appb-000022
488 conjugate,Invitrogen,#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℃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15结果显示，本申请的抗CD73抗体PR000846和PR001408能够特异性结合过表达人CD73的CHO-K1细胞(图15A)；本申请的CD73×PD-L1双特异性抗体(PR003569和PR003739)能够结合过表达人CD73的CHO-K1细胞(图15A)；本申请的抗CD73抗体PR003836能够特异性结合过表达人CD73的CHO-K1细胞(图15B)；本申请的CD73×PD-L1双特异性抗体PR004295能够结合过表达人CD73的CHO-K1细胞(图15B)；
本申请的抗CD73抗体PR000846能够特异性结合过表达人CD73的CHO-K1细胞(图15C、图15D)；本申请的CD73×PD-L1双特异性抗体(PR006268、PR006269、PR006270、PR006271、PR006661、PR006662、PR006663、PR006664、PR006667、PR006668)能够结合过表达人CD73的CHO-K1细胞(图15C、图15D)；本申请的抗PD-L1抗体PR002082能够结合过表达人PD-L1的CHO-K1细胞(图15E)；本申请的CD73×PD-L1双特异性抗体PR003569、PR003739和PR004295能够结合过表达人PD-L1的CHO-K1细胞(图15E、图15F)。
本申请的抗CD73抗体，抗PD-L1抗体以及CD73×PD-L1双特异性抗体的EC50值在0.3418nM-2.929nM的范围内。图15C中起始浓度为50nM，3倍梯度稀释，共八个浓度；图15D中起始浓度为100nM，3倍梯度稀释，共八个浓度。
实施例11.CD73×PD-L1双特异性抗体抑制CD73的酶活性
利用孔雀绿法测定抗体抑制可溶性重组CD73的酶活性，首先在384孔板(Corning,#3799)中加入12.5μL 1nM重组CD73蛋白和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盐的浓度，无机磷酸盐浓度的测定按照供应商的说明书操作。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。
图16结果显示，本申请的CD73×PD-L1双特异性抗体PR003569、PR003739、PR006268、PR006269、PR006270、PR006271、PR006663、PR006664、PR006667、PR006668能够非竞争性的抑制CD73蛋白酶活性，和亲本单克隆抗体PR000846活性相当。
实施例12.利用报告基因细胞系检测CD73×PD-L1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。在37℃5％CO 2环境下孵育过夜。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。在37℃5％CO 2环境下培养6小时。加入ONE-GloT荧光素酶试剂(Promega,#E6110)，室温孵育5分钟，酶标仪检测发光值。
图17结果显示，利用报告基因细胞系检测本申请的CD73×PD-L1双特异性抗体PR003569，PR003739以及PR004295对PD-1信号通路具有抑制作用。
实施例13.CD73×PD-L1双特异性抗体在混合淋巴细胞反应(MLR)试验中激活T细胞
购买AllCells PBMC细胞，分离CD14+单核细胞，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Pan-T细胞分离试剂盒(Miltenyibiotec,#130-096-535)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，加入稀释的抗原结合蛋白和及阴阳性对照抗体。在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。
图18结果显示，MLR法体外检测本申请的CD73×PD-L1双特异性抗体PR003569，PR003739和PR004295对T细胞具有激活作用。与人同种型IgG相比，单独的抗PD-L1单抗PR002082相比，以及与单独的抗CD73单抗PR000846，PR001408和PR003836相比，以及与抗PD-L1单抗PR002082与抗CD73的单抗PR000846的组合相比，以及与抗PD-L1单抗PR002082与抗CD73的单抗PR003836的组合相比，CD73×PD-L1双特异性抗体PR003569，PR003739和PR004295在10nM的浓度下对于激活T细胞分泌IL-2更显著有效。而且基于CD73×PD-L1双特异性抗体PR003569，PR003739和PR004295与抗PD-L1单抗PR002082与抗CD73的单抗PR000846的组合相比，以及与抗PD-L1单抗PR002082与抗CD73的单抗PR003836的组合相比更显著的效果，可以认为CD73×PD-L1双特异性抗体PR003569，PR003739和PR004295具有协同效果。
类似地，与人同种型IgG相比，与单独的抗PD-L1单抗PR002082相比，以及与单独的抗CD73单抗PR000846，PR001408和PR003836相比，以及与抗PD-L1单抗PR002082与抗CD73的单抗PR000846的组合相比，以及与抗PD-L1单抗PR002082与抗CD73的单抗PR003836的组合相比，CD73×PD-L1双特异性抗体PR003569，PR003739和PR004295在10nM的浓度下对于激活T细胞分泌IFN-γ显著更有效。而且基于CD73×PD-L1双特异性抗体PR003569，PR003739和PR004295与抗PD-L1单抗PR002082与抗CD73的单抗PR000846的组合相比，以及与抗PD-L1单抗PR002082与抗CD73的单抗PR003836的组合相比更显著的效果，可以认为CD73×PD-L1双特异性抗体PR003569，PR003739和PR004295具有协同效果。

Claims (69)

  1. 特异性结合PD-L1和/或其片段的分离的抗原结合蛋白，其包含抗体重链可变区VH，所述VH包含以下的互补决定区或其突变体：
    SEQ ID NO：9的氨基酸序列所示的HCDR1；
    SEQ ID NO：23的氨基酸序列所示的HCDR2；和/或
    SEQ ID NO：36的氨基酸序列所示的HCDR3。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其中，所述突变体为在所述VH的HCDR1、HCDR2、HCDR3的氨基酸序列上分别具有1、2、3或4个氨基酸的插入、缺失或取代，
    优选地，所述HCDR1的氨基酸序列为GFX 1FSX 2Y，
    所述HCDR2的氨基酸序列为X 3YX 4GX 5X 6，
    所述HCDR3的氨基酸序列为NRAX 7FGVX 8PDX 9SDI，
    其中，X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8或X 9为选自N、T、D、S、W、R、K、E、I、A、V和L中的任一种；
    更优选地，X 1为T、D或N，
    X 2为N或S，
    X 3为W或R，
    X 4为D或T，
    X 5为T或S，
    X 6为K、R或E，
    X 7为I或L，
    X 8为V或I，和/或
    X 9为A或D。
  3. 根据权利要求1或2所述的分离的抗原结合蛋白，其中，所述HCDR1的突变体具有SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：13和SEQ ID NO：14中任一项所示的氨基酸序列。
  4. 根据权利要求1或2所述的分离的抗原结合蛋白，其中，所述HCDR2的突变体具有SEQ ID NO：24或SEQ ID NO：27所示的氨基酸序列。
  5. 根据权利要求1或2所述的分离的抗原结合蛋白，其中，所述HCDR3的突变体具有SEQ ID NO：39、SEQ ID NO：40和SEQ ID NO：41中任一项所示的氨基酸序列。
  6. 根据权利要求1所述的分离的抗原结合蛋白，其中，所述VH包含以下的互补决定区或其突变体：
    SEQ ID NO：9-11和SEQ ID NO：13-14中任一项的氨基酸序列所示的HCDR1；
    SEQ ID NO：23-24和SEQ ID NO：27中任一项的氨基酸序列所示的HCDR2；和/或
    SEQ ID NO：36和SEQ ID NO：39-41中任一项的氨基酸序列所示的HCDR3。
  7. 根据权利要求1或2所述的分离的抗原结合蛋白，其中，所述VH包含以下互补决定区：
    分别如SEQ ID NO：9，SEQ ID NO：23和SEQ ID NO：36所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：36所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：11，SEQ ID NO：24和SEQ ID NO：36所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：11，SEQ ID NO：23和SEQ ID NO：36所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：13，SEQ ID NO：23和SEQ ID NO：36所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：40所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：23和SEQ ID NO：41所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：36所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：40所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：10，SEQ ID NO：27和SEQ ID NO：41所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：36所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：39所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：40所示的HCDR1，HCDR2和HCDR3；或
    分别如SEQ ID NO：14，SEQ ID NO：27和SEQ ID NO：41所示的HCDR1，HCDR2和HCDR3。
  8. 根据权利要求1或2所述的分离的抗原结合蛋白，其中，所述VH还包含重链可变区框架区VH FWR，所述VH FWR包含VH FWR1，VH FWR2，VH FWR3和VH FWR4，所述VH FWR1具有SEQ ID NO：3-5中任一项所示的氨基酸序列，所述VH FWR2具有SEQ ID NO：17或SEQ ID NO：20所示的氨基酸序列，所述VH FWR3具有SEQ ID NO：30或SEQ ID NO：31所示的氨基酸序列，以及所述VH FWR4具有SEQ ID NO：43所示的氨基酸序列。
  9. 根据权利要求1或2所述的分离的抗原结合蛋白，其中，所述VH包含SEQ ID NO：74-77，SEQ ID NO：79-80和SEQ ID NO：82-92中任一项所示的氨基酸序列或与其具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。
  10. 根据权利要求1或2所述的分离的抗原结合蛋白，其还包含Fc区，或者与免疫球蛋白的Fc区等同的区域，优选地，所述Fc区为人Fc，更优选地，所述人Fc为人IgG1 Fc。
  11. 根据权利要求10所述的分离的抗原结合蛋白，其中，所述Fc包含L234A、L235A和P329G突变，或L234A和L235A突变，或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地包含S298A，E333A和K334A突变。
  12. 根据权利要求1或2所述的分离的抗原结合蛋白，其包含氨基酸序列与SEQ ID NO：99、100、101、102、104、105、107、108、109、110、111、112、113、114、115、116或117具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。
  13. 根据权利要求1或2所述的分离的抗原结合蛋白，其是HCAb形式或纳米抗体形式的抗原结合片段。
  14. 分离的核酸，其编码根据权利要求1-13中任一项所述的分离的抗原结合蛋白。
  15. 表达载体，其包含根据权利要求14所述的分离的核酸。
  16. 宿主细胞，其包含根据权利要求14所述的分离的核酸，或根据权利要求15所述的表达载体。
  17. 抗体药物偶联物，其包含：根据权利要求1-13中任一项所述的分离的抗原结合蛋白；和，与所述抗原结合蛋白共价连接的药物。
  18. 根据权利要求17所述的抗体药物偶联物，所述药物选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物。
  19. 嵌合抗原受体，其包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，其中所述胞外抗原结合结构域包含权利要求1-13中任一项所述的分离的抗原结合蛋白。
  20. 修饰的免疫细胞，其包含根据权利要求19所述的嵌合抗原受体。
  21. 多特异性抗体，其包含两个或两个以上的抗原结合结构域，其中一个抗原结合结构域包含根据权利要求1-13中任一项所述的分离的抗原结合蛋白。
  22. 药物组合物，其包含：根据权利要求1-13中任一项所述的分离的抗原结合蛋白，根据权利要求17或18所述的抗体药物偶联物，根据权利要求19所述的嵌合抗原受体，根据权利要求20所述的修饰的免疫细胞或根据权利要求21所述的多特异性抗体；以及，药学上可接受的载体。
  23. 根据权利要求22所述的药物组合物，还包含选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物的治疗剂。
  24. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白、根据权利要求14所述的分离的核酸、根据权利要求17或18所述的抗体药物偶联物、根据权利要求19所述的嵌合抗原受体、根据权利要求20所述的修饰的免疫细胞或根据权利要求21所述的多特异性抗体、根据权利要求22或23所述的药物组合物在制备用于预防、治疗和/或诊断免疫性疾病，急性和慢性炎性疾病，以及肿瘤疾病的药物中的应用。
  25. 根据权利要求24所述的应用，所述肿瘤为乳腺癌、肾细胞癌、黑色素瘤、结肠癌，以及B细胞淋巴瘤，黑色素瘤，头颈癌，膀胱癌，胃癌，卵巢癌，恶性肉瘤，尿路上皮癌，肝癌，食道癌，胃食管交界癌，鼻咽癌，小细胞肺癌，宫颈癌，子宫内膜癌，胰腺癌，前列腺癌，胶质瘤，非小细胞肺癌，急性粒细胞白血病，霍奇金淋巴瘤，皮肤鳞状细胞癌，局部晚期和转移性恶性肿瘤中的一种或多种；
    所述炎性疾病为特应性皮炎或溃疡性结肠炎中的一种或多种；
    所述免疫性疾病为移植物抗宿主病、类风湿性关节炎、系统性红斑狼疮或哮喘中的一种或多种。
  26. 用于检测样品中的PD-L1的方法，所述方法包括用根据权利要求1-13中任一项所述的分离的抗原结合蛋白检测样品中的PD-L1的步骤，所述样品为全血，红血细胞浓缩物，血小板浓缩物，白细胞浓缩物，组织，骨髓吸出物，血浆，血清，脑脊液，粪便，尿液，培养的细胞，唾液，口腔分泌物和/或鼻腔分泌物；优选地，所述方法为非诊断目的的。
  27. 特异性结合蛋白，其包含至少两个结构域，并且能够结合PD-L1或其片段，和/或CD73或其片段。
  28. 根据权利要求27所述的特异性结合蛋白，其包含第一结构域和第二结构域，所述第一结构域结合CD73或其片段，并且所述第二结构域结合PD-L1或其片段，所述第一结构域与所述第二结构域连接形成双特异性结合蛋白。
  29. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域为CD73抗体或其抗原结合片段，所述第二结构域为PD-L1抗体或其抗原结合片段，所述第一结构域和/或所述第二结构域选自IgG、Fab、Fab’、F(ab’) 2、Fv、scFv、VH、或者HCAb的形式，优选地，所述Fab、Fab’、F(ab’) 2、Fv、scFv、VH的数量为一个或多个。
  30. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域为IgG的形式，优选地，所述IgG的重链恒定区为人重链恒定区，更优选为人IgG1、人IgG2、人IgG3或人IgG4重链恒定区。
  31. 根据权利要求30所述的特异性结合蛋白，其中，IgG包含L234A、L235A和P329G中的一个、两个或三个突变，更优选地包含L234A和L235A的突变；或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地包含S298A，E333A和K334A突变；
    优选地，IgG的Fc为人IgG1的Fc。
  32. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域为Fab的形式，更优选地，所述第一结构域包括2个Fab。
  33. 根据权利要求28所述的特异性结合蛋白，其中，所述第二结构域为VH，
    优选地，所述VH为人VH，更优选地，所述第二结构域具有两个VH。
  34. 根据权利要求28所述的特异性结合蛋白，其中，所述第二结构域为HCAb形式，优选地，所述HCAb的恒定区包含L234A、L235A和P329G突变，或L234A和L235A突变，或者包含S298A，E333A和K334A中的一个、两个或三个突变，更优选地包含S298A，E333A和K334A突变。
  35. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域与所述第二结构域直接连接或经连接肽L连接形成双特异性结合蛋白，所述第二结构域连接在所述第一结构域的C末端或N末端。
  36. 根据权利要求35所述的特异性结合蛋白，其中，所述双特异性结合蛋白包含短链和长链，所述短链具有N’-VL 1-CL 1-C’所示的结构；所述长链具有N’-VH 1-CH 1-h-CH 2-CH 3-L-VH 2-C’所示的结构；
    或者，所述短链具有N’-VL 1-CL 1-C’所示的结构；所述长链具有N’-VH 2-L-VH 1-CH 1-h-CH 2-CH 3-C’所示的结构；
    或者，所述短链具有N’-VH 1-CH 1-C’所示的结构；所述长链具有N’-VL 1-CL 1-L-VH 2-CH 2-CH 3-C’所示的结构，
    其中，所述VL 1和VH 1分别为第一结构域的VL和VH，所述VH 2为第二结构域的VH，所述h为铰链区，所述的L为连接肽，所述CL 1是第一结构域的CL。
  37. 根据权利要求35或36所述的特异性结合蛋白，其中，所述L为长度为0-30个氨基酸长度的肽，优选地，其氨基酸序列包括SEQ ID NO：136-157任一所示。
  38. 根据权利要求35或36所述的特异性结合蛋白，其中，所述长链的CH 3经由连接肽L连接到VH 2；L为0-30个氨基酸的多肽，优选地，L包括SEQ ID NO.136-157中任一项所示的氨基酸序列；或者长链的CH3与VH 2直接连接。
  39. 根据权利要求35或36所述的特异性结合蛋白，其中，所述长链的VH 1经由连接肽L连接到VH 2；L为长度为0-30个氨基酸长度的肽，优选地，L包括SEQ ID NO.136-157中任一项所示的氨基酸序列；或者所述长链的VH 1与VH 2直接连接。
  40. 根据权利要求35或36所述的特异性结合蛋白，其中，所述长链的VH 2经由连接肽L连接到CL 1；L是如SEQ ID NO.136-157中任一项所示的氨基酸序列；或者所述长链的VH 2与CL 1直接连接。
  41. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域包含轻链可变区VL和重链可变区VH，所述VL包含选自如下的互补决定区：
    SEQ ID NO：51，SEQ ID NO：52和SEQ ID NO：53中任一项的氨基酸序列所示的LCDR1；
    SEQ ID NO：57，SEQ ID NO：58和SEQ ID NO：59中任一项的氨基酸序列所示的LCDR2；和/或
    SEQ ID NO：65，SEQ ID NO：66和SEQ ID NO：67中任一项的氨基酸序列所示的LCDR3；
    所述VH包含选自如下的互补决定区：
    SEQ ID NO：8，SEQ ID NO：12和SEQ ID NO：10中任一项的氨基酸序列所示的HCDR1；
    SEQ ID NO：22，SEQ ID NO：25和SEQ ID NO：26中任一项的氨基酸序列所示的HCDR2；和/或
    SEQ ID NO：35，SEQ ID NO：37和SEQ ID NO：38中任一项的氨基酸序列所示的HCDR3。
  42. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域包含轻链可变区VL和重链可变区VH，所述VL和VH包含选自如下的互补决定区：
    分别如SEQ ID NO：51，SEQ ID NO：57和SEQ ID NO：65所示的LCDR1，LCDR2和LCDR3；和，分别如SEQ ID NO：8，SEQ ID NO：22和SEQ ID NO：35所示的HCDR1，HCDR2和HCDR3；
    分别如SEQ ID NO：52，SEQ ID NO：58和SEQ ID NO：66所示的LCDR1，LCDR2和LCDR3；和，分别如SEQ ID NO：12，SEQ ID NO：25和SEQ ID NO：37所示的HCDR1，HCDR2和HCDR3；
    分别如SEQ ID NO：53，SEQ ID NO：59和SEQ ID NO：67所示的LCDR1，LCDR2和LCDR3；和，分别如SEQ ID NO：10，SEQ ID NO：26和SEQ ID NO：38所示的HCDR1，HCDR2和HCDR3。
  43. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域包含VL和VH，所述VL的氨基酸序列与SEQ ID NO：94，SEQ ID NO：95或SEQ ID NO：96具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性；
    所述VH的氨基酸序列与SEQ ID NO：73，SEQ ID NO：78或SEQ ID NO：81具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性。
  44. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域包含VL和VH，所述VL和VH的氨基酸序列分别如SEQ ID NO:94和SEQ ID NO:73所示；或分别如SEQ ID NO:95和SEQ ID NO:78所示；或分别如SEQ ID NO:96和SEQ ID NO:81所示。
  45. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域包含氨基酸序列与SEQ ID NO:119，SEQ ID NO:120或SEQ ID NO:121具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的轻链；以及氨基酸序列与SEQ ID NO:98，SEQ ID NO:103或SEQ ID NO:106具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的重链。
  46. 根据权利要求28所述的特异性结合蛋白，其中，所述第一结构域包含：
    SEQ ID NO:119所示的轻链和SEQ ID NO:98所示的重链；或
    SEQ ID NO:120所示的轻链和SEQ ID NO:103所示的重链；或
    SEQ ID NO:121所示的轻链和SEQ ID NO:106所示的重链。
  47. 根据权利要求28所述的特异性结合蛋白，其中，所述第二结构域为权利要求1-13任一项的分离的抗原结合蛋白。
  48. 根据权利要求28所述的特异性结合蛋白，其中，所述特异性结合蛋白的长链包含与SEQ ID NO：122、123、124、125、126、127、128、129、130、132或133具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列；以及所述特异性结合蛋白的短链包含与SEQ ID NO：119、120、121或131具有至少80％、85％、88％、90％、92％、95％、97％、98％、99％或100％一致性的氨基酸序列。
  49. 根据权利要求28所述的特异性结合蛋白，其中，所述特异性结合蛋白具有：
    SEQ ID NO：122所示的长链以及SEQ ID NO：119所示的短链；或
    SEQ ID NO：123所示的长链以及SEQ ID NO：120所示的短链；或
    SEQ ID NO：124所示的长链以及SEQ ID NO：121所示的短链；或
    SEQ ID NO：125所示的长链以及SEQ ID NO：119所示的短链；或
    SEQ ID NO：126所示的长链以及SEQ ID NO：119所示的短链；或
    SEQ ID NO：127所示的长链以及SEQ ID NO：119所示的短链；或
    SEQ ID NO：128所示的长链以及SEQ ID NO：119所示的短链；或
    SEQ ID NO：129所示的长链以及SEQ ID NO：119所示的短链；或
    SEQ ID NO：132所示的长链以及SEQ ID NO：131所示的短链；或
    SEQ ID NO：130所示的长链以及SEQ ID NO：119所示的短链；或
    SEQ ID NO：133所示的长链以及SEQ ID NO：131所示的短链。
  50. 根据权利要求28所述的特异性结合蛋白，其中，所述双特异性结合蛋白为包含两条第一多肽链以及两条第二多肽链形成二价结构或四价对称结构。
  51. 分离的核酸，其编码根据权利要求28-50中任一项所述的特异性结合蛋白或其片段。
  52. 表达载体，其包含根据权利要求51所述的分离的核酸。
  53. 宿主细胞，其包含根据权利要求51所述的分离的核酸，或根据权利要求52所述的表达载体。
  54. 抗体药物偶联物，其包含：根据权利要求28-50中任一项所述的分离的抗原结合蛋白；和，与所述抗原结合蛋白共价连接的药物。
  55. 根据权利要求54所述的抗体药物偶联物，所述药物选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物。
  56. 药物组合物，其包含：根据权利要求28-50中任一项所述的特异性结合蛋白，或根据权利要求54或55所述的抗体药物偶联物；以及，药学上可接受的载体。
  57. 根据权利要求56所述的药物组合物，其还包含选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物的治疗剂。
  58. 根据权利要求28-50中任一项所述的特异性结合蛋白、根据权利要求51所述的分离的核酸、根据权利要求54或55所述的抗体药物偶联物或根据权利要求56或57所述的药物组合物在制备用于预防、治疗和/或诊断免疫性疾病，急性和慢性炎性疾病，以及肿瘤疾病的药物中的应用。
  59. 根据权利要求58所述的应用，所述肿瘤为乳腺癌、肾细胞癌、黑色素瘤、结肠癌，以及B细胞淋巴瘤，黑色素瘤，头颈癌，膀胱癌，胃癌，卵巢癌，恶性肉瘤，尿路上皮癌，肝癌，食道癌，胃食管交界癌，鼻咽癌，小细胞肺癌，宫颈癌，子宫内膜癌，胰腺癌，前列腺癌，胶质瘤，非小细胞肺癌，急性粒细胞白血病，霍奇金淋巴瘤，皮肤鳞状细胞癌，局部晚期和转移性恶性肿瘤中的一种或多种；
    所述炎性疾病为特应性皮炎或溃疡性结肠炎中的一种或多种；
    所述免疫性疾病为移植物抗宿主病、类风湿性关节炎、系统性红斑狼疮或哮喘中的一种或多种。
  60. 用于检测样品中的PD-L1和CD73的方法，所述方法包括用根据权利要求28-50中任一项所述的特异性结合蛋白检测样品中的PD-L1和CD73的步骤，所述样品为全血，红血细胞浓缩物，血小板浓缩物，白细胞浓缩物，组织，骨髓吸出物，血浆，血清，脑脊液，粪便，尿液，培养的细胞，唾液，口腔分泌物和/或鼻腔分泌物。
  61. 套装药盒，其包括一个或多个药盒，所述药盒包含根据权利要求1-13中任一项所述的抗原结合蛋白， 根据权利要求17或18所述的抗体药物偶联物，或根据权利要求22或23所述的药物组合物。
  62. 根据权利要求61所述的套装药盒，所述套装药盒包括第一药盒和第二药盒，其中，所述第一药盒包括根据权利要求1-13中任一项所述的抗原结合蛋白，根据权利要求17或18所述的抗体药物偶联物，或根据权利要求22或23所述的药物组合物；其中，所述第二药盒包含选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物的治疗剂。
  63. 套装药盒，其包括一个或多个药盒，所述药盒包含根据权利要求28-50中任一项所述的特异性结合蛋白，根据权利要求54或55所述的抗体药物偶联物，或根据权利要求56或57所述的药物组合物。
  64. 根据权利要求63所述的套装药盒，所述套装药盒包括第一药盒和第二药盒，其中，所述第一药盒包括根据权利要求28-50中任一项所述的抗原结合蛋白，根据权利要求54或55所述的抗体药物偶联物，或根据权利要求56或57所述的药物组合物；其中，所述第二药盒包含选自化学治疗剂、放射治疗剂、免疫抑制剂和细胞毒性药物的治疗剂。
  65. 给药装置，其包含根据权利要求1-13中任一项所述的抗原结合蛋白，根据权利要求17或18所述的抗体药物偶联物，或根据权利要求22或23所述的药物组合物，
    优选地，所述给药装置是预填充注射器。
  66. 给药装置，其包含根据权利要求28-50中任一项所述的特异性结合蛋白，根据权利要求54或55所述的抗体药物偶联物，或根据权利要求56或57所述的药物组合物，
    优选地，所述给药装置是预填充注射器。
  67. 预防、治疗和/或诊断肿瘤疾病、急性和慢性炎性疾病和/或免疫性疾病的方法，其包括向受试者施用治疗有效量的根据权利要求1-13中任一项所述的抗原结合蛋白，根据权利要求17或18所述的抗体药物偶联物，或根据权利要求22或23所述的药物组合物。
  68. 预防、治疗和/或诊断肿瘤疾病、急性和慢性炎性疾病和/或免疫性疾病的方法，其包括向受试者施用治疗有效量的根据权利要求28-50中任一项所述的抗原结合蛋白，根据权利要求54或55所述的抗体药物偶联物，或根据权利要求56或57所述的药物组合物。
  69. 根据权利要求67或68所述的方法，其中所述肿瘤为乳腺癌、肾细胞癌、黑色素瘤、结肠癌，以及B细胞淋巴瘤，黑色素瘤，头颈癌，膀胱癌，胃癌，卵巢癌，恶性肉瘤，尿路上皮癌，肝癌，食道癌，胃食管交界癌，鼻咽癌，小细胞肺癌，宫颈癌，子宫内膜癌，胰腺癌，前列腺癌，胶质瘤，非小细胞肺癌，急性粒细胞白血病，霍奇金淋巴瘤，皮肤鳞状细胞癌，局部晚期和转移性恶性肿瘤中的一种或多种；
    所述炎性疾病为特应性皮炎或溃疡性结肠炎中的一种或多种；
    所述免疫性疾病为移植物抗宿主病、类风湿性关节炎、系统性红斑狼疮或哮喘中的一种或多种。
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