WO2018023976A1 - 抗人ifnar1的抗体及其用途 - Google Patents

抗人ifnar1的抗体及其用途 Download PDF

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WO2018023976A1
WO2018023976A1 PCT/CN2017/075933 CN2017075933W WO2018023976A1 WO 2018023976 A1 WO2018023976 A1 WO 2018023976A1 CN 2017075933 W CN2017075933 W CN 2017075933W WO 2018023976 A1 WO2018023976 A1 WO 2018023976A1
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seq
sequence
antibody
variable region
chain variable
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PCT/CN2017/075933
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French (fr)
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刘志刚
刘玉兰
郭晶晶
郝小勃
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北京智仁美博生物科技有限公司
智翔（上海）医药科技有限公司
重庆智翔金泰生物制药有限公司
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Publication of WO2018023976A1 publication Critical patent/WO2018023976A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates generally to the field of antibody drugs, and in particular, the present application provides antibodies against human type I interferon receptor subunit 1 (IFNAR1) and their medical and biological uses.
  • IFNAR1 human type I interferon receptor subunit 1
  • Interferons include type I interferons, type II interferons, and type III interferons.
  • human type I interferons include IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ .
  • fibroblasts and monocytes in the human body secrete various type I interferons to prevent and interfere with DNA and RNA replication of the virus.
  • type I interferons also have anti-tumor and immunomodulatory functions (Capobianchi MR, et al., 2015, Cytokine Growth Factor Rev., 26:103; Zitvogel L., et al., 2015, Nat Rev Immunol., 15:405).
  • IFNAR1 has a weak ability to bind type I interferon alone (KD is about 10 -7 M), while IFNAR2 has a stronger ability to bind type I interferon alone (KD is about 10 -9 M).
  • type I interferons Early studies on the function of type I interferon have focused on innate immunity such as antiviral infection. However, in recent years, more studies have suggested that type I interferons also have strong immunomodulatory effects in adaptive immunity, including promoting antibody secretion and supporting the functional activity and survival of T memory cells. In particular, studies have shown that IFN- ⁇ can promote the maturation or activation of dendritic cells (DC) (Santini S. M., et al., 2000, J. EXP. Med., 191:1777). Moreover, studies have found that a variety of autoimmune diseases exhibit type I interferon overexpression.
  • DC dendritic cells
  • IDM insulin-dependent diabetes mellitus
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • TMPD is effective in inducing systemic lupus erythematosus in normal mice, but not in specific IFNAR1 gene-deficient mice (Nacionales DC, et al., 2007, Arthritis Rheum, 56: 3770).
  • anti-IFNAR1 antibodies showed significant therapeutic effects early in the disease (Baccala R., et al., 2012, J. Immunol, 189: 5876). Therefore, clinically, inhibition of type I interferon receptor (IFNAR) may benefit patients with certain autoimmune diseases, and clinically, some drugs that can effectively inhibit type I interferon receptors are required for various self. Treatment of immune diseases, including systemic lupus erythematosus.
  • the application provides an antibody that specifically binds to human type I interferon receptor subunit 1 (IFNAR1), comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences, characterized in that said HCDR1
  • the sequence is NYWVA (SEQ ID NO: 29)
  • the HCDR2 sequence is IIYPGDSDTRYSPSFQG (SEQ ID NO: 30)
  • the HCDR3 sequence is HDVTGYDY (SEQ ID NO: 31); or the HCDR1 sequence is NYWMA (SEQ ID) NO: 32)
  • the HCDR2 sequence is IIYPSDSDTRYSPSFQG (SEQ ID NO: 33)
  • the HCDR3 sequence is HDVEGYDY (SEQ ID NO: 34); or the HCDR1 sequence is NYWVA (SEQ ID NO: 29)
  • the HCDR2 sequence is IIYPSDSDTRYSPSFQG (SEQ ID NO: 33)
  • the HCDR3 sequence is HDVHG
  • the heavy chain variable region sequence of the antibody is set forth in the amino acid sequence of SEQ ID NO: 19, 20, 23, 24 or 25.
  • the application provides an antibody that specifically binds to human IFNAR1, comprising a light chain variable region comprising a sequence of LCDR1, LCDR2 and LCDR3, characterized in that the LCDR1 sequence is RASQNVGNYLN (SEQ ID NO: 41),
  • the LCDR2 sequence is RASNLAS (SEQ ID NO: 42), the LCDR3 sequence is QQMEHAPPT (SEQ ID NO: 43); or the LCDR1 sequence is RASQSVIGYYLA (SEQ ID NO: 44), and the LCDR2 sequence is SVSTLAS (SEQ ID NO: 45), the LCDR3 sequence is QQYYRFPIT (SEQ ID NO: 46); alternatively, the LCDR1 sequence is RASQNVSNYLN (SEQ ID NO: 47), and the LCDR2 sequence is RASNLQS (SEQ ID NO: 48)
  • the LCDR3 sequence is QQMMDAPPT (SEQ ID NO: 49); alternatively, the LCDR1 sequence is SGSSSNIGTNAVN
  • the light chain variable region sequence of the antibody is set forth in the amino acid sequence of SEQ ID NO: 21, 22, 26, 27 or 28.
  • the application provides an antibody that specifically binds to human IFNAR1, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, characterized by:
  • the HCDR1 sequence is NYWVA (SEQ ID NO: 29)
  • the HCDR2 sequence is IIYPGDSDTRYSPSFQG (SEQ ID NO: 30)
  • the HCDR3 sequence is HDVTGYDY (SEQ ID NO: 31)
  • the LCDR1 sequence is RASQNVGNYLN (SEQ ID NO: 41)
  • the LCDR2 sequence is RASNLAS (SEQ ID NO: 42)
  • the LCDR3 sequence is QQMEHAPPT (SEQ ID NO: 43); or
  • the HCDR1 sequence is NYWVA (SEQ ID NO: 29)
  • the HCDR2 sequence is IIYPGDSDTRYSPSFQG (SEQ ID NO: 30)
  • the LCDR2 sequence is RASNLAS (SEQ ID NO: 42), the LCDR3 sequence is QQMEHAPPT (SEQ ID NO: 43); alternatively, the HCDR1 sequence is NYWMA (SEQ ID NO: 32), and the HCDR2 sequence is IIYPSDSDTRYSPSFQG (SEQ ID NO: 33), the HCDR3 sequence is HDVEGYDY (SEQ ID NO: 34), the LCDR1 sequence is RASQSVIGYYLA (SEQ ID NO: 44), and the LCDR2 sequence is SVSTLAS (SEQ ID NO: 45),
  • the LCDR3 sequence is QQYYRFPIT (SEQ ID NO: 46); alternatively, the HCDR1 sequence is NYWVA (SEQ ID NO: 29), the HCDR2 sequence is IIYPSDSDTRYSPSFQG (SEQ ID NO: 33), and the HCDR3 sequence is HDVHGYDY (SEQ) ID NO: 35), the LCDR1 sequence is RASQNVSNYLN (SEQ
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO: 19 and the light chain variable region sequence is SEQ ID NO:21.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO: 19 and the light chain variable region sequence is SEQ ID NO: 22.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:20 and the light chain variable region sequence is SEQ ID NO:21.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:20 and the light chain variable region sequence is SEQ ID NO:22.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:23 and the light chain variable region sequence is SEQ ID NO:26.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:24 and the light chain variable region sequence is SEQ ID NO:27.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:25 and the light chain variable region sequence is SEQ ID NO:28.
  • the antibody that specifically binds to human IFNAR1 is a full length antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment.
  • the antibody that specifically binds to human IFNAR1 is a fully human antibody.
  • the antibody that specifically binds human IFNAR1 further comprises an IgG1 subtype (SEQ ID NO: 7), an IgG2 subtype (SEQ ID NO: 8) or an IgG4 subtype (SEQ ID NO: 9)
  • the heavy chain constant region and/or comprises a light chain constant region selected from the kappa subtype (SEQ ID NO: 10) or the lambda subtype (SEQ ID NO: 11).
  • the present application provides a pharmaceutical composition comprising the antibody of the first to third aspects which specifically binds to human IFNAR1.
  • the present application provides the antibody of the first aspect to the third aspect, which specifically binds to human IFNAR1, or the pharmaceutical composition of the fourth aspect, for the preparation of a medicament for preventing or treating a disease mediated by human IFNAR1 Use in.
  • the disease is an autoimmune disease.
  • the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, psoriasis, multiple sclerosis, rheumatoid arthritis.
  • the present application also provides a method of treating a disease mediated by human IFNAR1, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody of the first to third aspects of the antibody that specifically binds to human IFNAR1, or the fourth aspect The pharmaceutical composition.
  • the disease is an autoimmune disease.
  • the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, psoriasis, multiple sclerosis, rheumatoid arthritis.
  • Figure 1 shows a schematic representation of the structure of the phage display vector pADSCFV-S.
  • Figure 2 shows inhibition of phage display single chain antibodies S3A5, S3H8 and S5B4 binding to human IFNAR1 by the control monoclonal antibodies anti-IFNAR1-C1 and anti-IFNAR1-C2.
  • Figure 3 shows the ability of ELISA to analyze the binding ability of anti-human IFNAR1 monoclonal antibody to different species of IFNAR1 protein.
  • Figure 4 shows the stability analysis of different anti-human IFNAR1 mAbs in human serum.
  • Figure 5 shows the inhibition of IFN ⁇ -2b-HSA at a concentration of 0.67 nM by three different anti-human IFNAR1 mAbs (H19B7+L16C11, H19B7+L8C3 or anti-IFNAR1-C2 (chimeric)) based on Daudi cells.
  • Figure 6 shows on HEK-Blue TM IFN ⁇ / ⁇ -cell analysis of three different anti-human IFNAR1 mAb (H19B7 + L16C11, H19B7 + L8C3 or anti-IFNAR1-C2 (chimeric)) to a concentration of 0.1nM of inhibition IFN ⁇ .
  • Figure 7 shows a melting curve/derivative plot of heat stability analysis of different anti-human IFNAR1 mAbs (H19B7+L16C11, H19B7+L8C3, H15D10+L16C11, H15D10+L8C3 or anti-IFNAR1-C1) (20160405-DSF) .
  • SEQ ID NO: 1 is the amino acid sequence of the human IFNAR1 extracellular domain (hIFNAR1).
  • SEQ ID NO: 2 is the amino acid sequence of the mouse IFNAR1 extracellular domain (mIFNAR1).
  • SEQ ID NO: 3 is the amino acid sequence of the extracellular domain of cynomolgus IFNAR1 (mmIFNAR1).
  • SEQ ID NO: 4 is the amino acid sequence of human IFN ⁇ (IFN ⁇ ).
  • SEQ ID NO: 5 is the amino acid sequence of the His tag.
  • SEQ ID NO: 6 is the amino acid sequence of the Fc fragment (mFc) of murine antibody IgG2a.
  • SEQ ID NO: 7 is the amino acid sequence of the antibody heavy chain constant region IgGl subtype.
  • SEQ ID NO: 8 is the amino acid sequence of the antibody heavy chain constant region IgG2 subtype.
  • SEQ ID NO: 9 is the amino acid sequence of the antibody heavy chain constant region IgG4 subtype.
  • SEQ ID NO: 10 is the amino acid sequence of the light chain constant region of the antibody kappa subtype.
  • SEQ ID NO: 11 is the amino acid sequence of the constant region of the light chain of the antibody lambda subtype.
  • SEQ ID NO: 12 is the amino acid sequence of single-chain antibody S3A5.
  • SEQ ID NO: 13 is the amino acid sequence of the single chain antibody S3H8.
  • SEQ ID NO: 14 is the amino acid sequence of the single chain antibody S5B4.
  • SEQ ID NO: 15 is the amino acid sequence of the VH of the control recombinant antibody against IFNAR1-C1.
  • SEQ ID NO: 16 is the amino acid sequence of the VL of the control recombinant antibody against IFNAR1-C1.
  • SEQ ID NO: 17 is the amino acid sequence of the VH of the control recombinant antibody against IFNAR1-C2.
  • SEQ ID NO: 18 is the amino acid sequence of the VL of the control recombinant antibody against IFNAR1-C2.
  • SEQ ID NO: 19 is the amino acid sequence of heavy chain mutant H15D10.
  • SEQ ID NO: 20 is the amino acid sequence of heavy chain mutant H19B7.
  • SEQ ID NO: 21 is the amino acid sequence of the light chain mutant L8C3.
  • SEQ ID NO: 22 is the amino acid sequence of the light chain mutant L16C11.
  • SEQ ID NO: 23 is the amino acid sequence of the heavy chain variable region sequence of the single chain antibody S3A5.
  • SEQ ID NO: 24 is the amino acid sequence of the heavy chain variable region sequence of the single chain antibody S3H8.
  • SEQ ID NO: 25 is the amino acid sequence of the heavy chain variable region sequence of the single chain antibody S5B4.
  • SEQ ID NO:26 is the amino acid sequence of the light chain variable region of the single chain antibody S3A5.
  • SEQ ID NO:27 is the amino acid sequence of the light chain variable region of the single chain antibody S3H8.
  • SEQ ID NO:28 is the amino acid sequence of the light chain variable region of single chain antibody S5B4.
  • SEQ ID NO:29 is the amino acid sequence of one HCDR1 identified herein.
  • SEQ ID NO:30 is the amino acid sequence of one HCDR2 identified herein.
  • SEQ ID NO: 31 is the amino acid sequence of one HCDR3 identified herein.
  • SEQ ID NO:32 is the amino acid sequence of one HCDR1 identified herein.
  • SEQ ID NO: 33 is the amino acid sequence of one HCDR2 identified herein.
  • SEQ ID NO:34 is the amino acid sequence of one HCDR3 identified herein.
  • SEQ ID NO: 35 is the amino acid sequence of one HCDR3 identified herein.
  • SEQ ID NO: 36 is the amino acid sequence of one HCDR1 identified herein.
  • SEQ ID NO:37 is the amino acid sequence of one HCDR2 identified herein.
  • SEQ ID NO: 38 is the amino acid sequence of one HCDR3 identified herein.
  • SEQ ID NO: 39 is the amino acid sequence of one HCDR2 identified herein.
  • SEQ ID NO: 40 is the amino acid sequence of one HCDR3 identified herein.
  • SEQ ID NO: 41 is the amino acid sequence of an LCDR1 identified herein.
  • SEQ ID NO: 42 is the amino acid sequence of an LCDR2 identified herein.
  • SEQ ID NO: 43 is the amino acid sequence of an LCDR3 identified herein.
  • SEQ ID NO: 44 is the amino acid sequence of an LCDR1 identified herein.
  • SEQ ID NO: 45 is the amino acid sequence of an LCDR2 identified herein.
  • SEQ ID NO: 46 is the amino acid sequence of an LCDR3 identified herein.
  • SEQ ID NO: 47 is the amino acid sequence of an LCDR1 identified herein.
  • SEQ ID NO: 48 is the amino acid sequence of an LCDR2 identified herein.
  • SEQ ID NO: 49 is the amino acid sequence of an LCDR3 identified herein.
  • SEQ ID NO: 50 is the amino acid sequence of an LCDR1 identified herein.
  • SEQ ID NO: 51 is the amino acid sequence of an LCDR2 identified herein.
  • SEQ ID NO: 52 is the amino acid sequence of an LCDR3 identified herein.
  • SEQ ID NO:53 is the amino acid sequence of an LCDR1 identified herein.
  • SEQ ID NO: 54 is the amino acid sequence of an LCDR2 identified herein.
  • SEQ ID NO: 55 is the amino acid sequence of an LCDR3 identified herein.
  • SEQ ID NO: 56 is the nucleotide sequence of human antibody heavy chain variable region gene VH1.
  • SEQ ID NO: 57 is the nucleotide sequence of human antibody heavy chain variable region gene VH3.
  • SEQ ID NO: 58 is the nucleotide sequence of human antibody heavy chain variable region gene VH5.
  • SEQ ID NO: 59 is the nucleotide sequence of human antibody light chain variable region gene VK1.
  • SEQ ID NO: 60 is the nucleotide sequence of human antibody light chain variable region gene Vl3.
  • SEQ ID NO: 61 is the nucleotide sequence of the gene encoding the PelB leader peptide.
  • SEQ ID NO: 62 is the nucleotide sequence of an unrelated sequence.
  • SEQ ID NO: 63 is the nucleotide sequence of the gene encoding the single-chain antibody S3A5.
  • SEQ ID NO: 64 is the nucleotide sequence of the gene encoding the single-chain antibody S3H8.
  • SEQ ID NO: 65 is the nucleotide sequence of the gene encoding the single chain antibody S5B4.
  • the inventors of the present application screened and optimized an anti-human IFNAR1 antibody having a desired property by constructing a large-capacity natural human phage antibody library.
  • a novel monoclonal antibody against human IFNAR1, or an antigen-binding fragment thereof a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof, a vector comprising the polynucleotide, Host cells of the polynucleotide or vector, methods of making and purifying the antibodies, and medical and biological applications of the antibodies or antigen-binding fragments thereof.
  • variable region of the antibody allows the construction of a full-length antibody molecule as a drug for the treatment of autoimmune diseases mediated clinically by IFNAR1.
  • diseases include, but are not limited to, systemic lupus erythematosus, psoriasis, multiple sclerosis, rheumatoid arthritis.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule.
  • Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like.
  • antibody includes not only intact (ie, full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') 2 , Fv), variants thereof, and antibody-containing portions thereof.
  • Fusion proteins humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (eg bispecific antibodies) and any other immunoglobulin containing an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • a full or full length antibody comprises two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variant region (VH) and first, second and third constant regions (CH1, CH2 and CH3).
  • Each light chain contains a light chain variant region (VL) and a constant region (CL).
  • the full length antibody can be any kind of antibody, such as IgD, IgE, IgG, IgA or IgM (or a subclass of the above), but the antibody does not need to belong to any particular class.
  • Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of the heavy chain.
  • immunoglobulins there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to different immunoglobulin classes are referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • antigen-binding fragment refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variant region (VH), a light chain variant region (VL), or both.
  • VH and VL typically contains three complementarity determining regions, CDR1, CDR2 and CDR3.
  • CDRs complementarity determining regions
  • VH or VL There are two common definitions for the CDR sequences of VH or VL, namely the kabat definition and the Chothia definition.
  • CDR sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In an embodiment of the present application, CDR sequences are defined using Kabat.
  • variable region sequences of a given antibody the CDR region sequences in the variable region sequences can be analyzed in a variety of ways, for example, using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be a monovalent fragment having VL-CL chain and a VH-CH1 chain; (2) F (ab ' ) 2 fragments, which may be a two a bivalent fragment of a Fab' fragment joined by a disulfide bridge of the hinge region (ie, a dimer of Fab'); (3) an Fv fragment of the VL and VH domains of the one arm of the antibody; 4) a single-chain Fv (scFv), which may be a single multi-peptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) 2 , which may comprise two linked by a peptide A coupled VH domain and two VL domains are combined with the two VH domains via a disulfide bridge.
  • Fab fragments which may be a monovalent fragment having VL-CL chain and a VH-CH1 chain
  • the term "specifically binds" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • the application provides an antibody that specifically binds to human IFNAR1, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences, characterized in that the HCDR1 sequence is NYWVA (SEQ ID NO: 29), The HCDR2 sequence is IIYPGDSDTRYSPSFQG (SEQ ID NO: 30), the HCDR3 sequence is HDVTGYDY (SEQ ID NO: 31); or the HCDR1 sequence is NYWMA (SEQ ID NO: 32), and the HCDR2 sequence is IIYPSDSDTRYSPSFQG (SEQ ID NO: 33), the HCDR3 sequence is HDVEGYDY (SEQ ID NO: 34); alternatively, the HCDR1 sequence is NYWVA (SEQ ID NO: 29), and the HCDR2 sequence is IIYPSDSDTRYSPSFQG (SEQ ID NO: 33) The HCDR3 sequence is HDVHGYDY (SEQ ID NO: 35); alternatively, the HCDR1
  • the heavy chain variable region sequence of the antibody is set forth in the amino acid sequence of SEQ ID NO: 19, 20, 23, 24 or 25.
  • the application provides an antibody that specifically binds to human IFNAR1, comprising a light chain variable region comprising a sequence of LCDR1, LCDR2 and LCDR3, characterized in that the LCDR1 sequence is RASQNVGNYLN (SEQ ID NO: 41),
  • the LCDR2 sequence is RASNLAS (SEQ ID NO: 42), the LCDR3 sequence is QQMEHAPPT (SEQ ID NO: 43); or the LCDR1 sequence is RASQSVIGYYLA (SEQ ID NO: 44), and the LCDR2 sequence is SVSTLAS (SEQ ID NO: 45), the LCDR3 sequence is QQYYRFPIT (SEQ ID NO: 46); alternatively, the LCDR1 sequence is RASQNVSNYLN (SEQ ID NO: 47), and the LCDR2 sequence is RASNLQS (SEQ ID NO: 48)
  • the LCDR3 sequence is QQMMDAPPT (SEQ ID NO: 49); alternatively, the LCDR1 sequence is SGSSSNIGTNAVN
  • the light chain variable region sequence of the antibody is set forth in the amino acid sequence of SEQ ID NO: 21, 22, 26, 27 or 28.
  • the application provides an antibody that specifically binds to human IFNAR1, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, characterized by:
  • the HCDR1 sequence is NYWVA (SEQ ID NO: 29)
  • the HCDR2 sequence is IIYPGDSDTRYSPSFQG (SEQ ID NO: 30)
  • the HCDR3 sequence is HDVTGYDY (SEQ ID NO: 31)
  • the LCDR1 sequence is RASQNVGNYLN (SEQ ID NO: 41)
  • the LCDR2 sequence is RASNLAS (SEQ ID NO: 42)
  • the LCDR3 sequence is QQMEHAPPT (SEQ ID NO: 43); or
  • the HCDR1 sequence is NYWVA (SEQ ID NO: 29)
  • the HCDR2 sequence is IIYPGDSDTRYSPSFQG (SEQ ID NO: 30)
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO: 19 and the light chain variable region sequence is SEQ ID NO:21.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO: 19 and the light chain variable region sequence is SEQ ID NO: 22.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:20 and the light chain variable region sequence is SEQ ID NO:21.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:20 and the light chain variable region sequence is SEQ ID NO:22.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:23 and the light chain variable region sequence is SEQ ID NO:26.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:24 and the light chain variable region sequence is SEQ ID NO:27.
  • the heavy chain variable region sequence of an antibody that specifically binds to human IFNAR1 is SEQ ID NO:25 and the light chain variable region sequence is SEQ ID NO:28.
  • the antibody that specifically binds to human IFNAR1 is a full length antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment.
  • the antibody that specifically binds to human IFNAR1 is a fully human antibody.
  • the antibody that specifically binds human IFNAR1 further comprises an IgG1 subtype (SEQ ID NO: 7), an IgG2 subtype (SEQ ID NO: 8) or an IgG4 subtype (SEQ ID NO: 9)
  • the heavy chain constant region and/or comprises a light chain constant region selected from the kappa subtype (SEQ ID NO: 10) or the lambda subtype (SEQ ID NO: 11).
  • an antibody that specifically binds to human IFNAR1 antagonizes at least one in vitro or in vivo biological activity associated with IFNAR1 or a portion thereof.
  • an antibody that specifically binds IFNAR1 is capable of specifically binding to the extracellular region of recombinant human IFNAR1.
  • the antibody is characterized by specifically binding human IFNAR1 at a level above background And cynomolgus IFNAR1, but not mouse IFNAR1.
  • the present application also provides polynucleotides encoding the above antibodies or antigen-binding fragments thereof, vectors comprising the polynucleotides, and host cells transfected with the vectors.
  • the present application provides a pharmaceutical composition comprising the antibody of the first to third aspects which specifically binds to human IFNAR1.
  • the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, excipient, diluent, and the like.
  • the pharmaceutical composition may further comprise a lubricant such as talc, magnesium stearate and mineral oil; a wetting agent; an emulsifier; a suspending agent; a preservative such as benzoic acid, sorbic acid and calcium propionate.
  • a lubricant such as talc, magnesium stearate and mineral oil
  • a wetting agent such as talc, magnesium stearate and mineral oil
  • an emulsifier such as talc, magnesium stearate and mineral oil
  • a suspending agent such as benzoic acid, sorbic acid and calcium propionate.
  • a preservative such as benzoic acid, sorbic acid and calcium propionate.
  • the pharmaceutical compositions of the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical compositions of the present application can be delivered using any physiologically acceptable administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration. Drug, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
  • a pharmaceutical composition for therapeutic use may be formulated in a lyophilized formulation or as an aqueous solution by mixing an agent having the desired purity with a pharmaceutically acceptable carrier, excipient, or the like, as appropriate. storage.
  • the pharmaceutical composition is for use in preventing or treating a disease mediated by human IFNAR1, such as an autoimmune disease, including, but not limited to, systemic lupus erythematosus, psoriasis, multiple sclerosis, Rheumatoid arthritis.
  • a disease mediated by human IFNAR1 such as an autoimmune disease, including, but not limited to, systemic lupus erythematosus, psoriasis, multiple sclerosis, Rheumatoid arthritis.
  • the present application provides the antibody of the first aspect to the third aspect, which specifically binds to human IFNAR1, or the pharmaceutical composition of the fourth aspect, for the preparation of a medicament for preventing or treating a disease mediated by human IFNAR1 Use in.
  • the disease is an autoimmune disease.
  • the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, psoriasis, multiple sclerosis, rheumatoid arthritis.
  • the affinity of an antibody of the present application, its binding to IFNARl of a different species, its stability in human serum, its pharmacological activity, its thermostability can be demonstrated in standard test methods.
  • the present application also provides a method of treating a disease mediated by human IFNAR1, comprising administering to a patient in need thereof a therapeutically effective amount of the antibody of the first to third aspects of the antibody that specifically binds to human IFNAR1, or the fourth aspect The pharmaceutical composition.
  • the disease is an autoimmune disease.
  • the autoimmune disease includes, but is not limited to, systemic lupus erythematosus, psoriasis, multiple sclerosis, rheumatoid arthritis.
  • the application further provides an isolated nucleic acid molecule encoding an antibody of the invention or a light chain thereof or a heavy chain thereof, and a vector comprising the nucleic acid molecule, a host cell comprising the vector, and a method of producing the antibody.
  • the nucleic acid molecule is operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.
  • a method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid.
  • the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.
  • antibodies specifically binding to human IFNAR1 described herein can also be used to detect biological samples.
  • Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.
  • the antibody library technology is an important method for preparing and screening human monoclonal antibodies, and the phage display-based antibody library technology is a mature antibody library technology, which has been successfully applied to the preparation of human monoclonal antibody drugs.
  • This example describes strategies and methods for constructing phage display antibody libraries using a variety of genetic engineering techniques today.
  • VH heavy chain variable region
  • VL light chain variable region
  • the blood of 19 normal volunteers (50 mL each) was collected, and the collected blood was provided by the inventors and their colleagues as volunteers, and all volunteers had signed informed consent.
  • the inclusion criteria for volunteers are:
  • the age is greater than 18 years old;
  • cDNA (Cat#: AT301-03) was prepared.
  • PCR was performed using the primer sets listed in Table 1 below to amplify the heavy chain variable region gene (VH) and light chain variable region genes (VL, including Vk and Vl) of the antibody, respectively.
  • the amplified PCR product (VH, VK or Vl) was purified and recovered by a conventional agarose gel electrophoresis method and stored at -20 ° C until use.
  • the basic strategy for the preparation of fully synthetic antibody genes is to introduce designed mutations into the CDRs of selected template antibody genes using degenerate primers.
  • To construct a fully synthetic human antibody library three human antibody heavy chain variable region templates (VH1, VH3, and VH5) and two human antibody light chain variable region templates (VK1 and Vl3) were selected in this example. Synthetic antibody library.
  • VH1 SEQ ID NO: 56
  • VH3 SEQ ID NO: 57
  • VH5 SEQ ID NO: 58
  • VK1 SEQ ID NO: 59
  • Vl3 Vl3
  • VH1, VH3, VH5 complete heavy chain variable regions
  • VK1, VL3 light chain variable region
  • a commonly used flexible linker consisting of 15 amino acids is added between the heavy chain variable region (VH) and the light chain variable region (VL).
  • the sequence of the linker peptide is GGGGSGGGGSGGGGS.
  • the coding sequence of the linker peptide is ggtggaggcggttctggcggaggtgggagcggaggcggaggttca.
  • the structure of the designed single-chain antibody is VH-linker peptide-VL.
  • Table 3 shows various heavy and light chain variable region genes.
  • a commonly used phage display vector is based on the lactose promoter (Plac), which affects the capacity and diversity of the antibody library due to its leaky expression and the like.
  • lac lactose promoter
  • the Plac promoter and the g3 protein signal peptide portion of the pCANTAB5E vector were replaced with the AraC gene, the arabinose promoter (Para) and the PelB leader peptide (PelB leader, SEQ ID NO: 61) by double digestion with AflIII and NotI. Fragment.
  • the AraC gene and ParaC are from the pBADhis vector of Invitrogen, and the PelB leader peptide sequence is a synthetic sequence.
  • a 750 bp unsuccessful sequence (SEQ ID NO: 62) was cloned between NcoI and NotI sites by NcoI and NotI digestion, and the final novel phage display vector pADSCFV-S was constructed. 1).
  • the NcoI site and the NotI site in the vector can be conveniently used to clone a single chain antibody (ScFv) gene.
  • the 12 ScFvs prepared in 1.2 were cloned into the vector pADSCFV-S by NcoI and NotI double digestion.
  • the ligation products were electroporated into TG1 electroporation, respectively.
  • Each sub-pool was rotated about 20 times, for a total of about 240 electrospins.
  • the dilution method is used to calculate the library capacity of each sub-library, and 30 to 40 clones are randomly selected from each sub-pool for sequence analysis to estimate the correct rate of each sub-library.
  • the library capacity and correct rate of the 12 sub-libraries are summarized. Table 4.
  • the total library capacity of the 12 sub-libraries is 1.0*10E9, and the average correct rate is over 75%.
  • the phage amplification was carried out by shaking at 220 rpm overnight, and then the purified phage (phage-ScFv) was prepared by PEG/NaCl precipitation method, and titer was measured.
  • the prepared phage-ScFv of the 12 sub-pools were mixed with reference to the ratio of the library capacity to prepare a phage display human antibody library, and the final titer of the phage was 6*10E12 cfu/mL, and frozen at -70 °C.
  • This phage display antibody library can be used to screen for specific human antibodies against various antigens of interest.
  • IFNAR1 mAb To prepare and test anti-human IFNAR1 mAb, a variety of recombinant proteins were synthesized, including the human IFNAR1 extracellular domain (hIFNAR1, SEQ ID NO: 1), mouse IFNAR1 extracellular domain (mIFNAR1, SEQ ID NO: 2) and macaque IFNAR1 extracellular domain (mmIFNAR1, SEQ ID NO: 3), human IFN ⁇ (IFN ⁇ , SEQ ID NO: 4), and control recombinant antibody anti-IFNAR1-C1 (VH sequence is SEQ ID NO: 15, VL sequence is SEQ ID NO: :16; see U.S. Patent No.
  • the non-antibody recombinant protein has a His tag (SEQ ID NO: 5) or an Fc portion of the murine antibody IgG2a (mFc, SEQ ID NO: 6) at the C-terminus.
  • the antibody heavy chain constant region can be an IgGl subtype (SEQ ID NO: 7), an IgG2 subtype (SEQ ID NO: 8) or an IgG4 subtype (SEQ ID NO: 9), and the light chain constant region can be a kappa subtype ( SEQ ID NO: 10) or lambda subtype (SEQ ID NO: 11).
  • the genes of the above various recombinant proteins were designed and synthesized based on the amino acid sequence of the recombinant protein for various purposes of the Uniprot database.
  • the recombinant recombinant protein gene is cloned into a suitable eukaryotic expression vector (such as invitrogen pcDNA3.1, etc.) by conventional molecular biology techniques, and then subjected to liposome (such as invitrogen 293fectin, etc.) or other transfer.
  • the prepared recombinant protein expression plasmid (such as PEI) is transfected into HEK293 cells (such as HEK293F of Invitrogen), cultured in serum-free suspension culture for 3 to 5 days, and then the culture supernatant is harvested by centrifugation or the like.
  • the recombinant protein expressed by the His-tag fusion is subjected to one-step purification of the recombinant protein in the culture supernatant by a metal chelate affinity chromatography column (such as GE's HisTrap FF).
  • the recombinant protein and the whole antibody expressed by the mFc fusion were subjected to one-step purification using a ProteinA/G affinity chromatography column (e.g., Mabselect SURE of GE).
  • the recombinant protein storage buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (eg, Hitrap desaulting, GE, etc.).
  • Example 2 The recombinant hIFNAR1-his prepared in Example 2 was used as an antigen, and the solid phase screening strategy was utilized (experimental program reference phage display: general experimental guide / (American) Clarkson (Clackson, T.), (United States) Lohman (Lowman, HB Edited by Ma Wei et al.
  • the recombinant hIFNAR1-his prepared in Example 2 was coated in a 96-well ELISA plate (concentration: 4 ⁇ g/mL, 100 ⁇ L/well) and coated overnight at 4 °C. After blocking with a blocking solution (2% milk-PBST) for 1 hour at 37 ° C, then add purified phage displaying the single-chain antibody (S3A5, S3H8 or S5B4) or phage + 5 ⁇ g/mL of the control monoclonal antibody (anti-IFNAR1-C1) Or anti-IFNAR1-C2, the heavy chain constant regions of both recombinant control antibodies are IgG1 subtype, the light chain constant region is ⁇ subtype), after 1 hour of binding at 37 ° C, routine washing with PBST, then adding blocking solution The diluted HRP-anti-M13 mAb was routinely washed with PBST after binding for 1 hour at 37 ° C, and finally OPD substrate solution was added for color development, and OD
  • anti-IFNAR1-C1 significantly inhibited the binding of phage-displayed S3A5 and S3H8 mAbs to hIFNAR1-his, while anti-IFNAR1-C2 had no significant inhibitory effect on all three phage display antibodies.
  • the antibody S3A5 monoclonal antibody is subjected to in vitro affinity maturation in the first step by using the heavy chain CDR (HCDRs) mutation strategy (for details, refer to Example 5 of the Chinese Patent No. 201510097117.0 filed by the present applicant).
  • HCDRs heavy chain CDR
  • the key primers required to introduce mutations into the three CDRs of the S3A5 heavy chain (S3A5-VH) are shown in Table 5.
  • a mutant library of S3A5-VH with a library capacity exceeding 2.0*10E7 was constructed using the classical overlap extension PCR method. The recombinant hIFNAR1-his was then used as an antigen, and the heavy chain mutant library was screened for three rounds.
  • two affinity-enhanced heavy chain mutants H15D10 SEQ ID NO: 19
  • H19B7 SEQ ID NO: 20
  • H15D10+L8C3, H19B7+L8C3, H15D10+L16C11, H19B7+L16C11 and the control antibody anti-IFNAR1-C2 (chimeric) for hIFNAR1-His was determined using a BIAcore 3000 Biomacromolecule Interaction Analyzer.
  • Amine coupling kit and human antibody capture kit and CM5 chip and 10 ⁇ HBS-EP at pH 7.4 were purchased from GE Healthcare.
  • the antibody against the human F C segment was conjugated to the surface of the chip CM5 according to the instructions in the kit, and the antibody protein was diluted to a suitable concentration to ensure that about 200 RU of the antibody was captured by the antibody against human Fc.
  • hIFNAR1-His was subjected to a series of concentration gradients (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0 nM) flowing through the surface of the stationary phase to determine the affinity of each monoclonal antibody.
  • concentration gradients 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0 nM
  • the prepared human IFNAR1 (hIFNAR1), murine IFNAR1 (mIFNAR1) and cynomolgus IFNAR1 (mmIFNAR1) were each coated on a 96-well ELISA plate (concentration: 4 ⁇ g/mL, 100 ⁇ L/well), and coated at 4 ° C overnight. After blocking for 1 hour at 37 ° C with blocking solution (2% milk-PBST), various anti-human IFNAR1 monoclonal antibodies (H15D10+L8C3, H19B7+L8C3, H15D10+L16C11 and H19B7+L16C11, control antibody anti-IFNAR1-C1 were added respectively.
  • the ELISA plate was washed with PBST, HRP-anti-human IgG (secondary antibody) was added, and binding was carried out for 1 hour at 37 °C.
  • the ELISA plate was washed with PBST, and the OPD substrate coloring solution was added. After 5 to 10 minutes, the color development was terminated with a 1 M H 2 SO 4 solution, and the optical density value was measured by a microplate reader at 490 nm/630 nm.
  • the monoclonal antibodies against human IFNAR1 of the present application (H15D10+L8C3, H15D10+L16C11, H19B7+L8C3 and H19B7+L16C11) and control antibodies (anti-IFNAR1-C1 and anti-IFNAR1-C2 (chimeric)) Both are capable of binding to human IFNAR1 and cynomolgus IFNAR1, but not to murine IFNAR1.
  • the heavy chain constant regions of the monoclonal antibodies H15D10+L8C3, H15D10+L16C11, H19B7+L8C3 and H19B7+L16C11 are all IgG1 subtypes, and the light chain constant regions are all kappa subtypes.
  • the heavy chain constant regions of the monoclonal antibodies H15D10+L8C3, H15D10+L16C11, H19B7+L8C3 and H19B7+L16C11 are all IgG1 subtypes, and the light chain constant regions are all kappa subtypes.
  • Daudi cells are human Burkkit lymphoma cells, and type I interferons (including IFN ⁇ / ⁇ / ⁇ , etc.) are effective in inhibiting the growth of the cells.
  • Functional anti-IFNA R1 monoclonal antibody should be able to effectively block the binding of type I interferon (including IFN ⁇ / ⁇ / ⁇ , etc.) and its receptor (IFNAR1/IFNAR2 complex), and inhibit type I interferon-induced Daudi cells. death.
  • Daudi cells were seeded at a density of 3.5 ⁇ 10 4 /well in 96-well cell plates, and then fixed at a suitable fixed concentration (eg 0.67 nM).
  • Type I interferons eg, IFN ⁇ -2b-HSA
  • anti-human IFNAR1 monoclonal antibodies at different concentrations (eg, 0-25 nM) (eg, H19B7+L16C11, H19B7+L8C3, or anti-IFNAR1-C2 (chimeric)) Daudi cells were simultaneously treated, and then cultured at 37 ° C for 2 to 3 days in a CO 2 incubator.
  • Figure 5 shows inhibition curves of different anti-human IFNAR1 mAbs against IFNa-2b-HSA at a concentration of 0.67 nM.
  • Table 9 lists the inhibitory potency (IC50) of three different antibodies to three different type I interferons based on the Daudi cell assay.
  • Example 8 Anti-human IFNAR1 monoclonal antibody (IgG1 subtype) inhibits type I interferon-mediated intracellular signaling pathway
  • the human STAT2 and IRF9 genes are integrated and expressed in this cell line, and thus the ISGF3 signaling pathway can be activated under the stimulation of type I interferons (including IFN ⁇ / ⁇ / ⁇ ).
  • a secreted alkaline phosphatase (SEAP) reporter gene was simultaneously integrated into the cell line, and the expression of the SEAP reporter gene was regulated by the ISG54 promoter.
  • the JAK/STAT/ISGF3 signaling pathway is activated and the SEAP reporter gene expression is induced, and the expression of the SEAP gene can utilize the accessory reagent QUANTI-Blue TM provided by InvivoGen. (Invivo Gen, Cat#repqb1) for convenient testing.
  • this cell strain can be used for analysis of activity analysis of antagonists (e.g., antibodies, etc.) of type I interferons (including IFN ⁇ / ⁇ / ⁇ ) and type I interferon receptor (IFNAR1/2).
  • HEK-BlueTM IFN ⁇ / ⁇ cells were seeded at a density of 5 ⁇ 10 4 cells/well in 96-well cell plates at different densities when tested for inhibition of type I interferon by different anti-human IFNAR1 monoclonal antibodies.
  • type I interferon eg IFN ⁇
  • anti-IFNAR1 monoclonal antibodies at different concentrations (eg 0-300 nM) (eg H19B7+L16C11, H19B7+L8C3 or anti-IFNAR1-C2 (chimeric)
  • the HEK-BlueTM IFN ⁇ / ⁇ cells were simultaneously treated, and then cultured in a CO 2 incubator at 37 ° C for 20 to 24 hours.
  • Figure 6 shows the inhibition curves of IFN[omega] by different anti-human IFNARl mAbs.
  • Table 10 lists based upon HEK-Blue TM IFN ⁇ analysis / ⁇ cells of three different antibodies on three different type I interferons inhibit (IC50).
  • the fluorescent probe can bind to the hydrophobic region of the protein to emit a fluorescent signal.
  • the protein changes from a folded state to an expanded state, and the fluorescence signal changes with the exposure of the hydrophobic region, and a temperature fluorescence curve can be obtained, and the Tm can be obtained according to the curve. Value, to determine the thermal stability of the protein.
  • Dilute anti-human IFNAR1 monoclonal antibody eg, H19B7+L16C11, H19B7+L8C3, H15D10+L16C11, H15D10+L8C3, or control antibody anti-IFNAR1-C1
  • IgG4 Dilute anti-human IFNAR1 monoclonal antibody
  • a certain concentration such as 1mg / mL
  • add a certain proportion Orange (Sigma, Cat# S5692-50UL).
  • the melting curve was run on a real-time PCR (ABI, 7500 Fast) instrument, heating program: heating to 95 ° C at 25 ° C, heating rate 1 ° C / min, each temperature equilibrated for 2 minutes, and finally using Protein Thermal Shift Software 1.2 for data analysis and Drawing.
  • the heavy chain constant regions of the monoclonal antibodies H19B7+L16C11, H19B7+L8C3, H15D10+L16C11 and H15D10+L8C3 were all IgG4 subtypes, and the light chain constant regions were all kappa subtypes.
  • Figure 7 shows the heat stability of different anti-human IFNAR1 mAbs. The results show that the thermal stability of the four novel anti-human IFNAR1 mAbs is better than the control antibody anti-IFNAR1-C1.

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Abstract

提供了抗人I型干扰素受体亚基1 (IFNAR1)的抗体及其片段，还提供了包含该抗体或片段的药物组合物及其制药用途。

Description

抗人IFNAR1的抗体及其用途 技术领域
本申请大体涉及抗体药物领域，具体而言，本申请提供了抗人I型干扰素受体亚基1(IFNAR1)的抗体及其医学和生物学用途。
背景技术
干扰素(IFN)包括I型干扰素、II型干扰素和III型干扰素。其中人的I型干扰素包括IFN-α、IFN-β、IFN-ε、IFN-κ和IFN-ω。通常，当有病毒侵入机体时，人体内的成纤维细胞和单核细胞就分泌各种I型干扰素，以阻止和干扰病毒的DNA和RNA复制。而且，I型干扰素还有抗肿瘤和免疫调节的功能(Capobianchi M.R.,et al.,2015,Cytokine Growth Factor Rev.,26:103；Zitvogel L.,et al.,2015,Nat Rev Immunol.,15:405)。
所有的人I型干扰素都共用一组细胞表面的受体复合物，即IFNα/β受体复合物，该IFNα/β受体复合物包括IFNAR1和IFNAR2两个跨膜蛋白亚基。其中IFNAR1单独结合I型干扰素的能力较弱(KD约为10-7M),而IFNAR2单独结合I型干扰素的能力较强(KD约为10-9M)。但此两个受体亚基对I型干扰素功能实现都必不可少，而且都影响受体复合物对不同I型干扰素的高亲和力(KD约为10-11M)和特异性(Bekisz J,et al.，2004，Growth Factors,22:243)。
早期对I型干扰素的功能研究主要都集中在抗病毒感染等先天性免疫。但近年来，更多的研究提示I型干扰素在适应性免疫也有很强的免疫调节作用，包括促进抗体分泌和支持T记忆细胞的功能活性和存活等。尤其有研究表明IFN-α可以促进树突状细胞(DC)的成熟或者激活(Santini S.M.,et al.,2000,J.EXP.Med.,191:1777)。而且，有研究发现，多种自身免疫性疾病都表现出I型干扰素过表达。其中，胰岛素依赖的糖尿病(IDDM)和系统性红斑狼疮(SLE)与IFN-α的表达升高相关，而IFN-β则可能与类风湿性关节炎(RA)相关。而且，有报道显示临床上I型IFN用药导致一些自身免疫性疾病(包括银屑病和多发性硬化症等)的恶化，而且可能在没有自身免疫病史的病人中诱导SLE样症状。此外，研究显示，TMPD在正常小鼠中能够有效诱导系统性红斑狼疮症状，但不能在IFNAR1基因缺陷的小鼠中诱导特异的自身抗体(Nacionales D.C.,et al.,2007,Arthritis Rheum,56:3770)。在BXSB模型小鼠体内，在疾病早期抗IFNAR1抗体表现出明显的治疗效果(Baccala R.,et al.,2012,J.Immunol,189:5876)。因而，在临床上，抑制I型干扰素受体(IFNAR)可能使某些自身免疫性疾病的病人受益，而且临床上也需要一些能够有效抑制I型干扰素受体的药物用于多种自身免疫性疾病(包括系统性红斑狼疮)的治疗。
因此，探索和开发能抑制IFNAR的物质(例如，抗体)具有重要的生物学和医学意义。
发明内容
第一方面，本申请提供了特异性结合人I型干扰素受体亚基1(IFNAR1)的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区，其特征在于：所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)；或者，所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)；或者，所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ  ID NO:33)，所述HCDR3序列为HDVHGYDY(SEQ ID NO:35)；或者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPSDSNTSYSPSFQG(SEQ ID NO:37)，所述HCDR3序列为DASSKTYDS(SEQ ID NO:38)；或者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPGDSYTRYSPSFQG(SEQ ID NO:39)，所述HCDR3序列为DGAPAKGDFDY(SEQ ID NO:40)；其中HCDR序列根据Kabat定义。
在一些实施方案中，所述抗体的重链可变区序列如SEQ ID NO:19、20、23、24或者25的氨基酸序列所示。
第二方面，本申请提供了特异性结合人IFNAR1的抗体，其包含含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于：所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述LCDR1序列为RASQNVSNYLN(SEQ ID NO:47)，所述LCDR2序列为RASNLQS(SEQ ID NO:48)，所述LCDR3序列为QQMMDAPPT(SEQ ID NO:49)；或者，所述LCDR1序列为SGSSSNIGTNAVN(SEQ ID NO:50)，所述LCDR2序列为SKNQRPP(SEQ ID NO:51)，所述LCDR3序列为AAWDDSQNGYVV(SEQ ID NO:52)；或者，所述LCDR1序列为RASEGIGNHLN(SEQ ID NO:53)，所述LCDR2序列为TASNLQS(SEQ ID NO:54)，所述LCDR3序列为QQTYITPLT(SEQ ID NO:55)；其中LCDR序列根据Kabat定义。
在一些实施方案中，所述抗体的轻链可变区序列如SEQ ID NO:21、22、26、27或者28的氨基酸序列所示。
第三方面，本申请提供了特异性结合人IFNAR1的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区及含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于：所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)，所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)，所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVHGYDY(SEQ ID NO:35)，所述LCDR1序列为RASQNVSNYLN(SEQ ID NO:47)，所述LCDR2序列为RASNLQS(SEQ ID NO:48)，所述LCDR3序列为QQMMDAPPT(SEQ ID NO:49)；或 者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPSDSNTSYSPSFQG(SEQ ID NO:37)，所述HCDR3序列为DASSKTYDS(SEQ ID NO:38)，所述LCDR1序列为SGSSSNIGTNAVN(SEQ ID NO:50)，所述LCDR2序列为SKNQRPP(SEQ ID NO:51)，所述LCDR3序列为AAWDDSQNGYVV(SEQ ID NO:52)；或者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPGDSYTRYSPSFQG(SEQ ID NO:39)，所述HCDR3序列为DGAPAKGDFDY(SEQ ID NO:40)，所述LCDR1序列为RASEGIGNHLN(SEQ ID NO:53)，所述LCDR2序列为TASNLQS(SEQ ID NO:54)，所述LCDR3序列为QQTYITPLT(SEQ ID NO:55)；其中HCDR和LCDR序列根据Kabat定义。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:19，轻链可变区序列为SEQ ID NO:21。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:19，轻链可变区序列为SEQ ID NO:22。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:20，轻链可变区序列为SEQ ID NO:21。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:20，轻链可变区序列为SEQ ID NO:22。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:23，轻链可变区序列为SEQ ID NO:26。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:24，轻链可变区序列为SEQ ID NO:27。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:25，轻链可变区序列为SEQ ID NO:28。
在上述三方面的一些实施方案中，特异性结合人IFNAR1的抗体为全长抗体、Fab片段、F(ab')2片段或单链Fv片段。
在一些实施方案中，特异性结合人IFNAR1的抗体是全人源抗体。
在一些实施方案中，特异性结合人IFNAR1的抗体还包含选自IgG1亚型(SEQ ID NO:7)、IgG2亚型(SEQ ID NO:8)或IgG4亚型(SEQ ID NO:9)的重链恒定区和/或包含选自κ亚型(SEQ ID NO:10)或者λ亚型(SEQ ID NO:11)的轻链恒定区。
第四方面，本申请提供了包含第一至第三方面所述的特异性结合人IFNAR1的抗体的药物组合物。
第五方面，本申请提供了第一至第三方面所述的特异性结合人IFNAR1的抗体，或者第四方面所述药物组合物在制备用于预防或治疗由人IFNAR1介导的疾病的药物中的用途。
在一些实施方案中，所述疾病为自身免疫性疾病。
在一些实施方案中，所述自身免疫性疾病包括但不限于，系统性红斑狼疮、银屑病、多发性硬化症、类风湿性关节炎。
第六方面，本申请还提供治疗由人IFNAR1介导的疾病的方法，包括给予有需要的患者治疗有效量的第一至第三方面所述的特异性结合人IFNAR1的抗体，或者第四方面所述药物组合物。
在一些实施方案中，所述疾病为自身免疫性疾病。
在一些实施方案中，所述自身免疫性疾病包括但不限于，系统性红斑狼疮、银屑病、多发性硬化症、类风湿性关节炎。
附图说明
图1显示了噬菌体展示载体pADSCFV-S的结构示意图。
图2显示了对照单抗抗IFNAR1-C1和抗IFNAR1-C2对噬菌体展示单链抗体S3A5、S3H8和S5B4结合人IFNAR1能力的抑制。
图3显示了ELISA分析抗人IFNAR1单抗与不同种属IFNAR1蛋白的结合能力。
图4显示了不同抗人IFNAR1单抗在人血清中的稳定性分析。
图5显示了基于Daudi细胞分析三种不同抗人IFNAR1单抗(H19B7+L16C11、H19B7+L8C3或抗IFNAR1-C2(嵌合))对浓度为0.67nM的IFNα-2b-HSA的抑制。
图6显示了基于HEK-BlueTM IFNα/β细胞分析三种不同抗人IFNAR1单抗(H19B7+L16C11、H19B7+L8C3或抗IFNAR1-C2(嵌合))对浓度为0.1nM的IFNω的抑制。
图7显示了不同抗人IFNAR1单抗(H19B7+L16C11、H19B7+L8C3、H15D10+L16C11、H15D10+L8C3或抗IFNAR1-C1)对热的稳定性分析的熔解曲线图/导数图(20160405-DSF)。
序列说明
SEQ ID NO:1为人IFNAR1胞外区(hIFNAR1)的氨基酸序列。
SEQ ID NO:2为小鼠IFNAR1胞外区(mIFNAR1)的氨基酸序列。
SEQ ID NO:3为猕猴IFNAR1胞外区(mmIFNAR1)的氨基酸序列。
SEQ ID NO:4为人IFNβ(IFNβ)的氨基酸序列。
SEQ ID NO:5为His标签的氨基酸序列。
SEQ ID NO:6为鼠抗体IgG2a的Fc段(mFc)的氨基酸序列。
SEQ ID NO:7为抗体重链恒定区IgG1亚型的氨基酸序列。
SEQ ID NO:8为抗体重链恒定区IgG2亚型的氨基酸序列。
SEQ ID NO:9为抗体重链恒定区IgG4亚型的氨基酸序列。
SEQ ID NO:10为抗体κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:11为抗体λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:12为单链抗体S3A5的氨基酸序列。
SEQ ID NO:13为单链抗体S3H8的氨基酸序列。
SEQ ID NO:14为单链抗体S5B4的氨基酸序列。
SEQ ID NO:15为对照重组抗体抗IFNAR1-C1的VH的氨基酸序列。
SEQ ID NO:16为对照重组抗体抗IFNAR1-C1的VL的氨基酸序列。
SEQ ID NO:17为对照重组抗体抗IFNAR1-C2的VH的氨基酸序列。
SEQ ID NO:18为对照重组抗体抗IFNAR1-C2的VL的氨基酸序列。
SEQ ID NO:19为重链突变体H15D10的氨基酸序列。
SEQ ID NO:20为重链突变体H19B7的氨基酸序列。
SEQ ID NO:21为轻链突变体L8C3的氨基酸序列。
SEQ ID NO:22为轻链突变体L16C11的氨基酸序列。
SEQ ID NO:23为单链抗体S3A5的重链可变区序列的氨基酸序列。
SEQ ID NO:24为单链抗体S3H8的重链可变区序列的氨基酸序列。
SEQ ID NO:25为单链抗体S5B4的重链可变区序列的氨基酸序列。
SEQ ID NO:26为单链抗体S3A5的轻链可变区的氨基酸序列。
SEQ ID NO:27为单链抗体S3H8的轻链可变区的氨基酸序列。
SEQ ID NO:28为单链抗体S5B4的轻链可变区的氨基酸序列。
SEQ ID NO:29为本申请鉴定的一种HCDR1的氨基酸序列。
SEQ ID NO:30为本申请鉴定的一种HCDR2的氨基酸序列。
SEQ ID NO:31为本申请鉴定的一种HCDR3的氨基酸序列。
SEQ ID NO:32为本申请鉴定的一种HCDR1的氨基酸序列。
SEQ ID NO:33为本申请鉴定的一种HCDR2的氨基酸序列。
SEQ ID NO:34为本申请鉴定的一种HCDR3的氨基酸序列。
SEQ ID NO:35为本申请鉴定的一种HCDR3的氨基酸序列。
SEQ ID NO:36为本申请鉴定的一种HCDR1的氨基酸序列。
SEQ ID NO:37为本申请鉴定的一种HCDR2的氨基酸序列。
SEQ ID NO:38为本申请鉴定的一种HCDR3的氨基酸序列。
SEQ ID NO:39为本申请鉴定的一种HCDR2的氨基酸序列。
SEQ ID NO:40为本申请鉴定的一种HCDR3的氨基酸序列。
SEQ ID NO:41为本申请鉴定的一种LCDR1的氨基酸序列。
SEQ ID NO:42为本申请鉴定的一种LCDR2的氨基酸序列。
SEQ ID NO:43为本申请鉴定的一种LCDR3的氨基酸序列。
SEQ ID NO:44为本申请鉴定的一种LCDR1的氨基酸序列。
SEQ ID NO:45为本申请鉴定的一种LCDR2的氨基酸序列。
SEQ ID NO:46为本申请鉴定的一种LCDR3的氨基酸序列。
SEQ ID NO:47为本申请鉴定的一种LCDR1的氨基酸序列。
SEQ ID NO:48为本申请鉴定的一种LCDR2的氨基酸序列。
SEQ ID NO:49为本申请鉴定的一种LCDR3的氨基酸序列。
SEQ ID NO:50为本申请鉴定的一种LCDR1的氨基酸序列。
SEQ ID NO:51为本申请鉴定的一种LCDR2的氨基酸序列。
SEQ ID NO:52为本申请鉴定的一种LCDR3的氨基酸序列。
SEQ ID NO:53为本申请鉴定的一种LCDR1的氨基酸序列。
SEQ ID NO:54为本申请鉴定的一种LCDR2的氨基酸序列。
SEQ ID NO:55为本申请鉴定的一种LCDR3的氨基酸序列。
SEQ ID NO:56为人抗体重链可变区基因VH1的核苷酸序列。
SEQ ID NO:57为人抗体重链可变区基因VH3的核苷酸序列。
SEQ ID NO:58为人抗体重链可变区基因VH5的核苷酸序列。
SEQ ID NO:59为人抗体轻链可变区基因VK1的核苷酸序列。
SEQ ID NO:60为人抗体轻链可变区基因Vl3的核苷酸序列。
SEQ ID NO:61为PelB前导肽的编码基因的核苷酸序列。
SEQ ID NO:62为无关序列的核苷酸序列。
SEQ ID NO:63为单链抗体S3A5的编码基因的核苷酸序列。
SEQ ID NO:64为单链抗体S3H8的编码基因的核苷酸序列。
SEQ ID NO:65为单链抗体S5B4的编码基因的核苷酸序列。
发明详述
本申请的发明人通过构建大容量天然人源噬菌体抗体库，筛选并优化得到了具有希望性质的抗人IFNAR1抗体。在本申请的多个方面，提供了新的抗人IFNAR1的单克隆抗体或其抗原结合片段，编码该单克隆抗体或其抗原结合片段的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体或其抗原结合片段的医学和生物学应用。根据本申请提供 的抗体的可变区的序列，可构建全长的抗体分子作为药物用于治疗临床上由IFNAR1介导的自身免疫系统疾病。这些疾病包括但不局限于系统性红斑狼疮、银屑病、多发性硬化症、类风湿性关节炎。
除非另外指明，本发明的实施将采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“抗体”，是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到标靶的免疫球蛋白分子。标靶包括但不限于碳水化合物、多聚核苷酸、脂质、多肽等。本文所使用的“抗体”不仅包括完整的(即全长的)抗体，而且还包括其抗原结合片段(例如Fab、Fab'、F(ab')2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置，包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常，完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链变异区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链变异区(VL)和恒定区(CL)。全长的抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ、以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。
如本文所用术语“抗原结合片段”，是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合域可以包含重链变异区(VH)、轻链变异区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义。(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
抗原结合片段的实例包括但不限于：(1)Fab片段，其可以是具有VL-CL链和VH-CH1链的单价片段；(2)F(ab')2片段，其可以是具有两个Fab'片段的二价片段，该两个Fab'片段由铰链区的二硫桥(即Fab'的二聚物)连接；(3)具有抗体的单臂的VL和VH域的Fv片段；(4)单链Fv(scFv)，其可以是由VH域和VL域经由胜肽连接符组成的单一多胜肽链；以及(5)(scFv)2，其可以包含两个由胜肽连接符连接的VH域和两个VL域，该两个VL域是经由二硫桥与该两个VH域组合。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
具体实施方式
第一方面，本申请提供了特异性结合人IFNAR1的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区，其特征在于：所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)；或者，所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)；或者，所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVHGYDY(SEQ ID NO:35)；或者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPSDSNTSYSPSFQG(SEQ ID NO:37)，所述HCDR3序列为DASSKTYDS(SEQ ID NO:38)；或者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPGDSYTRYSPSFQG(SEQ ID NO:39)，所述HCDR3序列为DGAPAKGDFDY(SEQ ID NO:40)；其中HCDR序列根据Kabat定义。
在一些实施方案中，所述抗体的重链可变区序列如SEQ ID NO:19、20、23、24或者25的氨基酸序列所示。
第二方面，本申请提供了特异性结合人IFNAR1的抗体，其包含含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于：所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述LCDR1序列为RASQNVSNYLN(SEQ ID NO:47)，所述LCDR2序列为RASNLQS(SEQ ID NO:48)，所述LCDR3序列为QQMMDAPPT(SEQ ID NO:49)；或者，所述LCDR1序列为SGSSSNIGTNAVN(SEQ ID NO:50)，所述LCDR2序列为SKNQRPP(SEQ ID NO:51)，所述LCDR3序列为AAWDDSQNGYVV(SEQ ID NO:52)；或者，所述LCDR1序列为RASEGIGNHLN(SEQ ID NO:53)，所述LCDR2序列为TASNLQS(SEQ ID NO:54)，所述LCDR3序列为QQTYITPLT(SEQ ID NO:55)；其中LCDR序列根据Kabat定义。
在一些实施方案中，所述抗体的轻链可变区序列如SEQ ID NO:21、22、26、27或者28的氨基酸序列所示。
第三方面，本申请提供了特异性结合人IFNAR1的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区及含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于：所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)，所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID  NO:34)，所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVHGYDY(SEQ ID NO:35)，所述LCDR1序列为RASQNVSNYLN(SEQ ID NO:47)，所述LCDR2序列为RASNLQS(SEQ ID NO:48)，所述LCDR3序列为QQMMDAPPT(SEQ ID NO:49)；或者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPSDSNTSYSPSFQG(SEQ ID NO:37)，所述HCDR3序列为DASSKTYDS(SEQ ID NO:38)，所述LCDR1序列为SGSSSNIGTNAVN(SEQ ID NO:50)，所述LCDR2序列为SKNQRPP(SEQ ID NO:51)，所述LCDR3序列为AAWDDSQNGYVV(SEQ ID NO:52)；或者，所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPGDSYTRYSPSFQG(SEQ ID NO:39)，所述HCDR3序列为DGAPAKGDFDY(SEQ ID NO:40)，所述LCDR1序列为RASEGIGNHLN(SEQ ID NO:53)，所述LCDR2序列为TASNLQS(SEQ ID NO:54)，所述LCDR3序列为QQTYITPLT(SEQ ID NO:55)；其中HCDR和LCDR序列根据Kabat定义。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:19，轻链可变区序列为SEQ ID NO:21。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:19，轻链可变区序列为SEQ ID NO:22。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:20，轻链可变区序列为SEQ ID NO:21。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:20，轻链可变区序列为SEQ ID NO:22。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:23，轻链可变区序列为SEQ ID NO:26。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:24，轻链可变区序列为SEQ ID NO:27。
在一些实施方案中，特异性结合人IFNAR1的抗体的重链可变区序列为SEQ ID NO:25，轻链可变区序列为SEQ ID NO:28。
在上述三方面的一些实施方案中，特异性结合人IFNAR1的抗体为全长抗体、Fab片段、F(ab')2片段或单链Fv片段。
在一些实施方案中，特异性结合人IFNAR1的抗体是全人源抗体。
在一些实施方案中，特异性结合人IFNAR1的抗体还包含选自IgG1亚型(SEQ ID NO:7)、IgG2亚型(SEQ ID NO:8)或IgG4亚型(SEQ ID NO:9)的重链恒定区和/或包含选自κ亚型(SEQ ID NO:10)或者λ亚型(SEQ ID NO:11)的轻链恒定区。
在一些实施方案中，特异性结合人IFNAR1的抗体拮抗至少一种与IFNAR1或其部分相关的体外或体内生物活性。
在一些实施方案中，特异性结合IFNAR1的抗体能够特异性结合重组人IFNAR1胞外区。
在一些实施方案中，抗体特征在于以高于背景的水平特异性结合人IFNAR1 和猕猴IFNAR1，而不结合小鼠IFNAR1。
本申请还提供了编码上述抗体或其抗原结合片段的多核苷酸、包含所述多核苷酸的载体以及用所述载体转染的宿主细胞。
第四方面，本申请提供了包含第一至第三方面所述的特异性结合人IFNAR1的抗体的药物组合物。
在一些实施方案中，药物组合物还包含药学可接受的载体、赋形剂、稀释剂等。
在一些实施方案中，药物组合物还可包含润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
在一些实施方案中，所述药物组合物用于预防或治疗由人IFNAR1介导的疾病，例如自身免疫性疾病，其包括但不限于，系统性红斑狼疮、银屑病、多发性硬化症、类风湿性关节炎。
第五方面，本申请提供了第一至第三方面所述的特异性结合人IFNAR1的抗体，或者第四方面所述药物组合物在制备用于预防或治疗由人IFNAR1介导的疾病的药物中的用途。
在一些实施方案中，所述疾病为自身免疫性疾病。
在一些实施方案中，所述自身免疫性疾病包括但不限于，系统性红斑狼疮、银屑病、多发性硬化症、类风湿性关节炎。
在一些实施方案中，本申请的抗体的亲和力、其与不同种属的IFNAR1的结合、其在人血清中的稳定性、其药理学活性、其热稳定性可以在标准测试方法中进行证明。
第六方面，本申请还提供治疗由人IFNAR1介导的疾病的方法，包括给予有需要的患者治疗有效量的第一至第三方面所述的特异性结合人IFNAR1的抗体，或者第四方面所述药物组合物。
在一些实施方案中，所述疾病为自身免疫性疾病。
在一些实施方案中，所述自身免疫性疾病包括但不限于，系统性红斑狼疮、银屑病、多发性硬化症、类风湿性关节炎。
第七方面，本申请还提供编码本发明抗体或其轻链或其重链的分离的核酸分子以及包含所述核酸分子的载体、包含所述载体的宿主细胞以及产生所述抗体的方法。
在一些实施方案中，所述核酸分子可操作地连接到调控序列，调控序列可以被用所述载体转化过的宿主细胞识别。
在一些实施方案中，产生抗体的方法包括培养宿主细胞以便于表达核酸。
在一些实施方案中，产生抗体的方法还包括从宿主细胞培养基中回收抗体。
此外，本文所述的特异性结合人IFNAR1的抗体也可用于检测生物样品中 IFNAR1的存在。基于抗体的检测方法在本领域是众所周知的，并且包括例如ELISA、免疫印迹、放射免疫试验、免疫荧光、免疫沉淀以及其它相关技术。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
以下实施例仅用于说明而非限制本申请范围的目的。
实施例
实施例1高质量噬菌体展示抗体库的构建
抗体库技术是制备和筛选人单克隆抗体的一个重要方法，而基于噬菌体展示的抗体库技术则是目前成熟的抗体库技术，已经成功地应用于人单抗药物的制备。本实施例描述利用当今多种基因工程技术构建噬菌体展示抗体库的策略和方法。
1.1制备抗体重链和轻链可变区基因(VH和VL)
为构建人抗体库，首先需要获得人抗体的重链可变区(VH)和轻链可变区(VL)基因。抗体可变区基因来自正常人外周血淋巴细胞以及全合成。
1.1.1天然人抗体可变区基因的制备
采集19个正常志愿者的血液(各50mL)，其中所采集的血液由发明人及其同事作为志愿者提供，所有志愿者均已签署知情同意书。志愿者的纳入标准为：
1.年龄大于18周岁；
2.无HIV、HBV感染；
3.血常规检测正常；
4.非孕妇或哺乳期妇女。
然后，用淋巴细胞分离液(MP Biomedicals公司,Cat#:0850494)分离淋巴细胞，利用Omega公司的总RNA提取试剂盒(Cat#:R6834-01)制备RNA，然后用TransGenBiotech公司的反转录试剂盒(Cat#:AT301-03)制备cDNA。用下表1所列的引物组进行PCR，分别扩增抗体的重链可变区基因(VH)和轻链可变区基因(VL，包括Vk和Vl)。利用常规的琼脂糖凝胶电泳方法纯化并回收扩增的PCR产物(VH,VK或Vl)，置于-20℃保存备用。
表1扩增天然人抗体重链和轻链基因所用引物
引物   引物序列
VH正向引物 VHF1 CAGRTGCAGCTGGTGCARTCTGG
  VHF2 SAGGTCCAGCTGGTRCAGTCTGG
  VHF3 CAGRTCACCTTGAAGGAGTCTGG
  VHF4 SAGGTGCAGCTGGTGGAGTCTGG
  VHF5 GAGGTGCAGCTGGTGGAGWCYGG
  VHF6 CAGGTGCAGCTACAGCAGTGGGG
  VHF7 CAGSTGCAGCTGCAGGAGTCSGG
  VHF8 GARGTGCAGCTGGTGCAGTCTGG
  VHF9 CAGGTACAGCTGCAGCAGTCAGG
VH反向引物 VHR1 TGAGGAGACGGTGACCAGGGTGCC
  VHR2 TGAAGAGACGGTGACCATTGTCCC
  VHR3 TGAGGAGACGGTGACCAGGGTTCC
  VHR4 TGAGGAGACGGTGACCGTGGTCCC
VK正向引物 VKF1 GACATCCAGWTGACCCAGTCTCC
  VKF2 GATGTTGTGATGACTCAGTCTCC
  VKF3 GAAATTGTGWTGACRCAGTCTCC
  VKF4 GATATTGTGATGACCCAGACTCC
  VKF5 GAAACGACACTCACGCAGTCTCC
  VKF6 GAAATTGTGCTGACTCAGTCTCC
VK反向引物 VKR1 ACGTTTGATCTCGAGCTTGGTCCCYTGGCCRAA
  VKR2 ACGTTTGATCTCGAGTTTGGTCCCAGGGCCGAA
  VKR3 ACGTTTGATCTCGAGCTTGGTCCCTCCGCCGAA
  VKR4 ACGTTTAATCTCGAGTCGTGTCCCTTGGCCGAA
Vl正向引物 VlF1 CAGTCTGTGYTGACKCAGCCRCC
  VlF2 CARTCTGCCCTGACTCAGCCT
  VlF3 TCCTATGWGCTGACTCAGCCA
  VlF4 TCTTCTGAGCTGACTCAGGACCC
  VlF5 CAGGCTGTGCTGACTCAGCCG
  VlF6 AATTTTATGCTGACTCAGCCCCA
  VlF7 CAGRCTGTGGTGACYCAGGAGCC
  VlF8 CWGCCTGTGCTGACTCAGCC
Vl反向引物 VlR1 ACCTAGGACGGTGACCTTGGTCCC
  VlR2 ACCTAGGACGGTCAGCTTGGTCCC
  VlR3 ACCTAAAACGGTGAGCTGGGTCCC
1.1.2全合成人抗体可变区基因的制备
全合成抗体基因制备的基本策略是利用简并引物在选定的模板抗体基因的CDR中引入设计的突变。为构建全合成人抗体库，本实施例中选择了3种人抗体重链可变区模板(VH1、VH3和VH5)和两种人抗体轻链可变区模板(VK1和Vl3)构建人全合成抗体库。
设计并委托明琛志远生物技术(北京)有限公司合成5种抗体可变区基因VH1(SEQ ID NO:56)，VH3(SEQ ID NO:57)，VH5(SEQ IDNO:58)，VK1(SEQ ID NO:59)以及Vl3(SEQ ID NO:60)。设计并合成表2所列的引物，分别用于在5种可变区基因的CDR1、CDR2和CDR3中引入设计的各种突变。利用常规PCR技术及各组含有设计突变的兼并引物，分别在对应的CDR中引入设计的突变，然后再利用2～3轮重叠延伸PCR构建得到完整的重链可变区(VH1、VH3、VH5)和轻链可变区(VK1、VL3)基因。琼脂糖凝胶电泳回收扩增的最终的可变区基因PCR产物，置于-20℃保存备用。
表2扩增全合成人抗体可变区基因所用引物
Figure PCTCN2017075933-appb-000001
Figure PCTCN2017075933-appb-000002
Figure PCTCN2017075933-appb-000003
1.2构建单链抗体(ScFv)基因
为构建单链抗体基因(ScFv)，在重链可变区(VH)和轻链可变区(VL)之间添加常用的由15个氨基酸组成的柔性连接肽，该连接肽的序列为GGGGSGGGGSGGGGS，该连接肽的编码序列为ggtggaggcggttctggcggaggtgggagcggaggcggaggttca。设计的单链抗体的结构为VH-连接肽-VL。
基于此实施例第一部分的方法，共获得如下表3所示的多种重链和轻链可变区基因，即四种不同的重链可变区基因和3种轻链可变区基因。
表3各种不同重链和轻链可变区基因。
Figure PCTCN2017075933-appb-000004
Figure PCTCN2017075933-appb-000005
基于上述单链抗体的设计以及目前已经成熟重叠延伸PCR技术，可以将此表所示的不同重链和轻链进行组合，共构建获得12种不同的单链抗体基因。利用琼脂糖凝胶电泳方法纯化并回收PCR扩增获得的12种单链抗体基因，置于-20℃保存备用。
1.3构建基于阿拉伯糖启动子的噬菌体展示载体
常用的噬菌体展示载体基于乳糖启动子(Plac)，而乳糖启动子由于其渗漏表达等特性，影响抗体库的容量和多样性。以常用的噬菌体展示载体pCANTAB5E(Amersham Biosciences/GE公司)为基础，对pCANTAB5E进行了如下改造。
利用AflIII和NotI双酶切，将pCANTAB5E载体上的Plac启动子及g3蛋白信号肽部分更换为包含AraC基因，阿拉伯糖启动子(Para)及PelB前导肽(PelB leader，SEQ ID NO:61)的片段。其中AraC基因和ParaC来自invitrogen公司的pBADhis载体，而PelB前导肽序列为人工合成序列。然后再利用NcoI和NotI双酶切，将一段约750bp的无关序列(stuff sequence，SEQ ID NO:62)克隆在NcoI和NotI位点之间，构建得到最终的新型噬菌体展示载体pADSCFV-S(图1)。该载体中的NcoI位点和NotI位点，可以方便的用于克隆单链抗体(ScFv)基因。
1.4人单链抗体库及噬菌体展示抗体库的制备
利用NcoI和NotI双酶切的策略，将1.2中制备的12种ScFv分别克隆至载体pADSCFV-S，将连接产物分别电转TG1电转感受态，每个子库约20个电转，总共约240次电转。利用稀释法对每个子库的库容量进行推算，随机从每个子库中取30～40个克隆进行序列分析，以推算每个子库的正确率，汇总的12个子库的库容量及正确率见表4。此12个子库的总库容量达到1.0*10E9，平均正确率超过75％。
表4 12个子库的库容量及正确率
子库 库容量 正确率
ScFv-VH1-VK1 4.79*10E7 81％
ScFv-VH1-VL3 3.72*10E7 76％
ScFv-VH1-VL 2.2*10E7 70％
ScFv-VH3-VK1 2.2*10E7 83％
ScFv-VH3-VL3 3.14*10E7 78％
ScFv–VH3-VL 7.7*10E7 70％
ScFv-VH5-VK1 2.68*10E7 74％
ScFv-VH5-VL3 2.5*10E7 76％
ScFv–VH5-VL 9.2*10E7 84％
ScFv-VH-VK1 5.9*10E7 75％
ScFv-VH-VL3 7.7*10E7 72％
ScFv–VH-VL 27.2*10E7 85％
将构建的12个子库分别接种于2YTAG液体培养基(A:氨苄青霉素，100μg/mL；G:葡萄糖，2％)，37℃，220rpm震荡培养至对数生长期(OD600＝0.8)，感染M13辅助噬菌体(M13KO7,NEB公司)，感染结束后置换为2YTAKA液体培养基(A:氨苄青霉素，100μg/mL；K:卡那霉素，70μg/mL；A:阿拉伯糖，0.001％)，28℃，220rpm震 荡培养过夜进行噬菌体扩增，然后利用PEG/Nacl沉淀方法制备纯化噬菌体(噬菌体-ScFv)，并进行滴度测定。然后参照库容量的比例将制备的12个子库的噬菌体-ScFv进行混合，制备噬菌体展示人抗体库，噬菌体的最终滴度为6*10E12cfu/mL，冻存于-70℃。此噬菌体展示抗体库可以用于筛选针对各种目的抗原的特异性人抗体。
实施例2各种重组抗原和抗体的制备
为了制备和测试抗人IFNAR1单抗，合成了多种重组蛋白，包括人IFNAR1胞外区(hIFNAR1，SEQ ID NO:1)，小鼠IFNAR1胞外区(mIFNAR1，SEQ ID NO:2)和猕猴IFNAR1胞外区(mmIFNAR1，SEQ ID NO:3)，人IFNβ(IFNβ，SEQ ID NO:4)，以及对照重组抗体抗IFNAR1-C1(VH序列为SEQ ID NO:15，VL序列为SEQ ID NO:16；参见美国专利US7662381B2中人单抗3F11)和抗IFNAR1-C2(VH序列为SEQ ID NO:17,VL序列为SEQ ID NO:18；参见美国专利US7619070B2中鼠单抗64G12)。这些蛋白都有大量的翻译后修饰(如:糖基化或二硫键等)，因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功能。此外，为了方便纯化，非抗体类的重组蛋白在C端添加了His标签(SEQ ID NO:5)或者鼠抗体IgG2a的Fc段(mFc，SEQ ID NO:6)。抗体重链恒定区可以是IgG1亚型(SEQ ID NO:7)，IgG2亚型(SEQ ID NO:8)或者IgG4亚型(SEQ ID NO:9)，轻链恒定区可以是κ亚型(SEQ ID NO:10)或者λ亚型(SEQ ID NO:11)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His标签或者mFc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或其它转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3～5天，然后通过离心等方式收获培养上清。
His标签融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。而mFc融合表达的重组蛋白和全抗体用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其它合适的缓冲液。
实施例3利用噬菌体展示抗体库技术筛选和优化抗人IFNAR1单抗
3.1抗人IFNAR1单抗的筛选
以实施例2制备的重组hIFNAR1-his为抗原，利用固相筛选策略(实验方案参考噬菌体展示：通用实验指南/(美)克拉克森(Clackson,T.),(美)洛曼(Lowman,H.B.)编；马岚等译。化学工业出版社，2008.5)筛选实施例1制备的展示人单链抗体库的噬菌体库，获得3株序列不同，但均能特异性结合人IFNAR1的人抗体，包括克隆S3A5(氨基酸序列为SEQ ID NO:12，编码序列为SEQ ID NO:63，VH序列为SEQ ID NO:23，VL序列为SEQ ID NO:26)，S3H8(氨基酸序列为SEQ ID NO:13，编码序列为SEQ ID NO:64，VH序列为SEQ ID NO:24，VL序列为SEQ ID NO:27)，S5B4(氨基酸序列为SEQ ID NO:14，编码序列为SEQ ID NO:65，VH序列为SEQ ID NO:25，VL序列为SEQ ID NO:28)。
3.2抗人IFNAR1单抗的初步功能分析
利用经典的噬菌体-ELISA方法分析两种功能性抗人IFNAR1单抗抗IFNAR1-C1和抗IFNAR1-C2与步骤3.1中筛选到的三种新型抗人IFNAR1抗体(S3A5、S3H8和S5B4)的表位的差异。
将实施例2制备的重组hIFNAR1-his包被于96孔ELISA板(浓度为4μg/mL，100μL/孔)，4℃包被过夜。利用封闭液(2％牛乳-PBST)在37℃封闭1小时后，然后分别加入展示单链抗体(S3A5、S3H8或S5B4)的纯化噬菌体或者噬菌体+5μg/mL的对照单抗(抗IFNAR1-C1或抗IFNAR1-C2，两种重组对照抗体的重链恒定区均为IgG1亚型，轻链恒定区均为κ亚型)，在37℃结合1小时后用PBST进行常规洗涤，然后加入封闭液稀释的HRP-抗M13单抗，在37℃结合1小时后用PBST进行常规洗涤，最后加入OPD底物液进行显色,终止显色后测定OD490。
结果如图2所示，抗IFNAR1-C1能够明显抑制噬菌体展示的S3A5和S3H8单抗与hIFNAR1-his的结合，而抗IFNAR1-C2对三种噬菌体展示抗体都没有明显的抑制效果。据此数据推测S3A5和S3H8单抗与hIFNAR1-his的结合表位与抗IFNAR1-C1完全或者部分重叠，与抗IFNAR1-C2不同；而S5B4单抗与hIFNAR1-his的结合表位与两种对照抗体(抗IFNAR1-C1和抗IFNAR1-C2)都不相同。
3.3基于重链CDR突变和轻链置换的策略对抗体S3A5进行体外亲和力成熟
基于双载体的噬菌体展示系统，首先利用重链CDR(HCDRs)突变的策略(具体操作可参照本申请人之前提交的中国专利第201510097117.0号中的实施例5)对抗体S3A5单抗进行体外亲和力成熟。其中在S3A5重链(S3A5-VH)的三个CDR中引入突变所需的关键引物见表5。利用经典的重叠延伸PCR(overlapping PCR)方法构建了库容量超过2.0*10E7的S3A5-VH的突变库。然后利用重组的hIFNAR1-his为抗原，对此重链突变库进行了三轮筛选。最后鉴定出两个亲和力提高的重链突变体H15D10(SEQ ID NO:19)和H19B7(SEQ ID NO:20)。
表5构建S3A5重链HCDRs突变库所需引物
Figure PCTCN2017075933-appb-000006
随后，以筛选到的重链H19B7为基础，利用轻链置换(具体操作可参照本申请人之前提交的中国专利第201510097117.0号中的实施例4.3)对抗体S3A5进行进一步的亲和力体外成熟研究，获得能够提高单抗亲和力的轻链突变体L8C3(SEQ ID No:21)和L16C11(SEQ ID No:22)。
最终，将筛选到的两种新重链突变体(H15D10和H19B7)和两种轻链突变体(L8C3和L16C11)进行组合，得到四种较亲本S3A5亲和力更好的四种抗体突变体，具体序列信息见表6。
表6亲和力成熟的抗人IFNAR1的单抗
Figure PCTCN2017075933-appb-000007
Figure PCTCN2017075933-appb-000008
实施例4抗人IFNAR1的全抗体(IgG1亚型)亲和力测定
利用BIAcore 3000生物大分子相互作用分析仪分别测定H15D10+L8C3、H19B7+L8C3、H15D10+L16C11、H19B7+L16C11和对照抗体抗IFNAR1-C2(嵌合)对hIFNAR1-His的亲和力。胺基偶联试剂(Amine coupling kit)和人抗体捕获试剂(human antibody capture kit)以及CM5芯片和pH7.4的10×HBS-EP均购自GE healthcare公司。根据试剂盒中的说明书，将抗人FC段的抗体偶联至芯片CM5的表面上，稀释抗体蛋白至合适浓度，保证200RU左右的抗体被抗人Fc的抗体捕获。将hIFNAR1-His设置一系列的浓度梯度(100nM、50nM、25nM、12.5nM、6.25nM、3.125nM、1.5625nM、0nM)流经固定相表面，测定各单克隆抗体的亲和力。其中单抗H15D10+L8C3、H15D10+L16C11、H19B7+L8C3和H19B7+L16C11的重链恒定区均为IgG1亚型，轻链恒定区均为κ亚型。结果如表7所示：
表7抗人IFNAR1的单克隆抗体的亲和力常数测定值
抗体 Kon(1/MS) Koff(1/S) KD(nM)
H15D10+L8C3 4.147*10E5 2.512*10E-4 6.058*10E-10
H19B7+L8C3 5.478*10E5 1.673*10E-4 3.053*10E-10
H15D10+L16C11 5.445*10E5 3.312*10E-4 6.082*10E-10
H19B7+L16C11 5.491*10E5 1.933*10E-4 3.52*10E-10
抗IFNAR1-C2(嵌合) 3.953*10E4 6.115*10E-5 1.547*10E-9
实施例5抗人IFNAR1单抗(IgG1亚型)与不同种属IFNAR1的结合
将制备的人IFNAR1(hIFNAR1)、鼠IFNAR1(mIFNAR1)和猕猴IFNAR1(mmIFNAR1)分别包被于96孔ELISA板(浓度为4μg/mL，100μL/孔)，4℃包被过夜。利用封闭液(2％牛乳-PBST)在37℃封闭1小时后，分别加入各种抗人IFNAR1单抗(H15D10+L8C3、H19B7+L8C3、H15D10+L16C11和H19B7+L16C11，对照抗体抗IFNAR1-C1和抗IFNAR1-C2(嵌合))，37℃结合1小时。用PBST洗涤ELISA板，加入HRP-抗人IgG(二抗)，37℃结合1小时。PBST洗涤ELISA板，加入OPD底物显色液，5～10分钟后用浓度为1M的H2SO4溶液终止显色，酶标仪490nm/630nm双波长测定光密度值。结果如图3所示，本申请的抗人IFNAR1的单抗(H15D10+L8C3、H15D10+L16C11、H19B7+L8C3和H19B7+L16C11)及对照抗体(抗IFNAR1-C1和抗IFNAR1-C2(嵌合))均能够结合人IFNAR1和猕猴IFNAR1，但不结合鼠IFNAR1。其中单抗H15D10+L8C3、H15D10+L16C11、H19B7+L8C3和H19B7+L16C11的重链恒定区均为IgG1亚型，轻链恒定区均为κ亚型。
实施例6抗人IFNAR1单抗(IgG1亚型)在人血清中的稳定性分析
为了初步分析不同抗人IFNAR1单抗分子的特异性及血清稳定性，进行了抗 人IFNAR1单抗在人血清中的稳定性分析。此研究包括五种不同抗人IFNAR1单抗，分别为H15D10+L8C3、H15D10+L16C11、H19B7+L8C3、H19B7+L16C11和抗IFNAR1-C1。取过滤去菌的单抗样品，分别稀释于200μL无菌的正常人混合血清或PBS至终浓度30μg/mL，混匀后置37℃水浴放置12天(288小时)。12天后，利用ELISA分析血清样品(A:正常人血清处理，37℃、12天)，PBS样品(B:PBS处理，37℃、12天)和4℃保存的单抗样品(C:4℃、12天)与hIFNAR1的结合(图4)，并分别比较各单抗样品结合hIFNAR1能力的变化(A/B)和(A/C)。其中单抗H15D10+L8C3、H15D10+L16C11、H19B7+L8C3和H19B7+L16C11的重链恒定区均为IgG1亚型，轻链恒定区均为κ亚型。
图4和表8中的结果表明上述五种抗人IFNAR1单抗都具有较好的血清稳定性。
表8不同处理条件下抗人IFNAR1单抗结合hIFNAR1能力的变化
Figure PCTCN2017075933-appb-000009
实施例7抗人IFNAR1单抗(IgG1亚型)抑制I型干扰素诱导的Daudi细胞死亡
Daudi细胞是人Burkkit淋巴瘤细胞，I型干扰素(包括IFNα/β/ω等)可有效抑制该细胞的生长。功能性抗IFNA R1单抗应该能够有效阻断I型干扰素(包括IFNα/β/ω等)和其受体(IFNAR1/IFNAR2复合物)的结合，并能够抑制I型干扰素诱导的Daudi细胞死亡。
在测试不同抗人IFNAR1单抗对I型干扰素的抑制时，将Daudi细胞以3.5×104个/孔的密度接种于96孔细胞板，然后分别用合适的固定浓度(如：0.67nM)的I型干扰素(如：IFNα-2b-HSA)和不同浓度(如：0～25nM)的抗人IFNAR1单抗(如：H19B7+L16C11、H19B7+L8C3或抗IFNAR1-C2(嵌合))同时处理Daudi细胞，然后置CO2培养箱37℃正常培养2～3天。然后用CCK8细胞检测试剂盒(Yeasen，Cat#40203ES80)检测细胞的增殖。最后利用GraphPad Prism 6进行数据分析和作图。其中单抗H19B7+L16C11和H19B7+L8C3重链恒定区均为IgG1亚型，轻链恒定区均为κ亚型。
图5展现了不同抗人IFNAR1单抗对浓度为0.67nM的IFNα-2b-HSA的抑制曲线。表9列举了基于Daudi细胞分析方法时三种不同抗体对三种不同I型干扰素的抑制能力(IC50)。
表9基于Daudi细胞比较三种不同抗人IFNAR1单抗对三种I型干扰素的抑制(IC50)
Figure PCTCN2017075933-appb-000010
Figure PCTCN2017075933-appb-000011
实施例8抗人IFNAR1单抗(IgG1亚型)抑制I型干扰素介导的细胞内信号通路
HEK-BlueTM IFNα/β细胞为InvivoGen公司基于HEK293细胞开发的一个工程细胞株(Cat#hkb-ifnab)。该细胞株中整合并表达人STAT2和IRF9基因，因而在I型干扰素(包括IFNα/β/ω)刺激下可以激活ISGF3信号通路。此外该细胞株中同时整合了分泌型碱性磷酸酶(SEAP)报告基因，而且该SEAP报告基因的表达受ISG54启动子的调控。当用IFNα/β/ω刺激此细胞株时，细胞内的JAK/STAT/ISGF3信号通路被激活，并诱导SEAP报告基因表达，而且SEAP基因的表达可以利用InvivoGen公司提供的配套试剂QUANTI-BlueTM(Invivo Gen，Cat#repqb1)进行方便的检测。因而此细胞株可以用于分析I型干扰素(包括IFNα/β/ω)和I型干扰素受体(IFNAR1/2)的拮抗剂(如抗体等)的活性分析。
在测试不同抗人IFNAR1单抗对I型干扰素的抑制时，将HEK-BlueTM IFN α/β细胞以5×104个/孔的密度接种于96孔细胞板，然后分别用合适的固定浓度(如：0.1nM)的I型干扰素(如：IFNω)和不同浓度(如：0～300nM)的抗IFNAR1单抗(如：H19B7+L16C11、H19B7+L8C3或抗IFNAR1-C2(嵌合))同时处理HEK-BlueTM IFNα/β细胞，然后置CO2培养箱37℃正常培养20～24小时。然后用Qu抗BlueTM染色剂(Invivo Gen，Cat#repqb1)对培养上清中SEAP的表达量进行分析(参照试剂说明书进行)。最后利用GraphPad Prism 6进行数据分析和作图。其中单抗H19B7+L16C11和H19B7+L8C3重链恒定区均为IgG1亚型，轻链恒定区均为κ亚型。
图6展现了不同抗人IFNAR1单抗对IFNω的抑制曲线。表10列举了基于HEK-BlueTM IFNα/β细胞分析方法时三种不同抗体对三种不同I型干扰素的抑制能力(IC50)。
表10基于HEK-BlueTM IFNα/β细胞比较三种不同抗人IFNAR1单抗对三种I型干扰素的抑制(IC50)
Figure PCTCN2017075933-appb-000012
实施例9抗人IFNAR1单抗(IgG4亚型)热稳定性分析
荧光探针可与蛋白的疏水区结合发射荧光信号，程序升温过程中，蛋白从折叠状态转变为展开状态，荧光信号随着疏水区的暴露改变，可以获得温度荧光曲线，可以根据曲线求得Tm值，判断蛋白热稳定性。
在测试不同抗人IFNAR1单抗(IgG4)热稳定性时，将抗人IFNAR1单抗(如：H19B7+L16C11、H19B7+L8C3、H15D10+L16C11、H15D10+L8C3或对照抗体抗 IFNAR1-C1)稀释至某一浓度(如1mg/mL)，加入一定比例的
Figure PCTCN2017075933-appb-000013
 Orange(Sigma，Cat#S5692-50UL)。在荧光定量PCR(ABI，7500Fast)仪器上运行熔解曲线，升温程序:25℃加热到95℃，升温速度1℃/分钟，每个温度平衡2分钟，最后利用Protein Thermal Shift Software 1.2进行数据分析和作图。其中单抗H19B7+L16C11、H19B7+L8C3、H15D10+L16C11和H15D10+L8C3的重链恒定区均为IgG4亚型，轻链恒定区均为κ亚型。
图7展现了不同抗人IFNAR1单抗对热的稳定性，结果显示四种新型抗人IFNAR1单抗的热稳定性都较对照抗体抗IFNAR1-C1好。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (12)

  1. 特异性结合人I型干扰素受体亚基1(IFNAR1)的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区，其特征在于：
    所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)；或者，
    所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)；或者，
    所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVHGYDY(SEQ ID NO:35)；或者，
    所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPSDSNTSYSPSFQG(SEQ ID NO:37)，所述HCDR3序列为DASSKTYDS(SEQ ID NO:38)；或者，
    所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPGDSYTRYSPSFQG(SEQ ID NO:39)，所述HCDR3序列为DGAPAKGDFDY(SEQ ID NO:40)；
    其中HCDR序列根据Kabat定义。
  2. 如权利要求1所述的抗体，其特征在于：所述抗体的重链可变区序列如SEQ ID NO:19、20、23、24或者25的氨基酸序列所示。
  3. 特异性结合人I型干扰素受体亚基1(IFNAR1)的抗体，其包含含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于：
    所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，
    所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述LCDR1序列为RASQNVSNYLN(SEQ ID NO:47)，所述LCDR2序列为RASNLQS(SEQ ID NO:48)，所述LCDR3序列为QQMMDAPPT(SEQ ID NO:49)；或者，
    所述LCDR1序列为SGSSSNIGTNAVN(SEQ ID NO:50)，所述LCDR2序列为SKNQRPP(SEQ ID NO:51)，所述LCDR3序列为AAWDDSQNGYVV(SEQ ID NO:52)；或者，
    所述LCDR1序列为RASEGIGNHLN(SEQ ID NO:53)，所述LCDR2序列为TASNLQS(SEQ ID NO:54)，所述LCDR3序列为QQTYITPLT(SEQ ID NO:55)；
    其中LCDR序列根据Kabat定义。
  4. 如权利要求3所述的抗体，其特征在于：所述抗体的轻链可变区序列如SEQ ID NO:21、22、26、27或者28的氨基酸序列所示。
  5. 特异性结合人I型干扰素受体亚基1(IFNAR1)的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区及含LCDR1、LCDR2和LCDR3序列的轻链可变区，其特征在于：
    所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)，所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，
    所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPGDSDTRYSPSFQG(SEQ ID NO:30)，所述HCDR3序列为HDVTGYDY(SEQ ID NO:31)，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)，所述LCDR1序列为RASQNVGNYLN(SEQ ID NO:41)，所述LCDR2序列为RASNLAS(SEQ ID NO:42)，所述LCDR3序列为QQMEHAPPT(SEQ ID NO:43)；或者，
    所述HCDR1序列为NYWMA(SEQ ID NO:32)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVEGYDY(SEQ ID NO:34)，所述LCDR1序列为RASQSVIGYYLA(SEQ ID NO:44)，所述LCDR2序列为SVSTLAS(SEQ ID NO:45)，所述LCDR3序列为QQYYRFPIT(SEQ ID NO:46)；或者，所述HCDR1序列为NYWVA(SEQ ID NO:29)，所述HCDR2序列为IIYPSDSDTRYSPSFQG(SEQ ID NO:33)，所述HCDR3序列为HDVHGYDY(SEQ ID NO:35)，所述LCDR1序列为RASQNVSNYLN(SEQ ID NO:47)，所述LCDR2序列为RASNLQS(SEQ ID NO:48)，所述LCDR3序列为QQMMDAPPT(SEQ ID NO:49)；或者，
    所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPSDSNTSYSPSFQG(SEQ ID NO:37)，所述HCDR3序列为DASSKTYDS(SEQ ID NO:38)，所述LCDR1序列为SGSSSNIGTNAVN(SEQ ID NO:50)，所述LCDR2序列为SKNQRPP(SEQ ID NO:51)，所述LCDR3序列为AAWDDSQNGYVV(SEQ ID NO:52)；或者，
    所述HCDR1序列为NYWIG(SEQ ID NO:36)，所述HCDR2序列为RIYPGDSYTRYSPSFQG(SEQ ID NO:39)，所述HCDR3序列为DGAPAKGDFDY(SEQ ID NO:40)，所述LCDR1序列为RASEGIGNHLN(SEQ ID NO:53)，所述LCDR2序列为TASNLQS(SEQ ID NO:54)，所述LCDR3序列为QQTYITPLT(SEQ ID NO:55)；
    其中HCDR和LCDR序列根据Kabat定义。
  6. 如权利要求5所述的抗体，其中：
    所述重链可变区序列为SEQ ID NO:19，所述轻链可变区序列为SEQ ID NO:21；或者，
    所述重链可变区序列为SEQ ID NO:19，所述轻链可变区序列为SEQ ID NO:22；或者，
    所述重链可变区序列为SEQ ID NO:20，所述轻链可变区序列为SEQ ID NO:21； 或者，
    所述重链可变区序列为SEQ ID NO:20，所述轻链可变区序列为SEQ ID NO:22；或者，
    所述重链可变区序列为SEQ ID NO:23，所述轻链可变区序列为SEQ ID NO:26；或者，
    所述重链可变区序列为SEQ ID NO:24，所述轻链可变区序列为SEQ ID NO:27；或者，
    所述重链可变区序列为SEQ ID NO:25，所述轻链可变区序列为SEQ ID NO:28。
  7. 如权利要求1-6中任一项所述的抗体，其特征在于：所述抗体为全长抗体、Fab片段、F(ab')2片段或单链Fv片段；优选地，所述抗体为全人源抗体。
  8. 如权利要求1-7中任一项所述的抗体，其特征在于：所述抗体还包含选自IgG1亚型(SEQ ID NO:7)、IgG2亚型(SEQ ID NO:8)或IgG4亚型(SEQ ID NO:9)的重链恒定区和/或包含选自κ亚型(SEQ ID NO:10)或者λ亚型(SEQ ID NO:11)的轻链恒定区。
  9. 药物组合物，其包含权利要求1-8中任一项所述的抗体以及药学可接受的载体、赋形剂、稀释剂。
  10. 如权利要求9所述的药物组合物，其用于预防或治疗由人IFNAR1介导的疾病；优选地，所述疾病为自身免疫性疾病，例如系统性红斑狼疮、银屑病、多发性硬化症或类风湿性关节炎。
  11. 权利要求1-8中任一项所述的抗体，或者权利要求9所述的药物组合物在制备用于预防或治疗由人IFNAR1介导的疾病的药物中的用途；优选地，所述疾病为自身免疫性疾病，例如系统性红斑狼疮、银屑病、多发性硬化症或类风湿性关节炎。
  12. 预防或治疗由人IFNAR1介导的疾病的方法，包括给予有需要的患者治疗有效量的权利要求1-8中任一项所述的抗体或者权利要求9所述的药物组合物；优选地，所述疾病为自身免疫性疾病，例如系统性红斑狼疮、银屑病、多发性硬化症或类风湿性关节炎。
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