WO2021017071A1 - 抗人st2抗体及其用途 - Google Patents

抗人st2抗体及其用途 Download PDF

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WO2021017071A1
WO2021017071A1 PCT/CN2019/102818 CN2019102818W WO2021017071A1 WO 2021017071 A1 WO2021017071 A1 WO 2021017071A1 CN 2019102818 W CN2019102818 W CN 2019102818W WO 2021017071 A1 WO2021017071 A1 WO 2021017071A1
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seq
amino acid
acid sequence
antibody
variable region
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French (fr)
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刘志刚
刘玉兰
郝小勃
郭晶晶
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北京智仁美博生物科技有限公司
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • 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
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    • 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
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application generally relates to the fields of genetic engineering and antibody drugs; specifically, to the field of anti-human ST2 antibodies and their uses.
  • This application has developed a new anti-human ST2 antibody and provided the use of the antibody in the prevention or treatment of ST2-mediated diseases.
  • ST2 Growth Stimulation Expressed Gene 2
  • ST2 also known as IL1RL1, T1, DER4 or Fit-1
  • TLR Toll-like receptor
  • TIR Toll-like receptor
  • the 1R (TIR) superfamily is a selection marker for Th2 lymphocytes and an important effector molecule for Th2 response.
  • the ST2 gene was first discovered in 1989 by Tominaga et al. in the study of the BALB/c-3T3 cell line, and has been regarded as an orphan receptor for a long time thereafter, until Schmitz et al. discovered that IL-33 can be specifically activated ST2, and confirmed that it is a functional ligand of ST2.
  • ST2 has 4 splice variants, one is the transmembrane protein ST2L, which is mainly expressed in mast cells, Th2 lymphocytes and eosinophils, and forms an IL-33 receptor complex with IL-1RAcP (also known as IL-1R3)
  • IL-1RAcP also known as IL-1R3
  • Receptor which inhibits IL-33-mediated signal transduction
  • ST2V the third variant whose function is not known in the stomach, small intestine and colon is called ST2V, only the third extracellular Ig domain is different from sST2;
  • the four types of ST2LV are similar to ST2L, but do not have a transmembrane domain.
  • IL-33 (also known as IL-1F11) is a cytokine of the IL-1 family. IL-33 is mainly expressed in barrier tissues such as skin, intestine, and lung, and is mainly present in non-hematopoietic cells, especially stromal cells, epithelial cells and Endothelial cells. Full-length IL-33 is a nuclear protein that functions as a transcription factor. There are nuclear localization sequences and chromatin binding domains at the N-terminus. Cell necrosis will lead to the release of functional full-length IL-33. Long IL-33 is cleaved by protease to form mature IL-33 and released to the outside of the cell, where it functions as a cytokine.
  • IL-33 signal transmission depends on the specific receptor ST2 and IL1-RAcP (IL-1 receptor accessory protein).
  • IL-33 combines ST2 and IL-1RAcP to form a ternary signal complex, recruiting MYD88 (myeloid differentiation primary-response protein88), IRAK (IL-1 receptor-associated kinase), TRAF6 (TNF receptor associated factor 6) and/or MAPK (mitogen-activated protein kinase), which activates downstream signal molecules such as NF- ⁇ B (nuclear factor ⁇ B), I ⁇ B ⁇ (inhibitor of NF- ⁇ B- ⁇ ), ERK (extracellular signal regulated kinase), p38, JNK1 (JUN N terminal kinase 1) And AP-1 (activation protein 1).
  • MYD88 myeloid differentiation primary-response protein88
  • IRAK IL-1 receptor-associated kinase
  • TRAF6 TRAF6
  • MAPK mitogen-activated
  • Th2 lymphocytes expressing ST2 secrete IL-5 and IL-13 IL-4, IL-5 and IL-6, keratinocytes express IL-6 and TNF ⁇ , microglia
  • Plasma cells express a large number of pro-inflammatory factors such as TNF ⁇ , IL-1 ⁇ and IL-10, chemokines such as CCL2, CCL3, CCL5 and CXCL10, oxidative stress molecules NO and iNOS.
  • IL-33/ST2L signaling pathway is a bad factor in autoimmune diseases and inflammatory diseases, which promotes the occurrence and development of diseases.
  • the present application provides an antibody that binds to human ST2, which comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 amino acid sequences and a light chain variable region containing LCDR1, LCDR2, and LCDR3 amino acid sequences, wherein
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 36
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 37
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 38
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 40
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 41
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 43
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 44
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 45
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 46
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 47
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 36
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 37
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 38
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 40
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 41
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 43
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 44
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 45
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 46
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 47
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35;
  • HCDR and LCDR are defined by Kabat.
  • amino acid of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 26, 27, 28, 29 or 30.
  • amino acid of the light chain variable region of the antibody is as shown in SEQ ID NO: 24 or 25.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 26
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25.
  • the present application provides an antibody that binds to human ST2, wherein the amino acid sequence of the variable region of the heavy chain of the antibody has at least 90 percent from any one of SEQ ID NOs: 26, 27, 28, 29, and 30. % Identity, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with any one of SEQ ID NOs: 24 and 25.
  • the antibody is a monoclonal antibody.
  • the antibody is a whole antibody, Fab fragment, F(ab') 2 fragment, or single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of IgG1 subtype, IgG2 subtype, or IgG4 subtype.
  • the antibody further comprises a light chain constant region selected from the group consisting of ⁇ subtype or ⁇ subtype.
  • the antibody can bind to recombinant human ST2 (SEQ ID NO: 1), recombinant mouse ST2 (SEQ ID NO: 2) and recombinant monkey ST2 (SEQ ID NO: 3). ).
  • the antibody is capable of binding to recombinant human ST2 (SEQ ID NO: 1) and recombinant monkey ST2 (SEQ ID NO: 3).
  • the antibody inhibits the binding of human ST2 to human IL-33.
  • this application provides a nucleic acid molecule that encodes the antibody or antigen-binding portion thereof as described in the first or second aspect.
  • this application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody described in the first or second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is used to prevent or treat ST2-mediated diseases.
  • the ST2-mediated disease is an autoimmune disease or an inflammatory disease, such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-nephritis syndrome, allergic pneumonia, atopic dermatitis, asthma, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjogren’s syndrome and multiple sclerosis.
  • an inflammatory disease such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-ne
  • this application provides the use of the antibody described in the first or second aspect in the preparation of drugs for preventing or treating ST2-mediated diseases.
  • the ST2-mediated disease is an autoimmune disease or an inflammatory disease, such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-nephritis syndrome, allergic pneumonia, atopic dermatitis, asthma, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjogren’s syndrome and multiple sclerosis.
  • an inflammatory disease such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-ne
  • this application provides a method for preventing or treating ST2-mediated diseases, including administering the antibody of the first or second aspect or the pharmaceutical composition of the fourth aspect to an individual in need.
  • the ST2-mediated disease is an autoimmune disease or an inflammatory disease, such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-nephritis syndrome, allergic pneumonia, atopic dermatitis, asthma, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjogren’s syndrome and multiple sclerosis.
  • an inflammatory disease such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-ne
  • Figure 1 shows the ELISA analysis of the ability of anti-human ST2 monoclonal antibody S1A4 to inhibit the binding of ST2 to IL-33.
  • Figure 2 shows the ELISA analysis of the ability of different anti-human ST2 monoclonal antibodies to inhibit the binding of ST2 to IL-33.
  • Figure 3 shows the detection result of anti-human ST2 monoclonal antibody blocking ST2 activity based on HEK-BlueTM IL-33 cells.
  • Figure 4 shows the detection result of anti-human ST2 monoclonal antibody inhibiting IL-33 and IL-12 co-stimulated PBMC secreting IFN ⁇ .
  • Figure 5 shows the ELISA analysis of the binding ability of recombinant anti-hST2 monoclonal antibodies to ST2 of different species.
  • SEQ ID NO:1 shows the amino acid sequence of the extracellular region of human (homo sapiens) ST2 (h ST2).
  • SEQ ID NO: 2 shows the amino acid sequence of the extracellular region of mouse (musculus) ST2 (m ST2).
  • SEQ ID NO: 3 shows the amino acid sequence of the ST2 extracellular region (mm ST2) of Macaca (Macaca mulatta).
  • SEQ ID NO: 4 shows the amino acid sequence of the extracellular domain of human (homo sapiens) IL-33 (hIL-33).
  • SEQ ID NO: 5 shows the amino acid sequence of His tag (His).
  • SEQ ID NO: 6 shows the amino acid sequence of the Fc segment (mFc1) of the human (homo sapiens) IgG1 antibody.
  • SEQ ID NO: 7 shows the amino acid sequence of the Fc segment (mFc) of the mouse (mus musculus) IgG2a antibody.
  • SEQ ID NO: 8 shows the amino acid sequence of the heavy chain constant region of human (homo sapiens) IgG1 subtype.
  • SEQ ID NO: 9 shows the amino acid sequence of the heavy chain constant region of human (homo sapiens) IgG2 subtype.
  • SEQ ID NO: 10 shows the amino acid sequence of the heavy chain constant region of the human (homo sapiens) IgG4 subtype.
  • SEQ ID NO: 11 shows the amino acid sequence of the heavy chain constant region of the mouse (mus musculus) IgG1 subtype.
  • SEQ ID NO: 12 amino acid sequence of the heavy chain constant region of mouse (mus musculus) IgG2a subtype.
  • SEQ ID NO: 13 shows the amino acid sequence of the human (homo sapiens) ⁇ subtype light chain constant region.
  • SEQ ID NO: 14 shows the amino acid sequence of the light chain constant region of human (homo sapiens) subtype lambda.
  • SEQ ID NO: 15 shows the amino acid sequence of the constant region of the mouse (mus musculus) k subtype light chain.
  • SEQ ID NO: 16 shows the amino acid sequence of the constant region of the murine (mus musculus) lambda light chain.
  • SEQ ID NO: 17 shows the amino acid sequence of the fully human single-chain antibody S1A4.
  • SEQ ID NO: 18 shows the amino acid sequence of the heavy chain variable region (S1A4 VH) of the fully human single-chain antibody S1A4.
  • SEQ ID NO: 19 shows the amino acid sequence of the light chain variable region (S1A4 VK) of the fully human single-chain antibody S1A4.
  • SEQ ID NO: 20 shows the amino acid sequence of the heavy chain variable region (MSTT1041A VH) of the anti-human ST2 control antibody MSTT1041A.
  • SEQ ID NO: 21 shows the amino acid sequence of the light chain variable region (MSTT1041A VK) of the anti-human ST2 control antibody MSTT1041A.
  • SEQ ID NO: 22 shows the amino acid sequence of the heavy chain variable region (GSK3772847 VH) of the anti-human ST2 control antibody GSK3772847.
  • SEQ ID NO: 23 shows the amino acid sequence of the light chain variable region (GSK3772847 VK) of the anti-human ST2 control antibody GSK3772847.
  • SEQ ID NO: 24 shows the amino acid sequence of the light chain variable region mutant L1D1, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, respectively.
  • SEQ ID NO: 25 shows the amino acid sequence of the light chain variable region mutant L2G2, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 34, SEQ ID NO: 32 and SEQ ID NO: 35, respectively.
  • SEQ ID NO: 26 shows the amino acid sequence of the heavy chain variable region mutant H5E10.
  • the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 39, SEQ ID NO: 42 and SEQ ID NO: 43, respectively.
  • SEQ ID NO: 27 shows the amino acid sequence of the heavy chain variable region mutant H5G3.
  • the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, respectively.
  • SEQ ID NO: 28 shows the amino acid sequence of the heavy chain variable region mutant H5G10, and the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively.
  • SEQ ID NO: 29 shows the amino acid sequence of the heavy chain variable region mutant H6D3.
  • the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 39, SEQ ID NO: 44 and SEQ ID NO: 45, respectively.
  • SEQ ID NO: 30 shows the amino acid sequence of the heavy chain variable region mutant H6D6.
  • the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 46, SEQ ID NO: 42 and SEQ ID NO: 47, respectively.
  • SEQ ID NO: 48 shows the amino acid sequence of human (homo sapiens) IL-12 ⁇ .
  • SEQ ID NO: 49 shows the amino acid sequence of human (homo sapiens) IFN ⁇ .
  • new anti-human ST2 antibodies or antigen-binding fragments thereof there are provided new anti-human ST2 antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, vectors containing the nucleic acid molecules, and nucleic acid molecules or vectors containing the nucleic acid molecules or vectors.
  • a full-length antibody molecule can be constructed as a medicine for the treatment of clinically ST2-mediated diseases.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule.
  • Targets include but are not limited to carbohydrates, polynucleotides, lipids, polypeptides, etc.
  • antibody not only includes complete (ie, full-length) antibodies, but also includes antigen-binding fragments thereof (such as Fab, Fab', F(ab') 2 , Fv), variants thereof, and antibody portions. Fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g. bispecific antibodies) and any other immunoglobulin containing antigen recognition sites of the desired specificity Modified configuration of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • a complete or full-length antibody contains two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3).
  • Each light chain contains a light chain variable region (VL) and constant region (CL).
  • the full-length antibody can be any kind of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses above), but the antibody does not need to belong to any specific class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to different immunoglobulin classes are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • antigen binding domain refers to a part or region of a complete antibody molecule that is responsible for binding antigen.
  • the antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • VH and VL usually contains three complementarity determining regions CDR1, CDR2, and CDR3.
  • CDR complementarity determining region
  • the CDR sequences in the VH and VL sequences can be determined according to the Chothia definition or the Kabat definition. In this article In the embodiments of the application, Chothia is used to define CDR sequences.
  • variable region sequence of a given antibody the middle CDR sequence of the variable region sequence can be analyzed in various ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which can be monovalent fragments with VL-CL chains and VH-CH1 chains; (2) F(ab') 2 fragments, which can have two A bivalent fragment of Fab' fragment, the two Fab' fragments are connected by a disulfide bridge in the hinge region (ie, a dimer of Fab'); (3) Fv fragments with VL and VH domains with one arm of an antibody; 4) Single-chain Fv (scFv), which can be a single multi-peptide chain composed of a VH domain and a VL domain via a peptide linker; and (5)(scFv) 2 , which can contain two peptides connected The VH domain and two VL domains connected by a symbol are combined with the two VH domains via a disulfide bridge.
  • Fab fragments which can be monovalent fragments with VL-CL chains and VH-CH1 chains
  • amino acid position numbering refers to the EU numbering definition of human IgG1 antibody, which is well known and easily found by those skilled in the art.
  • a mutation refers to a mutation generated relative to the natural antibody sequence.
  • Fc fragment refers to a part of the constant region of an antibody heavy chain, including the hinge region, the CH2 fragment and the CH3 fragment of the constant region.
  • the Fc fragment is the amino acid sequence of positions 216-447 in the constant region of the antibody.
  • binding refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is the same except for the naturally occurring mutations that may be present in a small number of individuals.
  • the monoclonal antibodies described herein particularly include “chimeric" antibodies, in which a part of the heavy chain and/or light chain is the same or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and The remaining part of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies as long as they can exhibit the desired (US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984).
  • autoimmune disease refers to a disease caused by the body's immune response to self-antigens leading to damage to its own tissues, including but not limited to scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg- Strauss syndrome, Wegener's granulomatosis (Wegener's granulomatosis) and pulmonary hemorrhage-nephritis syndrome.
  • inflammatory disease refers to a general term for diseases in which inflammation is the main destructive factor. Inflammation is the biological response of tissues to harmful stimuli, which is a kind of pathology, and is accompanied by three events of tissue degradation, circulatory disturbance and fluid exudation, as well as hypertrophy.
  • inflammatory diseases include acute and chronic diseases, including but not limited to allergic pneumonia, atopic dermatitis, asthma, rhinitis, Crohn’s disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis , Chronic nephritis, Sjogren’s syndrome, and multiple sclerosis.
  • the present application provides an antibody that binds to human ST2, which comprises a heavy chain variable region containing HCDR1, HCDR2, and HCDR3 amino acid sequences and a light chain variable region containing LCDR1, LCDR2, and LCDR3 amino acid sequences, wherein
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 36
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 37
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 38
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 40
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 41
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 43
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 44
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 45
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 46
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 47
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 31
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 33; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 36
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 37
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 38
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 40
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 41
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 43
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 39
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 44
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 45
  • the LCDR1 amino acid sequence is shown in SEQ ID NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; or
  • the HCDR1 amino acid sequence is shown in SEQ ID NO: 46
  • the HCDR2 amino acid sequence is shown in SEQ ID NO: 42
  • the HCDR3 amino acid sequence is shown in SEQ ID NO: 47
  • the LCDR1 amino acid sequence is shown in SEQ ID
  • the LCDR2 amino acid sequence is shown in NO: 34
  • the LCDR2 amino acid sequence is shown in SEQ ID NO: 32
  • the LCDR3 amino acid sequence is shown in SEQ ID NO: 35; wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.
  • amino acid of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 26, 27, 28, 29 or 30.
  • amino acid of the light chain variable region of the antibody is shown in SEQ ID NO: 24 or 25.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 26
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 24; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25; or
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25.
  • the present application provides an antibody that binds to human ST2, wherein the amino acid sequence of the variable region of the heavy chain of the antibody has at least 90 percent from any one of SEQ ID NOs: 26, 27, 28, 29, and 30. % Identity, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity with any one of SEQ ID NOs: 24 and 25.
  • the antibody is a monoclonal antibody.
  • the antibody is a whole antibody, Fab fragment, F(ab') 2 fragment, or single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of IgG1 subtype, IgG2 subtype, or IgG4 subtype.
  • the heavy chain constant region of the antibody may be of human IgG1 subtype, human IgG2 subtype, human IgG4 subtype, murine IgG1 subtype, or murine IgG2a subtype.
  • the heavy chain constant region is of the IgG4 subtype.
  • the heavy chain constant region of the antibody comprises the Fc segment sequence of the heavy chain constant region of the human IgG1 subtype and the positions 234, 235 and 331 of the Fc sequence
  • the amino acid sequences are F, E and S respectively, and the amino acid sequence of the antibody constant region is determined according to EU numbering.
  • the antibody further comprises a light chain constant region selected from the group consisting of ⁇ subtype or ⁇ subtype.
  • the light chain constant region of the antibody may be human kappa subtype, human lambda subtype, murine kappa subtype, or murine lambda subtype.
  • the antibody can bind to recombinant human ST2 (SEQ ID NO: 1), recombinant mouse ST2 (SEQ ID NO: 2) and recombinant monkey ST2 (SEQ ID NO: 3). ).
  • the antibody is capable of binding to recombinant human ST2 (SEQ ID NO: 1) and recombinant monkey ST2 (SEQ ID NO: 3).
  • the antibody inhibits the binding of human ST2 to human IL-33.
  • this application provides a nucleic acid molecule that encodes the antibody or antigen-binding portion thereof as described in the first or second aspect.
  • the nucleic acid molecule is operably linked to a regulatory sequence, which can be recognized by a host cell transformed with the vector.
  • this application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody described in the first or second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is used to prevent or treat ST2-mediated diseases.
  • the ST2-mediated disease is an autoimmune disease or an inflammatory disease, such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-nephritis syndrome, allergic pneumonia, atopic dermatitis, asthma, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjogren’s syndrome and multiple sclerosis.
  • an inflammatory disease such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-ne
  • the pharmaceutical composition may further include one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives Agents such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifiers such as talc, magnesium stearate, and mineral oil
  • suspending agents such as benzoic acid, sorbic acid and calcium propionate
  • preservatives Agents such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and/or flavoring agents such as benzoic acid, sorbic acid and calcium propionate
  • the pharmaceutical composition of the present application can be formulated into the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.
  • any physiologically acceptable mode of administration can be used to deliver the pharmaceutical composition of the present application.
  • These modes of administration include, but are not limited to: oral administration, parenteral administration, nasal administration, rectal administration Medicine, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • a pharmaceutical composition for therapeutic use can be formulated in the form of a lyophilized preparation or an aqueous solution by mixing reagents with the required purity with pharmaceutically acceptable carriers, excipients, etc., as appropriate. storage.
  • this application provides the use of the antibody described in the first or second aspect in the preparation of drugs for preventing or treating ST2-mediated diseases.
  • the ST2-mediated disease is an autoimmune disease or an inflammatory disease, such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-nephritis syndrome, allergic pneumonia, atopic dermatitis, asthma, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjogren’s syndrome and multiple sclerosis.
  • an inflammatory disease such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-ne
  • this application provides a method for preventing or treating ST2-mediated diseases, including administering the antibody of the first or second aspect or the pharmaceutical composition of the fourth aspect to an individual in need.
  • the ST2-mediated disease is an autoimmune disease or an inflammatory disease, such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-nephritis syndrome, allergic pneumonia, atopic dermatitis, asthma, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjogren’s syndrome and multiple sclerosis.
  • an inflammatory disease such as scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis ( Wegener's granulomatosis), pulmonary hemorrhage-ne
  • the application also provides an isolated nucleic acid molecule encoding the antibody of the invention or its light or heavy chain, a vector containing the nucleic acid molecule, a host cell containing the vector, and a method for producing the antibody.
  • the nucleic acid molecule is operably linked to a regulatory amino acid sequence, and the regulatory amino acid sequence can be recognized by a host cell transformed with the vector.
  • methods of producing antibodies include culturing host cells to facilitate expression of nucleic acids.
  • the method of producing an antibody further includes recovering the antibody from the host cell culture medium.
  • antibodies described herein that specifically bind to human ST2 can also be used to detect the presence of ST2 in biological samples.
  • Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.
  • anti-ST2 monoclonal antibodies requires a variety of different recombinant proteins, including human ST2 extracellular domain (hST2, SEQ ID NO: 1), mouse ST2 extracellular domain (mST2, SEQ ID NO: 2) , The extracellular region of macaque ST2 (mmST2, SEQ ID NO: 3) and the extracellular region of human IL-33 (hIL-33, SEQ ID NO: 4).
  • These proteins have a large number of post-translational modifications (such as glycosylation or disulfide bonds, etc.), so the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of the recombinant protein.
  • His tag His, SEQ ID NO: 5
  • Fc segment of human antibody IgG1 hFc, SEQ ID NO: 6
  • mFc, SEQ ID Fc segment of mouse antibody IgG2a
  • the heavy chain constant region of an antibody can be of human IgG1 subtype (SEQ ID NO: 8), human IgG2 subtype (SEQ ID NO: 9), human IgG4 subtype (SEQ ID NO: 10), or mouse IgG1 subtype (SEQ ID NO: 11), mouse IgG2a subtype (SEQ ID NO: 12), the light chain constant region can be human ⁇ subtype (SEQ ID NO: 13), human ⁇ subtype (SEQ ID NO: 14) or mouse ⁇ subtype Type (SEQ ID NO: 15), murine lambda subtype (SEQ ID NO: 16).
  • the genes of the above-mentioned various recombinant proteins are designed and synthesized.
  • the recombinant protein expressed by His tag fusion is purified by a metal chelating affinity chromatography column (such as HisTrap FF of GE Company, etc.) in the culture supernatant.
  • the recombinant protein expressed by the fusion of hFc and mFc is purified by a ProteinA/G affinity chromatography column (such as Mabselect SURE from GE). Then use a desalting column (such as Hitrap desaulting from GE) to replace the recombinant protein storage buffer with PBS (pH 7.0) or other suitable buffers. If necessary, the antibody sample can be filtered and sterilized, and then aliquoted and stored at -20°C.
  • Example 3 Screening of anti-human ST2 monoclonal antibodies using phage display antibody library technology
  • Example 2 Using the recombinant human ST2-his prepared in Example 2 as the antigen, using a solid-phase screening strategy (refer to the experimental protocol for phage display: General Experimental Guide / (United States) Clarkson (Clackson, T.), (United States) Lowman (Lowman, HB) edited; Ma Lan et al. Translated by Chemical Industry Press, 2008.5)
  • the phage library prepared in Example 1 was screened to display the human single-chain antibody library, and finally a fully human single-chain antibody S1A4 that specifically binds to human ST2 was obtained.
  • nucleic acid molecules encoding the heavy chain variable region (S1A4 VH, SEQ ID NO: 18) and the light chain variable region (S1A4VK, SEQ ID NO: 19) of S1A4 were cloned into the fusion
  • MSTT1041A MSTT1041A VH, SEQ ID NO: 20; MSTT1041A VK, SEQ ID NO: 21
  • GSK3772847 GSK3772847 VH, SEQ ID NO: 22; GSK3772847 ID VK, SEQ ID NO: 21
  • the recombinant protein hST2-his was coated on a 96-well plate (1 ⁇ g/mL, 100 ⁇ l/well), and coated overnight at 4°C. Then, the antibodies S1A4, MSTT1041A and GSK3772847 were serially diluted with PBST-2% milk containing 0.5 ⁇ g/mL recombinant protein hIL33-mFc. The initial concentration of the antibody is 200 ⁇ g/mL, and it is diluted by 3 times, a total of 9 concentration gradients.
  • the binding signal of hIL33-mFc was detected by HRP goat anti-mouse IgG ((Zhongshan Jinqiao, ZB2305), and then graphPad Prism 6 was used for data analysis and mapping.
  • Biacore X100 was used to determine the affinity of anti-ST2 antibodies by surface plasmon resonance technology.
  • Amino coupling kit (BR-1000-50), human antibody capture kit (BR-1008-39), CM5 chip (BR100012) and pH7.4 10 ⁇ HBS-EP (BR100669) and other related reagents and consumables are available Purchased from GE healthcare.
  • 1M ethanolamine was injected to block unreacted groups.
  • set a series of concentration gradients for hST2-his for example, 0.625nM, 1.25nM, 2.5nM, 5nM, 10nM
  • the binding time is 120s, and the dissociation The time is 1800s, and the surface of the chip is regenerated by injecting 3M MgCl 2 solution at 10 ⁇ L/min for 30s.
  • the heavy chain variable region of S1A4 (S1A4VH, SEQ ID NO: 18) and the light chain variable region mutant L1D1 (S1A4VK, SEQ ID NO: 24) or L2G2 (amino acid sequence is SEQ ID NO:
  • the nucleic acid sequence of 25 was cloned into the eukaryotic expression vector fused with the nucleic acid sequence encoding the human heavy chain constant region and the light chain constant region, and combined to express the full antibodies S1A4VH+L1D1 and S1A4VH+L2G2.
  • the affinity analysis of the new light chain variable region of S1A4 was performed using Biacore X100, and the results are shown in Table 3.
  • the heavy chain constant regions of S1A4, S1A4VH+L1D1 and S1A4VH+L2G2 are of the IgG4 subtype.
  • H5E10 SEQ ID NO: 26
  • H5G3 SEQ ID NO: 27
  • H5G10 SEQ ID NO: 28
  • H6D3 SEQ ID NO: 29
  • H6D6 SEQ ID NO: 30
  • Example 3.2 to encode the heavy chain variable region mutants H5E10 (amino acid sequence is SEQ ID NO: 26), H5G3 (amino acid sequence is SEQ ID NO: 27), H5G10 (amino acid sequence is SEQ ID NO: 28), H6D3 (Amino acid sequence is SEQ ID NO: 29) or H6D6 (amino acid sequence is SEQ ID NO: 30) and the nucleic acid molecules of the light chain variable region mutant L2G2 (amino acid sequence is SEQ ID NO: 25) are cloned into the fusion code A eukaryotic expression vector of human heavy chain constant region and light chain constant region nucleic acid molecules, combined to express whole antibodies.
  • the affinity analysis of S1A4 mutants was performed using Biacore X100, and the results are shown in Table 7.
  • the heavy chain constant regions of S1A4, H5G3+L2G2, H5E10+L2G2, H5G10+L2G2, H6D3+L2G2, and H6D6+L2G2 are of the IgG4 subtype.
  • the recombinant protein hST2-Fc was coated on a 96-well plate (3 ⁇ g/mL, 100 ⁇ l/well), and coated overnight at 4°C. Then, each anti-ST2 antibody protein (IgG4 subtype) was serially diluted with PBST-2% milk containing 0.4 ⁇ g/mL recombinant protein hIL33-his. The initial concentration of the antibody is 50 ⁇ g/mL, with a 3-fold dilution, a total of 10 concentration gradients. HRP mouse anti-his IgG (Kangwei Century, CW0285M) was used to detect the binding signal of IL33-his, and then GraphPad Prism 6 was used for data analysis and mapping. The ELISA analysis results (Figure 2) show that different anti-ST2 monoclonal antibodies can effectively inhibit the binding of IL33 to ST2. The IC 50 values of each antibody are shown in Table 8.
  • Anti-ST2 antibody inhibits the IC 50 value of ST2 and IL33 binding
  • Example 6 The anti-human ST2 monoclonal antibody blocks the effect of ST2 based on HEK-BlueTM IL-33 cells
  • HEK-Blue TM IL-33 cells Stimulate HEK-Blue TM IL-33 cells (InvivoGen) with IL-33, activate NF- ⁇ B and AP-1 signaling pathways, start cells to express secreted alkaline phosphatase (SEAP), and detect the effect of antibodies on the expression level of SEAP The blocking effect of anti-ST2 antibody can be judged.
  • HEK-Blue TM IL-33 cells express ST2L, which is obtained by transferring the IL1RL1 gene to human HEK cells, and integrates the SEAP reporter gene controlled by the IFN- ⁇ small promoter. The promoter is fused with 5 NF- ⁇ B and 5 AP-1 binding site.
  • HEK-Blue TM IL-33 cells simultaneously block TNF- ⁇ and IL-1 ⁇ responses, so that HEK-Blue TM IL-33 cells only respond to IL-33.
  • HEK cells express endogenous IL-1RAcP.
  • hIL-33 (GenScript, Z03198) solution with test medium (DMEM+10%FBS+1%PS+100 ⁇ g/mLNormocin), and use the solution to resist human ST2 monoclonal antibodies (MSTT1041A, GSK3772847, H5G3+L2G2 H5G10+L2G2, H5G10+L1D1 and H5G3+L1D1) for serial dilution (antibody concentration gradients are 1, 4, 16, 64, 256, 1024, 4096, 16384, 65536, 262144pM), antibody solution and equal volume of HEK-Blue IL-33 cells (cell density 5 ⁇ 10 5 cells/mL) were mixed and seeded in a 96-well cell plate with 200 ⁇ L per well.
  • test medium DMEM+10%FBS+1%PS+100 ⁇ g/mLNormocin
  • the cell plate was placed in a CO 2 incubator and cultured at 37°C. After 23 hours, the cell plate was centrifuged at 1200 rpm for 5 minutes, 20 ⁇ L of supernatant and 180 ⁇ L of QUANTI-Blue TM (InvivoGen) were mixed in a 96-well plate, developed at 37°C for 30 minutes, and OD630 was measured. Then, GraphPad Prism 6 was used for data analysis and operation Figure.
  • Example 7 Anti-human ST2 monoclonal antibody inhibits the secretion of IFN ⁇ from PBMC co-stimulated by IL-33 and IL-12
  • primary NK cells can also be used to detect the blocking effect of anti-ST2 antibodies.
  • IL-33 and IL-12 each cannot activate NK cells alone, but IL-12 can significantly increase the expression of ST2 on the surface of NK cells.
  • Use IL-33 and IL-12 to stimulate freshly isolated NK cells, IL-33 and NK cell surface ST2 binding can activate NK cells to release a large amount of IFN- ⁇ .
  • the blocking effect of anti-ST2 antibody can be judged by detecting the effect of antibody on the expression level of IFN- ⁇
  • Inoculate a 96-well cell plate (1.5 ⁇ 10 6 cells/mL, 1640 medium + 10% fetal bovine serum + 1% PS) with human PBMC cells, 100 ⁇ L per well, and add 1 ng/ml IL-12-His (Yiqiao Shenzhou, CT050-H08H) + 0.1 nM IL-33 (GenScript, Z03198) + 5-fold serial dilution antibodies (MSTT1041A, GSK3772847, H5G3+L1D1 and H5G10+L2G2, the antibody concentration gradients are 0.00256, 0.0128, 0.064, 0.32, 1.6, 8 , 40, 200, 1000, 5000pM), the total reaction volume is 200 ⁇ L/well, and the reaction is performed in a CO 2 incubator at 37°C.
  • the cell plate is centrifuged at 1200rpm for 5 minutes, and the IFN- ⁇ in the supernatant (human IFN- ⁇ ELISA kit, Daktronics, DKW12-1000-096), and then use GraphPad Prism 6 for data analysis and mapping.
  • Both H5G3+L1D1 and H5G10+L2G2 inhibited IL-33 and IL-12 co-stimulating PBMC secretion of IFN ⁇ in a dose-dependent manner ( Figure 4).
  • the IC 50 value of each antibody is shown in Table 10.
  • the heavy chain constant regions of MSTT1041A, GSK3772847, H5G3+L1D1 and H5G10+L2G2 are of the IgG4 subtype.
  • the prepared human ST2 (hST2-his), macaque ST2 (mmST2-his) and mouse ST2 (mST2-his) were respectively coated on a 96-well ELISA plate (1 ⁇ g/mL, 100 ⁇ l/well), while coating 10% As an irrelevant control, BSA was coated overnight at 4°C. After blocking with blocking solution PBS-0.1% Tween20-3% milk at 37°C for 1 hour, add the recombinant anti-ST2 monoclonal antibodies (S1A4, H5G3+L2G2, H5E10+L2G2, H5G10+L2G2, H6D3+L2G2, H6D6+ Combine L2G2 and H5G3+L1D1) at 37°C for 1 hour.
  • ELISA analysis results showed that six anti-ST2 monoclonal antibodies (S1A4, H5G3+L2G2, H5E10+L2G2, H6D3+L2G2, H6D6+L2G2, and H5G3+L1D1) can specifically recognize human ST2 and interact with monkey ST2.
  • Mouse ST2 has cross-reactivity, H5G10+L2G2 does not bind to mouse ST2.
  • the heavy chain constant regions of the seven monoclonal antibodies are all of the IgG4 subtype, and the light chain constant regions are of the ⁇ subtype.

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Abstract

本申请公开了结合人ST2的抗体或其抗原结合部分，编码所述抗体或其抗原结合部分的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体或其抗原结合部分的应用。

Description

抗人ST2抗体及其用途
相关申请的引用
本申请要求于2019年7月31日递交的中国专利申请第201910700722.0号的优先权，其全部内容通过引用整体并入本文。
技术领域
本申请大体涉及基因工程和抗体药物领域；具体而言，涉及抗人ST2抗体领域及其用途。本申请开发了新的抗人ST2抗体，并提供了该抗体在预防或治疗ST2介导的疾病中的用途。
背景技术
生长刺激表达基因2蛋白(Growth Stimulation Expressed Gene 2，ST2)也被称为IL1RL1、T1、DER4或Fit-1，是IL-1受体家族的成员，属于Toll样受体(TLR)/IL-1R(TIR)超家族，是Th2淋巴细胞的选择标记，是Th2反应的重要的效应分子。ST2基因于1989年由Tominaga等人在研究BALB/c-3T3细胞系时首次被发现，其后很长一段时间一直被认为是孤儿受体，直到Schmitz等人发现了IL-33可特异性激活ST2，并证实其为ST2的功能配体。ST2有4种剪切变体，一种是跨膜蛋白ST2L，主要表达于肥大细胞、Th2淋巴细胞和嗜酸性粒细胞，与IL-1RAcP(也称IL-1R3)形成IL-33受体复合物；一种是短的分泌的剪切变体sST2，C端比ST2L胞外区多9个氨基酸残基，主要表达于胚胎组织、乳腺肿瘤和成纤维细胞，是IL-33的拮抗型伪受体，抑制IL-33介导的信号传导；第三种在胃、小肠和结肠中表达的功能尚不清楚的变体称为ST2V，只有第三个胞外Ig结构域与sST2不同；第四种ST2LV与ST2L相似，但是没有跨膜结构域。
IL-33(也称IL-1F11)是IL-1家族的细胞因子，IL-33主要在皮肤、肠、肺等屏障组织表达，主要存在于非造血细胞中，尤其是基质细胞、上皮细胞和内皮细胞。全长IL-33是核蛋白，发挥转录因子作用，N 端有核定位序列和染色质结合结构域，细胞坏死会导致有功能的全长IL-33释放，而当有促炎因子刺激时全长IL-33经蛋白酶切割形成成熟的IL-33释放到胞外，发挥细胞因子功能。IL-33信号传递依赖于特异受体ST2和IL1-RAcP(IL-1receptor accessory protein)。IL-33结合ST2和IL-1RAcP形成三元信号复合物，募集MYD88(myeloid differentiation primary-response protein88)、IRAK(IL-1 receptor-associated kinase)、TRAF6(TNF receptor associated factor 6)和/或MAPK(mitogen-activated protein kinase)，激活下游信号分子如NF-κB(nuclear factorκB)、IκBα(inhibitor of NF-κB-α)、ERK(extracellular signal regulated kinase)、p38、JNK1(JUN N terminal kinase 1)和AP-1(activation protein 1)。在IL-33的作用下，表达ST2的Th2淋巴细胞分泌IL-5和IL-13，肥大细胞表达IL-4、IL-5和IL-6，角质细胞表达IL-6和TNFα，小神经胶质细胞表达大量促炎因子如TNFα、IL-1β和IL-10，趋化因子如CCL2、CCL3、CCL5和CXCL10，氧化应激分子NO和iNOS。
IL-33/ST2L信号通路在自身免疫性疾病和炎性疾病等中是不良因素，促进了疾病的发生发展。
基于临床需求，探索和研发可以结合ST2分子的药物具有重要的生物学和医学意义。
发明概述
第一方面，本申请提供了一种结合人ST2的抗体，其包含含HCDR1、HCDR2和HCDR3氨基酸序列的重链可变区和含LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区，其中
所述HCDR1氨基酸序列如SEQ ID NO:36、所述HCDR2氨基酸序列如SEQ ID NO:37所示、所述HCDR3氨基酸序列如SEQ ID NO:38所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示，所述HCDR2 氨基酸序列如SEQ ID NO:40所示、所述HCDR3氨基酸序列如SEQ ID NO:41所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:43所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:44所示、所述HCDR3氨基酸序列如SEQ ID NO:45所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:46所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:47所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:36所示、所述HCDR2氨基酸序列如SEQ ID NO:37所示、所述HCDR3氨基酸序列如SEQ ID NO:38所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:40所示、所述HCDR3氨基酸序列如SEQ ID NO:41所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2 氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:43所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:44所示、所述HCDR3氨基酸序列如SEQ ID NO:45所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:46所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:47所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；
其中HCDR和LCDR氨基酸氨基酸序列根据Kabat定义。
在一些实施方案中，所述抗体的重链可变区的氨基酸如SEQ ID NO:26、27、28、29或者30所示。
在一些实施方案中，所述抗体的轻链可变区的氨基酸如SEQ ID NO:24或者25所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:26所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:27所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:29所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:26所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:27所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:29所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示。
第二方面，本申请提供了一种结合人ST2的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:26、27、28、29和30中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:24和25中任何一项具有至少90％的一致性。
在第一方面和第二方面的一些实施方案中，所述抗体为单克隆抗体。
在第一方面和第二方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面和第二方面的一些实施方案中，所述抗体为全人源抗体。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自κ亚型或者λ亚型的轻链恒定区。
在第一方面和第二方面的一些实施方案中，所述抗体能够结合重组人ST2(SEQ ID NO:1)、重组鼠ST2(SEQ ID NO:2)和重组猴ST2(SEQ ID NO:3)。
在第一方面和第二方面的一些实施方案中，所述抗体能够结合重组人ST2(SEQ ID NO:1)和重组猴ST2(SEQ ID NO:3)。
在第一方面和第二方面的一些实施方案中，所述抗体抑制人ST2 与人IL-33结合。
第三方面，本申请提供了核酸分子，其编码第一方面或第二方面所述的抗体或其抗原结合部分。
第四方面，本申请提供了药物组合物，其包含第一方面或第二方面所述的抗体和药学上可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物用于预防或治疗ST2介导的疾病。
在一些实施方案中，所述ST2介导的疾病为自身免疫疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
第五方面，本申请提供了第一方面或第二方面所述的抗体在制备用于预防或治疗ST2介导的疾病的药物中的用途。
在一些实施方案中，所述ST2介导的疾病为自身免疫疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
第六方面，本申请提供了预防或治疗ST2介导的疾病的方法，包括向有需要的个体给予第一方面或第二方面所述的抗体或第四方面所述的药物组合物。
在一些实施方案中，所述ST2介导的疾病为自身免疫疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
附图说明
图1显示ELISA分析抗人ST2单克隆抗体S1A4抑制ST2与IL-33结合的能力。
图2显示ELISA分析不同抗人ST2单克隆抗体抑制ST2与IL-33结合的能力。
图3显示抗人ST2单克隆抗体基于HEK-BlueTM IL-33细胞阻断ST2活性的检测结果。
图4显示抗人ST2单克隆抗体抑制IL-33与IL-12共刺激的PBMC分泌IFNγ的检测结果。
图5显示ELISA分析重组抗hST2单克隆抗体与不同种属ST2的结合能力。
序列说明
SEQ ID NO:1显示人(homo sapiens)ST2胞外区(h ST2)的氨基酸序列。
SEQ ID NO:2显示小鼠(mus musculus)ST2胞外区(m ST2)的氨基酸序列。
SEQ ID NO:3显示猕猴(Macaca mulatta)ST2胞外区(mm ST2)的氨基酸序列。
SEQ ID NO:4显示人(homo sapiens)IL-33胞外区(hIL-33)的氨基酸序列。
SEQ ID NO:5显示His标签(His)的氨基酸序列。
SEQ ID NO:6显示人(homo sapiens)IgG1抗体的Fc段(mFc1)的氨基酸序列。
SEQ ID NO:7显示小鼠(mus musculus)IgG2a抗体的Fc段(mFc)的氨基酸序列。
SEQ ID NO:8显示人(homo sapiens)IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:9显示人(homo sapiens)IgG2亚型重链恒定区的氨基酸序列。
SEQ ID NO:10显示人(homo sapiens)IgG4亚型重链恒定区的氨基酸序列。
SEQ ID NO:11显示小鼠(mus musculus)IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:12小鼠(mus musculus)IgG2a亚型重链恒定区的氨基酸序列。
SEQ ID NO:13显示人(homo sapiens)κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:14显示人(homo sapiens)λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:15显示小鼠(mus musculus)κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:16显示小鼠(mus musculus)λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:17显示全人源单链抗体S1A4的氨基酸序列。
SEQ ID NO:18显示全人源单链抗体S1A4的重链可变区(S1A4 VH)的氨基酸序列。
SEQ ID NO:19显示全人源单链抗体S1A4的轻链可变区(S1A4 VK)的氨基酸序列。
SEQ ID NO:20显示抗人ST2对照抗体MSTT1041A的重链可变区(MSTT1041A VH)氨基酸序列。
SEQ ID NO:21显示抗人ST2对照抗体MSTT1041A的轻链可变区(MSTT1041A VK)氨基酸序列。
SEQ ID NO:22显示抗人ST2对照抗体GSK3772847的重链可变区(GSK3772847 VH)氨基酸序列。
SEQ ID NO:23显示抗人ST2对照抗体GSK3772847的轻链可变区(GSK3772847 VK)氨基酸序列。
SEQ ID NO:24显示轻链可变区突变体L1D1的氨基酸序列，其CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33所示。
SEQ ID NO:25显示轻链可变区突变体L2G2的氨基酸序列，其CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:34、SEQ ID NO:32和SEQ ID NO:35所示。
SEQ ID NO:26显示重链可变区突变体H5E10的氨基酸序列，其CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:42和SEQ ID NO:43所示。
SEQ ID NO:27显示重链可变区突变体H5G3的氨基酸序列，其CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:36、SEQ ID NO:37和SEQ ID NO:38所示。
SEQ ID NO:28显示重链可变区突变体H5G10的氨基酸序列，其CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:40和SEQ ID NO:41所示。
SEQ ID NO:29显示重链可变区突变体H6D3的氨基酸序列，其CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:39、SEQ ID NO:44和SEQ ID NO:45所示。
SEQ ID NO:30显示重链可变区突变体H6D6的氨基酸序列，其CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO:46、SEQ ID NO:42和SEQ ID NO:47所示。
SEQ ID NO:48显示人(homo sapiens)IL-12α的氨基酸序列。
SEQ ID NO:49显示人(homo sapiens)IFNγ的氨基酸序列。
发明详细描述
本申请的发明人通过抗体工程技术得到了新的抗人ST2抗体。在本申请的多个方面，提供了新的抗人ST2抗体或其抗原结合片段，编码该抗体或其抗原结合片段的核酸分子、包含所述核酸分子的载体、包含所述核酸分子或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体或其抗原结合片段的医学和生物学应用。根据本申请提供的抗体的可变区的序列，可构建全长的抗体分子作为药物用于治疗临床上由ST2介导的疾病。
除非另外指明，本申请的实施采用本领域常规的分子生物学、微 生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“抗体”，是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到标靶的免疫球蛋白分子。标靶包括但不限于碳水化合物、多聚核苷酸、脂质、多肽等。本文所使用的“抗体”不仅包括完整的(即全长的)抗体，而且还包括其抗原结合片段(例如Fab、Fab'、F(ab') 2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置，包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常，完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链变异区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链变异区(VL)和恒定区(CL)。全长的抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ、以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。
如本文所用术语“抗原结合片段或抗原结合部分”，是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合域可以包含重链变异区(VH)、轻链变异区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即Chothia定义和kabat定义。(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani等人，J.Mol.Biol.273:927-948(1997)；以及Martin等人，Proc.Natl.Acad.Sci.USA86:9268-9272(1989)。对于给定抗体的可变区序列，可以根据Chothia定义或者Kabat定义来确定VH和VL序列中的CDR序列。在本申请的实施方案中，利用Chothia定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列的中CDR序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
抗原结合片段的实例包括但不限于：(1)Fab片段，其可以是具有VL-CL链和VH-CH1链的单价片段；(2)F(ab') 2片段，其可以是具有两个Fab'片段的二价片段，该两个Fab'片段由铰链区的二硫桥(即Fab'的二聚物)连接；(3)具有抗体的单臂的VL和VH域的Fv片段；(4)单链Fv(scFv)，其可以是由VH域和VL域经由胜肽连接符组成的单一多胜肽链；以及(5)(scFv) 2，其可以包含两个由胜肽连接符连接的VH域和两个VL域，该两个VL域是经由二硫桥与该两个VH域组合。
在本文描述抗体结构时，涉及氨基酸位置编号的描述参照人IgG1抗体的EU numbering定义，这是本领域技术人员公知且容易查询到的。此外，在本文结合EU numbering位置描述突变时，是指相对于天然抗体序列产生的突变。
如本文所用术语“Fc片段”、“Fc结构域”、“Fc部分”或类似的术语是指抗体重链恒定区的一部分，包括铰链区(hinge)、恒定区的CH2片段和CH3片段。参照人IgG1抗体的EU numbering定义，Fc片段是抗体恒定区中第216-447位的氨基酸序列。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结 合反应，例如抗体至抗原表位的结合。
如本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(美国专利号4,816,567；和Morrison等人，Proc.Natl.Acad.Sci.USA 81:6851-6855(1984)。
如本文所用术语“自身免疫病”是指自机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病，包括但不限于硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)和肺出血-肾炎综合征等。
如本文所用术语“炎性疾病”是指炎症作为其主要破坏因子的疾病的统称。炎症为组织对有害刺激的生物反应，其是一种病变，并且伴随有组织退化、循环紊乱和液体渗出三种事件，以及肥大。炎性疾病的实例包括急性和慢性疾病，包括但不限于过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
第一方面，本申请提供了一种结合人ST2的抗体，其包含含HCDR1、HCDR2和HCDR3氨基酸序列的重链可变区和含LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区，其中
所述HCDR1氨基酸序列如SEQ ID NO:36、所述HCDR2氨基酸序列如SEQ ID NO:37所示、所述HCDR3氨基酸序列如SEQ ID NO:38所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示，所述HCDR2氨基酸序列如SEQ ID NO:40所示、所述HCDR3氨基酸序列如SEQ  ID NO:41所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:43所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:44所示、所述HCDR3氨基酸序列如SEQ ID NO:45所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:46所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:47所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:36所示、所述HCDR2氨基酸序列如SEQ ID NO:37所示、所述HCDR3氨基酸序列如SEQ ID NO:38所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:40所示、所述HCDR3氨基酸序列如SEQ ID NO:41所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ  ID NO:43所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:44所示、所述HCDR3氨基酸序列如SEQ ID NO:45所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
所述HCDR1氨基酸序列如SEQ ID NO:46所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:47所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；其中HCDR和LCDR氨基酸氨基酸序列根据Kabat定义。
在一些实施方案中，所述抗体的重链可变区的氨基酸如SEQ ID NO:26、27、28、29或者30所示。
在一些实施方案中，所述抗体的轻链可变区的氨基酸如SEQ ID NO:24或者25所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:26所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:27所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:29所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:26所示，所 述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:27所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:29所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
所述抗体的重链可变区的氨基酸序列如SEQ ID NO:30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示。
第二方面，本申请提供了一种结合人ST2的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:26、27、28、29和30中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:24和25中任何一项具有至少90％的一致性。
在第一方面和第二方面的一些实施方案中，所述抗体为单克隆抗体。
在第一方面和第二方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面和第二方面的一些实施方案中，所述抗体为全人源抗体。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区。
在第一方面和第二方面的一些实施方案中，所述抗体的重链恒定区可以是人IgG1亚型、人IgG2亚型、人IgG4亚型、鼠IgG1亚型或鼠IgG2a亚型。
在第一方面和第二方面的一些具体实施方案中，所述重链恒定区为IgG4亚型。
在第一方面和第二方面的一些具体实施方案中，所述抗体的重链恒定区包含人IgG1亚型重链恒定区的Fc段序列并且所述Fc序列的第234、235和331位的氨基酸序列分别为F、E和S，其中抗体恒定区氨基酸序列顺序按照EU numbering来确定。
在第一方面和第二方面的一些实施方案中，所述抗体还包含选自κ亚型或者λ亚型的轻链恒定区。
在第一方面和第二方面的一些实施方案中，所述抗体的轻链恒定区可以是人κ亚型、人λ亚型、鼠κ亚型或者鼠λ亚型。
在第一方面和第二方面的一些实施方案中，所述抗体能够结合重组人ST2(SEQ ID NO:1)、重组鼠ST2(SEQ ID NO:2)和重组猴ST2(SEQ ID NO:3)。
在第一方面和第二方面的一些实施方案中，所述抗体能够结合重组人ST2(SEQ ID NO:1)和重组猴ST2(SEQ ID NO:3)。
在第一方面和第二方面的一些实施方案中，所述抗体抑制人ST2与人IL-33结合。
第三方面，本申请提供了核酸分子，其编码第一方面或第二方面所述的抗体或其抗原结合部分。
在一些实施方案中，所述核酸分子可操作地连接到调控序列，调控序列可以被用所述载体转化过的宿主细胞识别。
第四方面，本申请提供了药物组合物，其包含第一方面或第二方面所述的抗体和药学上可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物用于预防或治疗ST2介导的疾病。
在一些实施方案中，所述ST2介导的疾病为自身免疫疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
在一些实施方案中，所述药物组合物还可包含下述中的一种或多种：润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、 丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
第五方面，本申请提供了第一方面或第二方面所述的抗体在制备用于预防或治疗ST2介导的疾病的药物中的用途。
在一些实施方案中，所述ST2介导的疾病为自身免疫疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
第六方面，本申请提供了预防或治疗ST2介导的疾病的方法，包括向有需要的个体给予第一方面或第二方面所述的抗体或第四方面所述的药物组合物。
在一些实施方案中，所述ST2介导的疾病为自身免疫疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
在其他方面，本申请还提供编码本发明抗体或其轻链或重链的分离的核酸分子以及包含所述核酸分子的载体、包含所述载体的宿主细胞以及产生所述抗体的方法。在一些实施方案中，所述核酸分子可操作地连接到调控氨基酸序列，调控氨基酸序列可以 被用所述载体转化过的宿主细胞识别。在一些实施方案中，产生抗体的方法包括培养宿主细胞以便于表达核酸。在一些实施方案中，产生抗体的方法还包括从宿主细胞培养基中回收抗体。
此外，本文所述的特异性结合人ST2的抗体也可用于检测生物样品中ST2的存在。基于抗体的检测方法在本领域是众所周知的，并且包括例为ELISA、免疫印迹、放射免疫试验、免疫荧光、免疫沉淀以及其它相关技术。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
实施例
以下实施例仅用于说明而非限制本申请范围的目的。
实施例1：噬菌体展示抗体库的构建
具体实验流程可参见中国专利申请第201610609651.X号的实施例1以及中国专利申请第201510097117.0号的实施例1，通过引用方式将上述两篇专利申请的全部内容并入本文。
实施例2：重组蛋白的制备
制备抗ST2单克隆抗体的过程中需要用到多种不同的重组蛋白，包括人ST2胞外区(hST2，SEQ ID NO:1)、小鼠ST2胞外区(mST2，SEQ ID NO:2)、猕猴ST2胞外区(mmST2，SEQ ID NO:3)以及人IL-33胞外区(hIL-33，SEQ ID NO:4)。这些蛋白具有大量的翻译后修饰(如糖基化或者二硫键等)，因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功能。此外，在这些重组蛋白的C端添加了His标签(His，SEQ ID NO:5)或者人抗体IgG1的Fc段(hFc，SEQ ID NO:6)或者鼠抗体IgG2a的Fc段(mFc，SEQ ID NO:7)，将更有利于重组蛋白的纯化和单克隆抗体功能的鉴定。抗体重链恒定区可以是人IgG1亚型(SEQ ID NO:8)、人IgG2亚型(SEQ ID NO:9)、人IgG4亚型(SEQ ID NO:10)或者鼠IgG1亚型(SEQ ID NO:11)、鼠IgG2a亚型(SEQ  ID NO:12)，轻链恒定区可以是人κ亚型(SEQ ID NO:13)、人λ亚型(SEQ ID NO:14)或者鼠κ亚型(SEQ ID NO:15)、鼠λ亚型(SEQ ID NO:16)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His标签或者hFc、mFc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或者其他阳离子转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-4天。然后通过离心等方式收获培养上清。
His标签融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。hFc和mFc融合表达的重组蛋白用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其他合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例3：利用噬菌体展示抗体库技术筛选抗人ST2单克隆抗体
3.1抗人ST2单克隆抗体的筛选
以实施例2制备的重组人ST2-his为抗原，利用固相筛选策略(实验方案参考噬菌体展示：通用实验指南/(美)克拉克森(Clackson,T.),(美)洛曼(Lowman,H.B.)编；马岚等译。化学工业出版社，2008.5)筛选实施例1制备的展示人单链抗体库的噬菌体库，最终获得1株特异性结合人ST2的全人源单链抗体S1A4。
3.2制备全人源抗体
利用常规的分子生物学手段，将编码S1A4的重链可变区(S1A4 VH，SEQ ID NO:18)和轻链可变区(S1A4VK，SEQ ID NO:19)的核酸 分子分别克隆至融合有编码人重链恒定区和轻链恒定区的核酸分子的真核表达载体，组合表达全抗体。同时，参照专利US9382318B2和US9090694B2，分别制备阳性对照抗体MSTT1041A(MSTT1041A VH，SEQ ID NO:20；MSTT1041A VK，SEQ ID NO:21)和GSK3772847(GSK3772847 VH，SEQ ID NO:22；GSK3772847 VK，SEQ ID NO:23)以用于研究。
3.3 S1A4抑制重组人IL-33与重组人ST2结合
将重组蛋白hST2-his包被于96孔板(1μg/mL，100μl/孔)，4℃包被过夜。然后用含有0.5μg/mL重组蛋白hIL33-mFc的PBST-2％牛奶对抗体S1A4、MSTT1041A和GSK3772847进行梯度稀释。抗体的起始浓度均为200μg/mL，3倍梯度稀释，共9个浓度梯度。利用HRP山羊抗鼠IgG((中杉金桥，ZB2305)检测hIL33-mFc的结合信号，然后利用GraphPad Prism 6进行数据分析和作图。ELISA分析结果(图1)显示，S1A4能够有效抑制ST2与IL-33的结合，并且抑制能力与对照抗体MSTT1041A和GSK3772847相当，各抗体的IC 50值如表1所示。其中，S1A4、MSTT1041A和GSK3772847的重链恒定区为IgG4亚型。
表1.S1A4抑制ST2与IL33结合的IC 50值
  S1A4-IgG4 MSTT1041A-IgG4 GSK3772847-IgG4
IC 50(nM) 4.149 3.794 5.524
3.4 S1A4亲和力测定
利用Biacore X100通过表面等离子共振技术测定抗ST2抗体的亲和力。氨基偶联试剂盒(BR-1000-50)、人抗体捕获试剂盒(BR-1008-39)、CM5芯片(BR100012)和pH7.4的10×HBS-EP(BR100669)等相关试剂和耗材均购自GE healthcare。依照试剂盒中的说明书，用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride，EDC)和N-羟基琥珀酰亚胺(N-Hydroxysuccinimide，NHS)对羧基化CM5芯 片表面进行活化，将抗人IgG(Fc)抗体(捕获抗体)用10mM pH5.0乙酸钠溶液稀释至25μg/mL，之后以流速10μL/min注射以实现大约多至10000个响应单位(RU)的偶联量。注射捕获抗体之后，注射1M乙醇胺以封闭未反应的基团。对于动力学测量，稀释抗ST2抗体至0.5-1μg/mL，10μL/min注射，保证100RU左右的抗体被抗人Fc的抗体捕获。然后将hST2-his设置一系列的浓度梯度(例如0.625nM、1.25nM、2.5nM、5nM、10nM)，于25℃下30μL/min从低浓度到高浓度进行注射，结合时间为120s，解离时间为1800s，以10μL/min注射3M的MgCl 2溶液30s对芯片表面进行再生。使用Biacore X100评估软件2.0.1版，通过1:1结合模型拟合结合和解离传感图来计算结合速率(Kon)和解离速率(Koff)。以比率Koff/Kon计算解离平衡常数(KD)，拟合结果如表2所示。
表2.S1A4结合ST2的亲和力常数
  Kon Koff KD
S1A4-IgG4 1.646*10E6 2.215*10E-4 1.346*10E-10
MSTT1041A-IgG4 3.941*10E5 9.452*10E-5 2.398*10E-10
GSK3772847-IgG4 1.979*10E6 2.159*10E-4 1.091*10E-10
实施例4 S1A4的亲和力成熟
4.1轻链置换
基于双载体噬菌体呈现系统，以单克隆抗体S1A4的重链可变区(S1A4VH)为基础，利用轻链置换(实验技术流程可参见中国专利第201510097117.0号中的实施例4)对抗体S1A4进行体外亲和力成熟，获得了两个高亲和力轻链可变区突变体L1D1(氨基酸序列为SEQ ID NO:24)和L2G2(氨基酸序列为SEQ ID NO:25)。
4.2轻链突变体亲和力测定
参照实施例3.2将编码S1A4的重链可变区(S1A4VH，SEQ ID NO:18)和轻链可变区突变体L1D1(S1A4VK，SEQ ID NO:24)或 L2G2(氨基酸序列为SEQ ID NO:25)的核酸序列分别克隆至融合有编码人重链恒定区和轻链恒定区核酸序列的真核表达载体，组合表达全抗体S1A4VH+L1D1和S1A4VH+L2G2。参照实施例3.4，利用Biacore X100对S1A4新轻链可变区进行亲和力分析，结果如表3所示。其中，S1A4、S1A4VH+L1D1和S1A4VH+L2G2的重链恒定区为IgG4亚型。
表3.轻链突变体结合ST2的亲和力常数
  Kon Koff KD
S1A4 1.862E+6 1.892E-4 1.106E-10
S1A4VH+L1D1 2.365E+6 3.59E-4 1.518E-10
S1A4VH+L2G2 2.45E+6 1.944E-4 7.935E-11
4.3 S1A4重链突变库的筛选
利用常规分子生物学手段，通过在重链S1A4VH的CDR3区引入突变，构建基于重链可变区S1A4VH的CDR3突变库，设计的突变方案如表4所示。基于双载体呈现系统(实验技术流程可参见中国专利申请第201510097117.0号中的实施例5)，通过固相筛选的方法，利用hST2-His抗原对构建的S1A4VH-CDR3突变库进行共3轮筛选富集。收集富集得到的第3轮突变库产物，吸取1OD菌液利用天根生物无内毒素质粒大提试剂盒(#DP117)提取质粒。以提取的质粒为模板，设计引物扩增S1A4VH-CDR3突变库的FR3-FR4部分序列备用，以构建的天然人抗体库可变区重链基因为模板(参见中国专利申请第201510097117.0号中的实施例1)，设计引物扩增人重链胚系基因的FR1-FR3部分序列备用。以PCR扩增制备的S1A4VH的FR3-FR4序列和人重链胚系基因的FR1-FR3序列为模板，利用重叠延伸PCR(Overlap PCR)的方法扩增构建基于S1A4VH-CDR3突变体和人重链胚系基因的组合库，构建库容1.5×10E8，正确率为80％。用于组合库设计的扩增引物如下表5、表6所示。
表4.基于S1A4重链可变区VH的CDR3突变库的突变方案
Figure PCTCN2019102818-appb-000001
Figure PCTCN2019102818-appb-000002
表5.扩增S1A4VH-CDR3突变库的FR3-FR4部分序列所需引物
引物名称 引物序列
PWM34-C1-CDR3-F11a ACAGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGATA
PWM34-C1-HCDR3R12 GGTGACCAGTGTGCCCTGGCCCCAG
表6.扩增重链天然胚系基因FR1-FR3部分序列所需引物
引物名称 引物序列
PhVH1F11a CAGGTTCAGCTGGTGCAGTC
PhVH1F11b CAGGTGCAGCTGGTGCAGTC
PhVH1F11c CARATGCAGCTGGTGCAGTC
PhVH1F11d SAGGTCCAGCTGGTACAGTC
PhVH1F11e CAGGTCCAGCTTGTGCAGTC
PhVH1R13a CTCAGCTCCATGTAGGCTGTG
PhVH1R13b GCTCAGCTCCATGTAGACTGTG
基于双载体噬菌体展示系统，用hST2-his抗原对上述组合库进行筛选，最终得到5个重链可变区突变体H5E10(SEQ ID NO:26)、H5G3(SEQ ID NO:27)、H5G10(SEQ ID NO:28)、H6D3(SEQ ID NO:29)、H6D6(SEQ ID NO:30)。
4.4 S1A4突变体亲和力测定
参照实施例3.2将编码重链可变区突变体H5E10(氨基酸序列 为SEQ ID NO:26)、H5G3(氨基酸序列为SEQ ID NO:27)、H5G10(氨基酸序列为SEQ ID NO:28)、H6D3(氨基酸序列为SEQ ID NO:29)或H6D6(氨基酸序列为SEQ ID NO:30)和轻链可变区突变体L2G2(氨基酸序列为SEQ ID NO:25)的核酸分子分别克隆至融合有编码人重链恒定区和轻链恒定区核酸分子的真核表达载体，组合表达全抗体。参照实施例3.4，利用Biacore X100对S1A4突变体进行亲和力分析，结果如表7所示。其中，S1A4、H5G3+L2G2、H5E10+L2G2、H5G10+L2G2、H6D3+L2G2和H6D6+L2G2的重链恒定区为IgG4亚型。
表7.S1A4突变体结合ST2的亲和力常数
  Kon Koff KD
S1A4 1.991*10E6 2.76*10E-4 1.386*10E-10
H5G3+L2G2 1.018*10E6 1.116*10E-4 1.146*10E-10
H5E10+L2G2 3.354*10E6 2.378*10E-3 7.091*10E-10
H5G10+L2G2 4.203*10E6 3.581*10E-4 8.522*10E-11
H6D3+L2G2 3.342*10E6 1.245*10E-4 3.726*10E-11
H6D6+L2G2 2.569*10E6 1.785*10E-3 6.949*10E-10
实施例5 抗人ST2单克隆抗体竞争IL-33结合ST2
分析不同抗人ST2单克隆抗体(H5G3+L2G2、H5E10+L2G2、H5G10+L2G2、H6D3+L2G2、H6D6+L2G2和H5G3+L1D1)抑制ST2和IL33结合的能力。
将重组蛋白hST2-Fc包被于96孔板(3μg/mL，100μl/孔)，4℃包被过夜。然后用含有0.4μg/mL重组蛋白hIL33-his的PBST-2％牛奶对各抗ST2抗体蛋白(IgG4亚型)进行梯度稀释。抗体的起始浓度为50μg/mL，3倍梯度稀释，共10个浓度梯度。利用HRP鼠抗his IgG(康为世纪，CW0285M)检测IL33-his的结合信号，然后利用GraphPad Prism 6进行数据分析和作图。ELISA分析结果(图2)显示不同抗ST2单克隆抗体都能够有效抑制IL33与ST2的结合，各抗体的IC 50值如表8所示。
表8.抗ST2抗体抑制ST2与IL33结合的IC 50值
Figure PCTCN2019102818-appb-000003
实施例6 抗人ST2单克隆抗体基于HEK-BlueTM IL-33细胞阻断ST2的作用
用IL-33刺激HEK-Blue TM IL-33细胞(InvivoGen)，激活NF-κB和AP-1信号通路，启动细胞表达分泌型碱性磷酸酶(SEAP)，通过检测抗体对SEAP表达水平的影响可以判断抗ST2抗体的阻断作用。HEK-Blue TMIL-33细胞表达ST2L，是对人HEK细胞转入IL1RL1基因得到的，同时整合了IFN-β小启动子控制的SEAP报告基因，启动子融合了5个NF-κB和5个AP-1结合位点。HEK-Blue TMIL-33细胞同时阻断TNF-α和IL-1β应答，从而使HEK-Blue TMIL-33细胞只对IL-33产生应答反应。HEK细胞表达内源性IL-1RAcP。
用测试培养基(DMEM+10％FBS+1％P.S.+100μg/mLNormocin)配制60pM的hIL-33(GenScript，Z03198)溶液，用该溶液对抗人ST2单克隆抗体(MSTT1041A、GSK3772847、H5G3+L2G2、H5G10+L2G2、H5G10+L1D1和H5G3+L1D1)进行系列稀释(抗体浓度梯度分别为1、4、16、64、256、1024、4096、16384、65536、262144pM)，抗体溶液与等体积HEK-Blue IL-33细胞(细胞密度5×10 5个/mL)混合，接种96孔细胞板，每孔200μL，细胞板置于CO 2培养箱于37℃正常培养。23小时后细胞板1200rpm离心5分钟，取20μL上清与180μL QUANTI-Blue TM(InvivoGen)在96孔板中混合，37℃显色30分钟，测定OD630，然后利用GraphPad Prism 6进行数据分析和作图。结果显示6种抗人ST2单克隆抗体(MSTT1041A、GSK3772847、H5G3+L2G2、H5G10+L2G2、H5G10+L1D1和H5G3+L1D1)都是剂量依赖型抑制IL-33活性(图3)，各抗体的IC 50值如表9所示。其中，MSTT1041A、GSK3772847、H5G3+L2G2、H5G10+L2G2、H5G10+L1D1和H5G3+L1D1的重链恒定区为IgG4亚型，轻链恒定区为κ亚型。
表9.抗人ST2单克隆抗体基于HEK-Blue TM IL-33细胞抑制ST2的IC 50值
Figure PCTCN2019102818-appb-000004
实施例7 抗人ST2单克隆抗体抑制IL-33与IL-12共刺激的PBMC分泌IFNγ
除了报告基因系统，用原代NK细胞也可以检测抗ST2抗体的阻断作用。IL-33和IL-12各自不能单独激活NK细胞，但IL-12可以显著增加NK细胞表面ST2的表达，用IL-33和IL-12刺激新鲜分离的NK细胞，IL-33与NK细胞表面ST2结合，可以激活NK细胞大量释放IFN-γ。通过检测抗体对IFN-γ表达水平的影响可以判断抗ST2抗体的阻断作用
用人PBMC细胞接种96孔细胞板(1.5×10 6个/mL，1640培养基+10％胎牛血清+1％P.S)，每孔100μL，加入1ng/ml IL-12-His(义翘神州，CT050-H08H)+0.1nM IL-33(GenScript，Z03198)+5倍梯度稀释抗体(MSTT1041A、GSK3772847、H5G3+L1D1和H5G10+L2G2，抗体浓度梯度分别为0.00256、0.0128、0.064、0.32、1.6、8、40、200、1000、5000pM)，反应总体积200μL/孔，37℃在CO 2培养箱中作用，24小时后细胞板1200rpm离心5分钟，按照说明书检测上清液中的IFN-γ(人IFN-γELISA试剂盒，达科为，DKW12-1000-096)，然后利用GraphPad Prism 6进行数据分析和作图。H5G3+L1D1和H5G10+L2G2都是剂量依赖型抑制IL-33与IL-12共刺激PBMC分泌IFNγ(图4)，各抗体的IC 50值如表10所示。其中，MSTT1041A、GSK3772847、H5G3+L1D1和H5G10+L2G2的重链恒定区为IgG4亚型。
表10.抗人ST2单克隆抗体抑制IL-33与IL-12共刺激PBMC分泌IFNγ的IC 50值
  MSTT1041A GSK3772847 H5G3+L1D1 H5G10+L2G2
IC 50(pM) 36.96 15.70 38.00 17.46
实施例8 抗人ST2单克隆抗体的种属特异性
将制备的人ST2(hST2-his)、猕猴ST2(mmST2-his)和小鼠ST2(mST2-his)分别包被于96孔ELISA板(1μg/mL，100μl/孔)，同时包被10％BSA作为无关对照，4℃包被过夜。利用封闭液PBS-0.1％Tween20-3％牛奶在37℃封闭1小时后，分别加入各重组抗ST2单克隆抗体(S1A4、H5G3+L2G2、H5E10+L2G2、H5G10+L2G2、H6D3+L2G2、H6D6+L2G2和H5G3+L1D1)，37℃结合1小时。用PBST缓冲液洗涤ELISA板，加入HRP鼠抗人IgG(bioss，bsm-0297M-HRP)，37℃结合1小时。PBST缓冲液洗涤ELISA板，加入OPD底物显色液，5-10分钟后用1M的H 2SO 4终止显色，酶标仪492nm/630nm双波长测定光密度值。ELISA分析结果(图5)显示，六种抗ST2单克隆抗体(S1A4、H5G3+L2G2、H5E10+L2G2、H6D3+L2G2、H6D6+L2G2和H5G3+L1D1)能特异识别人ST2，并且和猴ST2、鼠ST2存在交叉反应，H5G10+L2G2不结合鼠ST2。七种单克隆抗体的重链恒定区均为IgG4亚型，轻链恒定区为κ亚型。

Claims (12)

  1. 一种结合人ST2的抗体，其包含含HCDR1、HCDR2和HCDR3氨基酸序列的重链可变区和含LCDR1、LCDR2和LCDR3氨基酸序列的轻链可变区，其中
    所述HCDR1氨基酸序列如SEQ ID NO:36、所述HCDR2氨基酸序列如SEQ ID NO:37所示、所述HCDR3氨基酸序列如SEQ ID NO:38所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:39所示，所述HCDR2氨基酸序列如SEQ ID NO:40所示、所述HCDR3氨基酸序列如SEQ ID NO:41所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:43所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:44所示、所述HCDR3氨基酸序列如SEQ ID NO:45所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:46所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:47所示、所述LCDR1氨基酸序列如SEQ ID NO:31所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:33所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:36所示、所述HCDR2氨基酸序列如SEQ ID NO:37所示、所述HCDR3氨基酸序列如SEQ ID NO:38所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:40所示、所述HCDR3氨基酸序列如SEQ ID NO:41所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:43所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:39所示、所述HCDR2氨基酸序列如SEQ ID NO:44所示、所述HCDR3氨基酸序列如SEQ ID NO:45所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；或者
    所述HCDR1氨基酸序列如SEQ ID NO:46所示、所述HCDR2氨基酸序列如SEQ ID NO:42所示、所述HCDR3氨基酸序列如SEQ ID NO:47所示、所述LCDR1氨基酸序列如SEQ ID NO:34所示、所述LCDR2氨基酸序列如SEQ ID NO:32所示、所述LCDR3氨基酸序列如SEQ ID NO:35所示；
    其中HCDR和LCDR氨基酸氨基酸序列根据Kabat定义。
  2. 根据权利要求1所述的抗体，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:26、27、28、29或者30所示。
  3. 根据权利要求1所述的抗体，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24或者25所示。
  4. 根据权利要求1所述的抗体，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:26所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:27所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:29所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:24所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:26所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:27所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:28所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:29所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:30所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:25所示。
  5. 一种结合人ST2的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:26、27、28、29和30中任何一项具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:24和25中任何一项具有至少90％的一致性。
  6. 根据权利要求1-5中任一项所述的抗体，其中
    所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)，优选地，所述抗体为全人源抗体；和/或
    所述抗体为单克隆抗体；和/或
    所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区；优选地，所述重链恒定区包含人IgG1亚型重链恒定区的Fc段序列并且所述Fc序列的第234、235和331位的氨基酸序列分别为F、E和S，其中抗体恒定区氨基酸序列顺序按照EU numbering来确定；和/或
    所述抗体还包含选自κ亚型或者λ亚型的轻链恒定区。
  7. 根据权利要求1-6中任一项所述的抗体所示，其中
    所述抗体能够结合重组人ST2(SEQ ID NO:1)、重组鼠ST2(SEQ ID NO:2)和重组猴ST2(SEQ ID NO:3)；和/或
    所述抗体能够结合重组人ST2(SEQ ID NO:1)和重组猴ST2(SEQ ID NO:3)；和/或
    所述抗体抑制人ST2与人IL-33结合。
  8. 核酸分子，其编码权利要求1-7中任一项所述的抗体或其抗原结合部分。
  9. 药物组合物，其包含权利要求1-7中任一项所述的抗体和药学上可接受的赋形剂、稀释剂或载体。
  10. 如权利要求9所述的药物组合物，其用于预防或治疗ST2介导的疾病；优选地，所述ST2介导的疾病为自身免疫性疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合 征以及多发性硬化等。
  11. 权利要求1-7中任一项所述的抗体，或权利要求9或10所述的药物组合物在制备用于预防或治疗ST2介导的疾病的药物中的用途；优选地，所述ST2介导的疾病为自身免疫性疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
  12. 预防或治疗ST2介导的疾病的方法，其包括向有需要的个体给予权利要求1-7中任一项所述的双特异性抗体，或权利要求9或10所述的药物组合物；优选地，所述ST2介导的疾病为自身免疫性疾病或炎性疾病，例如硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener’s granulomatosis)、肺出血-肾炎综合征、过敏性肺炎、特应性皮炎、哮喘、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
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