WO2018010140A1 - I型干扰素受体抗体及其用途 - Google Patents

I型干扰素受体抗体及其用途 Download PDF

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WO2018010140A1
WO2018010140A1 PCT/CN2016/090018 CN2016090018W WO2018010140A1 WO 2018010140 A1 WO2018010140 A1 WO 2018010140A1 CN 2016090018 W CN2016090018 W CN 2016090018W WO 2018010140 A1 WO2018010140 A1 WO 2018010140A1
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seq
amino acid
ifnαr1
monoclonal antibody
antibody
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PCT/CN2016/090018
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French (fr)
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张立国
苏立山
李靖云
马建平
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中国科学院生物物理研究所
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Priority to CN201810473570.0A priority Critical patent/CN108409862B/zh
Priority to PCT/CN2016/090018 priority patent/WO2018010140A1/zh
Priority to CN201680002838.1A priority patent/CN107074943B/zh
Priority to US16/317,035 priority patent/US11136399B2/en
Publication of WO2018010140A1 publication Critical patent/WO2018010140A1/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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K2317/30—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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a type I interferon receptor antibody and use thereof.
  • Type I interferon is a family of structurally related cytokines with antiviral, antitumor and immunomodulatory effects (Hardy et al. (2001) Blood 97: 473; Cutrone and Langer (2001). J. Biol. Chem. 276: 17140). Human IFN-I includes five species, which are IFN- ⁇ (including 13 subtypes), IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ , respectively. Although interferon is a potent antiviral agent, some acute viral diseases, such as those characterized by respiratory infections, are caused by an excess of human IFN- ⁇ .
  • human IFN-[alpha] is associated with chronic viral infections in experimental animals and pathogenesis of human chronic viral diseases such as measles virus infection.
  • Human IFN- ⁇ is also a potent immunomodulatory molecule that stimulates B cell activation, enhances NK cell cytotoxicity, inhibits T cell function, and regulates the expression of major compatible complex (MHC) class I antigens.
  • MHC major compatible complex
  • IFN-I binds to cell surface receptors (IFN- ⁇ receptor, IFN ⁇ R), a heterodimer composed of two transmembrane proteins, IFN ⁇ R1 and IFN ⁇ R2 (Uze et al. (1990) Cell 60: 225; Novick et al. (1994) Cell 77: 391).
  • Human IFN ⁇ R1 is an important component of the IFN-I signaling pathway, which belongs to the type II helix cytokine receptor, an extracellular domain (ECD) of four type III fibronectin domains of approximately 100 amino acids, one transmembrane The domain consists of an intracellular domain of 100 amino acid residues.
  • IFN ⁇ R1 The four subdomains (SD) of IFN ⁇ R1 are folded into domain 1 (SD1+SD2) and domain 2 (SD3+SD4).
  • IFN ⁇ R1 and IFN ⁇ R2 form a ternary signal complex with a type I interferon ligand (Cohen B et al. (1995), Mol Cell Biol 15(8): 4208-14).
  • the formation of a ternary complex is the first step in the activation of a signal transduction pathway.
  • targeting this receptor as well as blocking kinase activation has the potential to prevent downstream biological effects of diseases associated with interferon activation.
  • US7465451 (2008) also discloses monoclonal antibodies against IFNAR for the preparation of a disease for the treatment or prevention of IFN-I overproduction, treatment of graft rejection, autoimmune diseases such as systemic lupus erythematosus, treatment of type I diabetes, psoriasis, Rheumatoid arthritis, multiple sclerosis, acquired immunodeficiency syndrome (AIDS), and severe immunodeficiency diseases; use in the treatment of acute viral diseases such as respiratory infections, chronic viral diseases such as measles virus infections.
  • autoimmune diseases such as systemic lupus erythematosus
  • AIDS acquired immunodeficiency syndrome
  • severe immunodeficiency diseases use in the treatment of acute viral diseases such as respiratory infections, chronic viral diseases such as measles virus infections.
  • US6713609 (2004) discloses anti-IFNAR1 monoclonal antibodies 2E1 and 4A7 which inhibit the antiviral activity of various type I interferons, wherein monoclonal antibody 2E1 binds to the SD1 domain of human IFNAR1, and 4A7 binds to SD1 and SD2 of human IFNAR1. Domain.
  • U.S. Patent Nos. US7662381 (2010) and US Pat. No. 8,460,668 (2013) disclose the incorporation of IFNAR1 And a monoclonal antibody 9D4 capable of inhibiting type I interferon activity, which binds to the SD3 domain of human IFNAR1.
  • a first object of the invention is to provide a monoclonal antibody to IFN ⁇ R1.
  • the monoclonal antibody of IFN ⁇ R1 provided by the present invention binds all or part of the SD2 domain of human IFN ⁇ R1 and/or all or part of the SD3 domain, and inhibits the biological activity of human type I interferon.
  • the monoclonal antibody or antigen-binding portion thereof exhibits one or more of the following characteristics of A)-E):
  • the coding gene of the heavy chain variable region of the monoclonal antibody or antigen-binding portion thereof is as follows 1) or 2):
  • the coding gene of the light chain variable region of the monoclonal antibody or antigen-binding portion thereof is as follows 3) or 4):
  • the heavy chain variable region of the monoclonal antibody or antigen-binding portion thereof is as follows 5) or 6):
  • a protein or polypeptide comprising a full-length or partially-encoded protein or polypeptide of the mouse IGHV9-3 gene, a full-length or partial-encoded protein or polypeptide of the mouse IGHD2-13 gene, and a full-length or partial-encoded protein or polypeptide of the mouse IGHJ1 gene;
  • a protein or polypeptide comprising a full-length or partially-encoded protein or polypeptide of the mouse IGHV9-3 gene, a full-length or partial-encoding protein or polypeptide of the mouse IGHD1-1 gene, and a full-length or partial-encoding protein or polypeptide of the mouse IGHJ3 gene;
  • the light chain variable region of the monoclonal antibody or antigen-binding portion thereof is as follows 7) or 8):
  • a protein or polypeptide comprising a full-length or partial-encoded protein or polypeptide of the mouse IGKV6-13 gene and a full-length or partial-encoding protein or polypeptide of the mouse IGKJ5 gene;
  • a protein or polypeptide comprising a full-length or partially-encoded protein or polypeptide of the mouse IGKV6-15 gene and a full-length or partial-encoding protein or polypeptide of the mouse IGKJ2 gene.
  • the heavy chain variable region of the monoclonal antibody or antigen-binding portion thereof of IFN ⁇ R1 in turn includes the protein encoded by the mouse IGHV9-3 gene, the protein encoded by the mouse IGHD2-13 gene, and the mouse IGHJ1 a gene-encoded protein; and the light chain variable region thereof in turn includes a protein encoded by the mouse IGKV6-13 gene and a protein encoded by the mouse IGKJ5 gene;
  • the heavy chain variable region of the monoclonal antibody or antigen-binding portion thereof of the IFN ⁇ R1 includes the protein encoded by the mouse IGHV9-3 gene, the protein encoded by the mouse IGHD1-1 gene, and the mouse IGHJ3.
  • the gene-encoded protein, and its light chain variable region includes the protein encoded by the mouse IGKV6-15 gene and the protein encoded by the mouse IGKJ2 gene.
  • the heavy chain variable region of the monoclonal antibody or antigen-binding portion thereof of IFN ⁇ R1 comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2 and a heavy chain variable region CDR3;
  • the light chain variable region of the monoclonal antibody of IFN ⁇ R1 or antigen-binding portion thereof comprises a light chain variable region CDR1, a light chain variable region CDR2 and a light chain variable region CDR3;
  • amino acid sequence of the heavy chain variable region CDR1 is as follows (1) or (2):
  • amino acid sequence of the heavy chain variable region CDR2 is as follows (3) or (4):
  • amino acid sequence of the heavy chain variable region CDR3 is as follows (5) or (6):
  • amino acid sequence of the light chain variable region CDR1 is as follows (7) or (8):
  • amino acid sequence of the light chain variable region CDR2 is as follows (9) or (10):
  • amino acid sequence of the light chain variable region CDR3 is as follows (11) or (12):
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody of IFN ⁇ R1 is a1) or a2):
  • A1 the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12.
  • amino acid sequence of the light chain variable region of the monoclonal antibody of IFN ⁇ R1 is b1) or b2):
  • nucleotide sequence of the heavy chain variable region of the monoclonal antibody of IFN ⁇ R1 is c1) or c2) or c3):
  • C2 a cDNA molecule or genomic DNA molecule having a nucleotide sequence defined by c1) having 75% or more identity and a heavy chain variable region of a monoclonal antibody encoding the above IFN ⁇ R1;
  • C3 a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by c1) or c2) and which encodes a heavy chain variable region of a monoclonal antibody of the above IFN ⁇ R1;
  • nucleotide sequence of the light chain variable region of the monoclonal antibody of IFN ⁇ R1 is d1) or d2) or d3):
  • D2 a cDNA molecule or genomic DNA molecule having a 75% or more identity with a nucleotide sequence defined by d1) and encoding a light chain variable region of a monoclonal antibody of the above IFN ⁇ R1;
  • D3 a cDNA molecule or genomic DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and which encodes the light chain variable region of the monoclonal antibody of IFN ⁇ R1 described above.
  • the antibody of the present invention comprises a heavy chain and a light chain variable region comprising an amino acid sequence homologous to the amino acid sequence of the above antibody, and wherein the antibody retains the desired functional properties of the IFN ⁇ R1 antibody of the present invention.
  • the invention provides an isolated monoclonal antibody or antigen binding portion thereof comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises an amino acid sequence at least 80% homologous to an amino acid sequence selected from the sequence 2 and the sequence 12;
  • the light chain variable region comprises an amino acid sequence at least 80% homologous to an amino acid sequence selected from the sequence 7 or the sequence 17;
  • the antibody specifically binds to IFN ⁇ R1 and exhibits at least one of the functional properties as described below:
  • the antibody VH or VL amino acid sequence of the present invention may be homologous to the above amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98% or 99%.
  • the percent homology between two amino acid sequences corresponds to the percent identity between the two sequences.
  • Antibodies of the invention are characterized by specific functional characteristics or characteristics of the antibody.
  • the antibody specifically binds to IFN ⁇ R1, preferably human IFN ⁇ R1.
  • the antibody can be cross-reactive with IFN ⁇ R1 from one or more non-human primates such as macaques and/or rhesus monkeys.
  • the antibodies of the invention are capable of inhibiting the biological activity of type I interferons. These antibodies inhibit the biological activity of at least one type I interferon, preferably inhibiting a plurality of type I interferons (ie, at least two, more preferably at least three, or at least four, or at least five, or at least six, Or at least seven, or at least eight, or at least nine, or at least ten, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15 different subtypes of type I The biological activity of interferon).
  • a plurality of type I interferons ie, at least two, more preferably at least three, or at least four, or at least five, or at least six, Or at least seven, or at least eight, or at least nine, or at least ten, or at least 11, or at least 12, or at least 13, or at least 14, or at least 15 different subtypes of type I The biological activity of interferon).
  • the antibody inhibits the biological activity of the following type I interferons: alpha 1, alpha 2a, alpha 2b, alpha 4, alpha 5, alpha 6, alpha 7, alpha 8, alpha 10, alpha 14, alpha 16, alpha 17, alpha 21, beta and alpha.
  • the antibody inhibits the activity of type I interferon on human PBMC.
  • an antibody to inhibit the biological activity of a Type I interferon can be detected by one or more assays established in the art. Including inhibition of IFN-I activity on type I interferon signaling reporter cells, inhibition of expression of interferon-responsive genes, such as mx2 and isg15, in peripheral blood mononuclear cells (PBMCs) stimulated by stimulators such as CpG, IFN ⁇ 2b or R848 .
  • PBMCs peripheral blood mononuclear cells
  • An antibody inhibits activity by at least 20%, more preferably at least 30%, even more preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or, if compared to a non-specific control antibody At least 90%, the antibody "inhibits the biological activity of type I interferon".
  • the antibody inhibits the activity of IFN ⁇ 2b in the type I interferon signaling reporter cell line HEK293T, and inhibits the expression of interferon effector genes, such as mx2 and isg15, in PbMC induced by CpGA, IFN ⁇ 2b or R848.
  • preferred antibodies of the invention are monoclonal antibodies 10C2 and 10C9 isolated and structurally characterized as described in the Examples.
  • the VH amino acid sequences of 10C2 and 10C9 are shown in Sequence 2 and Sequence 12, respectively; VL Amino Acids of 10C2 and 10C9, respectively.
  • the sequences are shown in Sequence 7 and Sequence 17, respectively.
  • the VH and VL sequences can be "mixed and matched" to produce additional anti-IFN ⁇ R1 binding molecules of the invention.
  • the IFN ⁇ R1 binding of these "mixed and matched" antibodies can be detected using the HEK293 reporter cell line stably expressing IFNAR1 as described in the Examples of the present invention.
  • the VH sequences from a particular VH/VL pairing are replaced with structurally similar VH sequences.
  • the VL sequences from a particular VH/VL pairing are replaced by structurally similar VL sequences.
  • VH and VL sequences of 10C2 and 10C9 are particularly suitable for mixing and matching, as these antibodies use VH and VL sequences derived from the same germline sequences (IGHV9-3 and IGKV6-13), thus exhibiting structural similarity.
  • the invention provides an antibody comprising 10C2 and 10C9 heavy and light chain CDR1, CDR2 and CDR3, or a combination thereof.
  • the amino acid sequences of VH CDR1 of 10C2 and 10C9 are sequence 3 and sequence 13, respectively.
  • the amino acid sequences of VH CDR2 of 10C2 and 10C9 are sequence 4 and sequence 14, respectively.
  • the amino acid sequences of VH CDR3 of 10C2 and 10C9 are sequence 5 and sequence 15, respectively. .
  • the CDR regions are indicated by VECTOR NTI.
  • the VH CDR1, CDR2, CDR3 sequences can be "mixed and matched" with the VL CDR1, CDR2, CDR3 sequences (ie, The CDRs from different antibodies can be mixed and matched, but each antibody must contain VH CDR1, CDR2, CDR3 and VL CDR1, CDR2, CDR3) to produce additional anti-IFN ⁇ R1 binding molecules of the invention.
  • the IFN ⁇ R1 binding of these "mixed and matched" antibodies can be detected using the reporter cell HEK293T stably expressing IFNAR1 as described in the Examples.
  • the CDR1, CDR2 and/or CDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequences.
  • the CDR1, CDR2 and/or CDR3 sequences from a particular VL sequence are preferably replaced with structurally similar CDR sequences.
  • the VH CDR1 of 10C2 and 10C9 has a certain structural similarity and is therefore suitable for mixing and matching. Substitution of one or more VH and/or VL CDR region sequences into structurally similar sequences from the monoclonal antibodies 10C2 and 10C9 CDR sequences disclosed herein can result in new VH and/or VL sequences.
  • an antibody of the invention comprises a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene.
  • the invention provides an isolated anti-IFNRR1 monoclonal antibody or antigen-binding portion thereof, wherein the antibody:
  • a heavy chain variable region comprising the mouse IGHV9-3, IGHD2-13 and IGHJ1 genes; or the IGHV9-3, IGHD1-1 and IGHJ3 genes;
  • An example of a heavy chain variable region having the IGHV9-3, IGHD2-13 and IGHJ1 genes and a light chain variable region of the IGKV6-13 and IGKJ5 genes is 10C2.
  • An example of a heavy chain variable region having the IGHV9-3, IGHD1-1 and IGHJ3 genes and a light chain variable region of the IGKV6-15 and IGKJ2 genes is 10C9.
  • variable region of a mouse antibody is obtained from a system using a mouse germline immunoglobulin gene, the antibody comprises a particular germline sequence or "derived” from it or its "product” Heavy or light chain variable region.
  • a mouse antibody of a mouse germline immunoglobulin sequence or "derived from” or its “product” can be identified as follows: the amino acid sequence of the mouse antibody and the amino acid sequence of the mouse germline immunoglobulin Comparisons were made and mouse germline immunoglobulin sequences closest in sequence to the mouse antibody sequence (ie, having the highest % identity) were selected.
  • a mouse antibody of a particular mouse germline immunoglobulin sequence or "derived from” or its "product” may contain amino acid differences compared to the germline sequence, for example due to natural In somatic mutations or intentional introduction of site-directed mutagenesis.
  • the selected mouse antibody is generally at least 90% identical in amino acid sequence to the amino acid sequence encoded by the mouse germline immunoglobulin gene and contains germline immunoglobulin amino acid sequences (eg, murine germline sequences) with other species.
  • the mouse antibody is determined to be an amino acid residue of a mouse antibody.
  • the mouse antibody can be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene.
  • a mouse antibody derived from a particular mouse germline sequence will show no more than 10 amino acids from the amino acid sequence encoded by the mouse germline immunoglobulin gene.
  • the mouse antibody can exhibit no more than 5, or even no more than 4, 3, 2 or 1 amino acids, encoded by the amino acid sequence encoded by the germline immunoglobulin gene.
  • a second object of the present invention is to provide a hybridoma cell strain which secretes a monoclonal antibody of the above IFN ⁇ R1.
  • the hybridoma cell strain of the monoclonal antibody secreting the above IFN ⁇ R1 provided by the present invention has the accession number of CGMCC No. 12542 or CGMCC No. 12543.
  • hybridoma cell lines secreting the monoclonal antibody against IFN ⁇ R1 provided by the present invention were designated as hybridoma cell line 10C2 and hybridoma cell line 10C9, respectively.
  • the above hybridoma cell line 10C2 is classified as a mouse hybridoma cell line which has been deposited on May 31, 2016 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Beijing No. 3, Beichen West Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, 100101), the deposit number is CGMCC No.12542.
  • the above hybridoma cell line 10C9 is classified as a mouse hybridoma cell line which has been deposited on May 31, 2016 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Beijing No. 3, Beichen West Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences, 100101), the deposit number is CGMCC No.12543.
  • a third object of the present invention is to provide a monoclonal antibody or antigen-binding portion thereof of the above IFN ⁇ R1 or a novel use of the above hybridoma cell line.
  • the present invention provides the use of the above monoclonal antibody of IFN ⁇ R1 or antigen-binding portion thereof or hybridoma cell strain in at least one of the following A)-G):
  • the biological activity of the type I interferon inhibiting the peripheral blood mononuclear cells stimulated by the stimulant is as follows (1)-(6):
  • inhibiting the expression level of the interferon effector gene isg15 in peripheral blood mononuclear cells induced by IFN ⁇ 2b;
  • the type I interferon is interferon alpha.
  • a fourth object of the present invention is to provide an antagonist of type I interferon.
  • the active ingredient of the antagonist of type I interferon provided by the present invention is a monoclonal antibody of the above IFN ⁇ R1 or an antigen-binding portion thereof.
  • a fifth object of the invention is to provide a composition.
  • composition provided by the present invention contains the above monoclonal antibody of IFN ⁇ R1 or an antigen-binding portion thereof.
  • compositions such as a pharmaceutical composition, comprising a monoclonal antibody of the present invention or an antigen-binding portion thereof in one or a combination formulated with a pharmaceutically acceptable carrier.
  • compositions may comprise one or a combination (e.g., two or more different) antibodies or immunoconjugates or bispecific molecules of the invention.
  • a pharmaceutical composition of the invention may contain an antibody (or immunoconjugate or bispecific) that binds to a different epitope on the target antigen or has a complementary activity.
  • compositions of the invention may also be administered in combination therapy, i.e., in combination with other agents.
  • a combination therapy can include an anti-IFNRR1 antibody of the invention in combination with at least one other immunosuppressive agent.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., antibody, immunoconjugate or bispecific molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions that would inactivate the compound.
  • compositions of the invention may comprise one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not cause any unwanted toxicological effects (see, e.g., Berge, SM et al. (1977) J. Pharm. Sci. 66:1. -19).
  • Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as non-toxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids.
  • Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chlorine Salt derived from procaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
  • compositions of the invention may also contain a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated hydroxyanisole (BHA), butylated hydroxytoluene (DHT), lecithin, propyl gallate, alpha-tocopherol, etc.; (3) Metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • aqueous or nonaqueous vehicles examples include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, And injectable organic esters such as ethyl oleate.
  • polyols e.g., glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the application of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the sterilization procedure described above or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars and sodium chloride, in the compositions. In addition, prolonged absorption of the injectable drug can be achieved by the inclusion of a delayed absorbent such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by the sterilization procedure described above or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents,
  • the pharmaceutically acceptable carrier includes a sterile aqueous solution or dispersion and a powder for the temporary preparation of a sterile injectable solution or dispersion.
  • a sterile aqueous solution or dispersion and a powder for the temporary preparation of a sterile injectable solution or dispersion.
  • Supplementary active compounds can also be incorporated into the compositions.
  • compositions must generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations.
  • the carrier can be a solvent or dispersing agent containing, for example, water, ethanol, polyol (for example, glycerol, polyethylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • polyol for example, glycerol, polyethylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof for example, proper fluidity can be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersing agent, and by the use of a surfactant.
  • isotonic agents for example, sugars, polyols such as mannitol, sorbitol or sodium oxide.
  • Prolonged absorption of the injectable drug can be achieved by the addition of a delay absorbent such as monostearate and gelatin to the composition.
  • Sterile injectable solutions are prepared by admixing the active compound in a suitable amount in the required amount, and, if necessary, one or a combination of the ingredients listed above, followed by sterile microfiltration.
  • the dispersing agent is prepared by incorporating the active compound into a sterile vehicle which contains the base dispersion medium and other ingredients from the ingredients enumerated above.
  • the preferred preparation methods are vacuum drying and lyophilization (lyophilization), from which the sterile filtered solution described above yields a powder of the active ingredient plus any additional desired ingredients.
  • the amount of active ingredient which may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient which may be combined with carrier materials to produce a single dosage form is generally the amount of the composition which produces a therapeutic effect. Typically, this amount will range from about 0.01% to about 99% active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, by weight of the active ingredient, and pharmaceutically acceptable The carrier is combined.
  • compositions of the invention may be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will appreciate that the route and/or manner of administration will depend on the desired result. different. Preferred routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion.
  • parenteral administration refers to a mode of administration that differs from enteral and topical administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, cardiac. Internal, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.
  • antibodies of the invention may also be administered by parenteral routes, such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • parenteral routes such as topical, epidermal or mucosal routes, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the antibodies (and immunoconjugates and bispecific molecules) of the invention have both in vitro and in vivo diagnostic and therapeutic applications.
  • these molecules can be administered to, for example, cells cultured in vitro or ex vivo, or administered to a human subject, for example, in vivo to treat, prevent or diagnose a variety of diseases.
  • subject includes both human and non-human animals.
  • Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. These methods are particularly suitable for treating human patients suffering from diseases associated with abnormal or inappropriate type I interferon expression (eg, overexpression).
  • an anti-IFNRR1 antibody of the invention may be used in combination with one or more of the following: an anti-IFN ⁇ antibody, an anti-IFN- ⁇ receptor antibody, a soluble IFN- ⁇ receptor, an anti-TNF antibody, an anti-TNF receptor antibody, and/or Or soluble TNF receptor.
  • the anti-IFN ⁇ R1 antibody of the present invention can be used in combination with a Flt3 ligand antagonist.
  • a sixth object of the invention is to provide an immunoconjugate.
  • the immunoconjugate provided by the present invention contains the above monoclonal antibody of IFN ⁇ R1 or an antigen-binding portion thereof.
  • the immunoconjugate comprises a pharmaceutically acceptable carrier.
  • the anti-IFN ⁇ R1 antibody or fragment thereof provided by the present invention can be conjugated to a therapeutic agent such as a cytotoxin, a drug (e.g., an immunosuppressive agent) or a radioactive toxin to give a conjugate.
  • a therapeutic agent such as a cytotoxin, a drug (e.g., an immunosuppressive agent) or a radioactive toxin to give a conjugate.
  • cytotoxin e.g., an immunosuppressive agent
  • radioactive toxin e.g., an immunosuppressive agent
  • conjugates are referred to as "immunoconjugates.”
  • An immunoconjugate comprising one or more cytotoxins is referred to as an "immunotoxin.”
  • Cytotoxins or cytotoxic agents include any agent that is detrimental to the cell (eg, kills).
  • Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecaine, mitomycin, epipodophyllotoxin, epipodophyllotoxin, vincristine, vinblastine , colchicine, doxorubicin, daunorubicin, dihydroxy anthrax dione, mitoxantrone, phosfomycin, actinomycin D, l-dehydrotestosterone, glucocorticoid, proca , tetracaine, lidocaine, propranolol and puromycin and their analogs or homologs.
  • Therapeutic agents also include, for example, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine, a burning agent ( For example, nitrogen mustard, thioepa chlorambucil, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, chain Oxazomycin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (eg, daunorubicin (formerly known as daunorubicin) and trichothecene Antibiotics (eg, actinomycin D, bleomycin, phlotomycin, and amphotericin (AMC)), And anti-mitotic agents (eg, vincristine and vinblastine).
  • antimetabolites eg
  • Cytotoxins can be conjugated to the antibodies of the invention using linker techniques available in the art.
  • types of linkers that have been used to couple cytotoxins to antibodies include, but are not limited to, guanidine, thioether, ester, disulfide, and peptide-containing linkers.
  • the antibodies of the invention may also be conjugated to a radioisotope to produce a cytotoxic radiopharmaceutical, also known as a radioimmunoconjugate.
  • a radioisotope that can be coupled to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine 131 , indium 111 , ⁇ 90, and ⁇ 177 .
  • Methods of preparing radioactive immunoconjugates have been established in the art. Examples of radioimmunoconjugates may be used as commercially available, including Zevalin TM (IDEC Pharmaceuticals) and Bexxar TM (Corixa Pharmaceuticals), using a similar method capable of preparing radioimmunoconjugates using the antibodies of the present invention.
  • the antibody conjugates of the invention can be used to modify a particular biological response, and the drug moiety should not be construed as being limited to classical chemotherapeutic agents.
  • the drug moiety can be a protein or polypeptide having the desired biological activity.
  • Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon- ⁇ ; or biological response regulators such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophages Cell colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM-CSF granulocyte macrophages Cell colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • a seventh object of the present invention is to provide an antibody genetically engineered antibody.
  • the antibody genetically engineered antibody provided by the present invention is an antibody obtained by modifying and modifying the monoclonal antibody or antigen-binding portion thereof of the above IFN ⁇ R1.
  • the present invention provides an isolated monoclonal antibody or antigen binding portion thereof comprising a heavy chain variable region of CDR1, CDR2 and CDR3 sequences and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein:
  • the chain variable region CDR3 sequence comprises an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 15 or a conservatively modified amino acid sequence thereof
  • the CDR3 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: A conservatively modified amino acid sequence
  • the antibody specifically binds to IFN ⁇ R1 and exhibits at least one of the above functional properties.
  • the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 14 or a conservatively modified amino acid sequence thereof; and the light chain variable region CDR2 sequence comprises a sequence selected from sequence 9 and The amino acid sequence shown in 19 or its conservatively modified amino acid sequence.
  • the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 13 or a conservatively modified amino acid sequence thereof; and the light chain variable region CDR1 sequence comprises a sequence selected from the group consisting of The amino acid sequence shown in SEQ ID NO: 18 or a conservatively modified amino acid sequence thereof.
  • conservative sequence modification refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by techniques well known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains has been defined in the art.
  • Antibodies of the invention can also be prepared by engineering an antibody with a antibody having one or more of the VH and/or VL sequences disclosed herein as a starting material, and the modified antibody may have The altered properties of the antibody.
  • An antibody can be engineered by modifying one or both of the variable regions (ie, VH and/or VL), eg, one or more residues within one or more CDR regions and/or one or more backbone regions.
  • antibodies can be engineered by modifying residues within the constant region, for example by altering the effector function of the antibody.
  • CDR grafting One type of variable region engineering that can be performed is CDR grafting.
  • the antibody interacts with the target antigen primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse between the individual antibodies than the sequences outside the CDRs. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of the particular naturally occurring antibody can be expressed by constructing an expression vector comprising a CDR sequence from a particular naturally occurring antibody, the CDR sequence being Transplantation onto backbone sequences from different antibodies with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332:323-327 Jones, P. et al. (1986) Nature 321 :522-525; Queen, C. Et al. (1989) Proc. Natl. Acad. Sci. USA 86: 10029-10033).
  • another embodiment of the invention relates to an isolated monoclonal antibody or antigen binding portion thereof comprising: a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences, the CDR1, CDR2 and CDR3 sequences comprising From Sequence 3 and Sequence 13, Sequence 4 and Sequence 14; and the amino acid sequences shown in Sequences 5 and 15, and the light chain variable regions comprising CDR1, CDR2 and CDR3 sequences, the CDR1, CDR2 and CDR3 sequences are each selected From Sequence 8 and Sequence 18, Sequence 9 and Sequence 19, and the amino acid sequences shown in Sequence 10 and Sequence 20.
  • these antibodies contain the VH and VL CDR sequences of monoclonal antibodies 10C2 and 10C9, but may contain different backbone sequences than these antibodies.
  • germline DNA sequences of human heavy and light chain variable region genes can be obtained from the following resources: "VBase” human germline sequence database (available from the Internet at www.mrc-cpe.cam.ac.uk/vbase Obtained), and Kabat, EA et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM et al.
  • variable region modification is to mutate the amino acid sequences within the VH and VL CDR1, CDR2 and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest.
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, effects on antibody binding, or Other target functional characteristics can be evaluated using the tests described herein and provided in the examples.
  • Conservative sequence modifications are preferably introduced.
  • the mutation may be an amino acid substitution, addition or deletion, but is preferably substituted. Moreover, typically no more than 5 residues are altered within the CDR regions.
  • the invention provides an isolated anti-IFNRR1 monoclonal antibody or antigen binding portion thereof comprising a heavy chain variable region comprising:
  • VH CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 13, or having 1, 2, 3, 4 or 5 amino acids as compared with the amino acid sequence selected from SEQ ID NO: 3 and SEQ ID NO: 13.
  • VH CDR2 region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 14 or having 1, 2, 3, 4 or 5 amino acids as compared with the amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NO: 14.
  • VH CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 15, or 1, 2, 3, 4 or 5 as compared with the amino acid sequence selected from SEQ ID NO: 5 and SEQ ID NO: 15. Amino acid substitution, deletion or addition of amino acid sequences;
  • VL CDR1 region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 18, or having 1, 2, 3, 4 or 5 amino acid substitutions compared to the amino acid sequence selected from SEQ ID NO: 8 and SEQ ID NO: 18. Missing or added amino acid sequence;
  • VL CDR2 region comprising an amino acid sequence selected from the group consisting of the sequence 9 and the sequence 19, or having 1, 2, 3, 4 or 5 as compared with the amino acid sequence selected from the sequence 9 and the sequence 19 Amino acid sequence substitution, deletion or addition of amino acids;
  • VL CDR3 region comprising an amino acid sequence selected from the group consisting of Sequence 10 and Sequence 20, or having 1, 2, 3, 4 or 5 amino acids compared to the amino acid sequence selected from Sequence 10 and Sequence 20. An amino acid sequence substituted, deleted or added.
  • Engineered antibodies of the invention include, for example, antibodies that modify the backbone residues within their VH and/or VL in order to improve the properties of the antibody. Such backbone modifications are generally made to reduce the immunogenicity of the antibody. For example, one method is to "backmutate" one or more backbone residues to the corresponding germline sequence. More particularly, an antibody that undergoes somatic mutation may contain a backbone residue that is different from the germline sequence from which the antibody is derived. These residues can be identified by comparing the antibody backbone sequence to the germline sequence from which the antibody is derived.
  • Another type of backbone modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody.
  • antibodies of the invention can be engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as Serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cytotoxicity.
  • modifications in the Fc region typically to alter one or more functional properties of the antibody, such as Serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cytotoxicity.
  • an antibody of the invention e.g., one or more chemical moieties can be attached to the antibody
  • Structurally related anti-IFNRR1 antibodies are produced using the anti-IFNRR1 antibodies of the invention, such as the structural features of 10C2 and 10C9, which retain at least one of the functional properties of the antibodies of the invention, such as binding to IFN ⁇ R1.
  • one or more CDR regions of 10C2 or 10C9 can be recombinantly combined with known framework regions and/or other CDRs to generate additional recombinantly engineered anti-IFNRR1 antibodies of the invention.
  • Other types of modifications include the modifications described in the previous section.
  • the starting material for the engineering method is one or more of the VH and/or VL sequences provided herein, or one or more CDR regions thereof.
  • an engineered antibody it is not necessary to actually prepare (ie, express as a protein) an antibody having one or more of the VH and/or VL sequences provided herein or one or more CDR regions thereof. Rather, using the information contained in the sequence as a starting material, a "second generation" sequence derived from the original sequence is generated, and then the "second generation” sequence is prepared and expressed as a protein.
  • the above method for preparing an engineered IFN ⁇ R1 antibody comprises the following steps:
  • (a) provides the following sequence: (i) a heavy chain variable region antibody sequence comprising a CDR1 sequence selected from sequence 3 and sequence 13, a CDR2 sequence of sequence 4 and sequence 14, and a CDR3 selected from sequence 5 and sequence 15. a sequence; and (ii) a light chain variable region antibody sequence comprising a CDR1 sequence selected from the sequence 8 and the sequence 18, a CDR2 sequence of the sequence 9 and the sequence 19, and a CDR3 sequence selected from the sequence 10 and the sequence 20;
  • Altered antibody sequences can be prepared and expressed using conventional molecular biology techniques.
  • the antibody encoded by the altered antibody sequence retains one, some or all of the functional properties of an anti-IFNRR1 antibody as described below: binding to IFN ⁇ R1; inhibiting binding of type I interferon to IFN ⁇ R1; binding to cells expressing human IFN ⁇ R1; Inhibition of the biological activity of type I interferon on IFNAR1 cells; inhibition of the biological activity of interferon I on stimulating agent-stimulated PBMC.
  • the functional properties of the altered antibodies can be assessed using assays that are available in the art and/or described herein.
  • mutations can be introduced randomly or selectively along all or part of an anti-IFNRR1 antibody coding sequence (eg, a 10C2 or 10C9 coding sequence), and can be directed to binding activity and/or as The other functional properties described in the present invention are screened to obtain a modified anti-IFNR1R1 antibody.
  • Mutation methods have been described in the art. For example, PCT Publication No. W002/092780 to Short describes methods for generating and screening for antibody mutations using saturation mutagenesis, synthetic ligation assembly, or a combination thereof.
  • An eighth object of the present invention is to provide a humanized antibody.
  • the humanized antibody provided by the present invention is an antibody obtained by humanizing the monoclonal antibody of the above IFN ⁇ R1 or an antigen-binding portion thereof.
  • the monoclonal antibodies of the invention can be humanized or prepared as chimeric antibodies by recombinant DNA techniques well known in the art.
  • the murine variable region can be ligated using methods well known in the art. On the human constant zone.
  • For methods of preparing chimeric antibodies for example, see Morrison et al, Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al, Nature 314:452, 1985, Cabilly et al, US Patent No. 4,816,567; Boss et al US Patent U.S. Patent No. 4,816,397; Humanization of antibodies involves the transplantation of non-human antibody binding sites onto human antibodies.
  • the CDRs of a non-human antibody can be grafted into the framework regions of human antibodies and constant regions of human antibodies, or the variable regions of the entire non-human antibody can be hidden by replacing certain exposed amino acid residues with adult antibody surfaces. Details of the preparation of humanized antibodies are described in U.S. Pat. No. 5,472,693.
  • Humanized antibodies can also be prepared from transgenic mice expressing human heavy and light chain genes, but capable of expressing the heavy and light chain genes of endogenous mouse immunoglobulin. Winter describes a CDR-grafting method that can be used to prepare humanized antibodies (U.S. Patent No. 5,225,539). All CDRs of a particular human antibody can be replaced with at least a portion of the non-human CDRs, or some human CDRs can be replaced with only non-human CDRs. It is only necessary to replace the humanized antibody with the CDRs required for a particular antigen.
  • a humanized antibody or fragment thereof can be prepared by replacing the sequence of the Fv region not directly involved in antigen binding with an equivalent sequence derived from the human Fv variable region.
  • Examples of the production of humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; US 5, 585, 089; US 5, 693, 761; US 5, 693, 762; US 5, 859, 205; and US 6,407, 213.
  • These methods comprise isolating, manipulating and expressing a nucleic acid sequence from all or part of an immunoglobulin Fv variable region encoding at least one heavy or light chain.
  • These nucleic acids can be obtained from the production of hybridomas or other sources directed against a particular target as described above.
  • Recombinant DNA encoding a humanized antibody molecule can be cloned into a suitable expression vector.
  • a humanized antibody can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and/or back mutations.
  • These altered immunoglobulin molecules can be obtained by techniques well known in the art (e.g., Teng et al, Proc. Natl. Acad. Sci USA, 80: 7308-7312, 1983; Kozbor et al, Immunology Today, 4: 7279, 1983; Olsson et al, Meth. Enzymol, 92: 3-16, 1982).
  • conservative substitutions can be made using amino acids commonly used in human germline antibody sequences.
  • Human antibodies can be produced using methods well known in the art, such as phage display technology.
  • Phage display technology mimics the mammalian immune system by cloning the antibody gene library and selecting for binding to the desired target, such as SD2 of IFNAR1, an epitope in SD3.
  • Libraries used in phage display technology can be prepared from a variety of sources. For example, an immunological library produced by exposure to an antigen of interest by vaccination or disease, even if relatively small, has a high level of circulating antibodies against the antigen.
  • a naive library of mRNA isolated from non-immune individuals can be used repeatedly to isolate antibodies against multiple antigens.
  • a synthetic gene encoding a complete VH and VL chain consisting of an in vitro cloned and combinedly arranged germline antibody gene fragment
  • the library has the advantage of producing antibodies against its own antigen.
  • Semi-synthetic libraries can be prepared by screening one or more antibody backbones and random sequences in a CDR loop.
  • phage display technology once a library is produced, it is fused to a phage surface protein.
  • the phage displaying the antibody specific for the antigen of interest is selectively adsorbed onto the solid phase antigen by panning. Subsequently, the bound phage can be eluted from the surface and expanded by infection with E. coli cells.
  • phage display technology for the production of humanized antibodies are also known in the art.
  • antibodies are displayed on microbial cells such as E. coli and Saccharomyces cerevisiae rather than on the surface of phage.
  • screening can be performed by incubation with fluorescently labeled ligands in buffer.
  • Cells displaying antibodies that bind to the ligand are fluorescently labeled and the cells can be isolated by fluorescence activated cell sorting.
  • Another modification, ribosome display relies on the formation of a ternary complex between the ribosome, mRNA, and polypeptide.
  • mice Another method known in the art for producing human antibodies is to use transgenic mice.
  • the immunoglobulin library in these mice was replaced by the human V gene in the murine chromosome.
  • These mice can be injected with the desired antigen, and the produced antibodies can be obtained by cloning and screening in an immunological library or by conventional hybridoma technology. These mice produce large amounts of complete human antibodies that differ only in the glycosylation pattern.
  • a ninth object of the present invention is to provide a bispecific molecule.
  • the bispecific molecule provided by the present invention contains a specific binding site of the monoclonal antibody or antigen-binding portion thereof of the above IFN ⁇ R1.
  • the bispecific molecule provided by the present invention comprising the specific binding site of the monoclonal antibody or antigen-binding portion thereof of the above IFN ⁇ R1 can derivatize or link the antibody or antigen-binding portion thereof of the present invention to another functional molecule, As another peptide or protein (e.g., a ligand for another antibody or receptor), a bispecific molecule can be generated that binds to at least two different binding sites or target molecules.
  • An antibody of the invention may be derivatized or linked to more than one other functional molecule to generate a multispecific molecule that binds to two or more different binding sites and/or target molecules; such multispecific molecules Also included within the term "bispecific molecule" as used herein.
  • an antibody of the invention can be functionally linked to one or more other binding molecules, such as other antibodies, antibody fragments, peptides or binding mimics (eg, by chemical coupling, gene fusion, non- Covalent binding, etc.), thereby obtaining a bispecific molecule.
  • other binding molecules such as other antibodies, antibody fragments, peptides or binding mimics (eg, by chemical coupling, gene fusion, non- Covalent binding, etc.), thereby obtaining a bispecific molecule.
  • a tenth object of the present invention is to provide a biological material related to the above monoclonal antibody of IFN ⁇ R1 or an antigen-binding portion thereof.
  • biomaterial provided by the present invention relating to the monoclonal antibody of IFN ⁇ R1 or the antigen-binding portion thereof is any one of the following A1) to A12):
  • A1 a nucleic acid molecule encoding a monoclonal antibody or antigen-binding portion thereof of the above IFN ⁇ R1;
  • A2) an expression cassette comprising the nucleic acid molecule of A1);
  • A3 a recombinant vector comprising the nucleic acid molecule of A1);
  • A4 a recombinant vector comprising the expression cassette of A2);
  • A5 a recombinant microorganism comprising the nucleic acid molecule of A1);
  • A6 a recombinant microorganism comprising the expression cassette of A2)
  • A7 a recombinant microorganism comprising the recombinant vector of A3);
  • A8 a recombinant microorganism comprising the recombinant vector of A4)
  • A9 a transgenic cell line comprising the nucleic acid molecule of A1);
  • A10 a transgenic cell line comprising the expression cassette of A2)
  • A11 a transgenic cell line comprising the recombinant vector of A3);
  • a transgenic cell line comprising the recombinant vector of A4).
  • nucleic acid molecules may be present in intact cells, cell lysates, or in partially purified or substantially purified form. When isolated and purified from other cellular components or other contaminants such as other cellular nucleic acids or proteins by conventional techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods known in the art.
  • the nucleic acid is "isolated” or “substantially pure”. See, F. Ausubel et al., eds. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York.
  • the nucleic acid of the invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.
  • nucleic acid molecules of the invention can be obtained using conventional molecular biology techniques.
  • cDNA encoding the light and heavy chains of antibodies prepared by hybridomas can be obtained by PCR amplification or cDNA cloning techniques.
  • nucleic acids encoding antibodies can be obtained from libraries.
  • Preferred nucleic acid molecules of the invention are nucleic acid molecules encoding VH and/or VL sequences of 10C2 and 10C9 monoclonal antibodies.
  • the DNA sequences encoding the VH and VL sequences of 10C2 are shown in Sequence 1 and Sequence 6, respectively.
  • the DNA sequences encoding the VH and VL sequences of 10C9 are shown in Sequence 11 and Sequence 16, respectively.
  • DNA fragments encoding VH and/or VL are obtained, these DNA fragments can be further manipulated by recombinant DNA techniques, such as transformation of the variable region gene into a full length antibody chain gene, into a Fab fragment gene or scFv gene.
  • a DNA fragment encoding VH and/or VL is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operatively linked" as used herein means that two DNA fragments are joined together such that the amino acid sequences encoded by the two DNA fragments remain in reading frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3).
  • the sequence of the human heavy chain constant region gene is well known in the art (see, for example, Kabat, BA et al. (1991) Sequences of Proteins of Immunologicl Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments including these regions can be obtained by PCR amplification.
  • the heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgGl or IgG2a constant region.
  • the DNA encoding VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.
  • the isolated DNA of the coding region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL.
  • the sequence of the human light chain constant region gene is well known in the art (see, for example, Kabat, BA et al. (1991) Sequences of Proteins of Immunologicl Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 ), DNA fragments including these regions can be obtained by PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but is most preferably a kappa constant region.
  • a DNA fragment encoding VH and VL is operably linked to another flexible fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser) 3, such that the VH and VL sequences can be expressed as a continuous single-stranded protein, Its VH and VL regions are joined by a flexible linker (see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al. (1990) Nature. 348:552-554).
  • a flexible linker such as the amino acid sequence (Gly4-Ser) 3
  • An eleventh object of the present invention is to provide a method for producing an anti-IFNR1R1 antibody.
  • the method for preparing an anti-IFN ⁇ R1 antibody provided by the invention comprises:
  • a heavy chain variable region antibody sequence comprising a CDR1 sequence selected from SEQ ID NO: 3 or SEQ ID NO: 13, a CDR2 sequence selected from SEQ ID NO: 4 or SEQ ID NO: 14, and a CDR3 sequence selected from SEQ ID NO: 5 or SEQ ID NO: 15;
  • a light chain variable region antibody sequence comprising a CDR1 sequence selected from SEQ ID NO: 8 or SEQ ID NO: 18, a CDR2 sequence selected from SEQ ID NO: 9 or SEQ ID NO: 19, and a CDR3 sequence selected from SEQ ID NO: 10 or SEQ ID NO:
  • the monoclonal antibodies of the present invention are prepared by an authoritative monoclonal antibody preparation method (for example, Paterson, H.M.V.a.Y. (Jone Wiley and Sons, Inc. New York, 1995). Production of Antibodies. Current Protocols in Immunology).
  • Preferred animals for the preparation of hybridomas are mice.
  • the production of hybridomas in mice is a well established procedure. Immune procedures and separation techniques for fusion spleen cells are well known in the art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also well known.
  • the mouse in the mouse immunization step of preparing the monoclonal antibody of the present invention, can be immunized with a purified or enriched IFN ⁇ R1 antigen preparation and/or a cell expressing IFN ⁇ R1.
  • the mouse is immunized with mouse cells expressing IFNAR1.
  • the mice were 6-8 weeks old at the first infusion.
  • a purified or enriched IFN ⁇ R1 antigen preparation (5-50 ⁇ g) can be used to immunize mice intraperitoneally, or a mouse cell expressing IFN ⁇ R1 can be used to immunize mice intraperitoneally, preferably using mouse cells expressing IFN ⁇ R1 and an immunoadjuvant.
  • mice CpG-B mixed intraperitoneally immunized mice to promote immune response
  • mice can be immunized multiple times, for example 3 times, 4 Mice were immunized 5 or 5 times, at 3 or 4 weeks intervals, and 3 or 4 days after the last immunization, mouse spleen cells or lymph node cells were fused with mouse myeloma cells.
  • spleen cells and/or lymph node cells are isolated from the immunized mouse and fused with a suitable immortalized cell line such as a mouse myeloma cell line.
  • a suitable immortalized cell line such as a mouse myeloma cell line.
  • Hybridomas are screened based on the production of antigen-specific antibodies. For example, a single cell suspension of spleen lymphocytes from immunized mice is fused with one-third of mouse myeloma cells SP2/0 using 50% polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Cells were seeded at approximately 2 x 10 5 in flat-bottomed microtiter plates followed by 10% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 50 units/mL penicillin, 50 mg/mL streptomycin , and incubated for 10 days in selective medium with 1XHAT (Sigma; HAT after 24 hours of fusion). After about 10 days, hybridoma cells secreting IFNAR1 antibodies were screened.
  • 1XHAT Sigma; HAT after 24 hours of fusion
  • a reporter cell line for screening for IFNAR1 antibody secreted by a hybridoma is obtained by constructing a HEK293T cell line by constructing a eukaryotic expression vector expressing the extracellular region of human IFNAR1. This cell line stably expresses GFP. If the hybridoma cell secretes an IFNAR1 antibody, the antibody binds to the extracellular region of the cell-expressed IFNAR1, and the fluorescein PE-labeled anti-mouse IgG antibody is added, the cell emits green fluorescence and yellow fluorescence.
  • Hybridomas secreting IFNAR1 antibodies can be screened by analyzing the GFP and PE signals of the cells using a flow cytometer. Monoclonal IgG antibodies from each well were screened by constructed reporter cell lines stably expressing the extracellular domain of IFNAR1. Once extensive hybridoma growth occurs, the hybridoma cells are typically screened after 10-14 days. Hybridoma cells secreting IFNAR1 antibodies were subcloned by limiting dilution and screened again. If still positive for human IFNAR1, the monoclonal antibody is at least subcloned twice by limiting dilution. Stable subclones are then cultured in vitro and a small amount of antibody is produced in the tissue culture medium for characterization.
  • selected hybridomas can be grown in two-liter rotating shake flasks for monoclonal antibody purification. The supernatant was filtered, concentrated, and then subjected to affinity chromatography using a NAb Protein G Spin Column (GE). The eluted IgG was checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The concentration was determined by BCA protein quantification (Pierce). Monoclonal antibodies were divided into aliquots and stored at -80 °C.
  • the hybridoma culture supernatant is reported with the type I interferon signal constructed in the present invention.
  • the cells are incubated at 37 ° C for 30 minutes to 2 hours, preferably 30 minutes, 1 hour, 2 hours, more preferably 1 hour, and then added in an appropriate amount, such as 1 ng / ml to 1 ⁇ g / ml, preferably 1 ng / ml to 500 ng / ml, further preferably 1 ng /ml to 100 ng/ml, specifically 1 ng/ml, 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml, 50 ng/ml, more preferably 10 ng/ml of type I interferon.
  • the culture is continued for a certain period of time, such as 18-36 hours, preferably 20-28 hours, more preferably 24 hours, and the expression of GFP in the cells is reported by flow cytometry analysis.
  • a sample in which GFP expression in a cell is low is an antibody which blocks a human type I interferon receptor, and antibodies 10C2 and 10C9 which bind to both human IFNAR1 and type I interferon signals are obtained by the method as described above.
  • the monoclonal antibody of the present invention is prepared by an authoritative monoclonal antibody preparation method, and the mouse anti-human IFNAR1 monoclonal antibodies 10C2 and 10C9 can be obtained by amplifying hybridoma cells in vivo or in vitro, and the antibody of the present invention can also be used.
  • the coding gene was cloned into a eukaryotic expression vector by a conventional molecular cloning method, and the antibody was obtained by eukaryotic expression purification. Specific steps are as follows:
  • the method for constructing a eukaryotic expression vector comprises the following steps:
  • the DNA molecule shown in SEQ ID NO: 1 is inserted into a eukaryotic expression vector (for example, pCMV or pcDNA3.1) containing a CMV promoter and a screening gene puromycin by a method of molecular cloning to obtain a plasmid expressing a heavy chain of the monoclonal antibody 10C2;
  • a eukaryotic expression vector for example, pCMV or pcDNA3.1
  • the DNA molecule shown in SEQ ID NO: 6 is inserted into a eukaryotic expression vector (for example, pCMV or pcDNA3.1) containing a CMV promoter and a screening gene puromycin by a method of molecular cloning to obtain a plasmid expressing a light chain of the monoclonal antibody 10C2;
  • a eukaryotic expression vector for example, pCMV or pcDNA3.1
  • the DNA molecule shown in SEQ ID NO: 11 is inserted into a eukaryotic expression vector (for example, pCMV or pcDNA3.1) containing a CMV promoter and a screening gene puromycin by a method of molecular cloning to obtain a plasmid expressing a light chain of the monoclonal antibody 10C2;
  • a eukaryotic expression vector for example, pCMV or pcDNA3.1
  • the DNA molecule shown in SEQ ID NO: 16 is inserted into a eukaryotic expression vector (for example, pCMV or pcDNA3.1) containing a CMV promoter and a screening gene puromycin by a method of molecular cloning to obtain a plasmid expressing a light chain of the monoclonal antibody 10C2;
  • a eukaryotic expression vector for example, pCMV or pcDNA3.1
  • the method for constructing recombinant cells comprises the steps of: transfecting a plasmid expressing a heavy chain of the monoclonal antibody 10C2 and a plasmid expressing a light chain of the monoclonal antibody 10C2 into a mammalian cell at a molar ratio of 1:1 using Lipofectamine 2000 (Invitrogen). (for example, CHO cells or HEK293T cells), and obtain a cell line stably expressing monoclonal antibody 10C2 by resistance gene screening;
  • a plasmid expressing a heavy chain of the monoclonal antibody 10C9 and a plasmid expressing a light chain of the monoclonal antibody 10C9 were transfected into mammalian cells (for example, CHO cells or HEK293T cells) at a molar ratio of 1:1 using Lipofectamine 2000 (Invitrogen). And obtaining a cell line stably expressing monoclonal antibody 10C9 by resistance gene screening;
  • the method for in vitro expression and purification of the monoclonal antibody comprises the steps of separately expanding a cell line expressing the IFNAR1 monoclonal antibody 10C2 and a cell line expressing the IFNAR1 monoclonal antibody 10C9, and collecting the culture supernatant; filtering the supernatant, and concentrating Affinity chromatography was performed on NAb Protein G Spin Column (GE) to obtain a purified monoclonal antibody.
  • GE NAb Protein G Spin Column
  • the eluted IgG was examined by gel electrophoresis and high performance liquid chromatography to ensure purity, and the concentration was determined by BCA protein quantification (Pierce), and the monoclonal antibody was divided into aliquots and stored at -80 °C.
  • a twelfth object of the present invention is to provide a method of inhibiting the biological activity of type I interferon on cells expressing IFNAR1.
  • the present invention provides a method of inhibiting the biological activity of a type I interferon against an IFNAR-expressing cell, comprising contacting the cell with a monoclonal antibody or antigen-binding portion thereof of the present invention such that the biological activity of the type I interferon is inhibited.
  • a thirteenth object of the present invention is to provide a method of treating a type I interferon mediated disease.
  • the method for treating a type I interferon-mediated disease comprises administering to a subject having a type I interferon-mediated disease a monoclonal antibody or antigen-binding portion thereof of the above IFN ⁇ R1, in the subject Type I interferon-mediated diseases are treated.
  • the type I interferon-mediated disease is an interferon alpha-mediated disease, the disease or disorder being any one of the following: systemic lupus erythematosus, insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple Sclerosing disorder, psoriasis, autoimmune thyroiditis, rheumatoid arthritis and glomerulonephritis, HIV infection or AIDS and plant rejection or graft versus host disease.
  • the antibodies (and immunoconjugates and bispecific molecules) of the invention can be used to detect the level of IFN ⁇ R1 or the level of cells expressing IFN ⁇ R1. For example, this can be accomplished by contacting a sample (such as an in vitro sample) and a control sample with an anti-IFNR1R1 antibody under conditions that allow for the formation of a complex between the antibody and IFN ⁇ R1. Any complex formed between the antibody and IFN ⁇ R1 was detected and compared between the sample and the control. For example, detection methods well known in the art, such as ELISA and flow cytometry, can be performed using the compositions of the invention.
  • the invention further provides a method of detecting the presence of IFN ⁇ R1 (e.g., human IFN ⁇ R1 antigen) in a sample, or determining the amount of IFN ⁇ R1, comprising contacting a sample and a control sample under conditions that allow for the formation of a complex between the antibody or a portion thereof and IFN ⁇ R1. An antibody of the invention or an antigen binding portion thereof that binds IFN ⁇ R1. The formation of the complex is then detected, wherein the difference in complex formation between the sample and the control sample indicates the presence of the IFN ⁇ R1 antigen in the sample.
  • IFN ⁇ R1 e.g., human IFN ⁇ R1 antigen
  • IFN ⁇ R1 is part of a cellular receptor for type I interferons, which are well known to be immunoregulatory cytokines involved in T cell differentiation, antibody production, and memory T cell activity and survival. Moreover, increased expression of type I interferons has been described in many autoimmune diseases, HIV infection, transplant rejection, and graft versus host disease (GVHD).
  • anti-IFNRR1 antibodies (and immunoconjugates and bispecific molecules) of the invention that inhibit type I interferon functional activity can be used in a variety of clinical indications associated with abnormal or undesirable type I interferon activity.
  • the invention provides a method of inhibiting a Type I interferon mediated disease or condition, wherein the method comprises administering an antibody of the invention or an antigen binding portion thereof (or an immunoconjugate or bispecific molecule of the invention) A type I interferon mediated disease or condition is treated.
  • autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD) (including Crohn's disease, ulceration) Colitis and celiac disease), multiple sclerosis (MS), psoriasis, autoimmune thyroiditis, rheumatoid arthritis (RA), and glomerulonephritis.
  • SLE systemic lupus erythematosus
  • IDDM insulin-dependent diabetes mellitus
  • IBD inflammatory bowel disease
  • MS multiple sclerosis
  • psoriasis autoimmune thyroiditis
  • RA rheumatoid arthritis
  • glomerulonephritis glomerulonephritis.
  • the antibody compositions of the invention can be used to inhibit or prevent transplant rejection or to treat graft versus host disease (GVHD) or to treat HIV infection/AIDS.
  • GVHD graft versus host
  • an anti-IFNRR1 antibody of the invention can be used to treat SLE by administering an antibody to a subject in need of treatment.
  • the antibody can be used alone or in combination with other anti-SLE drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids (such as prednisone, hydrocortisone), immunosuppressants (such as cyclophosphamide, sulfur) Azathioprine and methotrexate, an antimalarial drug such as hydroxychloroquine, and a biopharmaceutical that inhibits the production of dsDNA antibodies (eg, LJP 394) are used in combination.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • analgesics such as prednisone, hydrocortisone
  • immunosuppressants such as cyclophosphamide, sulfur
  • Azathioprine and methotrexate such as cyclophosphamide, sulfur
  • an antimalarial drug such as hydroxychloroquine
  • a biopharmaceutical that inhibits the production of dsDNA antibodies
  • an anti-IFNRR1 antibody of the invention can be used to treat Type I diabetes by administering an antibody to a subject in need of treatment.
  • the antibody can be used alone or in combination with other anti-diabetic drugs such as insulin.
  • Antibodies to IFN ⁇ R have been shown to be effective in animal models of inflammatory bowel disease (see U.S. Patent Application Serial No. 60/465,155).
  • the anti-IFNRR1 antibodies of the invention can be used to treat inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, by administering antibodies to a subject in need of treatment.
  • IBD inflammatory bowel disease
  • Crohn's disease inflammatory bowel disease
  • the antibody can be used alone or in combination with other anti-IBD drugs such as mesalazine (including sulfasalazine and other drugs containing 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide) , non-steroidal anti-inflammatory drugs (NSAID), analgesics, corticosteroids (eg, prednisone, hydrocortisone), TNF inhibitors (including adilimumab Enazep It is used in combination with infliximab (Remicade), immunosuppressive agents (such as 6-mercaptopurine, azathioprine and cyclosporin A) and antibiotics.
  • anti-IBD drugs such as mesalazine (including sulfasalazine and other drugs containing 5-aminosalicylic acid (5-ASA), such as olsalazine and balsalazide) , non-steroidal anti-inflammatory drugs (NSAID
  • an anti-IFNRR1 antibody of the invention can be used to treat autoimmune thyroid disease by administering an antibody to a subject in need of treatment, including autoimmune primary hypothyroidism, Graves Sickle disease, Hashimoto's thyroiditis, and destructive thyroiditis with hypothyroidism.
  • the antibody can be used alone or in combination with other drugs or treatments such as antithyroid drugs, radioactive iodine and subtotal thyroidectomy.
  • an anti-IFNRR1 antibody of the invention can be used to treat RA by administering an antibody to a subject in need of treatment.
  • the antibody may be used alone or in combination with one or more other anti-RA drugs such as non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, analgesics, corticosteroids (eg, prednisone, hydrocortisone) , immunosuppressants (e.g.
  • methotrexate B cell depleting agents (e.g., Rituxan TM), B cell agonist (e.g., LymphoStat-B TM), and anti-TNF- ⁇ agent (e.g. EMBREL TM, And REMICADE TM) combination.
  • B cell depleting agents e.g., Rituxan TM
  • B cell agonist e.g., LymphoStat-B TM
  • anti-TNF- ⁇ agent e.g. EMBREL TM, And REMICADE TM
  • an anti-IFNRR1 antibody of the invention can be used to treat psoriasis and psoriatic arthritis by administering an antibody to a subject in need of treatment.
  • the antibody may be used alone or in combination with one or more other anti-psoriatic treatments such as phototherapy, topical treatment (eg topical glucocorticoids) or systemic treatment (eg methotrexate, synthetic retinoids, cyclosporine), anti-TNF-a agents (e.g. EMBREL TM, HUMIRA8 and REMICADE TM) and T-cell inhibitor (e.g., Raptiva TM) combination.
  • topical treatment eg topical glucocorticoids
  • systemic treatment eg methotrexate, synthetic retinoids, cyclosporine
  • anti-TNF-a agents e.g. EMBREL TM, HUMIRA8 and REMICADE TM
  • T-cell inhibitor e.g., Raptiva TM
  • an anti-IFNRR1 antibody of the invention is used to treat HIV infection or AIDS by administering an antibody to a subject in need of treatment.
  • the antibody can be used alone or in combination with other anti-HIV agents such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors.
  • Antibodies against IFN ⁇ R1 have been shown to be effective in inhibiting allograft rejection and prolonging allogeneic survival (see, for example, Tovey et al. (1996) J. Leukoc. Biol. 59: 512-517; Benizri et al. (1998) J. Interferon Cytokine Res. 18: 273-284).
  • the anti-IFNRR1 antibodies of the invention can also be used in transplant recipients to inhibit allograft rejection and/or prolong allogeneic survival.
  • the present invention provides a method for inhibiting transplant rejection by administering an anti-IFNR1R1 antibody of the present invention to a transplant recipient in need of treatment.
  • tissue transplants that can be treated include, but are not limited to, liver, lung, kidney, heart, small intestine, and islet cells, as well as treatment of graft versus host disease (GVHD).
  • the antibody can be used alone or in combination with other drugs that inhibit transplant rejection such as immunosuppressants (eg cyclosporine, azathioprine, methylprednisolone, prednisolone, prednisone, mycophenolate mofetil, Sirolimus, rapamycin, tacrolimus), anti-infectives (eg acyclovir, clotrimazole, ganciclovir, nystatin, cotrimoxazole), diuretics (eg cloth Methanib, furosemide, Metolazon) and ulcer drugs (such as cimetidine, famotidine, lansoprazole, omeprazole, ranitidine, sucralfate) in combination.
  • immunosuppressants eg cyclo
  • the present invention relates to an isolated mouse monoclonal antibody that binds to IFN ⁇ R1 and is capable of blocking the action of type I interferon, and an immunoconjugate and bispecific molecule comprising the antibody of the present invention, an antibody comprising the same, an immunoconjugate Or a pharmaceutical composition of a bispecific molecule.
  • the present invention also relates to the use of these antibodies to bind to IFN ⁇ R1 on cells expressing IFN ⁇ R1, to inhibit the signaling of type I interferons, for the treatment of immunologically mediated diseases in patients, including autoimmune diseases, transplant rejection and graft versus host disease (GVHD) method.
  • interferon alpha receptor 1 interferon alpha receptor 1
  • IFN ⁇ R1 immunoglobulin ⁇ receptor 1
  • IFN ⁇ R1 antigen an antibody that is used interchangeably and include variants, isoforms, homologs of human IFN ⁇ R1, and similar to IFN ⁇ R1 having at least one common epitope. Things.
  • the antibody of the present invention may, in some cases, cross-react with IFN ⁇ R1 from a species other than human, or Other proteins associated with human IFN ⁇ R1 (eg, human IFN ⁇ R1 homolog) are cross-reactive. In other cases, antibodies may be completely specific to mouse IFN ⁇ R1 and do not exhibit species or other types of cross-reactivity.
  • the complete cDNA sequence of human IFN ⁇ R1 has the Genebank accession number NM_000629.
  • type I interferon refers to a member of the family of type I interferons which are ligands for IFN ⁇ R1 (ie, members of the type I interferon family of molecules capable of binding IFN ⁇ R1).
  • types of type I interferon ligands are IFN ⁇ 1, 2a, 2b, 4, 5, 6, 7, 8, 10, 14, 16, 17, 21, interferon beta and interferon ⁇ .
  • immune response refers to, for example, the action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complements) produced by such cells or liver, resulting in selective damage, destruction, or Removal of invading pathogens from the human body Infects cells or tissues of the pathogen, cancer cells, or in the case of autoimmune or pathological inflammation, normal human cells or tissues.
  • Signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that function in a portion of a cell that is transmitted from one part of a cell to another.
  • the phrase "cell surface receptor” includes, for example, molecular and molecular complexes that are capable of receiving signals and transmitting such signals across the plasma membrane of the cell.
  • An example of a “cell surface receptor” of the present invention is the IFN ⁇ R1 receptor.
  • antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by a disulfide bond, or an antigen binding portion thereof.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), which are interspersed in a more conserved region called the framework region (FR).
  • CDRs complementarity determining regions
  • FR framework region
  • Both VH and VL consist of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, IFNR1). It has been shown that the antigen binding function of an antibody can be exercised by a fragment of a full length antibody.
  • binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, ie, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment a bivalent fragment comprising two Fab fragments joined by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of a VH and CH domain; (iv) consisting of a VL and VH domain of the antibody's one arm Fv fragment; (V) a dAb fragment consisting of a VH domain (Ward et al.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds to IFN ⁇ R1 is substantially free of antibodies that specifically bind to an antigen other than IFN ⁇ R1).
  • an isolated antibody that specifically binds to IFN ⁇ R1 may be cross-reactive with other antigens such as IFN ⁇ R1 molecules from other species.
  • the isolated antibodies may be substantially free of other cellular materials and/or chemicals.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions display a single binding specificity and affinity for a particular epitope.
  • subject as used herein includes any human or non-human animal.
  • non-human animal includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.
  • Figure 1 shows human type I interferon reporter cells.
  • Figure 1A shows the constructed type I interferon reporter plasmid;
  • Figure 1B shows that the cell line stably transfected with type I interferon reporter plasmid expresses GFP protein after stimulation with type I interferon.
  • Figure 2 shows the binding of human type I interferon receptor IFNAR1 and screening of blocking antibodies.
  • Figure 3 shows the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the 10C2 heavy chain variable region of the IFN ⁇ R1 monoclonal antibody.
  • the black shaded regions in the figure indicate the CDR1 (SEQ ID NO: 3), CDR2 (SEQ ID NO: 4) and CDR3 (SEQ ID NO: 5) regions.
  • Figure 4 shows the nucleotide sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 7) of the 10C2 light chain variable region of the IFN ⁇ R1 monoclonal antibody.
  • the black shaded regions in the figure indicate the CDR1 (SEQ ID NO: 8), CDR2 (SEQ ID NO: 9) and CDR3 (SEQ ID NO: 10) regions.
  • Figure 5 shows the comparison of the amino acid sequence of the heavy chain variable region of IFN ⁇ R1 monoclonal antibody 10C2 (SEQ ID NO: 2) with the mouse V region amino acid sequence (SEQ ID NO: 21).
  • the heavy chain variable region composition of the IFN ⁇ R1 monoclonal antibody 10C2 the V gene is IGHV9-3, the D gene is IGHD2-13, and the J gene is IGHJ1.
  • the figure shows the alignment of the amino acid sequence of the heavy chain amino acid sequence of the IFN ⁇ R1 monoclonal antibody 10C2 with its original VDJ gene.
  • FIG. 6 shows the comparison of the amino acid sequence of the light chain variable region of the IFN ⁇ R1 monoclonal antibody 10C2 with the mouse V region amino acid sequence (SEQ ID NO: 22).
  • IFN ⁇ R1 monoclonal antibody 10C2 light chain composition V gene is IGKV6-13, the J gene is IGKJ5.
  • the figure shows the alignment of the amino acid sequence of the 10C2 light chain amino acid sequence of the IFN ⁇ R1 monoclonal antibody with its original VJ gene.
  • Figure 7 shows the nucleotide sequence (SEQ ID NO: 11) and amino acid sequence (SEQ ID NO: 12) of the heavy chain variable region of the IFN ⁇ R1 monoclonal antibody 10C9.
  • the black shaded regions in the figure indicate the CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 14) and CDR3 (SEQ ID NO: 15) regions.
  • Figure 8 shows the nucleotide sequence (SEQ ID NO: 16) and amino acid sequence (SEQ ID NO: 17) of the IFN ⁇ R1 monoclonal antibody 10C9 light chain variable region.
  • the black shaded regions in the figure indicate the CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19) and CDR3 (SEQ ID NO: 20) regions.
  • Figure 9 shows the comparison of the amino acid sequence of the heavy chain variable region of IFN ⁇ R1 monoclonal antibody 10C9 (SEQ ID NO: 12) with the mouse V region amino acid sequence (SEQ ID NO: 23).
  • IFN ⁇ R1 monoclonal antibody 10C9 heavy chain composition V gene is IGHV9-3, D gene is IGHD1-1, and J gene is IGHJ3.
  • the figure shows the alignment of the amino acid sequence of the heavy chain amino acid sequence of the IFN ⁇ R1 monoclonal antibody 10C9 with its original VDJ gene.
  • Figure 10 shows the comparison of the amino acid sequence of the light chain variable region of IFN ⁇ R1 monoclonal antibody 10C9 (SEQ ID NO: 17) with the mouse V region amino acid sequence (SEQ ID NO: 24).
  • IFN ⁇ R1 monoclonal antibody 10C9 heavy chain composition V gene is IGKV6-15, J gene is IGKJ2.
  • the figure shows the alignment of the amino acid sequence of the 10C9 light chain of the IFN ⁇ R1 monoclonal antibody with its original VJ gene.
  • Figure 11 shows that IFN ⁇ R1 monoclonal antibodies 10C2 and 10C9 bind to IFNAR1.
  • Figure 12 shows that the IFN ⁇ R1 monoclonal antibodies 10C2 and 10C9 bind to the SD2 and/or SD3 domains of IFNAR1.
  • FIG. 13 shows that the IFN ⁇ R1 monoclonal antibodies 10C2 and 10C9 inhibit the biological activity of IFN ⁇ 2b on interferon signaling reporter cells.
  • FIG 14 shows that IFN ⁇ R1 monoclonal antibodies 10C2 and 10C9 inhibit the biological activity of type I interferon on stimulator-stimulated human peripheral blood mononuclear cells.
  • the genomic sequence of the mouse interferon effector gene Mx2 was obtained by the method of "Bürgi et al. (Journal of Immunological Methods 381 (2012) 70-74)", and according to the cleavage site used therein (Nse I and Nhe I) identified the promoter sequence of Mx2. Based on the promoter sequence of Mx2, the following primers were designed:
  • R CCCAAGCTTCAAATGCCCTGCTGTACTTACCAGT.
  • the mouse blood genomic DNA was used as a template, and the primers containing the Kpn I and Nhe I cleavage sites designed in step 1 were used for PCR amplification to obtain a PCR amplification product, which is a mouse Mx2 promoter fragment;
  • the PCR reaction conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing 60 ° C, 30 seconds; extension, 72 ° C, 1 minute; a total of 30 cycles, and finally increased denaturation time of 10 minutes.
  • pGL4.2-EGFP vector was replaced with the luc2 gene in the pGL4 (luc2) vector by the eGFP gene.
  • the vector obtained by keeping the other sequences of the pGL4 (luc2) vector unchanged was ligated to obtain the human type I interferon reporter plasmid pGL4.2mouseMx2promoter-EGFP (the structure of the pGL4.2mouseMx2promoter-EGFP plasmid is shown in Fig. 1A).
  • the pGL4.2mouseMx2promoter-EGFP plasmid was transfected into the HEK293T cell line using Lipofectamine 2000 (Invitrogen) (ATCC, Cat.
  • a stable transfected cell line (type I interferon reporter cell line) was obtained using CHO-3216 (TM ) and using puromycin screening.
  • the stably transfected cell line obtained in step 4 was stimulated with 100 ⁇ l of human IFN ⁇ 2b (Cedarlane, Cat. No. CL106-04E-100UG) at a concentration of 5 ng/ml for 24 hours, and the cells were collected for detection of GFP expression by flow cytometry.
  • human IFN ⁇ 2b Cedarlane, Cat. No. CL106-04E-100UG
  • Type I interferon reporter cell lines do not express GFP when stimulated without type I interferon, but can be expressed by type I interferon, and can be detected by flow cytometry. It was confirmed that the cell line prepared in the above step 4 can be used as a type I interferon reporter cell line, which can be used as a screening for IFNAR1 blocking antibody.
  • IFNAR1-F CTAGCTAGCTCTAGAGCCACCATGATGGTCGTCCTCCTGGGC
  • IFNAR1-R GGGTCCGGAACCTCCTCCTCCCACAGCATAAATGACAAACGGGAGA.
  • step (1) Using the cDNA of ex vivo human PBMCs as a template, the primers designed in step (1) were used for PCR amplification to obtain PCR amplification products, which are the extracellular region and transmembrane region of human type I interferon receptor IFNAR1. Fragment.
  • the above PCR reaction conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing, 60 ° C, 30 seconds; extension, 72 ° C, 2 minutes; a total of 30 cycles, and finally an extension of 10 minutes.
  • the PCR amplification product obtained in the step (2) and the pEGFP vector were digested with restriction endonucleases Nhe I and Bspe I, and ligated to obtain a pEGFP-humanIFNAR1EC plasmid.
  • the GFP gene was fused downstream of the extracellular and transmembrane region sequences of IFNAR1, and when IFNAR1 was expressed, a GFP signal was observed.
  • the pEGFP-humanIFNAR1EC plasmid was transfected into mouse L cells using Lipofectamine 2000 (Invitrogen) (ATCC, article number: In CRL-2648 (TM ), 48 hours after transfection, mouse L cells transfected with the extracellular domain of human IFNAR1 were obtained. Transfection efficiency was determined by analyzing the expression of GFP in mouse L cells transfected with the extracellular region of human IFNAR1 and used in the following mouse immunization experiments.
  • mice The mouse L cells transfected with the extracellular region of human IFNAR1 obtained in step 1 were used as immunogens of human IFNAR1. 5000,000 mouse L cells transfected with human IFNAR1 were mixed with 20 ⁇ g of CpG1826 (TAKARA synthesis) to form a 0.5 ml suspension, and 6-week-old BalB/C mice were immunized by intraperitoneal injection. Immunize once a month for a total of 3 immunizations. Finally, booster immunization was performed using the same method, and hybridoma fusion was performed 4 days later.
  • CpG1826 TAKARA synthesis
  • the pEGFP-humanIFNAR1EC plasmid was transfected into HEK293T cell line using Lipofectamine2000 (Invitrogen), and cells stably transfected with the plasmid were obtained by puromycin screening, thereby stably expressing the extracellular and transmembrane regions of human IFNAR1 (fused expression of IFNAR1 and GFP). .
  • This cell line will be used in the following antibody screening experiments.
  • mice to be fused after immunization were sacrificed, and spleen cells were taken out.
  • the mouse spleen cells were mixed with the mouse myeloma cell line SP2/0 (ATCC, CRL1581) at a ratio of 1:3 by cell counting, and the cells were fused using 50% PEG (Sigma).
  • the fused cells were added to 60 ml of RPMI medium (Cellgro, Cat. No. 15-041-CV) containing 1X HAT (containing 10% fetal bovine serum), and added to 96-well cells in an amount of 1-2 drops per well. In the culture plate. Subsequently, the hybridoma cells were cultured at 37 ° C, 5% CO 2 , and half-quantized for 3-4 days. Antibody screening was performed after about 10 days.
  • the supernatant in the 96-well cell culture plate was aspirated from about 100 ⁇ l to a 96-well U-shaped bottom plate, and 100 ⁇ l of fresh IX HAT-containing RPMI medium was added to the original well to continue culturing the cells.
  • the HEK293T cell line stably cultured to express human IFNAR1 was collected and added to a 96-well U-bottom plate, 10000 cells/200 ⁇ l/well. After centrifugation at 2200 rpm / 3 minutes, the supernatant was decanted, and 100 ⁇ l of the hybridoma culture supernatant was added, and resuspended and incubated at 4 ° C for 30 minutes.
  • FACS buffer PBS containing 2% FBS and 2 mM EDTA
  • PE-labeled goat anti-mouse IgG antibody Biolegend
  • FACS buffer 200 ⁇ l of FACS buffer (PBS containing 2% FBS and 2 mM EDTA) was added to each well, and the cells were resuspended and centrifuged, and then the supernatant was decanted to achieve the purpose of washing.
  • PE-labeled goat anti-mouse IgG antibody Biolegend
  • diluted 1:400 in FACS buffer was added to each well, and the cells were resuspended and incubated at 4 ° C for 30 minutes in the dark.
  • the culture supernatant of the positive wells was used for the next blocking function experiment.
  • the specific steps were as follows: First, the human type I interferon reporter cell line was added to a 96-well cell culture plate, 30,000 cells/well one day before the experiment. On the day of the experiment, the cell culture supernatant was aspirated, and 100 ⁇ l of the hybridoma culture supernatant was added, and the mixture was incubated at 37 ° C for 1 hour. Subsequently, 100 ⁇ l of DMEM (containing 10% fetal bovine serum FBS) medium (Hyclone, Cat. No. SH30022.01) containing 10 ng/ml of IFN ⁇ 2b was added for 24 hours.
  • DMEM containing 10% fetal bovine serum FBS
  • the expression of the GFP expression was blocked by analyzing the expression of the GFP, and the antibody having a low expression of GFP was an antibody blocking the human type I interferon receptor (Fig. 2B).
  • 10C2 and 10C9 were selected to bind to human IFNAR1, and two monoclonal hybridoma cell lines stably secreting IFNAR1 monoclonal antibodies were blocked, and they were named as hybridoma cell line 10C2 and hybridized.
  • the antibody secreted by the hybridoma cell line 10C2 was named mouse anti-human IFNAR1 monoclonal antibody 10C2; the antibody secreted by the hybridoma cell line 10C9 was named mouse anti-human IFNAR1 monoclonal antibody 10C9.
  • the hybridoma cell line 10C2 was classified as a mouse hybridoma cell line, which was deposited on May 31, 2016 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: Chaoyang, Beijing) No. 3, No. 1 Courtyard, Beichen West Road, District Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101), the deposit number is CGMCC No.12542.
  • the hybridoma cell line 10C9 was classified as a mouse hybridoma cell line, which was deposited on May 31, 2016 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC, Address: Chaoyang, Beijing) No. 3, No. 1 Courtyard, Beichen West Road, District Institute of Microbiology, Chinese Academy of Sciences, 100101), the deposit number is CGMCC No.12543.
  • hybridoma cell line 10C2 and hybridoma cell line 10C9 were lysed by Trizol (Invitrogen), hybridoma cell line 10C2 and hybridoma cell line 10C9 were extracted. Specific steps for RNA extraction:
  • the cDNA template obtained in the step 2 was subjected to PCR amplification using the heavy chain primer F and the heavy chain primer R, the light chain primer F and the light chain primer R, respectively, and the fragments encoding the heavy chain and the light chain were respectively obtained and sequenced.
  • the primer sequences are as follows:
  • Heavy chain primer F CTAGCTAGCTCTAGAGCCACC ATGATGGTCGTCCTCCTGGGC;
  • Heavy chain primer R CTTGACCAGGCATCCTAGAGTCA
  • Light chain primer F GAYATTGTGMTSACMCARWCTMCA
  • Light chain primer R GGATACAGTTGGTGCAGCATC.
  • the above PCR reaction conditions pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing, 54 ° C; extension 72 ° C, 1 minute; a total of 35 cycles, and finally increased extension for 10 minutes.
  • the nucleotide and amino acid sequences of the heavy chain variable region of IFN ⁇ R1 monoclonal antibody 10C2 are shown in Figure 3.
  • the heavy chain variable region nucleotide sequence of IFN ⁇ R1 monoclonal antibody 10C2 is sequence 1
  • the weight of IFN ⁇ R1 monoclonal antibody 10C2 is The amino acid sequence of the variable region of the chain is sequence 2; the amino acid sequence (SEQ ID NO: 3) shown in positions 26 to 33 of the heavy chain variable region amino acid sequence of IFN ⁇ R1 monoclonal antibody 10C2 is named 10C2 heavy chain CDR1,
  • the amino acid sequence (SEQ ID NO: 4) at position 51 to position 58 of the heavy chain variable region amino acid sequence of IFN ⁇ R1 monoclonal antibody 10C2 is designated as 10C2 heavy chain CDR2, and the heavy chain variable region amino acid of IFN ⁇ R1 monoclonal antibody 10C2
  • the nucleotide and amino acid sequences of the light chain variable region of IFN ⁇ R1 monoclonal antibody 10C2 are shown in Figure 4.
  • the light chain variable region nucleotide sequence of IFN ⁇ R1 monoclonal antibody 10C2 is sequence 6, and the IFN ⁇ R1 monoclonal antibody 10C2 is light.
  • the amino acid sequence of the variable region of the chain is sequence 7; the amino acid sequence (SEQ ID NO: 8) shown in positions 27 to 32 of the light chain variable region amino acid sequence of IFN ⁇ R1 monoclonal antibody 10C2 is named 10C2 light chain CDR1, The amino acid sequence (SEQ ID NO: 9) at position 50 to position 52 of the light chain variable region amino acid sequence of IFN ⁇ R1 monoclonal antibody 10C2 is named 10C2 light chain CDR2, and the light chain variable region amino acid of IFN ⁇ R1 monoclonal antibody 10C2 The amino acid sequence (SEQ ID NO: 10) shown in position 89 to position 94 of the sequence was designated as 10C2 light chain CDR3.
  • the nucleotide and amino acid sequences of the heavy chain variable region of IFN ⁇ R1 monoclonal antibody 10C9 are shown in Figure 7.
  • the heavy chain variable region nucleotide sequence of IFN ⁇ R1 monoclonal antibody 10C9 is sequence 11, and the weight of IFN ⁇ R1 monoclonal antibody 10C9 is The amino acid sequence of the variable region of the chain is sequence 12; the amino acid sequence (SEQ ID NO: 13) shown in positions 26 to 33 of the heavy chain variable region amino acid sequence of IFN ⁇ R1 monoclonal antibody 10C9 is named 10C9 heavy chain CDR1,
  • the amino acid sequence (SEQ ID NO: 14) at position 51 to position 58 of the heavy chain variable region amino acid sequence of IFN ⁇ R1 monoclonal antibody 10C9 is designated as 10C9 heavy chain CDR2, and the heavy chain variable region amino acid of IFN ⁇ R1 monoclonal antibody 10C9
  • the amino acid sequence (SEQ ID NO: 15) shown at positions 97 to 112 of the sequence was designated
  • the nucleotide and amino acid sequences of the light chain variable region of IFN ⁇ R1 monoclonal antibody 10C9 are shown in Figure 8.
  • the light chain variable region nucleotide sequence of IFN ⁇ R1 monoclonal antibody 10C9 is sequence 16; IFN ⁇ R1 monoclonal antibody 10C9 is light.
  • the amino acid sequence of the variable region of the chain is sequence 17.
  • the amino acid sequence (SEQ ID NO: 18) shown in positions 27 to 32 of the light chain variable region amino acid sequence of IFN ⁇ R1 monoclonal antibody 10C9 was designated as 10C9 light chain CDR1, and the light chain variable region of IFN ⁇ R1 monoclonal antibody 10C9 was designated.
  • amino acid sequence shown in the 50th to 52nd position of the amino acid sequence (SEQ ID NO: 19) is designated as the 10C9 light chain CDR2, and the 89th to 96th position of the amino acid sequence of the light chain variable region of the IFN ⁇ R1 monoclonal antibody 10C9 is shown.
  • amino acid sequence (SEQ ID NO: 20) was designated as 10C9 light chain CDR3.
  • the nucleic acid fragments of IFN ⁇ R1 monoclonal antibody 10C2 and IFN ⁇ R1 monoclonal antibody 10C9 were analyzed in the antibody sequence analysis tool igBlast tool (http://www.ncbi.nlm.nih.gov/igblast/) and found:
  • the V gene, D gene and J gene of the IFN ⁇ R1 monoclonal antibody 10C2 heavy chain coding gene correspond to the amino acid sequence of the heavy chain variable region of mouse IGHV9-3 gene, IGHD2-13 gene and IGHJ1 gene, IFN ⁇ R1 monoclonal antibody 10C2, respectively.
  • the results of (SEQ ID NO: 2) and mouse V region amino acid sequence (SEQ ID NO: 21) are shown in Figure 5;
  • IFN ⁇ R1 monoclonal antibody 10C2 light chain coding gene V gene and J gene correspond to mouse IGKV6-13 gene and IGKJ5, respectively.
  • the amino acid sequence of the light chain variable region of the gene, IFN ⁇ R1 monoclonal antibody 10C2 and the amino acid sequence of the mouse V region (sequence 22) are shown in Fig. 6;
  • the V gene, D gene and J gene of the IFN ⁇ R1 monoclonal antibody 10C9 heavy chain coding gene correspond to the amino acid sequence of the heavy chain variable region of mouse IGHV9-3 gene, IGHD1-1 gene and IGHJ3 gene, IFN ⁇ R1 monoclonal antibody 10C9, respectively.
  • the results of (SEQ ID NO: 12) and mouse V region amino acid sequence (SEQ ID NO: 23) are shown in Figure 9; IFN ⁇ R1 monoclonal antibody 10C9 light chain coding gene V gene and J gene correspond to mouse IGKV6-15 and IGKJ2, respectively.
  • the results of comparison of the amino acid sequence of the light chain variable region of IFN ⁇ R1 monoclonal antibody 10C9 (SEQ ID NO: 17) with the amino acid sequence of mouse V region (SEQ ID NO: 24) are shown in FIG.
  • IFNAR1 monoclonal antibody can bind human type I interferon receptor IFNAR1
  • the HEK293T cells stably expressing human type I interferon receptor IFNAR1 obtained in step 3 of Example 1 were treated with 2 mM EDTA in PBS (containing 0.27 g of KH 2 PO 4 per 1 L of PBS, Na 2 HPO 4 1.42). g, NaCl 8 g, KCl 0.2 g, pH adjusted to 7.2-7.4, and made up to 1 L with water, and treated to make a single cell suspension.
  • FACS buffer PBS containing 2% FBS and 2 mM EDTA
  • 5 ⁇ g/ml of the mouse anti-human IFNAR1 monoclonal antibody 10C2 and 10C9 prepared in Example 1 was added to the precipitate obtained in the step 2, and resuspended. Incubate for 30 minutes at °C.
  • PE-labeled goat anti-mouse IgG antibody (Biolegend) diluted 1:400 in FACS buffer was added to each well, and the cells were resuspended and incubated at 4 ° C for 30 minutes in the dark. After the incubation, the plate was centrifuged and the supernatant was removed, and the cells were washed once as described above, and then resuspended by adding 200 ⁇ l of FACS buffer. Finally, cellular GFP and PE signals were analyzed using a flow cytometer Guava (Millipore).
  • IFNAR1 monoclonal antibody can bind to the SD2 and SD3 domains of human type I interferon receptor IFNAR1
  • 3F CAGTTGAAAATGAACTACCTTTCCTACTTCCTCCAGTCTTTAACA;
  • the PCR conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing 58 ° C, 30 seconds; extension, 72 ° C, 2 minutes; a total of 30 cycles, and finally increased denaturation time of 10 minutes;
  • the PCR conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing 60 ° C, 30 seconds; extension, 72 ° C, 2 minutes; a total of 30 cycles, and finally increased denaturation time of 10 minutes;
  • the SD1 truncated IFNAR1 expression plasmid replaces the coding gene of the extracellular SD1 domain of human IFNAR1 (ie, the coding gene of amino acids 32-126 of the IFNAR1 amino acid sequence) with the pEGFP-humanIFNAR1EC plasmid EcoR I and Bspe I restriction sites. Fragment, and the vector obtained by keeping the other sequences of the pEGFP-humanIFNAR1EC plasmid unchanged.
  • the PCR conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing 58 ° C, 30 seconds; extension, 72 ° C, 2 minutes; a total of 30 cycles, and finally increased denaturation time of 10 minutes;
  • the PCR conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing 60 ° C, 30 seconds; extension, 72 ° C, 2 minutes; a total of 30 cycles, and finally increased denaturation time of 10 minutes;
  • the SD2 truncated IFNAR1 expression plasmid is a sequence in which the gene encoding the extracellular SD2 domain of human IFNAR1 (i.e., the coding gene of amino acids 127-227 of the IFNAR1 amino acid sequence) is replaced with the pEGFP-humanIFNAR1EC plasmid EcoR I and Bspe I cleavage sites. A fragment between the spots, and the vector obtained by keeping the other sequences of the pEGFP-humanIFNAR1EC plasmid unchanged.
  • the PCR conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing 58 ° C, 30 seconds; extension, 72 ° C, 2 minutes; a total of 30 cycles, and finally increased 10 minutes of denaturation time, this time
  • the reaction template is the aforementioned IFNAR1 expression plasmid;
  • the PCR conditions were: pre-denaturation, 98 ° C, 2 minutes; denaturation, 98 ° C, 30 seconds; annealing 60 ° C, 30 seconds; extension, 72 ° C, 2 minutes; a total of 30 cycles, and finally increased denaturation time of 10 minutes;
  • the SD3 truncated IFNAR1 expression plasmid is a gene encoding the gene encoding the extracellular SD3 domain of human IFNAR1 (ie, the coding gene of amino acids 231-329 of the IFNAR1 amino acid sequence) is replaced with the pEGFP-humanIFNAR1EC plasmid EcoR I and Bspe I restriction sites. The fragment was interposed and the vector obtained by keeping the other sequences of the pEGFP-humanIFNAR1EC plasmid unchanged.
  • the wild type IFNAR1 (pEGFP-humanIFNAR1EC) and the truncated human expression plasmids of knockdown human IFNAR1 extracellular SD1, SD2 and SD3 were transfected into HEK293T cell line by Lipofectamine 2000 (Invitrogen), respectively, and transfected for 24 hours using 2 mM EDTA.
  • HEK293T cells (R1-WT) expressing human type I interferon receptor IFNAR1 and HEK293T cells expressing human type I interferon receptor IFNAR1-SD1 truncation were obtained by treatment with PBS and single cell suspension (R1- ⁇ ).
  • HEK293T cells (R1- ⁇ 2) expressing human type I interferon receptor IFNAR1-SD1 truncation and HEK293T cells expressing human type I interferon receptor IFNAR1-SD1 truncation (R1- ⁇ 3) .
  • IFNAR1 monoclonal antibodies 10C2 and 10C9 and HEK293T cells expressing human type I interferon receptor IFNAR1 (R1-WT) and human type I interferon receptor IFNAR1-SD1 truncated body were detected as in Example 2, respectively.
  • IFNAR1 monoclonal antibodies 10C2 and 10C9 do not bind to human type I interferon receptor IFNAR1-SD1 truncated HEK293T cells (R1- ⁇ 1), but both express human type I interferon
  • the recipient IFNAR1-SD1 truncated HEK293T cells (R1- ⁇ 2) bind to HEK293T cells (R1- ⁇ 3) expressing the human type I interferon receptor IFNAR1-SD1 truncation. This indicates that the IFNAR1 monoclonal antibody can specifically bind to the SD2 and/or SD3 domain of the human type I interferon receptor IFNAR1.
  • the type I interferon reporter cell line in step 1 of Example 1 was used as a tool to analyze the efficiency of blocking IFNAR1 monoclonal antibodies 10C2 and 10C9 for blocking type I interferons. Specific steps are as follows:
  • the supernatant of the cells was aspirated, and 100 ⁇ l of DMEM medium supplemented with different gradient dilution antibodies was added, and incubated at 37 ° C for 1 hour.
  • GFP was detected by flow cytometry to analyze the biological activity of the antibody blocking type I interferon signal.
  • Iso ctrl represents an isotype control group of antibodies (mIgG2a, Biolegend)
  • Neg ctrl represents a control group not stimulated with IFN ⁇ 2b
  • Pos ctrl represents only IFN ⁇ 2b.
  • Example 5 IFNAR1 monoclonal antibody in blocking the stimulation of type I interferon on human peripheral blood mononuclear cells
  • PBMC peripheral blood mononucleus
  • the following three groups were divided into three groups to stimulate the cultured PBMC, and the stimulated cells were obtained:
  • the cultured PBMC was directly stimulated with IFN ⁇ 2b (Cedarlane, Cat. No. CL106-04E-100UG) (500 pg/ml).
  • RNA levels of type I interferon effector genes Mx2 and ISG15 in each stimulated cell were detected by real-time PCR, and GAPDH was an internal reference gene.
  • the primer sequence of Mx2 is:
  • Mx2-F CAGAGGCAGCGGAATCGTAA;
  • Mx2-R TGAAGCTCTAGCTCGGTGTTC;
  • the primer sequence of ISG15 is:
  • ISG15-F CCCACAGCCCACAGCCAT
  • ISG15-R TTCTGGGTGATCTGCGCCTT
  • the primer sequence of GAPDH is:
  • GAPDH-F AGCCACATCGCTCAGACAC
  • GAPDH-R GCCCAATACGACCAAATCC.
  • Fig. 14 The results are shown in Fig. 14, in which mock represents the RPMI medium control group, and Isotype represents the isotype control group of the antibody (mIgG2a, Biolegend).
  • Isotype represents the isotype control group of the antibody (mIgG2a, Biolegend).
  • Fig. 14 shows that after the addition of the IFNAR1 monoclonal antibodies 10C2 and 10C9, after each stimulation Type I interferon effector genes Mx2 and ISG15 in cells The expression level of RNA was significantly decreased, indicating that IFNAR1 monoclonal antibodies 10C2 and 10C9 can effectively block the stimulation signals of endogenous type I interferon and exogenous type I interferon stimulated.
  • the antibodies (and immunoconjugates and bispecific molecules) of the invention may, in one aspect, be used to detect the level of IFN ⁇ R1 or the level of cells expressing IFN ⁇ R1, or to detect the presence of IFN ⁇ R1 in a sample, or to determine the amount of IFN ⁇ R1;
  • the antibodies (and immunoconjugates and bispecific molecules) of the invention also have in vitro and in vivo diagnostic and therapeutic applications, including administration of an anti-IFNRR1 antibody of the invention or an antigen binding portion thereof (or an immunoconjugate of the invention) Or a bispecific molecule), or in combination with other drugs, to treat, prevent or diagnose a plurality of type I interferon mediated diseases, for example, an antibody of the invention can be used to administer an antibody to a subject in need of treatment To treat inflammatory bowel disease (including ulcerative colitis and Crohn's disease), autoimmune thyroid disease (including autoimmune primary hypothyroidism, Graves' disease, Hashimoto's thyroiditis and associated Type I interfer

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Abstract

本发明公开了结合I型干扰素受体的单克隆抗体及其用途，所述抗体能够抑制I型干扰素的生物活性，可用于治疗、预防或诊断I型干扰素介导的疾病。还提供了包含所述抗体的免疫偶联物、双特异性分子和药物组合物。

Description

I型干扰素受体抗体及其用途 技术领域
本发明属于生物技术领域，具体涉及I型干扰素受体抗体及其用途。
背景技术
I型干扰素(Type I interferon，IFN-I)是具有抗病毒、抗肿瘤和免疫调节作用的结构上相关的细胞因子的一个家族(Hardy等(2001)Blood 97:473；Cutrone和Langer(2001)J.Biol.Chem.276:17140)。人IFN-I包括5种，它们分别是IFN-α(包括13个亚型)、IFN-β、IFN-ε、IFN-κ和IFN-ω。虽然干扰素是一种强效的抗病毒制剂，但是一些急性病毒病，如上呼吸道感染的特征症状是由人IFN-α的过量引起的。而且，人IFN-α与实验动物中的慢性病毒感染以及人慢性病毒病如麻疹病毒感染的病理发生相关。人IFN-α也是强效的免疫调节分子，刺激B细胞的活化，增强NK细胞的细胞毒性，抑制T细胞功能，以及调节主要相容性复合物(MHC)I类抗原的表达。人IFN-α的异常产生与许多自身免疫疾病，包括系统性红斑狼疮(SLE)、I型糖尿病、牛皮癣、风湿性关节炎、多发性硬化症、获得性免疫缺陷综合征(AIDS)和严重的混合免疫缺陷疾病等相关。
所有的人IFN-I都与细胞表面受体(IFN-α受体，IFNΑR)结合，IFNΑR是由两个跨膜蛋白IFNΑR1和IFNΑR2组成的异源二聚体(Uze等(1990)Cell 60:225；Novick等(1994)Cell 77:391)。人IFNΑR1是IFN-I信号途径的重要组分，它属于II型螺旋型细胞因子受体，由各约100个氨基酸的4个III型纤连蛋白结构域的胞外域(ECD)，一个跨膜域和100个氨基酸残基的胞内域组成。IFNΑR1的4个子域(subdomain，SD)折叠成结构域1(SD1+SD2)和结构域2(SD3+SD4)。IFNΑR1和IFNΑR2与I型干扰素配体形成三元信号复合物(Cohen B等(1995)，Mol Cell Biol 15(8):4208-14)。三元复合物的形成是信号转导途径活化的第一步。因此，针对这个受体以及阻断激酶的活化具有防止与干扰素活化相关疾病的下游生物学效应的潜力。
美国专利号US7179465(2007)，US7465451(2008)公开了一种针对人IFNAR1的单克隆抗体64G12，该抗体可识别人IFNAR1，并对人I型干扰素的生物活性具有中和能力。该抗体结合人IFNAR1的SD1胞外域。US7465451(2008)还公开了IFNAR的单克隆抗体用于制备治疗或预防与IFN-I过量产生相关的疾病，治疗移植物排斥，自身免疫病，如系统性红斑狼疮、治疗I型糖尿病、牛皮癣、风湿性关节炎、多发性硬化、获得性免疫缺陷综合征(AIDS)以及严重的免疫缺陷性疾病；治疗急性病毒病如上呼吸道感染，慢性病毒病如麻疹病毒感染疾病的药物中的用途。美国专利号US6713609(2004)公开了抑制多种I型干扰素抗病毒活性的抗IFNAR1单克隆抗体2E1和4A7，其中单克隆抗体2E1结合人IFNAR1的SD1结构域，4A7结合人IFNAR1的SD1和SD2结构域。美国专利号US7662381(2010)和US8460668(2013)公开了结合IFNAR1 并且能够抑制I型干扰素活性的单克隆抗体9D4，该抗体结合人IFNAR1的SD3结构域。
发明公开
本发明的第一个目的是提供一种IFNΑR1的单克隆抗体。
本发明提供的IFNΑR1的单克隆抗体结合人IFNΑR1的SD2结构域全部或部分和/或SD3结构域全部或部分，可抑制人I型干扰素的生物活性。
上述单克隆抗体中，所述单克隆抗体或其抗原结合部分表现如下A)-E)中一种或多种特性：
A)结合人IFNΑR1；
B)结合人IFNΑR1的SD2结构域全部或部分和/或SD3结构域全部或部分；
C)抑制I型干扰素的生物活性；
D)抑制I型干扰素信号报告细胞上I型干扰素的生物活性；
E)抑制在经过刺激剂刺激的人外周血单个核细胞上I型干扰素的生物活性。
上述单克隆抗体中，
所述单克隆抗体或其抗原结合部分的重链可变区的编码基因为如下1)或2)：
1)小鼠IGHV9-3，IGHD2-13和IGHJ1基因的产物或来源于该基因；
2)小鼠IGHV9-3，IGHD1-1和IGHJ3基因的产物或来源于该基因；
所述单克隆抗体或其抗原结合部分的轻链可变区的编码基因为如下3)或4)：
3)小鼠IGKV6-13和IGKJ5基因的产物或来源于该基因；
4)小鼠IGKV6-15和IGKJ2基因的产物或来源于该基因。
上述单克隆抗体中，
所述单克隆抗体或其抗原结合部分的重链可变区为如下5)或6)：
5)依次包括小鼠IGHV9-3基因全长或部分编码的蛋白或多肽、小鼠IGHD2-13基因全长或部分编码的蛋白或多肽和小鼠IGHJ1基因全长或部分编码的蛋白或多肽；
6)依次包括小鼠IGHV9-3基因全长或部分编码的蛋白或多肽、小鼠IGHD1-1基因全长或部分编码的蛋白或多肽和小鼠IGHJ3基因全长或部分编码的蛋白或多肽；
所述单克隆抗体或其抗原结合部分的轻链可变区为如下7)或8)：
7)依次包括小鼠IGKV6-13基因全长或部分编码的蛋白或多肽和小鼠IGKJ5基因全长或部分编码的蛋白或多肽；
8)依次包括小鼠IGKV6-15基因全长或部分编码的蛋白或多肽和小鼠IGKJ2基因全长或部分编码的蛋白或多肽。
上述单克隆抗体中，
所述IFNΑR1的单克隆抗体或其抗原结合部分的重链可变区依次包括所述小鼠IGHV9-3基因编码的蛋白、所述小鼠IGHD2-13基因编码的蛋白和所述小鼠IGHJ1 基因编码的蛋白；且其轻链可变区依次包括所述小鼠IGKV6-13基因编码的蛋白和所述小鼠IGKJ5基因编码的蛋白；
或所述IFNΑR1的单克隆抗体或其抗原结合部分的重链可变区依次包括所述小鼠IGHV9-3基因编码的蛋白、所述小鼠IGHD1-1基因编码的蛋白和所述小鼠IGHJ3基因编码的蛋白，且其轻链可变区依次包括所述小鼠IGKV6-15基因编码的蛋白和所述小鼠IGKJ2基因编码的蛋白。
上述单克隆抗体中，
所述IFNΑR1的单克隆抗体或其抗原结合部分的重链可变区包括重链可变区CDR1、重链可变区CDR2和重链可变区CDR3；
所述IFNΑR1的单克隆抗体或其抗原结合部分的轻链可变区包括轻链可变区CDR1、轻链可变区CDR2和轻链可变区CDR3；
所述重链可变区CDR1的氨基酸序列为如下(1)或(2)：
(1)序列3或序列13所示的氨基酸序列；
(2)将序列3或序列13所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
所述重链可变区CDR2的氨基酸序列为如下(3)或(4)：
(3)序列4或序列14所示的氨基酸序列；
(4)将序列4或序列14所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
所述重链可变区CDR3的氨基酸序列为如下(5)或(6)：
(5)序列5或序列15所示的氨基酸序列；
(6)或将序列5或序列15所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
所述轻链可变区CDR1的氨基酸序列为如下(7)或(8)：
(7)序列8或序列18所示的氨基酸序列；
(8)将序列8或序列18所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
所述轻链可变区CDR2的氨基酸序列为如下(9)或(10)：
(9)序列9或序列19所示的氨基酸序列；
(10)将序列9或序列19所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
所述轻链可变区CDR3的氨基酸序列为如下(11)或(12)：
(11)序列10或序列20所示的氨基酸序列；
(12)将序列10或序列20所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列。
上述单克隆抗体中，
所述IFNΑR1的单克隆抗体的重链可变区的氨基酸序列为a1)或a2)：
a1)序列2或序列12所示的氨基酸序列；
a2)将序列2或序列12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
所述IFNΑR1的单克隆抗体的轻链可变区的氨基酸序列为b1)或b2)：
b1)序列7或序列17所示的氨基酸序列；
b2)将序列7或序列17所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列。
上述单克隆抗体中，
所述IFNΑR1的单克隆抗体重链可变区的核苷酸序列为c1)或c2)或c3)：
c1)序列1或序列11所示的DNA分子；
c2)与c1)限定的核苷酸序列具有75％或75％以上同一性，且编码上述IFNΑR1的单克隆抗体重链可变区的cDNA分子或基因组DNA分子；
c3)在严格条件下与c1)或c2)限定的核苷酸序列杂交，且编码上述IFNΑR1的单克隆抗体重链可变区的cDNA分子或基因组DNA分子；
所述IFNΑR1的单克隆抗体轻链可变区的核苷酸序列为d1)或d2)或d3)：
d1)序列6或序列16所示的DNA分子；
d2)与d1)限定的核苷酸序列具有75％或75％以上同一性，且编码上述IFNΑR1的单克隆抗体轻链可变区的cDNA分子或基因组DNA分子；
d3)在严格条件下与d1)或d2)限定的核苷酸序列杂交，且编码上述IFNΑR1的单克隆抗体轻链可变区的cDNA分子或基因组DNA分子。
本发明的抗体包含的重链和轻链可变区含有与上述抗体的氨基酸序列同源的氨基酸序列，并且其中该抗体保留了本发明的IFNΑR1抗体的需要的功能特性。例如，本发明提供一种分离的单克隆抗体或其抗原结合部分，其包含重链可变区和轻链可变区，其中：
(a)重链可变区包含与选自序列2和序列12所示的氨基酸序列至少80％同源的氨基酸序列；
(b)轻链可变区包含与选自序列7或序列17所示的氨基酸序列至少80％同源的氨基酸序列；
(c)该抗体特异性结合IFNΑR1，并且表现至少一种如下所述的功能特性：
L1：在I型干扰素信号报告细胞中抑制IFNα2b的活性；
L2：抑制CpGA诱导的外周血单核细胞中干扰素效应基因mx2的表达；
L3：抑制IFNα2b诱导的外周血单核细胞中干扰素效应基因mx2的表达；
L4：抑制R848诱导的外周血单核细胞中干扰素效应基因mx2的表达；
L5：抑制CpGA诱导的外周血单核细胞中干扰素效应基因isg15的表达；
L6：抑制IFNα2b诱导的外周血单核细胞中干扰素效应基因isg15的表达；
L7：抑制R848诱导的外周血单核细胞中干扰素效应基因isg15的表达。
本发明的抗体VH或VL氨基酸序列可以与上述氨基酸序列85％、90％、95％、96％、97％、98％或99％同源。两个氨基酸序列之间的百分同源性相当于两个序列之间的百分同一性。两个序列之间的百分同一性是考虑了为两个序列之间进行最佳对比所需要引入的空位的数目和每个空位的长度后，这两个序列共有的相同位点的数目的函数(即％同源性＝相同位点数/位点总数X100％)。
本发明的抗体用抗体的特定功能特征或特性来表征。例如，该抗体特异性结合IFNΑR1，优选人IFNΑR1。另外，该抗体可以与来自一种或多种非人类灵长类动物如猕猴和/或恒河猴的IFNΑR1交叉反应。
此外，本发明的抗体能够抑制I型干扰素的生物活性。这些抗体抑制至少一种I型干扰素的生物活性，优选抑制多种I型干扰素(即，至少两种，更优选至少三种，或者至少四种，或者至少五种，或者至少六种，或者至少七种，或者至少八种，或者至少九种，或者至少十种，或者至少11种，或者至少12种，或者至少13种，或者至少14种，或者至少15种不同亚型的I型干扰素)的生物活性。在一个优选实施方案中，该抗体抑制以下I型干扰素的生物活性：α1、α2a、α2b、α4、α5、α6、α7、α8、α10、α14、α16、α17、α21、β和ω。在其他优选实施方案中，该抗体在人PBMC上抑制I型干扰素的活性。
抗体抑制I型干扰素的生物活性的能力可以通过一种或多种本领域建立的试验来检测。包括在I型干扰素信号报告细胞上抑制IFN-I的活性，抑制经刺激剂，如CpG，IFNα2b或R848刺激的外周血单核细胞(PBMCs)中干扰素效应基因，如mx2和isg15的表达。如果与非特异性对照抗体相比，一种抗体将活性抑制了至少20％，更优选至少30％，甚至更优选至少40％，至少50％，至少60％，至少70％，至少80％，或至少90％，则该抗体“抑制I型干扰素的生物活性”。
在优选实施方案中，该抗体在I型干扰素信号报告细胞系HEK293T中抑制IFNα2b的活性，抑制CpGA，IFNα2b或R848诱导的PBMC中干扰素效应基因，如mx2和isg15的表达。
一方面，本发明优选的抗体是如实施例所述分离并进行结构表征的单克隆抗体10C2和10C9。10C2和10C9的VH氨基酸序列分别显示在序列2和序列12中；10C2和10C9的VL氨基酸序列分别显示在序列7和序列17中。
假定这些抗体都能够结合IFNΑR1，则VH和VL序列可以“混合并匹配”，以产生本发明的其他的抗IFNΑR1结合分子。这些“混合并匹配”抗体的IFNΑR1结合可以用本发明实施例中所述的稳定表达IFNAR1的HEK293报告细胞系来检测。优选地，当VH和VL链混合并匹配时，来自特定VH/VL配对的VH序列被替代为结构上相似的VH序列。同样，优选地来自特定VH/VL配对的VL序列被替代为结构上相似的VL序列。例如，10C2和10C9的VH和VL序列特别适合混合并匹配，因为这些抗体使用源自相同种系序列(IGHV9-3和IGKV6-13)的VH和VL序列，因此显示结构相似性。
另一方面，本发明提供包含10C2和10C9的重链和轻链CDR1、CDR2和CDR3或其组合的抗体。10C2和10C9的VH CDRl的氨基酸序列分别为序列3和序列13。10C2和10C9的VH CDR2的氨基酸序列分别为序列4和序列14。10C2和10C9的VH CDR3的氨基酸序列分别为序列5和序列15。CDR区用VECTOR NTI标出。
假如这些抗体中的每一个都能够结合IFNΑR1并且抗原结合特异性主要由CDR1、CDR2、CDR3区提供，则VH CDR1、CDR2、CDR3序列与VL CDR1、CDR2、CDR3序列可以“混合并匹配”(即来自不同抗体的CDR可以混合并匹配，但是每个抗体必须含有VH CDRl、CDR2、CDR3和VL CDR1、CDR2、CDR3)，从而产生本发明的其他的抗IFNΑR1结合分子。这些“混合并匹配的”抗体的IFNΑR1结合可以用实施例中所述的稳定表达IFNAR1的报告细胞HEK293T检测。优选地，当VH CDR序列混合并匹配时，来自特定VH序列的CDRl、CDR2和/或CDR3序列被替代为结构上相似的CDR序列。同样，当VL CDR序列混合并匹配时，来自特定VL序列的CDR1、CDR2和/或CDR3序列优选地被替代为结构上相似的CDR序列。例如，10C2和10C9的VH CDR1具有一定的结构相似性，因此适合混合和匹配。将一个或多个VH和/或VL CDR区序列替代为来自单克隆抗体10C2和10C9此处公开的CDR序列的结构上相似的序列，可以产生新的VH和/或VL序列。
在某些实施方案中，本发明的抗体包含来自特定种系重链免疫球蛋白基因的重链可变区和/或来自特定种系轻链免疫球蛋白基因的轻链可变区。
例如，在一个优选实施方案中，本发明提供一种分离的抗IFNΑR1单克隆抗体或其抗原结合部分，其中该抗体：
(a)包含小鼠IGHV9-3，IGHD2-13和IGHJ1基因；或IGHV9-3，IGHD1-1和IGHJ3基因的重链可变区；
(b)包含小鼠IGKV6-13和IGKJ5基因；或IGKV6-15和IGKJ2基因的轻链可变区；
(c)特异性结合人IFNAR1。
具有IGHV9-3，IGHD2-13和IGHJ1基因的重链可变区和IGKV6-13和IGKJ5基因的轻链可变区的例子是10C2。具有IGHV9-3，IGHD1-1和IGHJ3基因的重链可变区和IGKV6-15和IGKJ2基因的轻链可变区的例子是10C9。
如果一种小鼠抗体的可变区是从使用小鼠种系免疫球蛋白基因的系统中获得的，则该抗体包含特定种系序列的或“源自”它的或是它的“产物”的重链或轻链可变区。小鼠种系免疫球蛋白序列的或“源自”它的或是它的“产物”的小鼠抗体可以如下鉴定：将该小鼠抗体的氨基酸序列与小鼠种系免疫球蛋白的氨基酸序列进行比较，并选择在序列上最接近于该小鼠抗体序列(即有最高％同一性)的小鼠种系免疫球蛋白序列。特定小鼠种系免疫球蛋白序列的或“源自”它的或是它的“产物”的小鼠抗体与该种系序列相比可能包含氨基酸差异，例如是由于天然存 在的体细胞突变或有意引入定点突变。但是，选择的小鼠抗体在氨基酸序列上与小鼠种系免疫球蛋白基因编码的氨基酸序列一般至少90％相同，并且含有当与其他物种的种系免疫球蛋白氨基酸序列(例如鼠种系序列)相比时决定该小鼠抗体为小鼠抗体的氨基酸残基。
在某些情况下，小鼠抗体在氨基酸序列上与该种系免疫球蛋白基因编码的氨基酸序列可以至少95％、或者甚至至少96％、97％、98％或99％相同。一般来说，源自特定小鼠种系序列的小鼠抗体将显示与该小鼠种系免疫球蛋白基因编码的氨基酸序列相差不超过10个氨基酸。在某些情况下，该小鼠抗体可显示与该种系免疫球蛋白基因编码的氨基酸序列相差不超过5个、或者甚至不超过4、3、2或1个氨基酸。
本发明的第二个目的是提供一株分泌上述IFNΑR1的单克隆抗体的杂交瘤细胞株。
本发明提供的分泌上述IFNΑR1的单克隆抗体的杂交瘤细胞株的保藏号为CGMCC No.12542或CGMCC No.12543。
将本发明提供的分泌上述IFNΑR1的单克隆抗体的杂交瘤细胞株分别命名为杂交瘤细胞株10C2和杂交瘤细胞株10C9。
上述杂交瘤细胞株10C2的分类命名为小鼠杂交瘤细胞系，该杂交瘤细胞株已于2016年5月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏号为CGMCC No.12542。
上述杂交瘤细胞株10C9的分类命名为小鼠杂交瘤细胞系，该杂交瘤细胞株已于2016年5月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏号为CGMCC No.12543。
本发明的第三个目的是提供上述IFNΑR1的单克隆抗体或其抗原结合部分或上述杂交瘤细胞株的新用途。
本发明提供了上述IFNΑR1的单克隆抗体或其抗原结合部分或杂交瘤细胞株在如下A)-G)中至少一种中的应用：
A)结合人IFNΑR1；
B)抑制I型干扰素的生物活性；
C)抑制IFNα2b的活性；
D)结合人IFNΑR1的SD2结构域和/或SD3结构域；
E)抑制I型干扰素信号报告细胞上I型干扰素的生物活性；
F)抑制在经过刺激剂刺激的人外周血单个核细胞上I型干扰素的生物活性；
G)治疗和/或预防I型干扰素介导和/或I型干扰素异常引起的疾病。
上述应用中，所述抑制在经过刺激剂刺激的人外周血单个核细胞上I型干扰素的生物活性体现在如下(1)-(6)：
(1)抑制CpGA诱导的外周血单核细胞中干扰素效应基因mx2的表达水平；
(2)抑制IFNα2b诱导的外周血单核细胞中干扰素效应基因mx2的表达水平；
(3)抑制R848诱导的外周血单核细胞中干扰素效应基因mx2的表达水平；
(4)抑制CpGA诱导的外周血单核细胞中干扰素效应基因isg15的表达水平；
(5)抑制IFNα2b诱导的外周血单核细胞中干扰素效应基因isg15的表达水平；
(6)抑制R848诱导的外周血单核细胞中干扰素效应基因isg15的表达水平。
上述应用中，所述I型干扰素为干扰素α。
本发明的第四个目的是提供一种I型干扰素的拮抗剂。
本发明提供的I型干扰素的拮抗剂的活性成分为上述IFNΑR1的单克隆抗体或其抗原结合部分。
本发明的第五个目的是提供一种组合物。
本发明提供的组合物含有上述IFNΑR1的单克隆抗体或其抗原结合部分。
本发明提供的组合物，例如药物组合物，其含有与药学上可接受的载体配制在一起的一种或组合的本发明的单克隆抗体或者其抗原结合部分。这样的组合物可以包含一种或组合的(例如两种或多种不同的)本发明的抗体或免疫偶联物或双特异性分子。例如，本发明的药物组合物可以含有可与靶抗原上不同表位结合或具有互补活性的组合的抗体(或免疫偶联物或双特异性剂)。
本发明的药物组合物也可在联合治疗中施用，即与其他药剂联用。例如，联合治疗可包括本发明的抗IFNΑR1抗体与至少一种其他的免疫抑制剂组合。文中所用的，“药学上可接受的载体”包括生理学相容的任何和所有溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗和吸收延迟剂等。优选地，该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即抗体、免疫偶联物或双特异性分子包被于一种材料中，以保护该化合物免于可使该化合物失活的酸和其他天然条件的作用。
本发明的药物组合物可包含一种或多种药学上可接受的盐。"药学上可接受的盐”是指保持了亲代化合物的所需生物活性且不引起任何不想要的毒理学作用的盐(参见如Berge,S.M.等(1977)J.Pharm.Sci.66:1-19)。这样的盐的例子包括酸加成盐和碱加成盐。酸加成盐包括那些由无毒性无机酸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等衍生的盐，以及由无毒性有机酸如脂族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳族酸、脂族和芳族磺酸等衍生的盐。碱加成盐包括那些由碱土金属如钠、钾、镁、钙等衍生的盐，以及由无毒性有机胺如N,N’-二苄基乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等衍生的盐。
本发明的药物组合物也可含有药学上可接受的抗氧化剂。药学上可接受的抗氧化剂的例子包括：(1)水溶性抗氧化剂，如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠，亚硫酸钠等；(2)油溶性抗氧化剂，如棕榈酸抗坏血酸酯、丁羟茴醚(BHA)、丁羟甲苯(DHT)、卵磷脂、没食子酸丙酯、α-生育酚等； (3)金属螯合剂，如柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等。
可用于本发明的药物组合物中的适当的水性或非水性载体的例子包括水，乙醇，多元醇(如甘油、丙二醇、聚乙二醇等)，及其适当的混合物，植物油如橄榄油，和可注射的有机酯如油酸乙酯。例如通过应用包衣材料如卵磷脂，在分散液的情况下通过维持所需的颗粒大小，以及通过应用表面活性剂，可维持适当的流动性。
这些组合物也可含有佐剂，如防腐剂、润湿剂、乳化剂和分散剂。可以通过上述的灭菌程序或通过包含各种抗细菌和抗真菌剂如对羟基苯甲酸酯、氯代丁醇、苯酚山梨酸等来确保防止存在微生物。也可能需要在组合物中包含等渗剂，例如，糖和氯化钠等。另外，通过包含延迟吸收剂，例如单硬脂酸铝和明胶，可实现注射型药物延长的吸收。
药学上可接受的载体包括无菌水溶液或分散液和用于临时制备无菌注射液或分散液的粉末剂。这些介质和试剂用于药学活性物质的应用是本领域公知的。还可以向组合物中掺入补充的活性化合物。
治疗组合物一般必须是无菌的并且在制备和贮存条件下稳定。可以将组合物配制成溶液、微乳状液、脂质体或其他适合高药物浓度的有序结构。载体可以是含有例如水、乙醇、多元醇(例如，丙三醇、聚乙二醇和液态聚乙二醇等)和它们的合适的混合物的溶剂或分散剂。例如，通过使用包衣，例如卵磷脂，在分散剂的情况下通过保持所需的颗粒大小，以及通过使用表面活性剂，可以保持适当的流动性。在很多情况下，组合物中优选包含等渗剂，例如，糖、多元醇例如甘露糖醇、山梨糖醇或氧化钠。通过在组合物中加入延迟吸收剂，例如单硬脂酸盐和明胶，可实现注射型药物延长的吸收。
通过将活性化合物以需要的量混入合适的溶剂中，并且根据需要加入以上列举的成分的一种或其组合，接着无菌微过滤，可制备无菌注射液。通常，通过将活性化合物掺入到含有基本分散介质和来自上面所列举的其他所需成分的无菌载体中制备分散剂。对于用于制备无菌注射液的无菌粉末剂，优选的制备方法是真空干燥和冷冻干燥(冻干)，由其前述无菌过滤的溶液得到活性成分加任何额外所需成分的粉末。
可以与载体材料组合以制备单一剂量形式的活性成分的量根据所治疗的受试者和特定的给药方式而不同。可以与载体材料组合以制备单一剂量形式的活性成分的量一般是产生治疗效果的组合物的量。通常，以100％计，这个量的范围是大约0.01％至大约99％的活性成分，优选大约0.1％至大约70％，最优选大约1％至大约30％的活性成分，与药学上可接受的载体相组合。
本发明的组合物可以利用本领域公知的一种或多种方法通过一种或多种施用途径施用。本领域技术人员应当明白，施用途径和/或方式根据需要的结果而 不同。用于本发明抗体的优选施用途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊柱或其他肠胃外施用途径，例如注射或输注。
文中所用的短语“肠胃外施用”是指不同于肠和局部施用的施用模式，通常是通过注射，包括但不局限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。
此外，本发明的抗体也可以通过非肠胃外途径施用，如局部、表皮或粘膜途径施用，例如，鼻内、经口、阴道、直肠、舌下或局部。
本发明的抗体(和免疫偶联物和双特异性分子)具有体外和体内诊断和治疗应用。例如，这些分子可以施用于例如在体外或离体培养的细胞，或者例如在体内施用于人类受试者，以治疗、预防或诊断多种疾病。
文中使用的术语“受试者”包括人和非人动物。非人动物包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物、绵羊、狗、猫、牛、马、鸡、两栖类动物和爬行类动物。这些方法特别适合治疗患有与异常或不当I型干扰素表达(例如过量表达)相关的疾病的人类患者。
当抗IFNΑR1抗体与另一种药物一起给药时，这两种药物可以依次或同时给药。例如，本发明的抗IFNΑR1抗体可以与一种或多种以下药物组合使用：抗IFNα抗体、抗IFN-γ受体抗体、可溶性IFN-γ受体、抗TNF抗体、抗TNF受体抗体和/或可溶性TNF受体。此外，本发明的抗IFNΑR1抗体可以与Flt3配体拮抗剂组合使用。
本发明的第六个目的是提供一种免疫偶联物。
本发明提供的免疫偶联物含有上述IFNΑR1的单克隆抗体或其抗原结合部分。
上述免疫偶联物中，所述免疫偶联物含有药学上可接受的载体。
本发明提供的抗IFNΑR1抗体或其片段可以与治疗性剂如细胞毒素、药物(例如免疫抑制剂)或放射性毒素偶联，得到偶联物。这些偶联物称为“免疫偶联物”。包括一个或多个细胞毒素的免疫偶联物称作“免疫毒素”。细胞毒素或细胞毒性剂包括对细胞有害(例如杀伤)的任何试剂。实例包括紫杉醇、细胞松弛素B、短杆菌肽D、溴化乙啶、吐根碱、丝裂霉素、表鬼臼毒吡喃葡糖苷、表鬼臼毒噻吩糖苷、长春新碱、长春碱、秋水仙素、阿霉素、柔红霉素、二羟基炭疽菌素二酮、米托蒽醌、光辉霉素、放线菌素D、l-脱氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普萘洛尔和嘌呤霉素和它们的类似物或同系物。治疗剂还包括，例如，抗代谢物(例如，氨甲喋呤、6-巯基嘌呤、6-硫代鸟嘌呤、阿糖胞苷、5-氟尿嘧唳、氨烯咪胺(decarbazine)，烧化剂(例如，氮芥、thioepa苯丁酸氮芥、苯丙氨酸氮芥、卡莫司汀(BSNU)和洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露糖醇、链唑霉素、丝裂霉素C和顺-二氯二胺铂(II)(DDP)顺铂)，氨茴霉素类(例如，柔红菌素(以前称为道诺霉素)和阿霉素)，抗生素(例如，放线菌素D、博来霉素、光辉霉素和安曲霉素(AMC))， 和抗有丝分裂剂(例如，长春新碱和长春碱)。能与本发明抗体偶联的治疗性细胞毒素的其他优选例子包括倍癌霉素、刺孢霉素、美坦生、auristatin，和它们的衍生物。
可以利用本领域可用的接头技术将细胞毒素与本发明的抗体偶联。已经用于将细胞毒素与抗体偶联的接头类型的实例包括但不限于腙、硫醚、酯、二硫化物和含肽的接头。可以选择，例如，在溶酶体室内易被低pH切割或易被蛋白酶切割的接头，该蛋白酶例如是在肿瘤组织中优先表达的蛋白酶，如组织蛋白酶(例如组织蛋白酶B、C、D)。
关于细胞毒素的类型、用于偶联治疗剂与抗体的接头和方法的进一步讨论，参见Saito,G.等(2003)Adv.Drug Deliv.Rev.55:199-215；Trail,P.A.等(2003)Cancer.Immunol.Immunother.52:328-337；Payne,G.(2003)Cancer Cell 3:207-212；Allen,T.M.(2002)Nat.Rev.Cancer 2:750-763；Pastan,I.和Kreitman,R.J.(2002)Curr.Opin.Investig.Drugs 3:1089-1091；Senter,P.D.和Springer,C.J.(2001)Adv.Drug Deliv.Rev.53:247-264。
本发明的抗体也可与放射性同位素偶联，产生细胞毒性放射性药物，也被称为放射性免疫偶联物。能够与诊断或治疗性使用的抗体偶联的放射性同位素的例子包括但不限于碘131、铟111、钇90和镥177。制备放射性免疫偶联物的方法在本领域中已经建立。放射性免疫偶联物的例子可以作为商品获得，包括ZevalinTM(IDEC Pharmaceuticals)和BexxarTM(Corixa Pharmaceuticals)，能够利用类似的方法使用本发明的抗体制备放射性免疫偶联物。
本发明的抗体偶联物可用于修饰特定的生物学反应，且药物部分不应理解为局限于经典的化学治疗剂。例如，药物部分可以是具有需要的生物活性的蛋白质或多肽。这样的蛋白质可包括，例如，具有酶活性的毒素或其活性片段，如相思豆毒蛋白、蓖麻毒蛋白A、假单胞菌外毒素或白喉毒素；蛋白质，如肿瘤坏死因子或干扰素-γ；或生物学反应调节物，如淋巴因子、白细胞介素-1(IL-1)、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)、粒细胞巨噬细胞集落刺激因子(GM-CSF)、粒细胞集落刺激因子(G-CSF)或其他生长因子。
用于将这种治疗剂与抗体偶联的技术是众所周知的，参见，例如，Arnon等，“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy(在癌症治疗中用于药物的免疫祀向的单克隆抗体)”，Monoclonal Antibodies And Cancer Therapy,Reisfeldt等(编)，pp.243-56(Alan R.Liss,Inc.1985)；Hellstrom等，“Antibodies For Drug Delivery(用于给药的抗体)”，Controlled Drug Delivery(第二版)，Robinson等(编)，pp.623-53(Marcel Dekker,Inc.1987)；Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review(癌症治疗中细胞毒剂的抗体载体：综述)”，Monoclonal Antibodies’84:Biological And Clinical Applications,Pinchera等(编)，pp.475-506(1985)“Analysis,Results,And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer  Therapy(放射性标记抗体在癌症治疗中的治疗用途的分析、结果和未来前景)”，Monoclonal Antibodies For Cancer Detection And Therapy,Baldwin等(编)，pp.303-16(Academic Press 1985),和Thorpe等,“The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates(抗体-毒素偶联物的制备和细胞毒性)”,Immunol.Rev.,62:119-58(1982)。
本发明的第七个目的是提供一种抗体基因工程改造的抗体。
本发明提供的抗体基因工程改造的抗体是将上述IFNΑR1的单克隆抗体或其抗原结合部分进行修饰和改造后得到的抗体。
在某些实施方案中，本发明的抗体包含CDR1、CDR2和CDR3序列的重链可变区和含CDR1、CDR2和CDR3序列的轻链可变区，其中这些CDR序列中的一个或多个包含基于本发明的抗体(例如10C2和10C9)的特定氨基酸序列或其保守修饰，并且其中该抗体保留本发明抗IFNΑR1抗体的需要的功能特性。因此，本发明提供一种分离的单克隆抗体或其抗原结合部分，其包含CDR1、CDR2和CDR3序列的重链可变区和含CDR1、CDR2和CDR3序列的轻链可变区，其中：重链可变区CDR3序列包含选自序列5和序列15所示的氨基酸序列或其保守修饰的氨基酸序列，且轻链可变区CDR3序列包含选自序列10和序列20所示的氨基酸序列或其保守修饰的氨基酸序列；且该抗体特异性结合IFNΑR1，并且表现至少一种上述功能特性。
在另一个实施方案中，重链可变区CDR2序列包含选自序列4和序列14所示的氨基酸序列或其保守修饰的氨基酸序列；且轻链可变区CDR2序列包含选自序列9和序列19所示的氨基酸序列或其保守修饰的氨基酸序列。在又另一个实施方案中，重链可变区CDRl序列包含选自序列3和序列13所示的氨基酸序列或其保守修饰的氨基酸序列；且轻链可变区CDRl序列包含选自序列8和序列18所示的氨基酸序列或其保守修饰的氨基酸序列。
文中所用的术语“保守序列修饰”是指不会显著影响或改变含该氨基酸序列的抗体的结合特征的氨基酸修饰。这样的保守修饰包括氨基酸替代、添加和缺失。可以通过本领域公知的技术，如定点诱变和PCR介导的诱变，向本发明的抗体中引入修饰。保守氨基酸替代是指将氨基酸残基替代为一个具有相似侧链的氨基酸残基。具有相似侧链的氨基酸残基家族在本领域中已经定义。这些家族包括：具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，本发明抗体的CDR区内的一个或多个氨基酸残基可以被替代为来自相同侧链家族的其他氨基酸残基，并且可以用本文所述的功能试验检测改变的抗体保留的上述功能。
本发明的抗体还可以如下制备：用具有此处所公开的一种或多种VH和/或VL序列的抗体作为起始材料，工程化一种修饰的抗体，该修饰的抗体可能具有与起始抗体相比改变的特性。可以通过修饰一个或两个可变区(即VH和/或VL)例如一个或多个CDR区内和/或一个或多个骨架区内的一个或多个残基来工程化一种抗体。另外，可以通过修饰恒定区内的残基来工程化抗体，例如改变该抗体的效应功能。
可以进行的一种类型的可变区工程化是CDR移植。抗体主要是通过位于六个重链和轻链互补决定区(CDR)中的氨基酸残基与靶抗原相互作用。由于这个原因，CDR内的氨基酸序列比CDR外的序列在各个抗体之间更加多样化。由于CDR序列负责大多数抗体-抗原相互作用，因此通过构建如下的表达载体可以表达模拟该特定天然存在抗体的特性的重组抗体：该表达载体包含来自特定天然存在抗体的CDR序列，该CDR序列被移植到来自具有不同特性的不同抗体的骨架序列上(参见，例如，Riechmann,L.等(1998)Nature 332:323-327Jones,P.等(1986)Nature 321:522-525；Queen,C.等(1989)Proc.Natl.Acad.Sci.U.S.A.86:10029-10033)。
因此，本发明的另一个实施方案涉及一种分离的单克隆抗体或其抗原结合部分，其包含：含CDR1、CDR2和CDR3序列的重链可变区，该CDR1、CDR2和CDR3序列分别包含选自序列3和序列13、序列4和序列14；以及序列5和序列15所示的氨基酸序列，以及含CDR1、CDR2和CDR3序列的轻链可变区，该CDR1、CDR2和CDR3序列分别包含选自序列8和序列18、序列9和序列19，以及序列10和序列20所示的氨基酸序列。因此，这些抗体含有单克隆抗体10C2和10C9的VH和VL CDR序列，但是可能含有与这些抗体不同的骨架序列。
这些骨架序列可以从包括种系抗体基因序列的公共DNA数据库或发表的参考文献中获得。例如，人重链和轻链可变区基因的种系DNA序列可以在以下资源中获得：“VBase”人种系序列数据库(可从因特网上www.mrc-cpe.cam.ac.uk/vbase获得)，以及Kabat,E.A.等(1991)Sequences of Proteins of Immunological Interest,第五版，U.S.Department of Health and Human Services,NIH出版号91-3242；Tomlinson,I.M.等(1992)“The Repertoire of Human Germline VH Sequences Reveals about Fifty Groups of VH Segments with Different Hypervariable Loops(人种系Vh序列的全部组成成分揭示了具有不同高变环的大约50个VH区段组)”J.Mol.Biol.227:776-798；Cox,J.P.L.等(1994)“A Directory of Human Germ-line Vh Segments Reveals a Strong Bias in their Usage(人种系Vh区段目录揭示其应用的强烈偏好)”Eur.J.Immunol.24:827-836。
另一种类型的可变区修饰是将VH和VL CDR1、CDR2和/或CDR3区内的氨基酸序列突变，从而改善目标抗体的一种或多种结合特性(例如亲和力)。可以进行定点诱变或PCR介导的诱变，以引入突变，对抗体结合的影响，或者 其他目标功能特性，可以用文中所述以及在实施例中提供的试验来评价。优选引入(如上文所述的)保守序列修饰。突变可以是氨基酸替代、添加或缺失，但是优选替代。而且，一般在CDR区内改变不超过5个残基。
因此，在另一个实施方案中，本发明提供分离的抗IFNΑR1单克隆抗体或其抗原结合部分，其包含的重链可变区含有：
(a)VH CDR1区，其包含选自序列3和序列13所示的氨基酸序列，或与选自序列3和序列13所示的氨基酸序列相比具有1、2、3、4或5个氨基酸替代、缺失或添加的氨基酸序列；
(b)VH CDR2区，其包含选自序列4和序列14所示的氨基酸序列，或与选自序列4和序列14所示的氨基酸序列相比具有1、2、3、4或5个氨基酸替代、缺失或添加的氨基酸序列；
(c)VH CDR3区，其包含选自序列5和序列15所示的的氨基酸序列，或与选自序列5和序列15所示的氨基酸序列相比具有1、2、3、4或5个氨基酸替代、缺失或添加的氨基酸序列；
(d)VL CDR1区，其包含选自序列8和序列18的氨基酸序列，或与选自序列8和序列18所示的氨基酸序列相比具有1、2、3、4或5个氨基酸替代、缺失或添加的氨基酸序列；
(e)VL CDR2区，其包含选自序列9和序列19所示的的氨基酸序列，或与选自序列9和序列19所示的的氨基酸序列相比具有1、2、3、4或5个氨基酸替代、缺失或添加的氨基酸序列；
(f)VL CDR3区，其包含选自序列10和序列20所示的氨基酸序列，或与选自序列10和序列20所示的氨基酸序列相比具有1、2、3、4或5个氨基酸替代、缺失或添加的氨基酸序列。
本发明的工程化抗体包括例如为了改善抗体性质而对其VH和/或VL内的骨架残基进行了修饰的抗体。进行这样的骨架修饰一般是为了降低抗体的免疫原性。例如，一种方法是将一个或多个骨架残基“回复突变”为相应的种系序列。更特别地，经历体细胞突变的抗体可含有与衍生该抗体的种系序列不同的骨架残基。通过比较抗体骨架序列与衍生该抗体的种系序列，可以鉴定这些残基。
另一种类型的骨架修饰涉及对骨架区内、或者甚至一个或多个CDR区内的一个或多个残基进行突变，以除去T细胞表位，从而降低该抗体的潜在的免疫原性。
除了在骨架区或CDR区内进行修饰以外，或者作为它的替代，本发明的抗体可以被工程化为包括Fc区内的修饰，一般是为了改变该抗体的一种或多种功能特性，如血清半衰期、补体固定、Fc受体结合和/或抗原依赖性细胞毒性。此外，也可以化学修饰本发明的抗体(例如一个或多个化学部分可以连接到该抗体上)，或者修饰改变其糖基化，以改变该抗体的一种或多种功能特性。
利用本发明的抗IFNΑR1抗体，如10C2和10C9的结构特征产生结构上相关的抗IFNΑR1抗体，该结构相关的抗体保留本发明抗体的至少一种功能特性，如结合IFNΑR1。例如，10C2或10C9的一个或多个CDR区，可以与已知的骨架区和/或其他CDR重组组合，产生另外的重组工程化的本发明的抗IFNΑR1抗体。其他类型的修饰包括以上部分所述的修饰。用于工程化方法的起始材料是此处提供的一种或多种VH和/或VL序列，或其一个或多个CDR区。为了产生工程化抗体，不一定实际制备(即表达为蛋白质)具有此处提供的一种或多种VH和/或VL序列或其一个或多个CDR区的抗体。而是用该序列中所含的信息作为起始材料，产生源自原始序列的“第二代”序列，然后制备该“第二代”序列，并将其表达为蛋白质。
上述制备工程化IFNΑR1抗体的方法，包括如下步骤：
(a)提供如下序列：(i)重链可变区抗体序列，其包含选自序列3和序列13的CDR1序列、序列4和序列14的CDR2序列，和选自序列5和序列15的CDR3序列；和(ii)轻链可变区抗体序列，其包含选自序列8和序列18的CDR1序列、序列9和序列19的CDR2序列，和选自序列10和序列20的CDR3序列；
(b)改变第一抗体序列和/或第二抗体序列内的至少一个氨基酸残基，从而产生至少一个改变的抗体序列；和
(c)制备该改变的抗体序列；和
(d)将该改变的抗体序列表达为蛋白质。
可以利用常规分子生物学技术制备和表达改变的抗体序列。优选地，由改变的抗体序列编码的抗体保留如下所述的抗IFNΑR1抗体的一种、一些或全部功能特性：结合IFNΑR1；抑制I型干扰素与IFNΑR1的结合；结合表达人IFNΑR1的细胞；在表达IFNAR1细胞上抑制I型干扰素的生物活性；在经过刺激剂刺激的PBMC上抑制I干扰素的生物活性。
改变的抗体的功能特性可以用本领域中可使用的和/或本发明所述的试验来评价。
在工程化本发明抗体的方法的某些实施方案中，可以沿全部或部分抗IFNΑR1抗体编码序列(例如10C2或10C9编码序列)随机或选择性地引入突变，并可以对结合活性和/或如本发明所述的其他功能特性筛选获得修饰的抗IFNΑR1抗体。突变方法在本领域中已经描述。例如，Short的PCT公布W002/092780记载了利用饱和诱变、合成连接装配或其组合产生和筛选抗体突变的方法。
本发明的第八个目的是提供一种人源化抗体。
本发明提供的人源化抗体是将上述IFNΑR1的单克隆抗体或其抗原结合部分进行人源化后得到的抗体。
可用本领域公知的重组DNA技术将本发明的单克隆抗体人源化，或制备成嵌合抗体。为了产生嵌合抗体，可以利用本领域公知的方法将鼠可变区连接到 人恒定区上。制备嵌合抗体的方法，例如，参见Morrison等,Proc.Natl.Acad.ScL U.S.A.81:6851,1985；Takeda等,Nature 314:452,1985,Cabilly等,U.S.Patent No.4,816,567；Boss等U.S.Patent No.4,816,397；Tanaguchi等,European Patent Publication EP171496；European Patent Publication 0173494,United Kingdom Patent GB 2177096B，在此全部引入作为参考。抗体的人源化涉及将非人抗体结合位点移植到人抗体上。可将非人类抗体的CDR移植到人抗体的骨架区和人抗体的恒定区，或者通过将某些暴露的氨基酸残基替代成人抗体表面而将整个非人类抗体的可变区隐藏。制备人源化抗体的细节在U.S.Pat.No.5,472,693中描述。
也可用表达人重链和轻链基因，但是能表达内源性小鼠免疫球蛋白的重链和轻链基因的转基因小鼠制备人源化抗体。Winter描述了可用于制备人源化抗体的CDR-移植方法(U.S.Patent No.5,225,539)。可以用至少部分非人CDR替代特定人抗体的所有CDRs，或者仅用非人CDRs替代一些人CDRs。仅需要将人源化抗体与特定抗原需要的CDRs替代。
可以用来源于人Fv可变区的等同序列替代没有直接涉及抗原结合的Fv区的序列制备人源化抗体或其片段。Morrison(1985)Science 229:1202-1207；Oi等(1986)BioTechniques 4:214；US 5,585,089；US 5,693,761；US 5,693,762；US 5,859,205；和US 6,407,213提供了产生人源化抗体或其片段的实例。这些方法包括从编码至少一个重链或轻链的所有或部分免疫球蛋白Fv可变区分离、操作和表达核酸序列。这些核酸可从产生针对如上所述特定靶的杂交瘤或其他来源获得。可以将编码人源化抗体分子的重组DNA克隆至合适的表达载体中。
在一些实施方案中，可以引入保守替代、共有序列替代、胚系替代和/或回复突变将人源化抗体进行优化。这些改变的免疫球蛋白分子可以通过本领域公知的技术获得(例如，Teng等，Proc.Natl.Acad.Sci U.S.A.,80:7308-7312,1983；Kozbor等，Immunology Today,4:7279,1983；Olsson等，Meth.Enzymol,92:3-16,1982)。一般地，进行保守替代，可以使用人胚系抗体序列中常用的氨基酸。Tomlinson等(1992)J.MoI Biol.227:776-798；Cook,G.P等(1995)Immunol.Today Vol.16(5):237-242；Chothia,D等(1992)J.MoI Biol.227:799-817；Tomlinson等(1995)EMBO J.14:4628-4638中公开了人胚系序列中常用的氨基酸。这些序列可以作为人序列如骨架区和CDR的来源。
可以使用本领域公知的方法，如噬菌体展示技术产生人抗体。噬菌体展示技术通过克隆抗体基因文库，并选择结合需要的靶，如IFNAR1的SD2，SD3中的表位模拟哺乳动物免疫系统。噬菌体展示技术中使用的文库可以从多种来源制备。例如，通过接种或疾病而暴露于目的抗原产生的免疫文库，即使相对较小，也具有针对抗原的高水平的循环抗体。从非免疫的个体中分离的mRNA制备的naive文库，可被重复用于分离针对多种抗原的抗体。另外，体外克隆和组合排列的胚系抗体基因片段组成的编码完全VH和VL链的重组基因的合成文 库，具有产生针对自身抗原的抗体的优势。通过在CDR环中筛选一个或多个抗体骨架和随机序列，可制备半合成文库。
在噬菌体展示技术中，一旦产生文库，就与噬菌体表面蛋白发生融合。通过淘筛，展示针对目的抗原特异的抗体的噬菌体被选择性的吸附到固相抗原上而被富集。随后，结合的噬菌体可从表面洗脱下并通过感染大肠杆菌细胞进行扩大培养。
产生人源化抗体的噬菌体展示技术的其他改进也是本领域已知的。例如，在微生物细胞例如大肠杆菌和酿酒酵母，而不是在噬菌体表面展示抗体。在这种情况下，可以与缓冲液中荧光标记的配体孵育进行筛选。展示结合配体的抗体的细胞被荧光标记，可通过荧光激活的细胞分选将细胞分离出来。另一种修饰，核糖体展示，依赖于在核糖体、mRNA和多肽之间形成三元复合物。
本领域已知的另一产生人抗体的方法是使用转基因小鼠。这些小鼠中的免疫球蛋白库被鼠染色体中人V基因替代。可以用需要的抗原注射这些小鼠，通过在免疫文库，或者通过常规的杂交瘤技术中克隆和筛选获得产生的抗体。这些小鼠产生大量的仅在糖基化图式中不同的完全的人抗体。
本发明的第九个目的是提供一种双特异性分子。
本发明提供的双特异性分子含有上述IFNΑR1的单克隆抗体或其抗原结合部分的特异性结合位点。
本发明提供的含有上述IFNΑR1的单克隆抗体或其抗原结合部分的特异性结合位点的双特异性分子可将本发明的抗体或其抗原结合部分衍生化或连接到另一功能性分子上，如另一种肽或蛋白质(例如另一种抗体或受体的配体)上，以生成可与至少两个不同结合位点或靶分子结合的双特异性分子。可以将本发明的抗体衍生化或连接到一种以上的其他功能性分子上，以生成可与两个以上不同结合位点和/或靶分子结合的多特异性分子；这样的多特异性分子也包括在此处所用的术语“双特异性分子”内。为了产生本发明的双特异性分子，本发明的抗体可与一种或多种其他结合分子如其他抗体、抗体片段、肽或结合模拟物功能性连接(如通过化学偶联、基因融合、非共价结合等)，从而得到双特异性分子。
本发明的第十个目的是提供与上述IFNΑR1的单克隆抗体或其抗原结合部分相关的生物材料。
本发明提供的与上述IFNΑR1的单克隆抗体或其抗原结合部分相关的生物材料为下述A1)至A12)中的任一种：
A1)编码上述IFNΑR1的单克隆抗体或其抗原结合部分的核酸分子；
A2)含有A1)所述核酸分子的表达盒；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述表达盒的重组载体；
A5)含有A1)所述核酸分子的重组微生物；
A6)含有A2)所述表达盒的重组微生物；
A7)含有A3)所述重组载体的重组微生物；
A8)含有A4)所述重组载体的重组微生物；
A9)含有A1)所述核酸分子的转基因细胞系；
A10)含有A2)所述表达盒的转基因细胞系；
A11)含有A3)所述重组载体的转基因细胞系；
A12)含有A4)所述重组载体的转基因细胞系。
上述核酸分子可以存在于完整细胞、细胞裂解物中，或以部分纯化或基本纯化的形式存在。当通过常规技术，包括碱/SDS处理、CsCl显带、柱层析、琼脂糖凝胶电泳和本领域公知的其他方法，从其他细胞成分或其他污染物例如其他细胞核酸或蛋白质中分离纯化时，该核酸是“分离的”或“基本上纯的”。参见，F.Ausubel等编著(1987)Current Protocols in Molecular Biology,Greene Publishing and Wiley Interscience,New York。本发明的核酸可以是例如DNA或RNA，并且可以含有或者可以不含内含子序列。在一个优选实施方案中，该核酸是cDNA分子。
本发明的核酸分子可以利用常规分子生物学技术获得。对于杂交瘤表达的抗体，编码通过杂交瘤制备的抗体轻链和重链的cDNA可以用PCR扩增或cDNA克隆技术获得。对于从免疫球蛋白基因文库中获得的抗体(例如使用噬菌体展示技术)，编码抗体的核酸可以从文库中获得。
本发明优选的核酸分子是编码10C2和10C9单克隆抗体的VH和/或VL序列的核酸分子。编码10C2的VH和VL序列的DNA序列分别显示在序列1和序列6中。编码10C9的VH和VL序列的DNA序列分别显示在序列11和序列16中。
一旦获得编码VH和/或VL的DNA片段，即可通过重组DNA技术进一步操作这些DNA片段，例如将可变区基因转化为全长抗体链基因，转化为Fab片段基因或scFv基因。在这些操作中，将编码VH和/或VL的DNA片段与编码另外一种蛋白质如抗体恒定区或柔性接头的另一种DNA片段可操作连接。如在本文中使用的术语“可操作连接(operatively linked)”意思是两个DNA片段连接在一起，使得这两个DNA片段编码的氨基酸序列保持在阅读框内。
通过将编码VH的DNA与编码重链恒定区(CH1、CH2和CH3)的另外一种DNA分子可操作连接，可以将编码VH区的分离的DNA转化为全长重链基因。人重链恒定区基因的序列在本领域中公知(参见，例如，Kabat,B.A.等(1991)Sequences of Proteins of Immunologicl Interest，第五版，U.S.Department of Health and Human Services,NIH出版号91-3242)，包括这些区域的DNA片段可以通过PCR扩增获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是最优选的是IgG1或IgG2a恒定区。对于Fab片段重链基因， 编码VH的DNA可以与只编码重链CH1恒定区的另外一种DNA分子可操作连接。
通过将编码VL的DNA与编码轻链恒定区CL的另外一种DNA分子可操作连接，可以将编码区的分离的DNA转化为全长轻链基因(以及Fab轻链基因)。人轻链恒定区基因的序列在本领域中公知(参见，例如，Kabat,B.A.等(1991)Sequences of Proteins of Immunologicl Interest,第五版，U.S.Department of Health and Human Services,NIH出版号91-3242)，包括这些区域的DNA片段可以通过PCR扩增获得。轻链恒定区可以是κ或λ恒定区，但是最优选的是κ恒定区。
为了产生scFv基因，将编码VH和VL的DNA片段与编码柔性接头例如编码氨基酸序列(Gly4-Ser)3的另外一种片段可操作连接，使得VH和VL序列可以表达为连续的单链蛋白质，其VH和VL区通过柔性接头连接(参见，例如Bird等(1988)Science 242:423-426；Huston等(1988)Proc.Natl.Acad.Sci.USA85:5879-5883；McCafferty等(1990)Nature 348:552-554)。
本发明的第十一个目的是提供一种制备抗IFNΑR1抗体的方法。
本发明提供的制备抗IFNΑR1抗体的方法，包括：
1)提供：
(i)重链可变区抗体序列，其含有选自序列3或序列13的CDR1序列，选自序列4或序列14的CDR2序列，和选自序列5或序列15的CDR3序列；
或(ii)轻链可变区抗体序列，其含有选自序列8或序列18的CDR1序列，选自序列9或序列19的CDR2序列，和选自序列10或序列20的CDR3序列；
2)改变至少一个可变区抗体序列内的至少一个氨基酸残基，所述序列选自所述重链可变区抗体序列和所述轻链可变区抗体序列，从而产生至少一个改变的抗体序列；
3)将所述改变的抗体序列表达为蛋白质。
本发明的单克隆抗体通过权威的单克隆抗体制备方法(例如，Paterson,H.M.V.a.Y.(Jone Wiley and Sons,Inc.New York,1995).Production of Antibodies.Current Protocols in Immunology)制备得到。制备杂交瘤优选的动物是小鼠。用小鼠产生杂交瘤是非常完善确立的程序。免疫程序和用于融合的免疫脾细胞的分离技术是本领域公知的。融合配偶体(例如鼠骨髓瘤细胞)和融合方法也是公知的。
在制备本发明的单克隆抗体的小鼠免疫步骤中，可以用纯化的或富集的IFNΑR1抗原制剂和/或表达IFNΑR1的细胞免疫小鼠。优选地，用表达IFNAR1的小鼠细胞免疫小鼠。第一次输注时小鼠为6-8周龄。例如，可以使用纯化的或富集的IFNΑR1抗原制剂(5-50μg)腹膜内免疫小鼠，或使用表达IFNΑR1的小鼠细胞腹膜内免疫小鼠，优选使用表达IFNΑR1的小鼠细胞与免疫佐剂，例如，CpG-B混合腹膜内免疫小鼠，促进免疫应答，可以多次免疫小鼠，例如3次，4 次或5次免疫小鼠，时间间隔3周或4周，最后一次免疫后3天，4天或5天后取小鼠脾细胞或淋巴结细胞与小鼠骨髓瘤细胞进行融合。
在制备产生本发明的单克隆抗体的杂交瘤细胞的步骤中，从接受免疫的小鼠中分离脾细胞和/或淋巴结细胞，并且与合适的无限增殖细胞系例如小鼠骨髓瘤细胞系融合。根据抗原特异性抗体的产生筛选得到杂交瘤。例如，使用50％聚乙二醇(PEG)，将来自被免疫小鼠的脾淋巴细胞的单细胞悬液与三分之一数目的小鼠骨髓瘤细胞SP2/0融合。细胞以大约2×105接种于平底微量滴定板，接着在含有10％胎牛血清，4mM L-谷氨酰胺，1mM丙酮酸钠，5mM HEPES，50单位/mL青霉素，50mg/mL链霉素，和1XHAT(Sigma；融合24小时后加入HAT)的选择性培养基中温育10天。大约10天之后，筛选分泌IFNAR1抗体的杂交瘤细胞。
在制备产生本发明的单克隆抗体的杂交瘤细胞筛选的步骤中，通过构建表达人IFNAR1胞外区的真核表达载体转染HEK293T细胞系得到用于筛选杂交瘤分泌的IFNAR1抗体的报告细胞系，该细胞系稳定表达GFP。如果杂交瘤细胞分泌IFNAR1抗体，抗体与细胞表达的IFNAR1胞外区结合，再加入荧光素PE标记的抗鼠IgG抗体，则该细胞可发出绿色荧光和黄色荧光。使用流式细胞仪分析细胞的GFP与PE信号，可筛选到分泌IFNAR1抗体的杂交瘤。通过构建的稳定表达IFNAR1胞外区的报告细胞系筛选各孔的单克隆IgG抗体。一旦发生广泛的杂交瘤生长，则通常在10-14天之后对杂交瘤细胞进行筛选。对分泌IFNAR1抗体的杂交瘤细胞利用有限稀释法进行亚克隆，再次筛选。如果对于人IFNAR1仍然是阳性，则通过有限稀释将单克隆抗体至少亚克隆两次。然后体外培养稳定的亚克隆，在组织培养基中产生少量抗体用于表征。
为了纯化单克隆抗体，选择的杂交瘤可以在用于单克隆抗体纯化的两升旋转摇瓶中生长。过滤上清液，浓缩，之后用NAb Protein G Spin Column(GE)进行亲和层析。洗脱下来的IgG通过凝胶电泳和高效液相色谱法检查以确保纯度。通过BCA蛋白定量法(Pierce)测定浓度。将单克隆抗体分成等份并且在-80℃下保存。
为了测定筛选到的结合IFNAR1表达细胞的单克隆抗体是否阻止I型干扰素的结合并阻断其下游信号转导的能力，将杂交瘤培养上清液与本发明构建的I型干扰素信号报告细胞37℃孵育30分钟至2小时，优选30分钟，1小时，2小时，更优选1小时，然后加入适量，如1ng/ml至1μg/ml，优选1ng/ml至500ng/ml，进一步优选1ng/ml至100ng/ml，具体为1ng/ml，10ng/ml，20ng/ml，30ng/ml，40ng/ml，50ng/ml，更优选10ng/ml的I型干扰素。继续培养一定时间，如18-36小时，优选20-28小时，更优选24小时后，通过流式细胞仪分析报告细胞中GFP的表达。使细胞中GFP表达低的样品为阻断人I型干扰素受体的抗体，通过如上所述的方法，获得了既结合人IFNAR1，又可以阻断I型干扰素信号的抗体10C2和10C9。
本发明的单克隆抗体是通过权威的单克隆抗体制备方法制备得到的，除了通过在体内或体外扩增杂交瘤细胞获得鼠抗人IFNAR1单克隆抗体10C2和10C9，还可以将本发明的抗体的编码基因利用传统的分子克隆方法克隆到真核表达载体，通过真核表达纯化的方法获得抗体。具体步骤如下：
真核表达载体构建的方法包括如下步骤：
将序列1所示的DNA分子利用分子克隆的方法插入含有CMV启动子和筛选基因puromycin的真核表达载体(例如pCMV或pcDNA3.1)，得到表达单克隆抗体10C2重链的质粒；
将序列6所示的DNA分子利用分子克隆的方法插入含有CMV启动子和筛选基因puromycin的真核表达载体(例如pCMV或pcDNA3.1)，得到表达单克隆抗体10C2轻链的质粒；
或，
将序列11所示的DNA分子利用分子克隆的方法插入含有CMV启动子和筛选基因puromycin的真核表达载体(例如pCMV或pcDNA3.1)，得到表达单克隆抗体10C2轻链的质粒；
将序列16所示的DNA分子利用分子克隆的方法插入含有CMV启动子和筛选基因puromycin的真核表达载体(例如pCMV或pcDNA3.1)，得到表达单克隆抗体10C2轻链的质粒；
重组细胞的构建方法包括如下步骤：将表达单克隆抗体10C2重链的质粒和表达单克隆抗体10C2轻链的质粒按照摩尔比为1:1的比例，使用Lipofectamine2000(Invitrogen)转染入哺乳动物细胞(例如CHO细胞或HEK293T细胞)，并通过抗性基因筛选获得稳定表达单克隆抗体10C2的细胞株；
或，
将表达单克隆抗体10C9重链的质粒和表达单克隆抗体10C9轻链的质粒按照摩尔比为1:1的比例，使用Lipofectamine2000(Invitrogen)转染入哺乳动物细胞(例如CHO细胞或HEK293T细胞)，并通过抗性基因筛选获得稳定表达单克隆抗体10C9的细胞株；
单克隆抗体的体外表达与纯化的方法包括如下步骤：分别扩大培养表达IFNAR1单克隆抗体10C2的细胞株和表达IFNAR1单克隆抗体10C9的细胞株，并收集培养上清；过滤上清，浓缩之后用NAb Protein G Spin Column(GE)进行亲和层析，得到纯化后的单克隆抗体。洗脱下来的IgG通过凝胶电泳和高效液相色谱法检查以确保纯度，并通过BCA蛋白定量法(Pierce)测定浓度，将单克隆抗体分成等份并且在-80℃下保存。
本发明的第十二个目的是提供一种抑制I型干扰素对表达IFNAR1的细胞的生物活性的方法。
本发明提供一种抑制I型干扰素对表达IFNAR的细胞的生物活性的方法，包括使所述细胞接触本发明的单克隆抗体或其抗原结合部分，使得I型干扰素的生物活性受到抑制。
本发明的第十三个目的是提供一种治疗I型干扰素介导的疾病的方法。
本发明提供的治疗I型干扰素介导的疾病的方法包括向患有I型干扰素介导的疾病的受试者施用上述IFNΑR1的单克隆抗体或其抗原结合部分，使该受试者中I型干扰素介导的疾病得到治疗。
上述方法中，所述I型干扰素介导的疾病是干扰素α介导的疾病，所述疾病或病症为如下任一种：系统性红斑狼疮、胰岛素依赖型糖尿病、炎性肠病、多发性硬化症、牛皮癣、自身免疫性甲状腺炎、类风湿性关节炎和肾小球肾炎、HIV感染或AIDS和植排斥或移植物抗宿主疾病。
本发明的抗体(和免疫偶联物和双特异性分子)可以用于检测IFNΑR1的水平或表达IFNΑR1的细胞的水平。例如，这可以如下实现：使样品(如体外样品)和对照样品在允许该抗体和IFNΑR1之间形成复合物的条件下接触抗IFNΑR1抗体。检测在抗体和IFNΑR1之间形成的任何复合物，并在样品和对照之间进行比较。例如，可以用本发明的组合物进行本领域公知的检测方法，如ELISA和流式细胞试验。
本发明进一步提供检测样品中IFNΑR1(例如人IFNΑR1抗原)的存在，或测定IFNΑR1的量的方法，包括在允许抗体或其部分与IFNΑR1之间形成复合物的条件下使样品和对照样品接触特异性结合IFNΑR1的本发明的抗体或其抗原结合部分。然后检测复合物的形成，其中样品与对照样品之间复合物形成的不同表示样品中存在IFNΑR1抗原。
IFNΑR1是I型干扰素的细胞受体的一部分，众所周知I型干扰素是与T细胞分化、抗体产生和记忆T细胞活性和存活有关的免疫调节细胞因子。而且，在许多自身免疫病、HIV感染、移植排斥和移植物抗宿主病(GVHD)中已经描述了I型干扰素的表达提高。因此，抑制I型干扰素功能活性的本发明的抗IFNΑR1抗体(和免疫偶联物和双特异性分子)可以用于与异常或不希望的I型干扰素活性有关的多种临床适应症。因此，本发明提供一种抑制I型干扰素介导的疾病或病症的方法，其中该方法包括施用本发明的抗体或其抗原结合部分(或本发明的免疫偶联物或双特异性分子)，使I型干扰素介导的疾病或病症得到治疗。
可以应用本发明的抗体治疗的自身免疫病的具体例子包括但不限于：系统性红斑狼疮(SLE)、胰岛素依赖型糖尿病(IDDM)、炎性肠病(IBD)(包括克罗恩病、溃疡性结肠炎和乳糜泻)、多发性硬化症(MS)、牛皮癣、自身免疫性甲状腺炎、类风湿性关节炎(RA)和肾小球肾炎。此外，本发明的抗体组合物能够用于抑制或预防移植排斥或用于治疗移植物抗宿主疾病(GVHD)或治疗HIV感染/AIDS。在系统性红斑狼疮(SLE)患者的血清中观察到高水平的IFNα (参见，例如，Kim等(1987)Clin.Exp.Immunol.70:5662-569)。而且，例如在癌症或病毒性感染治疗中的IFNα给药已经显不诱发SLE(Garcia-Porrua等(1998)Clin.Exp.Rheumatol.16:107-108)。因此，在另一个实施方案中，本发明的抗IFNΑR1抗体可以通过对需要治疗的受试者施用抗体用于治疗SLE。该抗体可以单独使用或者与其他抗SLE药物如非留体抗炎药(NSAID)、镇痛药、皮质类固醇(例如强的松、氢化可的松)、免疫抑制剂(如环磷酰胺、硫唑嘌呤和氨甲喋呤)、抗疟药(如羟氯喹)和抑制产生dsDNA抗体的生物药物(例如LJP394)组合使用。
IFNα也与I型糖尿病的病理学有关。例如，已经报道了在I型糖尿病患者的胰岛β细胞中存在免疫反应性IFNα(Foulis等(1987)Lancet2:1423-1427)。在抗病毒治疗中长期使用IFNα也显不诱发I型糖尿病(Waguri等(1994)Diabetes Res.Clin.Pract.23:33-36)。因此，在另一个实施方案中，本发明的抗-IFNΑR1抗体可以用于通过对需要治疗的受试者施用抗体来治疗I型糖尿病。该抗体可以单独使用或者与其他抗糖尿病药物如胰岛素组合使用。
IFNΑR的抗体已经证明在炎性肠病动物模型中有效(参见美国专利申请60/465,155)。因此，本发明的抗IFNΑR1抗体可以用于通过对需要治疗的受试者施用抗体来治疗炎性肠病(IBD)，包括溃疡性结肠炎和克罗恩病。该抗体可以单独使用或者与其他抗IBD药物如含有美沙拉秦的药物(包括柳氮磺吡啶和含有5-氨基水杨酸(5-ASA)的其他药物，如奥沙拉秦和巴柳氮)、非留体抗炎药(NSAID)、镇痛药、皮质类固醇(例如强的松、氢化可的松)、TNF抑制剂(包括adilimumab
Figure PCTCN2016090018-appb-000001
依那西普
Figure PCTCN2016090018-appb-000002
和英夫利昔单抗(Remicade))、免疫抑制剂(如6-巯基嘌呤、硫唑嘌呤和环孢素A)和抗生素组合使用。
已经发现用IFNα治疗可诱发自身免疫性甲状腺炎(Monzani等(2004)Clin.Exp Med.3:199-210；Prummel和Laurberg(2003)Thyroidl3:547-551)。因此，在另一个实施方案中，本发明的抗IFNΑR1抗体可以用于通过对需要治疗的受试者施用抗体来治疗自身免疫性甲状腺病，包括自身免疫性原发性甲状腺功能减退症、格雷夫斯病、桥本氏甲状腺炎和伴有甲状腺功能减退的破坏性甲状腺炎。该抗体可以单独使用或者与其他药物或治疗如抗甲状腺药物、放射性碘和甲状腺次全切除术组合使用。
在RA患者血清中已经观察到水平升高的I型干扰素，特别是IFN-β(参见例如Hertzog等(1988)Clin.Immunol.Immunopath.48:192)。因此，在一个实施方案中，本发明的抗IFNΑR1抗体可以用于通过对需要治疗的受试者施用抗体来治疗RA。该抗体可以单独使用或者与一种或多种其他抗RA药物如非留体抗炎药(NSAID)、C0X-2抑制剂、镇痛药、皮质类固醇(例如强的松、氢化可的松)、免疫抑制剂(如氨甲喋呤)、B细胞消耗剂(例如RituxanTM)、B 细胞激动剂(例如LymphoStat-BTM)和抗TNF-α剂(例如EMBRELTM、
Figure PCTCN2016090018-appb-000003
Figure PCTCN2016090018-appb-000004
和REMICADETM)组合使用。
曾经报道IFNα给药使牛皮癣恶化。因此，在另一个实施方案中，本发明的抗IFNΑR1抗体可以用于通过对需要治疗的受试者施用抗体来治疗牛皮癣和牛皮癣性关节炎。该抗体可以单独使用或者与一种或多种其他抗牛皮癣治疗如光疗、局部治疗(例如局部糖皮质激素)或全身治疗(例如氨甲喋呤、合成类维生素A、环孢素)、抗TNF-a剂(例如EMBRELTM、HUMIRA⑧和REMICADETM)和T细胞抑制剂(例如RaptivaTM)组合使用。在HIV感染患者的循环中也曾经观察到高水平的IFNα，并且它的存在是AIDS进展的预测性标记(DeStefano等(1982)J.Infec.Diseasel46:451；Vadhan_Raj等(1986)Cancer Res.46:417)。因此，在另一个实施方案中，本发明的抗-IFNΑR1抗体用于通过对需要治疗的受试者施用抗体来治疗HIV感染或AIDS。该抗体可以单独使用或者与其他抗HIV剂如核苷逆转录酶抑制剂、非核苷逆转录酶抑制剂、蛋白酶抑制剂和融合抑制剂组合使用。
已经证明IFNΑR1的抗体在抑制同种异体移植排斥和延长同种异体移植存活期中有效(参见，例如，Tovey等(1996)J.Leukoc.Biol.59:512-517；Benizri等(1998)J.Interferon Cytokine Res.18:273-284)。因此，本发明的抗IFNΑR1抗体也可以用于移植物接受者，以抑制同种异体移植排斥和/或延长同种异体移植存活期。本发明提供一种通过对需要治疗的移植接受者施用本发明的抗IFNΑR1抗体来抑制移植排斥的方法。可以治疗的组织移植的例子包括但不限于：肝、肺、肾、心脏、小肠和胰岛细胞，以及移植物抗宿主病(GVHD)的治疗。该抗体可以单独使用或者与其他抑制移植排斥的药物如免疫抑制剂(例如环孢素、硫唑嘌呤、甲基强的松龙、强的松龙、强的松、麦考酚酸莫酯、西罗莫司、雷帕霉素、他克莫司)、抗感染剂(例如阿昔洛韦、克霉唑、更昔洛韦、制霉菌素、复方新诺明)、利尿剂(例如布美他尼、呋塞米、美托拉宗)和溃疡药物(例如西咪替丁、法莫替丁、兰索拉唑、奥美拉唑、雷尼替丁、硫糖铝)组合使用。
本发明涉及结合IFNΑR1并且能够阻断I型干扰素作用的分离的小鼠单克隆抗体，以及含有本发明的抗体的免疫偶联物和双特异性分子、含有本发明的抗体、免疫偶联物或双特异性分子的药物组合物。本发明还涉及使用这些抗体与表达IFNΑR1的细胞上的IFNΑR1结合，抑制I型干扰素的信号，用于治疗患者的免疫介导的疾病，包括自身免疫病、移植排斥和移植物抗宿主病(GVHD)的方法。
为了使本发明更容易理解，首先定义了一些术语。术语“干扰素α受体1”、“IFNΑR1”和“IFNΑR1抗原”可互换使用，包括人IFNΑR1的变体、同种型、种同源物，和与IFNΑR1具有至少一个共同表位的类似物。因此，本发明的抗体在某些情况下可以与来自人类以外的物种的IFNΑR1交叉反应，或者与在结构 上与人IFNΑR1相关的其他蛋白质(例如人IFNΑR1同源物)交叉反应。在其他情况下，抗体可能对小鼠IFNΑR1是完全特异性的，不表现物种或其他类型的交叉反应性。
人IFNΑR1的完整cDNA序列的Genebank登记号为NM_000629。
文中所用的术语“I型干扰素”是指I型干扰素分子家族的成员，它们是IFNΑR1的配体(即，能够结合IFNΑR1的分子的I型干扰素家族的成员)。I型干扰素配体的例子有IFNαl、2a、2b、4、5、6、7、8、10、14、16、17、21、干扰素β和干扰素ω。
术语“免疫应答”是指例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝脏产生的可溶性大分子(包括抗体、细胞因子和补体)的作用，导致选择性损伤、破坏或从人体中清除侵入的病原体感染了病原体的细胞或组织、癌细胞，或在自身免疫或病理性炎症的情况下，正常人细胞或组织。
“信号转导途径”是指在信号从细胞的一部分传送到细胞的另一部分中起作用的多种信号转导分子之间的生化关系。文中所用的，短语“细胞表面受体”包括，例如，能够接收信号和跨细胞质膜传播这种信号的分子和分子复合物。本发明的“细胞表面受体”的一个例子是IFNΑR1受体。
这里提到的术语“抗体”包括完整抗体及其任何抗原结合片段(即“抗原结合部分”)或单链。“抗体”是指包含通过二硫键互相连接在一起的至少两条重(H)链和两条轻(L)链的糖蛋白，或其抗原结合部分。每条重链由重链可变区(在此缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(在此缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可进一步再分为高变区，称为互补决定区(CDR)，CDR散布在被称为骨架区(FR)的更加保守的区域中。VH和VL均由三个CDR和四个FR组成，它们从氨基端至羧基端以如下顺序排列：FR1，CDRl，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有可与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子，包括与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成分(C1q)的结合。
文中所用的术语抗体的“抗原结合部分”(或简称为“抗体部分”)是指保留特异性结合抗原(例如IFNΑR1)的能力的抗体的一个或多个片段。已显示抗体的抗原结合功能可由全长抗体的片段来行使。术语抗体的“抗原结合部分”中所包括的结合片段的例子包括：(i)Fab片段，即由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab’)2片段，即包含在铰链区处通过二硫键连接的两个Fab片段的双价片段；(iii)由VH和CH结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)由VH结构域组成的dAb片段(Ward等(1989)Nature 341:544-546)；和(vi)分离的互补决定区(CDR)。此外，尽管Fv片段的两个结构域VL和VH由分别的基因编码，但是它们可以利用重组方法通 过合成接头连接在一起，从而能够将它们制成一条蛋白质链，其中VL和VH区配对构成单价分子(称为单链Fv(scFv)；参见，例如Bird等(1988)Science 242:423-426；和Huston等(1988)Proc.Natl.Acad.Sci.USA 85:5879-5883)。这种单链抗体也包括在术语抗体的“抗原结合部分”内。这些抗体片段用本领域技术人员公知的常规技术获得，并用与完整抗体相同的方法对这些片段进行实用性筛选。
文中所用的，“分离的抗体”是指基本不含具有不同抗原特异性的其他抗体的抗体(例如，特异性结合IFNΑR1的分离的抗体基本不含特异性结合IFNΑR1以外抗原的抗体)。但是，特异性结合IFNΑR1的分离的抗体与其他抗原如来自其他物种的IFNΑR1分子可能具有交叉反应性。而且，分离的抗体可能基本不含其他细胞材料和/或化学物质。
文中所用的术语“单克隆抗体”或“单克隆抗体组合物”是指具有单一分子组成的抗体分子的制剂。单克隆抗体组合物显示对特定表位的单一的结合特异性和亲和性。
文中所用的术语“受试者”包括任何人或非人类动物。术语“非人类动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，如非人类灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖类动物、爬行类动物等。
本发明进一步通过下面的实施例进行阐述，不应将该实施例理解为进一步的限制。全部附图和在本申请中引用的全部参考文献、专利和公开专利申请的内容均引用作为参考。
附图说明
图1显示人I型干扰素报告细胞。图1A显示构建的I型干扰素报告质粒；图1B显示稳定转染I型干扰素报告质粒的细胞系在经I型干扰素刺激后，表达GFP蛋白。
图2显示人I型干扰素受体IFNAR1的结合和阻断抗体的筛选。
图3显示IFNΑR1单克隆抗体10C2重链可变区的核苷酸序列(序列1)和氨基酸序列(序列2)。图中黑色阴影区域标出了CDR1(序列3)、CDR2(序列4)和CDR3(序列5)区。
图4显示IFNΑR1单克隆抗体10C2轻链可变区的核苷酸序列(序列6)和氨基酸序列(序列7)。图中黑色阴影区域标出了CDR1(序列8)、CDR2(序列9)和CDR3(序列10)区。
图5显示IFNΑR1单克隆抗体10C2的重链可变区的氨基酸序列(序列2)与小鼠V区氨基酸序列(序列21)的对比。IFNΑR1单克隆抗体10C2的重链可变区组成：V基因为IGHV9-3，D基因为IGHD2-13，J基因为IGHJ1。图中所示为IFNΑR1单克隆抗体10C2重链氨基酸序列与其原始VDJ基因氨基酸序列比对结果。
图6显示IFNΑR1单克隆抗体10C2的轻链可变区的氨基酸序列与小鼠V区氨基酸序列(序列22)的对比。IFNΑR1单克隆抗体10C2轻链组成：V基因为 IGKV6-13，J基因为IGKJ5。图中所示为IFNΑR1单克隆抗体10C2轻链氨基酸序列与其原始VJ基因氨基酸序列比对结果。
图7显示IFNΑR1单克隆抗体10C9重链可变区的核苷酸序列(序列11)和氨基酸序列(序列12)。图中黑色阴影区域标出了CDR1(序列13)、CDR2(序列14)和CDR3(序列15)区。
图8显示IFNΑR1单克隆抗体10C9轻链可变区的核苷酸序列(序列16)和氨基酸序列(序列17)。图中黑色阴影区域标出了CDR1(序列18)、CDR2(序列19)和CDR3(序列20)区。
图9显示IFNΑR1单克隆抗体10C9的重链可变区的氨基酸序列(序列12)与小鼠V区氨基酸序列(序列23)的对比。IFNΑR1单克隆抗体10C9重链组成:V基因为IGHV9-3，D基因为IGHD1-1，J基因为IGHJ3。图中所示为IFNΑR1单克隆抗体10C9重链氨基酸序列与其原始VDJ基因氨基酸序列比对结果。
图10显示IFNΑR1单克隆抗体10C9的轻链可变区的氨基酸序列(序列17)与小鼠V区氨基酸序列(序列24)的对比。IFNΑR1单克隆抗体10C9重链组成:V基因为IGKV6-15，J基因为IGKJ2。图中所示为IFNΑR1单克隆抗体10C9轻链氨基酸序列与其原始VJ基因氨基酸序列比对结果。
图11显示IFNΑR1单克隆抗体10C2和10C9结合IFNAR1。
图12显示IFNΑR1单克隆抗体10C2和10C9结合IFNAR1的SD2和/或SD3结构域。
图13显示IFNΑR1单克隆抗体10C2和10C9在干扰素信号报告细胞上抑制IFNα2b的生物活性。
图14显示IFNΑR1单克隆抗体10C2和10C9在经过刺激剂刺激的人外周血单个核细胞上抑制I型干扰素的生物活性。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、抗人IFNΑR1单克隆抗体的制备
一、I型干扰素报告细胞系的制备
1、引物的设计
参照文献“Bürgi等人(Journal of Immunological Methods 381(2012)70–74)”中的方法获取小鼠干扰素效应基因Mx2的基因组序列(NCBI genebank:AB086958)，并根据其使用的酶切位点(Nse I和Nhe I)确定了Mx2的启动子有效序列。根据Mx2的启动子序列，设计如下引物：
F：CTAGCTAGCAAGTCTAAGGGCTCTGAGGACAGAC；
R：CCCAAGCTTCAAATGCCCTGCTGTACTTACCAGT。
2、PCR扩增
以小鼠血液基因组DNA为模板，采用步骤1中设计的含Kpn I和Nhe I酶切位点的引物进行PCR扩增，得到PCR扩增产物，即为小鼠Mx2启动子片段；
PCR反应条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火60℃，30秒；延伸，72℃，1分钟；共进行30循环，最后增加10分钟变性时间。
3、pGL4.2mouseMx2promoter-EGFP的获得
用限制性内切酶Kpn I和Nhe I分别酶切步骤2获得的PCR扩增产物和pGL4.2-EGFP载体(pGL4.2-EGFP载体是将eGFP基因替换pGL4(luc2)载体中luc2基因，并保持pGL4(luc2)载体的其它序列不变得到的载体)，连接，得到人I型干扰素报告质粒pGL4.2mouseMx2promoter-EGFP(pGL4.2mouseMx2promoter-EGFP质粒的结构如图1A所示)。
4、I型干扰素报告细胞系的获得
使用Lipofectamine2000(Invitrogen)将pGL4.2mouseMx2promoter-EGFP质粒转染进入HEK293T细胞系(ATCC，货号：
Figure PCTCN2016090018-appb-000005
CRL-3216TM)中，并使用puromycin筛选获得稳定转染细胞系(I型干扰素报告细胞系)。
5、I型干扰素报告细胞系的功能验证
使用100μl的浓度为5ng/ml的人IFNα2b(Cedarlane公司，货号：CL106-04E-100UG)刺激步骤4获得的稳定转染细胞系24小时，收取细胞使用流式细胞仪检测GFP表达情况。
结果如图1B所示：I型干扰素报告细胞系在未经I型干扰素刺激时不会表达GFP，但通过I型干扰素刺激后，可以表达GFP蛋白，并可被流式细胞仪检测，证实上述步骤4制备得到的细胞系可以作为I型干扰素报告细胞系，该细胞系可以用作IFNAR1阻断抗体的筛选。
二、人I型干扰素受体IFNAR1抗体的制备
1、人IFNAR1抗原的制备
(1)根据人IFNAR1序列，设计如下引物：IFNAR1-F：CTAGCTAGCTCTAGAGCCACCATGATGGTCGTCCTCCTGGGC；IFNAR1-R：GGGTCCGGAACCTCCTCCTCCCACAGCATAAATGACAAACGGGAGA。
(2)以离体人PBMCs的cDNA为模板，采用步骤(1)设计的引物进行PCR扩增，得到PCR扩增产物，即为人I型干扰素受体IFNAR1的胞外区和跨膜区的片段。
上述PCR反应条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火，60℃，30秒；延伸，72℃，2分钟；共进行30循环，最后增加延伸10分钟。
(3)用限制性内切酶Nhe I和Bspe I酶切步骤(2)获得的PCR扩增产物和pEGFP载体，连接，得到pEGFP-humanIFNAR1EC质粒。在pEGFP-humanIFNAR1EC质粒中，IFNAR1胞外和跨膜区序列下游融合有GFP基因，当IFNAR1表达时，可以观察到GFP信号。
(4)将pEGFP-humanIFNAR1EC质粒使用Lipofectamine2000(Invitrogen)转染进入小鼠L细胞(ATCC，货号：
Figure PCTCN2016090018-appb-000006
CRL-2648TM)中，转染48小时后，得到转染人IFNAR1胞外区的小鼠L细胞。通过分析转染人IFNAR1胞外区的小鼠L细胞中GFP的表达确定转染效率并用于下述小鼠免疫实验。
2、BalB/C小鼠的免疫
将步骤1获得的转染人IFNAR1胞外区的小鼠L细胞作为人IFNAR1的免疫原。将5000,000个转染人IFNAR1的小鼠L细胞与20μg CpG1826(TAKARA合成)混合，形成0.5ml的悬液，通过腹腔注射的方式免疫6周龄BalB/C小鼠。每个月免疫一次，共免疫3次。最后，使用同样方法进行加强免疫，并在4天后进行杂交瘤融合。
3、稳定表达人IFNAR1胞外区细胞系的构建
将pEGFP-humanIFNAR1EC质粒使用Lipofectamine2000(Invitrogen)转染进入HEK293T细胞系，并通过puromycin筛选获得稳定转染该质粒的细胞，从而使其稳定表达人IFNAR1胞外与跨膜区(IFNAR1与GFP融合表达)。该细胞系将用于下述抗体筛选实验。
4、杂交瘤的融合
将免疫后待融合的小鼠处死，并取出脾脏细胞。通过细胞计数，将小鼠脾脏细胞与小鼠骨髓瘤细胞系SP2/0(ATCC，CRL1581)按1:3的比例进行混合，并使用50％PEG(Sigma)融合细胞。将融合后的细胞加入到60ml含1X HAT的RPMI培养基(Cellgro,货号：15-041-CV)中(含10％胎牛血清)，按每孔1-2滴的量加入到96孔细胞培养板中。随后，将杂交瘤细胞在37℃，5％CO2的条件下进行培养，3-4天进行半量换液。约10天后进行抗体筛选。
5、人I型干扰素受体IFNAR1抗体的筛选
筛选前一天，将96孔细胞培养板中的上清吸出约100μl至96孔U形底的培养板中，并在原孔补入100μl新鲜的含1X HAT的RPMI培养基，继续培养细胞。收取已经培养好的稳定表达人IFNAR1的HEK293T细胞系，将其加至96孔U形底培养板中，10000个细胞/200μl/孔。2200rpm/3分钟离心后，甩出上清，并加入100μl杂交瘤培养上清，重悬后4℃孵育30分钟。孵育完毕，进行离心并甩出上清，每孔加入200μl FACS buffer(含2％FBS和2mM EDTA的PBS)，重悬细胞并进行离心，之后甩出上清以达到清洗的目的。每孔加入1:400稀释于FACS buffer的PE标记的羊抗鼠IgG抗体(Biolegend)，将细胞重悬后在4℃避光孵育30分钟。孵育后，将培养板进行离心并除去上清，按前述方法清洗细胞一次后加入200μl FACS buffer重悬细胞。最后，使用流式细胞仪Guava(Millipore)分析细胞GFP与PE信号。GFP与PE双阳性孔为IFNAR1结合抗体的阳性孔(图2A)。
将阳性孔的培养上清用于下一步的阻断功能实验。具体步骤为：首先，在实验前一天将人I型干扰素报告细胞系加至96孔细胞培养板，30000个细胞/孔。 实验当天，将细胞培养上清吸出，加入100μl杂交瘤培养上清，37℃孵育1小时。随后，加入100μl含10ng/ml IFNα2b的DMEM(含10％胎牛血清FBS)培养基(Hyclone,货号：SH30022.01)培养24小时。培养后通过分析细胞GFP表达情况，确定抗体阻断I型干扰素信号的活性，GFP表达低的样品为阻断人I型干扰素受体的抗体(图2B)。根据上述步骤，最终选择了10C2和10C9既结合人IFNAR1，又可以阻断其信号的两个稳定分泌IFNAR1单克隆抗体的单克隆杂交瘤细胞株，分别将其命名为杂交瘤细胞株10C2和杂交瘤细胞株10C9。杂交瘤细胞株10C2分泌的抗体命名为鼠抗人IFNAR1单克隆抗体10C2；杂交瘤细胞株10C9分泌的抗体命名为鼠抗人IFNAR1单克隆抗体10C9。
杂交瘤细胞株10C2的分类命名为小鼠杂交瘤细胞系，该杂交瘤细胞株已于2016年5月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏号为CGMCC No.12542。
杂交瘤细胞株10C9的分类命名为小鼠杂交瘤细胞系，该杂交瘤细胞株已于2016年5月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏号为CGMCC No.12543。
三、鼠抗人IFNAR1单克隆抗体10C2和10C9的序列
1、RNA的提取
利用Trizol(Invitrogen)裂解杂交瘤细胞株10C2和杂交瘤细胞株10C9后，提取了杂交瘤细胞株10C2和杂交瘤细胞株10C9的RNA。RNA的提取的具体操作步骤：
每1毫升Trizol加入200微升三氯甲烷，充分震荡后静置10分钟；13000rpm/4℃/15分钟离心；吸取400微升上清加至400微升预冷异丙醇中，混匀后-20℃静置过夜；13000rpm/4℃/15分钟离心；去除上清，加入70％乙醇；13000rpm/4℃/10分钟离心；去除上清，加入70％乙醇；去除上清，加入40微升水溶解，得到RNA溶液；
2、cDNA的获得
吸取16微升步骤1制备的RNA溶液，加入1微升100nM的Oligo dT(Invitrogen)；70℃反应5分钟；立即放置冰上；分别加入1微升RNA酶抑制剂(Takara)，1微升10mM的dNTP(Takara)，1微升MLV反转录酶和5微升5X缓冲液(Promega)；42度反应60分钟；80℃处理10分钟；
3、PCR扩增及测序
以步骤2获得的cDNA模板，分别采用重链引物F和重链引物R、轻链引物F和轻链引物R进行PCR扩增，分别获得编码重链和轻链的片段并对其进行测序。引物序列如下：
重链引物F：CTAGCTAGCTCTAGAGCCACC ATGATGGTCGTCCTCCTGGGC；
重链引物R：CTTGACCAGGCATCCTAGAGTCA；
轻链引物F：GAYATTGTGMTSACMCARWCTMCA；
轻链引物R：GGATACAGTTGGTGCAGCATC。
上述PCR反应条件：预变性，98℃，2分钟；变性，98℃，30秒；退火，54℃；延伸72℃，1分钟；共35循环，最后增加延伸10分钟。
测序结果表明：
IFNΑR1单克隆抗体10C2的重链可变区的核苷酸和氨基酸序列显示于图3中，IFNΑR1单克隆抗体10C2的重链可变区核苷酸序列为序列1，IFNΑR1单克隆抗体10C2的重链可变区的氨基酸序列为序列2；将IFNΑR1单克隆抗体10C2的重链可变区氨基酸序列的第26位-第33位所示的氨基酸序列(序列3)命名为10C2重链CDR1，将IFNΑR1单克隆抗体10C2的重链可变区氨基酸序列的第51位-第58位所示的氨基酸序列(序列4)命名为10C2重链CDR2，将IFNΑR1单克隆抗体10C2的重链可变区氨基酸序列的第97位-第112位所示的氨基酸序列(序列5)命名为10C2重链CDR3。
IFNΑR1单克隆抗体10C2的轻链可变区的核苷酸和氨基酸序列显示于图4中，IFNΑR1单克隆抗体10C2的轻链可变区核苷酸序列为序列6，IFNΑR1单克隆抗体10C2的轻链可变区的氨基酸序列为序列7；将IFNΑR1单克隆抗体10C2的轻链可变区氨基酸序列的第27位-第32位所示的氨基酸序列(序列8)命名为10C2轻链CDR1，将IFNΑR1单克隆抗体10C2的轻链可变区氨基酸序列的第50位-第52位所示的氨基酸序列(序列9)命名为10C2轻链CDR2，将IFNΑR1单克隆抗体10C2的轻链可变区氨基酸序列的第89位-第94位所示的氨基酸序列(序列10)命名为10C2轻链CDR3。
IFNΑR1单克隆抗体10C9的重链可变区的核苷酸和氨基酸序列显示于图7中，IFNΑR1单克隆抗体10C9的重链可变区核苷酸序列为序列11，IFNΑR1单克隆抗体10C9的重链可变区的氨基酸序列为序列12；将IFNΑR1单克隆抗体10C9的重链可变区氨基酸序列的第26位-第33位所示的氨基酸序列(序列13)命名为10C9重链CDR1，将IFNΑR1单克隆抗体10C9的重链可变区氨基酸序列的第51位-第58位所示的氨基酸序列(序列14)命名为10C9重链CDR2，将IFNΑR1单克隆抗体10C9的重链可变区氨基酸序列的第97位-第112位所示的氨基酸序列(序列15)命名为10C9重链CDR3。
IFNΑR1单克隆抗体10C9的轻链可变区的核苷酸和氨基酸序列显示于图8中，IFNΑR1单克隆抗体10C9的轻链可变区核苷酸序列为序列16；IFNΑR1单克隆抗体10C9的轻链可变区的氨基酸序列为序列17。将IFNΑR1单克隆抗体10C9的轻链可变区氨基酸序列的第27位-第32位所示的氨基酸序列(序列18)命名为10C9轻链CDR1，将IFNΑR1单克隆抗体10C9的轻链可变区氨基酸序列的第50位-第52位所示的氨基酸序列(序列19)命名为10C9轻链CDR2，将IFNΑR1单克隆抗体10C9的轻链可变区氨基酸序列的第89位-第96位所示 的氨基酸序列(序列20)命名为10C9轻链CDR3。
4、抗体序列分析
将IFNΑR1单克隆抗体10C2和IFNΑR1单克隆抗体10C9的核酸片段在抗体序列分析工具igBlast tool(http://www.ncbi.nlm.nih.gov/igblast/)中进行分析发现：
IFNΑR1单克隆抗体10C2重链编码基因的V基因，D基因和J基因分别对应于小鼠IGHV9-3基因，IGHD2-13基因和IGHJ1基因，IFNΑR1单克隆抗体10C2的重链可变区的氨基酸序列(序列2)与小鼠V区氨基酸序列(序列21)的对比结果如图5所示；IFNΑR1单克隆抗体10C2轻链编码基因的V基因和J基因分别对应于小鼠IGKV6-13基因和IGKJ5基因，IFNΑR1单克隆抗体10C2的轻链可变区的氨基酸序列与小鼠V区氨基酸序列(序列22)的对比结果如图6所示；
IFNΑR1单克隆抗体10C9重链编码基因的V基因，D基因和J基因分别对应于小鼠IGHV9-3基因，IGHD1-1基因和IGHJ3基因，IFNΑR1单克隆抗体10C9的重链可变区的氨基酸序列(序列12)与小鼠V区氨基酸序列(序列23)的对比结果如图9所示；IFNΑR1单克隆抗体10C9轻链编码基因的V基因和J基因分别对应于小鼠IGKV6-15和IGKJ2，IFNΑR1单克隆抗体10C9的轻链可变区的氨基酸序列(序列17)与小鼠V区氨基酸序列(序列24)的对比结果如图10所示。
实施例2、IFNAR1单克隆抗体可以结合人I型干扰素受体IFNAR1
1、将实施例1步骤二的3中获得的稳定表达人I型干扰素受体IFNAR1的HEK293T细胞使用含2mM EDTA的PBS溶液(每1L PBS含KH2PO4 0.27g、Na2HPO4 1.42g、NaCl 8g、KCl 0.2g、调节pH至7.2-7.4，用水定容至1L)进行处理，使之成为单细胞悬液。
2、将稳定表达人I型干扰素受体IFNAR1的单细胞悬液加至96孔U形底培养板中，10000个细胞/200μl/孔。通过2200rpm/3分钟离心后，弃上清，收集沉淀。
3、向步骤2获得的沉淀中分别加入含有5μg/ml的实施例1制备的鼠抗人IFNAR1单克隆抗体10C2和10C9的FACS buffer(含2％FBS和2mM EDTA的PBS)，重新悬浮后4℃孵育30分钟。
4、孵育完毕，进行离心，弃上清，收集沉淀，每孔加入200μl FACS buffer，重悬细胞并进行离心，弃上清，收集沉淀并进行清洗。
5、每孔加入1:400稀释于FACS buffer的PE标记的羊抗小鼠IgG抗体(Biolegend)，将细胞重新悬浮后在4℃避光孵育30分钟。孵育后，将培养板进行离心并除去上清，按前述方法清洗细胞一次后加入200μl FACS buffer重悬细胞。最后，使用流式细胞仪Guava(Millipore)分析细胞GFP与PE信号。
结果如图11所示。由于IFNAR1与GFP融合表达，GFP表达量高的细胞其IFNAR1表达量也较高。因此，GFP表达高的细胞结合抗人IFNAR1抗体也较多，会结合更多的PE标记的羊抗小鼠IgG抗体，也就具有更高的PE信号。从图中可以看出：鼠抗人IFNAR1单克隆抗体10C2和10C9均可以结合人IFNAR1受体。
实施例3、IFNAR1单克隆抗体可以结合人I型干扰素受体IFNAR1的SD2和SD3结构域
一、敲除人IFNAR1胞外SD1(aa32-126)，SD2(aa127-227)和SD3(aa231-329)的截短体表达质粒的构建
1、引物的设计
根据人IFNAR1和其质粒的序列，设计引物：
F：ATTGACGCAAATGGGCGGTA(位于IFNAR1表达质粒的CMV启动子内)；
1F：CAGGTGGAAAAAATCTAAAACAGATTGGTCCTCCAGAAGTACATT；
1R：ACTTCTGGAGGACCAATCTGTTTTAGATTTTTTCCACCTGCGGC；
2F：TTACACCATTTCGCAAAGCTGAACTACCTCCACCAGAAAATATA；
2R：TTTTCTGGTGGAGGTAGTTCAGCTTTGCGAAATGGTGTAAATGAG；
3F:CAGTTGAAAATGAACTACCTTTCCTACTTCCTCCAGTCTTTAACA；
3R：ACTGGAGGAAGTAGGAAAGCAGGTAGTTCATTTTCAACTGTGGTC；
R：GGGTCCGGAACCTCCTCCTCCCACAGCATAAATGACAAACGGGAGA
2、敲除人IFNAR1胞外SD1，SD2和SD3的截短体表达质粒的构建
(1)敲除人IFNAR1胞外SD1的截短体表达质粒的构建
A)以实施例1中的pEGFP-humanIFNAR1EC质粒为模板，使用F和1R，1F和R进行第一轮PCR，得到第一轮PCR产物；
PCR条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火58℃，30秒；延伸，72℃，2分钟；共进行30循环，最后增加10分钟变性时间；
B)回收第一轮PCR产物，并将其作为第二轮PCR模板，使用引物F和R进行第二轮PCR，得到第二轮PCR产物；
PCR条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火60℃，30秒；延伸，72℃，2分钟；共进行30循环，最后增加10分钟变性时间；
C)回收第二轮PCR产物，使用EcoR I和Bspe I酶切第二轮PCR产物和pEGFP-humanIFNAR1EC质粒，进行回收连接，获得SD1截短的IFNAR1表达质粒。
SD1截短的IFNAR1表达质粒为将人IFNAR1胞外SD1结构域的编码基因(即IFNAR1氨基酸序列的第32-126位氨基酸的编码基因)替换pEGFP-humanIFNAR1EC质粒EcoR I和Bspe I酶切位点间的片段，且保持pEGFP-humanIFNAR1EC质粒的其他序列不变得到的载体。
(2)敲除人IFNAR1胞外SD2的截短体表达质粒
A)以实施例1中的pEGFP-humanIFNAR1EC质粒为模板，使用F和2R，2F和R进行第一轮PCR，得到第一轮PCR产物；
PCR条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火58℃，30秒；延伸，72℃，2分钟；共进行30循环，最后增加10分钟变性时间；
B)回收第一轮PCR产物，并将其作为第二轮PCR模板，使用引物F和R进行第二轮PCR，得到第二轮PCR产物；
PCR条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火60℃，30秒；延伸，72℃，2分钟；共进行30循环，最后增加10分钟变性时间；
C)回收得到第二轮PCR产物，使用EcoR I和Bspe I酶切第二轮PCR产物和pEGFP-humanIFNAR1EC质粒，进行回收连接，获得SD2截短的IFNAR1表达质粒。
SD2截短的IFNAR1表达质粒为将序列将人IFNAR1胞外SD2结构域的编码基因(即IFNAR1氨基酸序列的第127-227位氨基酸的编码基因)替换pEGFP-humanIFNAR1EC质粒EcoR I和Bspe I酶切位点间的片段，且保持pEGFP-humanIFNAR1EC质粒的其他序列不变得到的载体。
(3)敲除人IFNAR1胞外SD3的截短体表达质粒
A)以实施例1中的pEGFP-humanIFNAR1EC质粒为模板，使用F和3R，3F和R进行第一轮PCR，得到第一轮PCR产物；
PCR条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火58℃，30秒；延伸，72℃，2分钟；共进行30循环，最后增加10分钟变性时间，此次反应模板为前述IFNAR1表达质粒；
B)回收第一轮PCR产物，并将其作为第二轮PCR模板，使用引物F和R进行第二轮PCR，得到第二轮PCR产物；
PCR条件为：预变性，98℃，2分钟；变性，98℃，30秒；退火60℃，30秒；延伸，72℃，2分钟；共进行30循环，最后增加10分钟变性时间；
C)回收得到第二轮PCR产物，使用EcoR I和Bspe I酶切第二轮PCR产物和pEGFP-humanIFNAR1EC质粒，进行回收连接，获得SD3截短的IFNAR1表达质粒。
SD3截短的IFNAR1表达质粒为将将人IFNAR1胞外SD3结构域的编码基因(即IFNAR1氨基酸序列的第231-329位氨基酸的编码基因)替换pEGFP-humanIFNAR1EC质粒EcoR I和Bspe I酶切位点间的片段，且保持pEGFP-humanIFNAR1EC质粒的其他序列不变得到的载体。
3、表达人IFNAR1胞外SD1，SD2和SD3的截短体的HEK293T细胞的获得
将野生型IFNAR1(pEGFP-humanIFNAR1EC)与敲除人IFNAR1胞外SD1，SD2和SD3的截短体表达质粒分别通过Lipofectamine2000(Invitrogen)转染进入HEK293T细胞系，转染24小时后使用含2mM EDTA的PBS处理并收取单细胞悬液，分别得到表达人I型干扰素受体IFNAR1的HEK293T细胞(R1-WT)、表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△1)、表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△2)和表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△3)。
二、IFNAR1单克隆抗体10C2和10C9与各截短体的结合情况
按实施例2中的方法，分别检测IFNAR1单克隆抗体10C2和10C9与表达人I型干扰素受体IFNAR1的HEK293T细胞(R1-WT)、表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△1)、表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△2)和表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△3)的结合情况。
结果如图12所示：IFNAR1单克隆抗体10C2和10C9均不结合表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△1)，但均与表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△2)和表达人I型干扰素受体IFNAR1-SD1截短体的HEK293T细胞(R1-△3)结合。说明IFNAR1单克隆抗体可以特异结合人I型干扰素受体IFNAR1的SD2和/或SD3结构域。
实施例4、抗人IFNΑR1单克隆抗体在抑制IFNα2b的生物活性中的应用
将实施例1步骤一中的I型干扰素报告细胞系作为工具，检测分析IFNAR1单克隆抗体10C2和10C9阻断I型干扰素的效率。具体步骤如下：
1、将人I型干扰素报告细胞加至96孔细胞培养板，30000个细胞/200μl/孔。24小时后。
2、将梯度稀释的抗体(浓度分别为20μg/ml、10μg/ml、5μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml、0.313μg/ml、0.156μg/ml、0.078μg/ml和0μg/ml)分别加至DMEM培养基(含10％FBS)。
3、将细胞上清吸出，分别加入不同梯度稀释抗体的DMEM培养基100μl，37℃孵育1小时。
4、将100μl含10ng/ml人IFNα2b(Cedarlane公司，货号：CL106-04E-100UG)的DMEM培养基加至孔中，刺激培养24小时(37℃，5％CO2)。
5、利用流式细胞仪检测GFP表达情况，以此分析抗体阻断I型干扰素信号的生物活性。
结果如图13所示，其中，Iso ctrl代表抗体的同型对照组(mIgG2a,Biolegend)，Neg ctrl代表未用IFNα2b刺激对照组，Pos ctrl代表仅用IFNα2b 刺激对照组，从图中可以看出，加入不同浓度的IFNAR1单克隆抗体10C2和10C9后，GFP表达水平均有所降低，且随着浓度的增加，表达水平逐渐降低，说明IFNAR1单克隆抗体10C2和10C9均可以有效阻断IFNα2b的信号，进而说明抗人IFNΑR1单克隆抗体可以抑制IFNα2b的生物活性。
实施例5、IFNAR1单克隆抗体在阻断I型干扰素对人外周血单核细胞刺激作用中的应用
1、分别将不同浓度(1μg/ml和10μg/ml)的IFNAR1单克隆抗体10C2和10C9加入到RPMI 1640培养基(Cellgro公司，货号：15-041-CV)中，再加入人外周血单核细胞(PBMC)(通过常规方案利用Ficoll分离从新鲜血液中制备)，孵育(37℃，1小时)，得到培养后的PBMC。
2、按照刺激剂的不同，分为如下三组对培养后的PBMC进行刺激，分别得到刺激后的细胞：
(1)用CpGA(1μM)(Invivogen公司，货号：tlrl-2216)对培养后的PBMC进行刺激，使其中的浆细胞样树突状细胞(pDC，体内主要的I型干扰素产生细胞)产生I型干扰素。
(2)用R848(1μg/ml)(Invivogen公司，货号：tlrl-r848)对培养后的PBMC进行刺激，使其中的浆细胞样树突状细胞(pDC，体内主要的I型干扰素产生细胞)产生I型干扰素。
(3)用IFNα2b(Cedarlane公司，货号：CL106-04E-100UG)(500pg/ml)直接对培养后的PBMC进行刺激。
3、刺激14小时后分别收集刺激后的细胞，提取RNA并逆转录合成cDNA。
4、利用荧光定量PCR检测各个刺激后的细胞中I型干扰素效应基因Mx2和ISG15的RNA水平，其中GAPDH为内参基因。
Mx2的引物序列为：
Mx2-F：CAGAGGCAGCGGAATCGTAA；
Mx2-R：TGAAGCTCTAGCTCGGTGTTC；
ISG15的引物序列为：
ISG15-F：CCCACAGCCCACAGCCAT；
ISG15-R：TTCTGGGTGATCTGCGCCTT；
GAPDH的引物序列为：
GAPDH-F：AGCCACATCGCTCAGACAC；
GAPDH-R：GCCCAATACGACCAAATCC。
结果如图14所示，其中，mock代表RPMI培养基对照组，Isotype代表抗体的同型对照组(mIgG2a,Biolegend)，从图中可以看出，加入IFNAR1单克隆抗体10C2和10C9后，各个刺激后的细胞中I型干扰素效应基因Mx2和ISG15 的RNA表达水平明显降低，说明IFNAR1单克隆抗体10C2和10C9均可以有效阻断经刺激产生的内源I型干扰素和外源I型干扰素的刺激信号。
工业应用
本发明的抗体(和免疫偶联物和双特异性分子)一方面，可以用于检测IFNΑR1的水平或表达IFNΑR1的细胞的水平，或检测样品中IFNΑR1的存在，或测定IFNΑR1的量；另一方面，本发明的抗体(和免疫偶联物和双特异性分子)还具有体外和体内诊断和治疗应用，包括施用本发明的抗IFNΑR1抗体或其抗原结合部分(或本发明的免疫偶联物或双特异性分子)，或与其他药物组合使用，以治疗、预防或诊断多种I型干扰素介导的疾病，例如，本发明的抗体可以用于通过对需要治疗的受试者施用抗体来治疗炎性肠病(包括溃疡性结肠炎和克罗恩病)、自身免疫性甲状腺病(包括自身免疫性原发性甲状腺功能减退症、格雷夫斯病、桥本氏甲状腺炎和伴有甲状腺功能减退的破坏性甲状腺炎)、RA、牛皮癣和牛皮癣性关节炎、HIV感染或AIDS等I型干扰素介导的疾病，本发明的抗体也可以用于移植物接受者，以抑制同种异体移植排斥和/或延长同种异体移植存活期。

Claims (23)

  1. 一种IFNΑR1的单克隆抗体或其抗原结合部分，其结合人IFNΑR1的SD2结构域全部或部分和/或SD3结构域全部或部分，可抑制人I型干扰素的生物活性。
  2. 根据权利要求1所述的IFNAR1的单克隆抗体或其抗原结合部分，其特征在于：所述单克隆抗体或其抗原结合部分表现如下A)-E)中一种或多种特性：
    A)结合人IFNΑR1；
    B)结合人IFNΑR1的SD2结构域全部或部分和/或SD3结构域全部或部分；
    C)抑制I型干扰素的生物活性；
    D)抑制I型干扰素信号报告细胞上I型干扰素的生物活性；
    E)抑制在经过刺激剂刺激的人外周血单个核细胞上I型干扰素的生物活性。
  3. 根据权利要求1所述的IFNAR1的单克隆抗体或其抗原结合部分，其特征在于：
    所述单克隆抗体或其抗原结合部分的重链可变区的编码基因为如下1)或2)：
    1)小鼠IGHV9-3，IGHD2-13和IGHJ1基因的产物或来源于该基因；
    2)小鼠IGHV9-3，IGHD1-1和IGHJ3基因的产物或来源于该基因；
    所述单克隆抗体或其抗原结合部分的轻链可变区的编码基因为如下3)或4)：
    3)小鼠IGKV6-13和IGKJ5基因的产物或来源于该基因；
    4)小鼠IGKV6-15和IGKJ2基因的产物或来源于该基因。
  4. 根据权利要求3所述的IFNAR1的单克隆抗体或其抗原结合部分，其特征在于：
    所述单克隆抗体或其抗原结合部分的重链可变区为如下5)或6)：
    5)依次包括小鼠IGHV9-3基因全长或部分编码的蛋白或多肽、小鼠IGHD2-13基因全长或部分编码的蛋白或多肽和小鼠IGHJ1基因全长或部分编码的蛋白或多肽；
    6)依次包括小鼠IGHV9-3基因全长或部分编码的蛋白或多肽、小鼠IGHD1-1基因全长或部分编码的蛋白或多肽和小鼠IGHJ3基因全长或部分编码的蛋白或多肽；
    所述单克隆抗体或其抗原结合部分的轻链可变区为如下7)或8)：
    7)依次包括小鼠IGKV6-13基因全长或部分编码的蛋白或多肽和小鼠IGKJ5基因全长或部分编码的蛋白或多肽；
    8)依次包括小鼠IGKV6-15基因全长或部分编码的蛋白或多肽和小鼠IGKJ2基因全长或部分编码的蛋白或多肽。
  5. 根据权利要求4所述的单克隆抗体或其抗原结合部分，其特征在于：
    所述IFNΑR1的单克隆抗体或其抗原结合部分的重链可变区依次包括所述小鼠IGHV9-3基因编码的蛋白、所述小鼠IGHD2-13基因编码的蛋白和所述小鼠IGHJ1 基因编码的蛋白；且其轻链可变区依次包括所述小鼠IGKV6-13基因编码的蛋白和所述小鼠IGKJ5基因编码的蛋白；
    或所述IFNΑR1的单克隆抗体或其抗原结合部分的重链可变区依次包括所述小鼠IGHV9-3基因编码的蛋白、所述小鼠IGHD1-1基因编码的蛋白和所述小鼠IGHJ3基因编码的蛋白，且其轻链可变区依次包括所述小鼠IGKV6-15基因编码的蛋白和所述小鼠IGKJ2基因编码的蛋白。
  6. 根据权利要求1所述的IFNAR1的单克隆抗体或其抗原结合部分，其特征在于：
    所述IFNΑR1的单克隆抗体或其抗原结合部分的重链可变区包括重链可变区CDR1、重链可变区CDR2和重链可变区CDR3；
    所述IFNΑR1的单克隆抗体或其抗原结合部分的轻链可变区包括轻链可变区CDR1、轻链可变区CDR2和轻链可变区CDR3；
    所述重链可变区CDR1的氨基酸序列为如下(1)或(2)：
    (1)序列3或序列13所示的氨基酸序列；
    (2)将序列3或序列13所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    所述重链可变区CDR2的氨基酸序列为如下(3)或(4)：
    (3)序列4或序列14所示的氨基酸序列；
    (4)将序列4或序列14所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    所述重链可变区CDR3的氨基酸序列为如下(5)或(6)：
    (5)序列5或序列15所示的氨基酸序列；
    (6)或将序列5或序列15所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    所述轻链可变区CDR1的氨基酸序列为如下(7)或(8)：
    (7)序列8或序列18所示的氨基酸序列；
    (8)将序列8或序列18所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    所述轻链可变区CDR2的氨基酸序列为如下(9)或(10)：
    (9)序列9或序列19所示的氨基酸序列；
    (10)将序列9或序列19所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    所述轻链可变区CDR3的氨基酸序列为如下(11)或(12)：
    (11)序列10或序列20所示的氨基酸序列；
    (12)将序列10或序列20所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列。
  7. 根据权利要求6所述的IFNΑR1的单克隆抗体或其抗原结合部分，其特征在于：
    所述IFNΑR1的单克隆抗体的重链可变区的氨基酸序列为a1)或a2)：
    a1)序列2或序列12所示的氨基酸序列；
    a2)将序列2或序列12所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列；
    所述IFNΑR1的单克隆抗体的轻链可变区的氨基酸序列为b1)或b2)：
    b1)序列7或序列17所示的氨基酸序列；
    b2)将序列7或序列17所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的氨基酸序列。
  8. 根据权利要求7所述的抗体，其特征在于：
    所述IFNΑR1的单克隆抗体重链可变区的核苷酸序列为c1)或c2)或c3)：
    c1)序列1或序列11所示的DNA分子；
    c2)与c1)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求6中所述IFNΑR1的单克隆抗体重链可变区的cDNA分子或基因组DNA分子；
    c3)在严格条件下与c1)或c2)限定的核苷酸序列杂交，且编码权利要求6中所述IFNΑR1的单克隆抗体重链可变区的cDNA分子或基因组DNA分子；
    所述IFNΑR1的单克隆抗体轻链可变区的核苷酸序列为d1)或d2)或d3)：
    d1)序列6或序列16所示的DNA分子；
    d2)与d1)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求6中所述IFNΑR1的单克隆抗体轻链可变区的cDNA分子或基因组DNA分子；
    d3)在严格条件下与d1)或d2)限定的核苷酸序列杂交，且编码权利要求6中所述IFNΑR1的单克隆抗体轻链可变区的cDNA分子或基因组DNA分子。
  9. 一株分泌权利要求1-8中任一所述IFNΑR1的单克隆抗体的杂交瘤细胞株，其保藏号为CGMCC No.12542或CGMCC No.12543。
  10. 权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分或权利要求9所述的杂交瘤细胞株在如下A)-F)中至少一种中的应用：
    A)结合人IFNΑR1；
    B)抑制I型干扰素的生物活性；
    C)结合人IFNΑR1的SD2结构域全部或部分和/或SD3结构域全部或部分；
    D)抑制I型干扰素信号报告细胞上I型干扰素的生物活性；
    E)抑制在经过刺激剂刺激的人外周血单个核细胞上I型干扰素的生物活性；
    F)治疗和/或预防I型干扰素介导和/或I型干扰素异常引起的疾病。
  11. 一种I型干扰素的拮抗剂，其活性成分为权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分。
  12. 一种组合物，其含有权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分。
  13. 一种免疫偶联物，其含有权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分。
  14. 根据权利要求13的免疫偶联物，其特征在于：其含有药学上可接受的载体。
  15. 一种抗体基因工程改造的抗体，是将权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分进行修饰和/或改造后得到的抗体。
  16. 一种人源化抗体，是将权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分进行人源化后得到的抗体。
  17. 一种双特异性分子，其含有权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分的特异性结合位点。
  18. 与权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分相关的生物材料，为下述A1)至A12)中的任一种：
    A1)编码权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分的核酸分子；
    A2)含有A1)所述核酸分子的表达盒；
    A3)含有A1)所述核酸分子的重组载体；
    A4)含有A2)所述表达盒的重组载体；
    A5)含有A1)所述核酸分子的重组微生物；
    A6)含有A2)所述表达盒的重组微生物；
    A7)含有A3)所述重组载体的重组微生物；
    A8)含有A4)所述重组载体的重组微生物；
    A9)含有A1)所述核酸分子的转基因细胞系；
    A10)含有A2)所述表达盒的转基因细胞系；
    A11)含有A3)所述重组载体的转基因细胞系；
    A12)含有A4)所述重组载体的转基因细胞系。
  19. 一种制备抗IFNΑR1抗体的方法，包括：
    1)提供：
    (i)重链可变区抗体序列，其含有选自序列3或序列13的CDR1序列，选自序列4或序列14的CDR2序列，和选自序列5或序列15的CDR3序列；
    或(ii)轻链可变区抗体序列，其含有选自序列8或序列18的CDR1序列，选自序列9或序列19的CDR2序列，和选自序列10或序列20的CDR3序列；
    2)改变至少一个可变区抗体序列内的至少一个氨基酸残基，所述序列选自所述重链可变区抗体序列和所述轻链可变区抗体序列，从而产生至少一个改变的抗体序列；
    3)将所述改变的抗体序列表达为蛋白质。
  20. 一种抑制I型干扰素对表达IFNAR1的细胞的生物活性的方法，包括如下步骤：使表达IFNAR1的细胞接触权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分。
  21. 一种治疗I型干扰素介导的疾病的方法，包括向患有I型干扰素介导的疾病的受试者施用权利要求1-8中任一所述IFNΑR1的单克隆抗体或其抗原结合部分，使该受试者中I型干扰素介导的疾病得到治疗。
  22. 根据权利要求21的方法，其特征在于：所述I型干扰素介导的疾病是干扰素α介导的疾病或病症。
  23. 根据权利要求22的方法，其特征在于：所述疾病或病症为如下任一种：系统性红斑狼疮、胰岛素依赖型糖尿病、炎性肠病、多发性硬化症、牛皮癣、自身免疫性甲状腺炎、类风湿性关节炎和肾小球肾炎、HIV感染或AIDS和植排斥或移植物抗宿主疾病。
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