WO2020119707A1 - 抗il-17a抗体及其应用 - Google Patents

抗il-17a抗体及其应用 Download PDF

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WO2020119707A1
WO2020119707A1 PCT/CN2019/124486 CN2019124486W WO2020119707A1 WO 2020119707 A1 WO2020119707 A1 WO 2020119707A1 CN 2019124486 W CN2019124486 W CN 2019124486W WO 2020119707 A1 WO2020119707 A1 WO 2020119707A1
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seq
antibody
antigen
binding fragment
amino acid
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PCT/CN2019/124486
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English (en)
French (fr)
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姚剑
蒙丹
冯辉
姚盛
武海
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Priority to BR112021011257-0A priority Critical patent/BR112021011257A2/pt
Application filed by 上海君实生物医药科技股份有限公司, 苏州君盟生物医药科技有限公司 filed Critical 上海君实生物医药科技股份有限公司
Priority to EP19897274.7A priority patent/EP3896086A4/en
Priority to SG11202106128TA priority patent/SG11202106128TA/en
Priority to US17/413,152 priority patent/US20230159632A1/en
Priority to JP2021534214A priority patent/JP2022513224A/ja
Priority to MX2021007028A priority patent/MX2021007028A/es
Priority to CA3123124A priority patent/CA3123124A1/en
Priority to AU2019397309A priority patent/AU2019397309A1/en
Priority to KR1020217021674A priority patent/KR20210102946A/ko
Priority to CN201980082789.0A priority patent/CN113166239B/zh
Publication of WO2020119707A1 publication Critical patent/WO2020119707A1/zh
Priority to IL283881A priority patent/IL283881A/en
Priority to ZA2021/04048A priority patent/ZA202104048B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/06—Antipsoriatics
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    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • 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]
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to antibodies and antigen-binding fragments that specifically bind IL-17A.
  • the present invention more specifically relates to antibodies and antigen-binding fragments thereof that inhibit IL-17A-mediated biological activity, and compositions and methods for treating related diseases using the antibodies or antigen-binding fragments thereof.
  • the present invention more specifically relates to the use of anti-IL-17A antibodies or antigen-binding fragments thereof for the treatment of immunopathological diseases, including autoimmune and inflammatory diseases, such as rheumatoid arthritis, psoriasis, obligatory Spondylitis, multiple sclerosis, systemic lupus erythematosus (SLE), lupus nephritis or chronic obstructive pulmonary disease, asthma, infectious granuloma, cystic fibrosis or cancer.
  • autoimmune and inflammatory diseases such as rheumatoid arthritis, psoriasis, obligatory Spondylitis, multiple sclerosis, systemic lupus erythematosus (SLE), lupus nephritis or chronic obstructive pulmonary disease, asthma, infectious granuloma, cystic fibrosis or cancer.
  • Interleukin 17 also known as CTLA-8 or IL-17A
  • CTLA-8 Interleukin 17A
  • IL-17A also known as CTLA-8 or IL-17A
  • IL-17A also known as CTLA-8 or IL-17A
  • IL-17A A total of six members of the IL-17 family, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F, all contain four critically conserved cysteine residues
  • the biological effects vary greatly among members. Among them, the homology and biological functions of IL-17A and IL-17F are the closest, and the current research is also the most in-depth.
  • IL-17A expressed in vivo has a 23 amino acid N-terminal signal peptide, which is cleaved to produce mature IL-17A.
  • IL-17A or IL-17 refers to the IL-17A homodimer protein, which is mainly produced by helper T cells 17 (T helper 17, Thl7), and can also be produced by other immune cells such as ⁇ T cells, LTi (Lymphoid Tissue inducer cells), ILCs (Innate Lymphoid Cells) and NKT (Natural Killer T) cells (Cua DJ, Tato CM., Nature reviews. 2010, 10:479–489).
  • helper T cells 17 T helper 17, Thl7
  • LTi Lymphoid Tissue inducer cells
  • ILCs Innate Lymphoid Cells
  • NKT Natural Killer T
  • IL-17A The expression and regulation of IL-17A is very complicated. Studies have found that the cytokines IL-6, IL-1 ⁇ and TGF ⁇ induce the differentiation of initial CD4 + T cells into Th17, but at this time Th17 cells are weak and secrete a small amount of IL-17A. The damage effect is small; when IL-23 is present, it promotes the stability of Th17 cells and continuously secretes IL-17A, up-regulates pro-inflammatory factors (IL-22, CSF-2 and IFN- ⁇ ) and down-regulates inflammatory factors (IL- 2. IL-27 and IL-12) expression and other pathways, causing inflammatory outbreaks and tissue damage (McGeachy MJ, et al., Nature immunology. 2009, 10:314-324). Therefore, when IL-23 is abnormally expressed in tissues, the IL-17A pathway plays a key role in tissue damage.
  • IL-22, CSF-2 and IFN- ⁇ pro-inflammatory factors
  • IL-17 is generally secreted at a specific site and binds to the IL-17 receptor (IL-17recptor, IL-17R) on the surface of target cells in local tissues to exert an effect.
  • the IL-17R family includes five members, IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE, which are widely expressed on a variety of cell membranes (Iwakura Y, et al., Immunity. 2011; 34:149 –162).
  • IL-17 mainly combines with the IL-17RA/IL-17RC complex on the surface of non-hematopoietic cells (such as epithelial cells and mesenchymal cells) (Ishigame H, et al., Immunity.
  • cytokines such as IL-6, G-CSF, GM-CSF, IL-10, TGF- ⁇ , TNF- ⁇
  • chemokines including IL-8, CXCL1 and MCP-1
  • prostaglandins eg, PGE2
  • ROS reactive oxygen species
  • IL-17 secretion disorders are closely related to the occurrence and development of such diseases.
  • Antibodies targeting IL-17 can effectively relieve the symptoms of autoimmune diseases by inhibiting the IL-17-IL-17R signaling pathway (Sarah L, et al., Nat Rev Immunol. 2014, 14(9): 585– 600).
  • Cosentyx (secukinumab), developed by Novartis, is the world's first IL-17 monoclonal antibody.
  • Approved indications include moderate to severe plaque psoriasis (plaque psoriasis), providing an important frontline for the majority of psoriasis groups Biological treatment options.
  • plaque psoriasis plaque psoriasis
  • anti-IL-17 antibodies with different characteristics, such as different structures, better efficacy, and wider indications, are developed to treat autoimmune-related disorders such as psoriasis, rheumatoid joints, ankylosing spondylitis, and other IL-17-related Diseases have urgent needs and important significance.
  • the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human IL-17A, which comprises at least one complementarity determining region (CDR) sequence selected from SEQ ID NO: 1-24, 60-65.
  • CDR complementarity determining region
  • the antibody or antigen-binding fragment thereof of the present invention comprises at least one heavy chain CDR structure selected from SEQ ID NO: 1-3, 4-6, 7-9, 10-12, or 60-62
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), characterized in that the heavy chain variable region comprises selected from SEQ ID NO: 1, 4, 7 , HCDR1 of 10 or 60, selected from HCDR2 of SEQ ID NO: 2, 5, 8, 11, or 61, and HCDR3 selected from SEQ ID NO: 3, 6, 9, 12, or 62.
  • VH heavy chain variable region
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), characterized in that the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 amino acid sequences selected from the following Any one of Group A to Group E:
  • VH heavy chain variable region
  • SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6
  • SEQ ID NO: 7 SEQ ID NO: 8
  • SEQ ID NO: 9 D
  • SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12
  • E SEQ ID NO: 60
  • SEQ ID NO: 61 SEQ ID NO: 62
  • the antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), characterized in that the light chain variable region comprises SEQ ID NO: 13, 16, 19 , LCDR1 of 22 or 63, is selected from LCDR2 of SEQ ID NO: 14, 17, 20, 23 or 64, and LCDR3 of SEQ ID NO: 15, 18, 21, 24 or 65.
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), characterized in that the amino acid sequence of LCDR1, LCDR2 and LCDR3 contained in the light chain variable region is selected from Any one of the following groups F to J:
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), characterized by the 6 CDRs contained in the variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the amino acid sequence is selected from any of the following groups I to VI:
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), which is characterized by the amino acid of the heavy chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the sequence is selected from SEQ ID NO: 25, 26, 27, 28, 33, 35, 38 or 40
  • LV light chain variable region amino acid sequence is selected from SEQ ID NO: 29, 30, 31 , 32, 34, 36, 38, 39 or 41.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), characterized in that the amino acid sequence of the variable region is selected from the following Any one of Group 1 to Group 7:
  • the antibody or antigen-binding fragment thereof of the present invention comprises a light chain (LC) and/or a heavy chain (HC), characterized in that the amino acid sequence of the heavy chain (HC) is selected from the sequence number SEQ ID NO: 42, 44, 46 or 49, and/or the light chain (LC) amino acid sequence is selected from the sequence numbers SEQ ID NO: 43, 45, 47, 48 or 50.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a light chain (LC) and a heavy chain (HC), and is characterized in that the amino acid sequence of the light chain is as shown in SEQ ID NO: 43
  • the amino acid sequence of the heavy chain is shown in SEQ ID NO: 42 or 44; or the amino acid sequence of the light chain is shown in SEQ ID NO: 45, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 44; or
  • the amino acid sequence of the light chain is shown in SEQ ID NO: 47 or 48, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 46; or the amino acid sequence of the light chain is shown in SEQ ID NO: 50, and the heavy chain
  • the amino acid sequence is shown in SEQ ID NO: 49.
  • the antibody of the present invention is a complete antibody, preferably IgG, and more preferably IgG4 type.
  • the antibody of the present invention is 1F8, 2B2, 2F5, ch1, ch2, ch16, hu31, hu43, hu44, hu59, hu60, or hu250.
  • the antibody or antigen-binding fragment of the present invention is characterized by: a) specifically binding IL-17A homodimer and IL-17AF heterodimer; b) blocking IL- 17A binds to its receptor; and/or c) inhibits IL-17A-mediated biological activity.
  • the IL-17A, IL-17AF or IL-17F of the present invention is selected from one or more of cynomolgus monkey, mouse or human.
  • the antibody or antigen-binding fragment of the present invention does not specifically bind a) human IL-17F homodimer, IL-17B homodimer, IL-17C homodimer, IL-17D Any one or more of homodimer, IL-17E homodimer, and/or b) any one of cynomolgus monkey IL-17F homodimer, mouse IL-17F homodimer or Multiple.
  • the antibody or antigen-binding fragment thereof of the present invention has the function of inhibiting the binding between IL-17A and its receptor and/or reducing the cell signal transduction and/or biological activity mediated by IL-17A .
  • the antibody or antigen-binding protein of the present invention can inhibit IL-17A-induced secretion of CXCL1 by epithelial cells during in vitro activity evaluation.
  • the antibody or antigen-binding fragment of the present invention can inhibit IL-17A-induced secretion of CXCL1 in mice during in vivo activity evaluation.
  • the antibody or antigen binding protein of the present invention can resist the onset of mice in the experimental model of psoriasis induced by imiquimod in the in vivo activity evaluation of mice, and the clinical onset of psoriasis in mice The score and the degree of ear swelling were significantly reduced.
  • the isolated antibody or antigen-binding fragment thereof of the present invention can inhibit knee joint swelling in an antigen-induced arthritis experimental model such as the cynomolgus monkey AIA-model.
  • the present invention also provides the antibody or antigen-binding fragment thereof, preferably the use of hu31, hu43, hu44, hu59, hu60 or hu250 as a medicine, more preferably as a treatment for pathological diseases mediated by IL-17A and/or by Use of drugs that inhibit IL-17A signal transduction to treat pathological diseases.
  • the pathological disease mediated by IL-17A is an inflammatory condition or condition, such as arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic Lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.
  • an inflammatory condition or condition such as arthritis, rheumatoid arthritis, psoriasis, ankylosing spondylitis, chronic obstructive pulmonary disease, systemic Lupus erythematosus (SLE), lupus nephritis, asthma, multiple sclerosis or cystic fibrosis.
  • the present invention provides a method for treating pathological diseases mediated by IL-17A, the method comprising administering an effective amount of the isolated antibody or antigen-binding fragment thereof according to the present invention, preferably hu31, hu43, hu44, hu59 , Hu60 or hu250 antibodies to alleviate the condition.
  • the invention also relates to a method for producing the antibody or antigen-binding fragment of the invention.
  • Such methods include isolated nucleic acid molecules encoding at least the heavy and/or light chain variable regions of the antibodies or proteins of the invention, or clonal expression vectors containing such nucleic acids, especially for recombinant production in host cells
  • the antibody or protein of the present invention is, for example, hu31, hu43, hu44, hu59, hu60, or hu250.
  • the present invention also relates to host cells containing one or more of the above-mentioned clonal vectors or expression vectors and proteins used to produce the antibodies of the present invention or antigen-binding fragments thereof, specifically, such as the production of hu31, hu43, hu44, hu59, hu60 Or hu250 antibody method, the method comprising culturing the host cell, purifying and recovering the antibody or protein.
  • the isolated antibody or protein comprising an antigen-binding fragment of the invention is conjugated to other active moieties.
  • the antibody or antigen-binding fragment thereof of the present invention may be a monoclonal antibody or antigen-binding fragment thereof, preferably a chimeric antibody, a humanized antibody, or a human antibody or a part thereof.
  • composition comprising the antibody of any embodiment of the invention in combination with one or more pharmaceutically acceptable excipients, diluents or carriers or comprising antigen binding thereof Fragment of protein.
  • the pharmaceutical composition contains one or more additional active ingredients.
  • the pharmaceutical composition is a lyophilized powder.
  • the pharmaceutical composition is a stable liquid formulation containing a therapeutically acceptable amount of the antibody or molecule of the invention.
  • the present invention relates to the use of the antibody or antigen-binding fragment thereof for the preparation of a medicament for treating any pathological condition mediated by IL-17A.
  • the present invention provides isolated nucleic acid molecules encoding the above antibodies or antigen-binding fragments thereof, expression vectors or recombinant vectors containing the above nucleic acid molecules, and host cells that transform the vectors.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, vector or host cell, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides the use of the aforementioned antibody or antigen-binding fragment thereof, nucleic acid molecule, vector or host cell or the aforementioned pharmaceutical composition in the preparation of a medicament for treating and/or preventing IL-17A-mediated diseases or disorders.
  • the above drugs are used to treat arthritis, rheumatoid arthritis, psoriasis, mandatory spondylitis, chronic obstructive pulmonary disease, systemic lupus erythematosus (SLE), lupus nephritis, asthma, multiple Drugs for sexual sclerosis or cystic fibrosis.
  • Figure 1 SDS-PAGE electrophoresis of human IL-17A-mFc recombinant protein.
  • Figure 2 FACS detection of human IL-17A-mFc binding to IL-17RA on 293F cells (MFI indicates average fluorescence intensity, experimental group vs. control: 7695vs308).
  • FIG. 3 ELISA detection of hybridoma antibodies blocking IL-17A-mediated biological activity in vivo.
  • the hybridoma antibody significantly inhibited IL-17A-induced mice to express CXCL1.
  • Figure 4 ELISA detects the binding effect of chimeric antibody to human IL-17A.
  • the chimeric antibodies ch1, ch2, ch4, ch7, and ch16 have high specificity for binding to human IL-17A, and their EC 50 is 6.62ng/ml, 5.17ng/ml, 88.48ng/ml, 39.96ng/mL, respectively. And 15.42ng/mL.
  • Figure 5 FACS detection of chimeric antibodies blocks the binding of human IL-17A to IL-17RA on 293F cells.
  • the chimeric antibodies ch1, ch2, ch7 and ch16 have a high-efficiency blocking effect, with IC 50 of 4.46ug/ml, 3.145ug/ml, 1.220ug/ml and 1.445ug/ml, respectively.
  • FIG. 6 ELISA detects the binding of humanized antibody to human IL-17A.
  • antibodies hu31, hu43, hu44, hu59, hu60 and hu250 have high specificity for binding to human IL-17A, and their EC 50 was 8.13ng/mL, 8.64ng/mL, 6.764ng/ml, 6.102ng/mL, respectively. , 5.776ng/mL and 6.351ng/mL.
  • Figure 7 FACS detection of humanized antibodies blocks the binding of human IL-17A to IL-17RA on 293F cells.
  • antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all have high-efficiency blocking effects, with IC 50 of 867.6ng/mL, 780.8ng/mL, 828.5ng/ml, 467.4ng/mL, 482.8ng/mL, respectively. And 577.8ng/mL.
  • Figure 8 ELISA detects the effect of humanized antibodies on blocking IL-17A-mediated epithelial cell secretion of CXCL1.
  • Antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all effectively inhibited the expression of CXCL1 induced by IL-17A in epithelial cells, and had a stronger blocking effect than the control antibody.
  • Figure 9 ELISA detection of humanized antibodies blocks IL-17A-mediated biological activity in vivo.
  • the antibodies hu31, hu43, hu44, hu60 and hu250 can effectively inhibit the expression of CXCL1 induced by IL-17A in mice and have a stronger blocking effect than the control antibody.
  • Figure 10-1 Effect of humanized antibody administration on clinical scores of imiquimod-induced psoriasis mice.
  • the administration of hu31 and hu44 can significantly inhibit imiquimod-induced skin psoriasis, induration, redness and swelling in the mouse model of psoriasis, that is, the clinical score decreases (*P ⁇ 0.05 vs KLH).
  • Figure 10-2 Effect of humanized antibodies on the degree of ear swelling induced by imiquimod in psoriasis mice.
  • Administration of hu31 and hu44 can significantly improve the degree of ear swelling. (*P ⁇ 0.05vsKLH,**P ⁇ 0.01vsKLH).
  • Figure 11-1 Effect of humanized antibodies on the body weight of female cynomolgus monkeys induced by type II collagen. hu31 and hu59 have a certain improvement effect on weight loss caused by arthritis (**P ⁇ 0.01, ****P ⁇ 0.0001, compared with "G2: vehicle group”; One-way ANOVA/Dunnett).
  • Figure 11-2 The effect of humanized antibodies on type II collagen-induced female cynomolgus monkey arthritis score.
  • Hu31 significantly inhibited the increasing trend of cynomolgus monkey arthritis clinical score (***P ⁇ 0.001, #P ⁇ 0.05, compared with G2: vehicle group; One-way ANOVA/Dunnett).
  • Figure 12 Effect of humanized antibodies on mouse joint swelling induced by NIH3T3-IL17 cells.
  • Figure 13 Effect of humanized antibodies on mouse air bladder inflammation induced by NIH3T3-IL17 cells.
  • the present invention relates to antibodies that specifically bind to human IL-17A and block the biological activity mediated by IL-17A participation.
  • the present invention relates to full-length IgG format antibodies and antigen-binding fragments thereof described further below.
  • IL-17 or IL-17A is interleukin-17. Unless otherwise stated, the IL-17 generally refers to human IL-17A.
  • IL-17A expressed in vivo is an N-terminal signal peptide with 23 amino acids, NCBI accession number NP_443104.1, which is cleaved to produce mature IL-17A.
  • IL-17A in the present disclosure refers to mature IL-17A, but does not include an N-terminal signal peptide, and its amino acid sequence is SEQ ID NO: 66, and its nucleotide sequence is SEQ ID NO: 67.
  • IL-17F expressed in vivo is an N-terminal signal peptide with 30 amino acids, NCBI accession number NP_443104.1, which is cleaved to produce mature IL-17F.
  • IL-17F herein refers to mature IL-17F, but does not include an N-terminal signal peptide, and its amino acid sequence is SEQ ID NO: 68.
  • IL-17AF is a heterodimer of the IL-17A subunit and the IL-17F subunit, as understood by those of ordinary skill in the art.
  • Immuno response refers to the production of soluble macromolecules (including antibodies, cytokines, and complement) by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and by the above-mentioned cells or liver, which results in selective damage from the human body , Destroy or remove invading pathogens, pathogen-infecting cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmunity or pathological inflammation.
  • lymphocytes including antibodies, cytokines, and complement
  • Signal transduction pathway or “signal transduction activity” refers to a biochemical causal relationship usually initiated by protein-protein interactions such as the binding of growth factors to receptors, which results in the transmission of signals from one part of the cell to another part of the cell .
  • delivery includes specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that cause signal transduction.
  • the penultimate process usually involves nuclear events, which lead to changes in gene expression.
  • the terms "activity” or “biological activity”, or the terms “biological properties” or “biological characteristics” are used interchangeably herein and include but are not limited to epitope/antigen affinity And specificity, ability to neutralize or antagonize IL-17A activity in vivo or in vitro, IC50, in vivo stability of antibodies, and immunogenic properties of antibodies.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., non-human homologues that are usually targeted to the peptide, or cross-reactivity with other proteins or tissues), and retention The ability to express high levels of proteins in mammalian cells.
  • techniques known in the art including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptors Binding, production and/or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources, including humans, primates, or any other source.
  • Antibody refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the bound compound, and in this case means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium.
  • isolated does not mean the complete absence of such substances or the absence of water, buffers or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic application of the binding compounds described herein.
  • “Monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies that make up this population are identical except for possible naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific and target a single epitope. In contrast, conventional (polyclonal) antibody preparations usually include a large number of antibodies directed against (or specific for) different epitopes. The modifier “monoclonal” indicates the characteristics of antibodies obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the production of antibodies by any particular method.
  • Full-length antibody an immunoglobulin molecule containing four peptide chains in nature, two heavy (H) chains (approximately 50-70 kDa at full length) and two light (L) chains (approximately 25 kDa at full length) Connected to each other through disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region is composed of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • VH and VL regions can be further subdivided into complementarity determining regions (CDRs) with high variability and regions whose spacing is more conservatively referred to as framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arranged from amino terminus to carboxy terminus.
  • 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 the binding of the immunoglobulin to host tissues or factors (including various cells of the immune system (eg, effector cells)) and the first component (Clq) of the classical complement system.
  • Antigen-binding fragments of antibodies include fragments or derivatives of antibodies, usually including at least one fragment of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody, which retains the parent antibody At least some of the binding specificities.
  • antibody binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies (nanobody) formed from antibody fragments And multispecific antibodies.
  • the binding fragment or derivative When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen binding activity.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen binding affinity of the parent antibody.
  • the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • binding compound refers to both antibodies and binding fragments thereof.
  • Single-chain Fv or “scFv” antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.
  • Fv polypeptides generally also include a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • Domain antibody is an immunofunctional immunoglobulin fragment containing only the heavy chain variable region or the light chain variable region.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • bivalent antibody contains two antigen binding sites. In some cases, the two binding sites have the same antigen specificity. However, bivalent antibodies can be bispecific.
  • “Diabody” refers to a small antibody fragment with two antigen binding sites, which fragment contains a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH) Variable domain (VH).
  • VL light chain variable domain
  • VH-VL or VL-VH Variable domain
  • a “chimeric antibody” is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, where the first antibody and the second antibody are from different species.
  • variable domains are obtained from antibodies of experimental animals such as rodents ("parent antibodies”), while constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibodies are in human subjects compared to parental rodent antibodies The possibility of inducing an adverse immune response is low.
  • Humanized antibody refers to an antibody format that contains sequences from human and non-human (eg, murine, rat) antibodies. Generally speaking, humanized antibodies comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops are equivalent to the hypervariable loops of non-human immunoglobulins, and all or substantially all
  • the framework (FR) region is the framework region of the human immunoglobulin sequence.
  • the humanized antibody optionally may comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody that contains only human immunoglobulin protein sequences. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, fully human antibodies may contain murine sugar chains. Similarly, “mouse antibody” refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, the fully human antibody may contain rat sugar chains. Similarly, “rat antibody” refers to an antibody that contains only rat immunoglobulin sequences.
  • Isotype refers to the type of antibody provided by the heavy chain constant region gene (eg, IgM, IgE, IgG such as IgG1 or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been made to alter Fc function, for example to enhance or decrease effector function or binding to Fc receptors.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single-stranded or double-stranded form. Unless specifically limited, the term includes nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. (See, U.S. Patent No. 8,278,036 belonging to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo).
  • modified mRNA can be used, for example, those disclosed in US Patent No. 8,278,036 belonging to Kariko et al. and Patent Application WO2013/090186A1 belonging to Moderna Corporation.
  • specific nucleic acid sequences also implicitly include conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid. Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossilini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construct refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA polynucleotide molecule) derived from Any source, capable of integrating with the genome or autonomously replicating, constitutes a polynucleotide molecule in which one or more polynucleotide molecules have been linked (ie, operably linked) in a functionally operative manner.
  • Recombinant constructs will typically contain polynucleotides of the invention operably linked to transcription initiation regulatory sequences, which will direct the transcription of the polynucleotide in the host cell.
  • transcription initiation regulatory sequences which will direct the transcription of the polynucleotide in the host cell.
  • Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct expression of the nucleic acids of the invention.
  • Vector refers to any recombinant polynucleotide construct that can be used for transformation purposes (ie, introducing heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • viral vector Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origin of replication and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell, and thus replicate together with the host genome. In addition, certain vectors can direct the expression of operatively linked genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell.
  • Expression vectors contain one or more phenotypic selection markers and origins of replication to ensure maintenance of the vector and provide amplification within the host if needed.
  • IL-17A antagonist or "IL-17A blocking molecule” means an antibody that inhibits IL-17A-induced signal transduction activity through IL-17R, thereby reducing or neutralizing IL-17A activity Or its antigen binding protein. This can be demonstrated in human cell assays such as IL-17A-dependent CXCL1 production assays for human cells. Such assays are described in more detail in the examples below.
  • Activation may have the same meaning, for example, the cell or receptor is activated, stimulated, or treated with a ligand, unless the context indicates otherwise or explicitly.
  • Ligand includes natural and synthetic ligands such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies.
  • Ligand also includes small molecules such as peptide mimetics of cytokines and peptide mimetics of antibodies.
  • Activation may refer to the activation of cells regulated by internal mechanisms and external or environmental factors.
  • Response/Response such as the response of a cell, tissue, organ, or organism, including changes in biochemical or physiological behaviors (such as concentration, density, adhesion or migration, gene expression rate, or differentiation status) within a biological zone, where the change It is related to activation, stimulation or treatment, or to internal mechanisms such as genetic programming.
  • treatment refers to ameliorating the disease or disorder (ie, slowing or preventing or reducing the progression of the disease or at least one of its clinical symptoms).
  • treatment refers to relieving or improving at least one physical parameter, including those physical parameters that may not be discernable by the patient.
  • treatment or “healing” refers to regulating the disease or condition physically (eg, the stabilization of discernable symptoms), physiologically (eg, the stabilization of physical parameters), or both.
  • methods for assessing the treatment and/or prevention of diseases are generally known in the art.
  • Subject 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, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to cynomolgus monkey.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” mean that the IL-17A antibody or antigen-binding fragment thereof of the present invention is effectively prevented when administered to cells, tissues or subjects alone or in combination with other therapeutic drugs Or the amount that improves the symptoms of one or more diseases or conditions or the development of that disease or condition.
  • Therapeutically effective dose also refers to an amount of antibody or antigen-binding fragment thereof sufficient to cause improvement of symptoms, for example, an amount to treat, cure, prevent or ameliorate related medical conditions or increase the speed of treatment, cure, prevention or improvement of such conditions. When an individual is given an active ingredient administered alone, the therapeutically effective dose refers only to that ingredient.
  • a therapeutically effective dose refers to the combined amount of active ingredients that cause a therapeutic effect, whether in combination, sequential administration or simultaneous administration.
  • the effective amount of the therapeutic agent will result in an increase in diagnostic criteria or parameters of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%, most preferably at least 50%.
  • Any suitable method for producing antibodies can be used to produce the antibodies of the present invention.
  • Any suitable form of human IL-17A can be used as the immunogen (antigen) for antibody production.
  • any human IL-17A isotype or fragment thereof can be used as the immunogen. Examples include, but are not limited to, the natural mature human IL-17A (amino acid sequence is SEQ ID NO: 66) described herein.
  • the hybridoma cells producing murine monoclonal anti-human IL-17A antibody can be produced by methods known in the art. These methods include but are not limited to the hybridoma technology originally developed by Kohler et al. (1975) (Nature 256:495-497).
  • mouse splenocytes are isolated and fused with mouse myeloma cell lines using PEG or by electrofusion.
  • the resulting hybridomas that produce antigen-specific antibodies can then be screened.
  • a single cell suspension derived from splenic lymphocytes of immunized mice can be fused with 1/6 number of mouse myeloma cells SP20 (ATCC).
  • Cells can be seeded in flat-bottom microtiter plates at approximately 2 ⁇ 10 5 cells/mL, followed by selective culture in complete medium containing 20% fetal bovine serum and 1 ⁇ HAT (Sigma; HAT added 24 hours after fusion) Incubate in Jizhong for 2 weeks. After 2 weeks, the cells can be cultured in a medium where HAT is replaced with HT. Each well can then be screened for anti-human IL-17A monoclonal IgG antibody by ELISA. Once large-scale hybridoma growth occurs, the medium can usually be observed after 10-14 days. Antibody-secreting hybridomas can be vaccinated and screened again.
  • anti-human IL-17A monoclonal antibody hybridomas can be subcloned at least twice by limiting dilution. Stable subclones can then be cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization.
  • the monoclonal hybridoma cells obtained by the present invention are 1F8, 2F5, and 2B2, and the antibodies secreted by them bind to IL-17A with high specificity, block the binding of IL-17A to IL-17RA, and inhibit IL- 17A-mediated biological activity, such as inhibition of CXCL1 secretion.
  • the present invention utilizes a method based on degenerate primer PCR to determine the DNA sequence of the variable regions of immunoglobulin 1F8, 2F5 and 2B2 from candidate hybridoma cells.
  • the hybridoma cell 1F8 has two antibody light chain genes and one antibody heavy chain gene
  • 2F5 has two antibody heavy chain genes and one antibody light chain gene. Therefore, the antibodies secreted by 1F8 and 2F5 may contain two mixed complete antibodies.
  • the antibodies secreted by 1F8 are called 1F8-1 and 1F8-2
  • the antibodies secreted by 2F5 are called 2F5-1 and 2F5-2.
  • Antibodies derived from rodents can cause unwanted antibody immunogenicity when used as therapeutic drugs in the body, and repeated use will cause the body to produce immune responses against therapeutic antibodies. Such immune responses will at least cause loss of therapeutic efficacy , And the highest leads to a potentially lethal allergic reaction.
  • One method of reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies, in which a mouse variable region is fused to a human constant region (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439- 43). However, the retention of intact rodent variable regions in chimeric antibodies can still cause harmful immunogenicity in patients.
  • CDR complementarity determining region
  • the chimeric or humanized antibody of the present invention can be prepared based on the sequence of the prepared mouse monoclonal hybridoma antibody.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from target murine hybridomas and engineered using standard molecular biology techniques to include non-mouse (eg, human) immunoglobulin sequences.
  • the chimeric antibodies of the present invention can use methods known in the art to effectively link immunoglobulin-encoded heavy and light chain variable regions derived from hybridomas to human IgG constant regions (see, for example, U.S. Patent No. 4,816,567, belonging to Cabilly et al., obtains a chimeric heavy chain and a chimeric light chain.
  • the human IgG can be selected from any subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric antibody of the present invention can be obtained by transfecting an expression cell with a chimeric light chain and a chimeric heavy chain expression plasmid, such "mixing and matching"
  • the IL-17A binding of the antibody can be tested using the above binding assay and other conventional binding assays (eg, ELISA).
  • the preferred ch1, ch2, ch4, ch7, and ch16 have optimal binding and blocking activity, which can vary
  • the amino acid sequence of the region is shown in Table 2.
  • variable region CDRs of the antibodies of the present invention can be determined using any of a number of well-known schemes, including by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest, No. 5 Version. Public Health, National Institutes of Health, Bethesda, MD ("Kabat” numbering plan) described Kabat plan and Lefranc M.-P. et al. described IMGT plan (1999NucleicAcidsResearch, 27,209-212).
  • Table 3 the specific definition scheme and amino acid sequence of the CDR of the preferred murine antibody variable region of the present invention are shown in Table 3.
  • the humanized antibodies of the present invention can use a method known in the art to insert the murine CDR region into the human germline framework region. See US Patent No. 5,225,539 for Winter et al. and US Patent Nos. 5,530,101 for Queen et al., 5,585,089; 5,693,762 and 6,180,370.
  • the inventors searched for the homology with the cDNA sequence of the variable region of the murine antibody by using the human immunoglobulin gene database on the NCBI (http://www.ncbi.nlm.nih.gov/igblast/) website
  • human germline IgG genes are humanized by grafting selected CDRs.
  • CDR loop exchange still cannot uniformly produce antibodies with the same binding properties as the starting antibodies.
  • framework residue (FR) residues involved in CDR loop support
  • changes are often required to maintain antigen binding affinity.
  • C. Use computer simulation technology to analyze the framework amino acid sequence of the variable region and its surroundings by molecular docking, and examine the spatial three-dimensional binding method.
  • the preferred humanized antibodies obtained by the present invention are hu31, hu43, hu44, hu59, hu60, and hu250.
  • variable regions of humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 and their corresponding CDR amino acid sequences are shown in Table 4.
  • antibodies of the present invention include those that have been mutated by amino acid deletion, insertion or substitution, but are still at least 70%, 75%, 80%, 85%, with the above antibody (particularly in the CDR regions depicted in the above sequence), Those antibodies with an amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99% or 100% identity.
  • the antibody of the present invention is a mutant of any one of hu31, hu43, hu44, hu59, hu60, and hu250, wherein the mutant includes a mutant amino acid sequence, and in the mutant amino acid sequence, when When comparing the CDR regions depicted in the sequence, no more than 1, 2, 3, 4, or 5 amino acid mutations have been deleted, inserted, or replaced by amino acids in the CDR regions.
  • nucleic acids encoding antibodies of the invention include those that have been mutated by nucleotide deletion, insertion, or substitution, but still have a coding region corresponding to the CDR depicted in the sequence described above with at least 60, 70, 80, 90, 95, or Nucleic acids with 100% identity.
  • the present invention relates to a host cell comprising one or more expression vectors or expression vectors and a method for producing the antibody of the present invention or comprising an antigen-binding fragment thereof, the method comprising culturing the host cell, The antibody or antigen-binding fragment is purified and recovered.
  • the antibody of the present invention can be produced in a host expression cell using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (for example, Morrison, S. 1985, Science 229: 1202).
  • DNA encoding partial or full-length light and heavy chains can utilize standard molecular biology or biochemical techniques (eg, DNA chemical synthesis, PCR amplification, or use of hybridomas expressing the target antibody CDNA clone), and the DNA can be inserted into an expression vector so that the gene is effectively linked to transcription and translation control sequences.
  • operably linked means that the antibody genes are linked into the vector so that the transcription and translation control sequences within the vector perform their predetermined functions of regulating the transcription and translation of the antibody genes.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into different vectors, or more generally, both genes are inserted into the same expression vector.
  • the antibody gene is inserted into the expression vector by standard methods (e.g., ligation of the antibody gene fragment and the complementary restriction site on the vector, or blunt-end ligation if there is no restriction site).
  • the light chain and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting them into the heavy chain constant region and light chain constant region of the desired isotype
  • the VH segment is effectively linked to the CH segment in the vector
  • the VL segment is effectively linked to the CL segment in the vector.
  • the recombinant expression vector may encode a signal peptide (also called a leader sequence), which facilitates secretion of the antibody chain from the host cell.
  • the antibody chain gene can be cloned into a vector so that the signal peptide and the amino terminus of the antibody chain gene are connected in the same reading frame.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).
  • the mammalian host cells used to express the recombinant antibodies of the present invention include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include in particular Chinese hamster ovary (CHO) cells, NS0, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocyte cancer cells, A549 cells, 293T cells and many others Cell line. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse and hamster cells. A particularly preferred cell line is selected by determining which cell line has a high expression level.
  • ATCC American Type Culture Collection
  • a recombinant expression vector encoding a heavy chain or an antigen-binding fragment or fragment thereof, a light chain and/or an antigen-binding fragment into a mammalian host cell, by culturing the host cell for a sufficient period of time to allow the antibody in the host cell Medium expression, or more preferably, the antibody is secreted into the growth medium of the host cell to produce the antibody.
  • Antibodies can be recovered from the culture medium using standard protein purification methods.
  • antibodies expressed by different cell lines or expressed in transgenic animals have different glycosylation from each other.
  • all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein are an integral part of the present invention, regardless of the glycosylation of the antibody.
  • non-fucosylated antibodies are advantageous because they generally have more potent efficacy than their fucosylated counterparts in vitro and in vivo, and cannot be immunogenic Because their sugar structure is a normal component of natural human serum IgG.
  • the antibody or antigen-binding fragment of the present invention has diagnostic and therapeutic uses in vitro and in vivo.
  • humanized antibodies hu31, hu43, hu44, hu59, hu60 or hu250 antibodies can be used to treat diseases associated with IL-17A.
  • the isolated antibody or antigen-binding fragment of the present invention can resist the onset of mice in the experimental model of psoriasis induced by imiquimod in the in vivo activity evaluation, and the clinical score and the incidence of psoriasis in mice Ear swelling is significantly reduced.
  • humanized antibodies hu31 and hu44 can significantly resist the onset of mice, the clinical score of psoriasis on mice and ear swelling The degree is reduced.
  • the isolated antibody or antigen-binding fragment of the present invention can inhibit knee joint swelling in an antigen-induced arthritis experimental model, such as the cynomolgus monkey AIA-model, when evaluated in vivo.
  • the humanized antibody hu31 significantly inhibits the increasing trend of cynomolgus monkey arthritis clinical scores.
  • a method of treating pathological diseases mediated by IL-17A comprising administering an effective amount of an isolated antibody or antigen-binding fragment thereof according to the invention, in particular hu31, hu43, hu44 , Hu59, hu60, or hu250 antibodies to alleviate the condition.
  • the isolated antibody or protein comprising an antigen-binding fragment of the invention is conjugated to other active moieties.
  • the isolated antibody or protein comprising an antigen-binding fragment thereof according to the present invention may be a monoclonal antibody or an antigen-binding fragment thereof, preferably a chimeric antibody, a humanized antibody or a human antibody or a part thereof.
  • a pharmaceutical composition comprising the antibody of the embodiment of the present invention or an antigen-binding fragment thereof in combination with one or more pharmaceutically acceptable excipients, diluents or carriers protein.
  • the pharmaceutical composition contains one or more additional active ingredients.
  • the pharmaceutical composition is a lyophilized powder.
  • the pharmaceutical composition is a stable liquid formulation containing a therapeutically acceptable amount of the antibody or molecule of the invention.
  • the present invention provides methods for treating IL-17A-related disorders and/or autoimmune and inflammatory disorders.
  • the method includes the step of administering the isolated antibody or antigen-binding fragment thereof according to the present invention to a subject in need thereof.
  • the present invention also provides a method for reducing or inhibiting signal transduction induced by IL-17A or IL-17AF in target cells or tissues by contacting the cells with a composition comprising a therapeutically effective dose of the antibody of the present invention answer.
  • IL-17A-mediated disease or "IL-17A-related disorder” includes all diseases where IL-17A or IL-17AF plays a role (either directly or indirectly) in a disease or medical condition And condition, including the cause, development, progression, persistence or pathology of the disease or condition. Therefore, these terms include conditions that are related to or characterized by abnormal IL-17A or IL-17AF levels and/or can be reduced or inhibited in target cells or tissues by reducing or inhibiting IL-17A/AF Induced activity (eg CXCL1) to treat diseases or conditions.
  • CXCL1 IL-17A/AF Induced activity
  • Such diseases or conditions include inflammatory conditions and autoimmune diseases such as arthritis, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis or psoriasis.
  • autoimmune diseases such as arthritis, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis or psoriasis.
  • diseases also include allergies and allergic conditions, hypersensitivity reactions, chronic obstructive pulmonary disease, cystic fibrosis, and organ or tissue transplant rejection.
  • inhibition or “treatment” or “treatment” includes delayed development of symptoms associated with a disorder and/or reduction in the severity of symptoms of such disorders.
  • the term also includes improving existing uncontrolled or harmful symptoms, preventing other symptoms, and improving or preventing the underlying causes of such symptoms. Therefore, the term indicates that a vertebrate subject suffering from a disorder, disease, or symptom or a vertebrate subject who may have such a disorder, disease, or symptom may have provided beneficial results.
  • therapeutically effective amount refers to the IL-17 binding compound of the present invention when administered to cells, tissues or subjects alone or in combination with other therapeutic agents, effectively prevent Or the amount that improves the symptoms of one or more diseases or conditions or the development of that disease or condition.
  • Therapeutically effective dose also refers to an amount of binding compound sufficient to cause improvement of symptoms, for example, an amount to treat, cure, prevent or ameliorate related medical conditions or increase the rate of treatment, cure, prevention or improvement of such conditions.
  • the therapeutically effective dose refers only to that ingredient.
  • a therapeutically effective dose refers to the combined amount of active ingredients that cause a therapeutic effect, whether in combination, sequential administration or simultaneous administration.
  • the effective amount of the therapeutic agent will result in an increase in diagnostic criteria or parameters of at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40%; most preferably at least 50%.
  • RA Rheumatoid arthritis
  • RA is a progressive systemic disease characterized by inflammation of the synovial joint, affecting approximately 0.5% of the global population. See Emery (2006) BMJ332:152-155. Inflammation of the joints can lead to deformities, pain, stiffness, and swelling, and eventually irreversible degradation of the joints.
  • the affected joints include knee, elbow, neck and hand-foot joints.
  • Conventional treatment includes the use of NSAIDs to relieve symptoms, followed by administration of anti-rheumatic drugs (DMRD) such as gold, penicillamine, sulfasalazine, and methotrexate.
  • DMRD anti-rheumatic drugs
  • TNF- ⁇ inhibitors including monoclonal antibodies such as infliximab, adalimumab, and golimumab, and receptor fusion proteins, such as etanercept. Treatment with these TNF- ⁇ inhibitors significantly reduces structural damage due to disease.
  • the anti-IL-17A antibody of the present invention can be used to treat RA in a subject in need of such treatment.
  • the anti-IL-17A antibodies of the invention can also be combined with other treatments for RA, such as methotrexate, azathioprine, cyclophosphamide, mycophenolate mofetil, NSAID or TNF- ⁇ inhibitors.
  • Skin is an important barrier between the internal environment and the outside world, preventing contact with potentially harmful antigens.
  • T cells, polymorphonuclear cells, and macrophages at the skin contact site infiltrate and initiate an inflammatory response to eliminate the antigen.
  • this inflammatory response caused by the pathogen is closely monitored and stopped when the pathogen is eliminated. In some cases, this inflammatory response occurs without external stimulation and without proper control, resulting in skin inflammation.
  • the present invention provides methods for treating and diagnosing skin inflammation.
  • Inflammation of the skin includes several inflammatory conditions such as scarchy pemphigoid, scleroderma, suppurative sweat gland inflammation, toxic epidermal necrolysis, acne, Osteitis, graft-versus-host disease (GVHD), gangrenous pyoderma (pyroderma gangrenosum) and Behcet's syndrome (Behcet's Syndrome) (see for example Williams and Griffiths, (2002) Clin. Exp. Dermatol., 27: 585-590).
  • GVHD graft-versus-host disease
  • Behcet's syndrome Behcet's Syndrome
  • Psoriasis is characterized by excessive proliferation of keratinocytes mediated by T cells with inflammation and infiltration.
  • the disease has certain clearly overlapping clinical phenotypes, including chronic plaque lesions, rashes, and pustular lesions (see, for example, Gudjonsson et al. (2004) Clin Exp. Immunol. 135: 1-8).
  • Approximately 10% of patients with psoriasis develop arthritis.
  • the disease has a strong and complex genetic cause, with 60% consistency among single egg twins.
  • Typical psoriasis lesions are erythema with very clear borders, which are covered by thick silver scales.
  • inflammation and hyperproliferation of psoriatic tissues are associated with different histological, antigenic, and cytokine profiles.
  • the cytokines associated with psoriasis are: TNF- ⁇ , IL-19, IL-18, IL-15, IL-12, IL-7, IFN- ⁇ , IL-17A and IL-23 (see Gudjonsson et al., Ibid.).
  • the anti-IL-17A antibody of the present invention can also be used to prevent, treat, diagnose, and predict the onset of psoriasis.
  • the antibody is mixed with a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient See, for example, Remington’s Pharmaceuticals and U.S. Pharmaceuticals: National Formaly, Mack Publishing, Company, Easton, PA (1984).
  • Dosage forms of therapeutic and diagnostic agents in the following forms can be prepared by mixing with acceptable carriers, excipients, or stabilizers: for example, lyophilized powder, ointment, aqueous solution, or suspension.
  • the IL-17A antibody of the present invention is diluted to an appropriate concentration in sodium acetate solution (pH 5-6), and NaCl or sucrose is added to facilitate osmotic pressure.
  • Other substances such as polysorbate 20 or polysorbate 80 may be added to improve stability.
  • the dosing regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody, the symptom level, the immunogenicity of the therapeutic antibody, and the availability of target cells in the biological matrix.
  • the preferred dosing regimen delivers sufficient therapeutic antibodies to achieve an improvement in the target disease state, while minimizing undesirable side effects. Therefore, the amount of biologic delivered depends in part on the specific therapeutic antibody and the severity of the condition to be treated. Guidance on selecting appropriate doses of therapeutic antibodies is available. For example, using parameters or factors known or speculated in the art that affect treatment, the clinician can determine the appropriate dose. In general, the dose starts with an amount slightly less than the optimal dose, and then increases in small increments until the desired or optimal effect is achieved relative to any negative side effects.
  • diagnostic methods include diagnostic methods such as symptoms of inflammation or levels of inflammatory cytokines produced.
  • the biological agent that can be used is derived from the same species as the animal targeted for treatment, thereby minimizing the inflammation, autoimmunity, or proliferation response to the agent.
  • chimeric, humanized, and fully human antibodies are preferred.
  • the amplified fragment was digested with BSPQI and cloned into an eukaryotic expression plasmid system (MXT1-Fc, which contains the Fc domain of the murine-derived IgG heavy chain), thereby generating recombinant fusion protein expression plasmid IL-17A-mFc.
  • MXT1-Fc eukaryotic expression plasmid system
  • the identified correct plasmids were transfected into expression cells 293F, expressed and purified to obtain human IL-17A-mFc recombinant protein.
  • Figure 1 shows the SDS-PAGE electrophoresis of human IL-17A-mFc recombinant protein.
  • the plasmid HG10895-G containing the cDNA sequence encoding human full-length IL-17RA was purchased from Yiqiao Shenzhou, and the DNA sequence encoding the full-length human IL-17RA (SEQ ID NO: 69) was amplified by conventional PCR. Using conventional cloning techniques, the amplified fragments were cloned into a self-built eukaryotic expression plasmid system (HXP), which contains puromycin screening system. The successfully constructed IL-17RA recombinant expression plasmid was transfected into 293F (ATCC) cells.
  • HXP self-built eukaryotic expression plasmid system
  • the cells were screened by puromycin (2 ⁇ g/ml) until 293F IL-17RA stable transfected cell bank was formed. Isolate single clones by conventional methods, such as limiting dilution method, 0.8 cells per well, spread 96-well plates, after 15 days, select IL-17RA-293F monoclonal and passaging to form 293F IL-17RA stable transfected cells Strains, all the clones were screened by FACS analysis, and the top expression clones were selected for FACS binding assay to screen hybridoma monoclonal antibodies, or used in functional assays.
  • the FACS experiment detected the binding specificity of recombinant protein IL-17A-mFc to IL-17RA on 293F cells.
  • the cells (293F IL-17RA stably transformed cell line) were prepared into a cell suspension of 1 ⁇ 10 6 /ml, and added to a 96-well plate at 20ul per well, the actual number of cells per well was 2 ⁇ 10 4 Cells, mix the recombinant protein IL-17A-mFc (3ug/ml, 20ul/well; experimental group) or 1% BSA (20ul/well; negative control group) with the cell suspension, and incubate for 30 minutes at 37 degrees Celsius with FACS buffer After the solution was eluted three times, anti-mouse IgG (1:200) was added and incubated at room temperature for 30 minutes.
  • the recombinant protein IL-17A-mFc can specifically bind to IL-17RA on 293F cells.
  • Standard molecular biology techniques are used to produce hybridoma antibodies. Briefly, the natural human IL-17A protein purchased from HumanZyme was mixed as an antigen with an equal amount of immune adjuvant, and five 6-week-old female FVB mice were immunized. After the initial immunization, a booster immunization was conducted once a week, for a total of seven immunizations. After the last booster immunization, mice with high anti-IL-17A antibody titers in the serum were selected for cell fusion experiments. Using standard hybridoma techniques, spleen cells are isolated and fused with the murine myeloma cell line SP2/0 cells (ATCC). The fused cells were resuspended in RPMI-1640 complete medium containing HAT and plated in wells with peritoneal cell feeder layer.
  • antibody/antigen binding characteristics such as binding affinity for IL-17A, ability to block IL-17A binding to its receptor, species cross-reactivity, and blocking IL-17A-mediated in vitro assays Ability of biological effects
  • Example 5 Effect of hybridoma antibody blocking IL-17A biological activity in vivo
  • IL-17A promotes the expression and release of cytokine CXCL1 in vivo, so the quantitative changes of CXCL1 expression in mouse serum can be detected by ELISA to determine the effect of hybridoma antibodies on IL-17A-mediated biological activity in mice.
  • 40 female 10-week-old Balb/c mice were selected and divided into 8 groups of 5 mice.
  • serum was collected, and the expression level of CXCL1 was detected as the base value.
  • the candidate hybridoma antibody, physiological saline (cntrol) or reference antibody mAb317 commercial anti-IL-17A antibody, purchased from R&D
  • cntrol physiological saline
  • mAb317 commercial anti-IL-17A antibody, purchased from R&D
  • the dosage of kg was administered by subcutaneous injection of natural human IL-17A (HumanZyme); 2 hours after injection of human IL-17A, serum was collected to detect the concentration of CXCL1 in the blood, and compared with the basic value, calculating before and after administration of each group CXCL1 concentration change multiple (mean ⁇ standard error (mean ⁇ SEM)).
  • the hybridoma antibody obtained in Example 4 and the commercial antibody mAb317 can significantly inhibit IL-17A-induced CXCL1 expression in mice.
  • the DNA sequence of the antibody variable regions expressed by hybridomas 1F8, 2B2, and 2F5 was determined. Briefly, the hybridoma cell lines 1F8, 2B2 and 2F5 were separately expanded and cultured, and the cells were collected by centrifugation at 1000 rpm, and the total RNA was extracted with Trizol. Using this as a template, after synthesizing the first-strand cDNA, the first-strand cDNA is used as the subsequent template to PCR amplify the corresponding variable region DNA sequence.
  • the PCR primers used are based on the Ig-primer set.
  • hybridoma cells 1F8 have two antibody light chain variable region gene sequences and one antibody heavy chain variable region gene sequence
  • 2F5 have two antibody heavy chain variable region gene sequences and one antibody light chain variable region gene sequence. Therefore, the antibodies secreted by 1F8 and 2F5 may contain two kinds of mixed complete antibodies.
  • the antibodies secreted by 1F8 are called 1F8-1 and 1F8-2, and the antibodies secreted by 2F5 are called 2F5-1 and 2F5-2.
  • amino acid sequences of the heavy chain variable region and light chain variable region of the antibodies expressed by 1F8, 2F5, and 2B2 are shown in Table 1 (see detailed description in the specification).
  • Human IgG4 heavy chain constant region Fc fragment and light chain kappa constant region were cloned from human blood cells (Beijing Blood Research Institute), and were inserted into pCDNA3.1 plasmid for modification.
  • the above heavy chain and light chain variable region sequence fragments were synthesized by Genscript Corporation.
  • the heavy chain was digested with Bspq I, and the light chain was digested with Bspq I, and then connected to the corresponding modified pCDNA3.1 plasmid. After sequencing, IgG4 was embedded.
  • the binding specificity of chimeric antibody to human IL-17A was detected by conventional ELISA detection method. That is, the human IL-17A-mFc of 0.5 ⁇ g/ml is coated on a 96-well microplate and incubated at 37°C for 60-90 minutes at a constant temperature. Then, the solution in the well was discarded, washed 3 times with washing buffer, and added with PBS solution containing 2% BSA to block for 60 minutes.
  • the chimeric antibodies ch1, ch2, ch4, ch7, and ch16 have high specificity for binding to human IL-17A, with EC 50 of 6.62ng/mL, 5.17ng/mL, and 88.48ng/ml, respectively. , 39.96ng/mL and 15.42ng/mL.
  • Example 9 Chimeric antibodies block the binding of human IL-17A to IL-17RA
  • a competitive cell-based flow cytometry (FACS) assay was used to detect chimeric antibody blocking IL-17A binding to IL-17RA on cells. Briefly, different concentrations of chimeric antibody dilutions (initial 10ug/ml, 3 times titration) were mixed with human IL-17A-mFC (3ug/ml) obtained in Example 1 pre-biotinylated at room temperature Incubate for 30 minutes.
  • the mixture and the cell suspension (293F IL-17RA obtained in Example 2 stably transformed cell line, 1.5 ⁇ 10 5 cells/well) were incubated for 15 minutes at 37 degrees Celsius, after eluting with PBS three times, 5 ⁇ g/ml of Anti-mouse IgG and incubate at room temperature for 30 minutes. After eluting three times with PBS, the inhibitory effect of chimeric antibody on the binding of IL-17A to IL-17RA on the surface of 293F cells was detected by flow cytometry.
  • the chimeric antibodies ch1, ch2, ch7, and ch16 can significantly inhibit the binding of human IL-17 to IL-17RA on the surface of 293F cells. Based on the comprehensive experimental results, ch1 and ch16 were selected to continue the humanization transformation.
  • humanization of antibodies For humanization of antibodies, first search the human immunoglobulin gene database on the NCBI (http://www.ncbi.nlm.nih.gov/igblast/) website for homology to the cDNA sequence of the murine antibody variable region Human germline IgG gene.
  • the Kabat numbering system or IMGT numbering system is used to define the amino acid sequence of the variable region CDR and its precise boundaries.
  • human IGVH and IGVk with high homology to the murine antibody variable region are selected as humanization templates, and humanization is performed by CDR grafting.
  • the humanization process involves the following steps: A. Compare the gene sequence of each candidate antibody with the human embryonic antibody gene sequence to find a sequence with high homology; B.
  • chimeric antibodies ch1 and ch16 were selected to continue humanization. After primary screening of a series of antibody/antigen binding characteristics (such as binding affinity for IL-17A, ability to block IL-17A binding to its receptor), humanized antibodies hu31, hu43, hu44, hu59, hu60 were selected And hu250 continue to follow-up verification.
  • the humanized antibody hu31, hu43, hu44, hu59, hu60 and hu250 variable regions and their CDR amino acid sequences are shown in Table 4 of the Detailed Description section of this disclosure.
  • humanized antibodies hu31, hu43, hu44, hu59, hu60 and hu250 all specifically bind to IL-17A. Its EC 50 was 8.13 ng/mL, 8.64 ng/mL, 6.76 ng/ml, 6.10 ng/mL, 5.78 ng/mL and 6.35 ng/mL, respectively.
  • Example 12 Humanized antibodies block the binding of human IL-17A to IL-17RA
  • Example 13 Humanized antibody antagonizes IL-17A to induce CXCL1 expression in epithelial cells
  • IL-17A can stimulate the expression and release of cytokine CXCL1 secreted by a variety of epithelial cells and other cells.
  • the expression of CXCL1 in the cell supernatant can be quantitatively detected by ELISA to determine the biological effects of humanized antibodies on IL-17A in cells. Effect of activity.
  • HT-29 cells human colorectal adenocarcinoma epithelial cells, ATCC
  • ATCC human colorectal adenocarcinoma epithelial cells
  • humanized antibodies hu31, hu59, hu60, and hu250 have a stronger antagonistic effect on IL-17A stimulation of epithelial cells to release CXCL1 than the reference antibody Secukinumab.
  • Example 14 Humanized antibody antagonizes IL-17A to induce mice to express CXCL1
  • Example 5 the effect of humanized antibodies on IL-17A-mediated biological activity in vivo was determined by detecting changes in serum CXCL1 levels in mice.
  • 40 female 10-week-old Balb/c mice were selected and divided into 8 groups of 5 mice.
  • serum was collected, and the expression level of CXCL1 was detected as the base value.
  • the candidateized antibodies hu31, hu43, hu44, hu60, and hu250 have a stronger antagonistic effect on IL-17A-stimulated release of CXCL1 in mice.
  • Example 15 Humanized antibody to improve the efficacy of imiquimod-induced mouse psoriasis model
  • Applying imiquimod to the skin of the mouse ear can induce psoriasis-like pathological features, that is, hyperplasia of keratinocytes, aggregation of inflammatory cells and vascular hyperplasia of the dermal papilla, etc., to construct a psoriasis mouse model.
  • the clinical score and ear swelling degree were used as indicators to judge the therapeutic effect of the drug on psoriasis mice.
  • mice Forty-eight C57BL/6 female mice (purchased by the Institute of Model Animal Research of Nanjing University, animal certification number 201605578), 6 to 8 weeks, were removed from the back. Except for the sham operation group, they were sensitized two days later.
  • mice of group II-VI applied approximately 62.5 mg of imiquimod cream (adalole, 5%, 3M Health Care Limited) on the skin of the right ear and back for 4 consecutive days.
  • the thickness of the right ear of the mouse was measured with a spiral micrometer every day, and the thickness of the ear swelling of the mouse was calculated based on the thickness of the right ear of day 1.
  • the mice were weighed every day, and the skin scales, induration, and erythema were observed and scored using a 4-level scoring method: 0 points, no disease; 1 point, mild; 2 points, moderate; 3 points, severe; 4 points ,very serious. The results were expressed as mean ⁇ SEM.
  • One-way analysis of variance (ANOVA) was used first. The differences between the two groups were compared using Student’s-t test. P ⁇ 0.05 was considered to be significant.
  • administration of the humanized antibody of the present invention can significantly inhibit imiquimod-induced skin psoriasis, induration, and redness in a mouse psoriasis model, that is, the score is small.
  • the humanized antibody of the present invention can obviously resist the onset of mice, and the phenotype is the clinical score of mice and the reduction of the degree of ear swelling.
  • Example 16 Effect of humanized antibody on improving type II collagen-induced female cynomolgus monkey arthritis
  • Type II collagen-induced arthritis is an animal model widely used in the study of rheumatoid arthritis (RA), and has the same histopathological characteristics as human RA, characterized by inflammation of the facet joints and progressive erosion of cartilage and bone .
  • Human/humanized biomacromolecules, including antibodies often have better cross-reactivity with antigens in cynomolgus monkeys, so the cynomolgus monkey arthritis model is one of the anti-rheumatic effects of humanized antibody IL-17A in the present invention Effective system.
  • the efficacy of candidate antibodies was evaluated on a cynomolgus monkey rheumatoid arthritis model.
  • Type II bovine collagen (CII, Sichuan University) was dissolved in acetic acid (Catalog No. 10000218; Sinopharm; Shanghai; China) and placed in a 4°C refrigerator overnight, and then equal volume of complete Freund's adjuvant (Catalog No. F5881, Sigma- Aldrich, USA) emulsified collagen, the final concentration of emulsion collagen is 2 mg/ml.
  • Shutai 1.5-5mg/kg, i.m
  • anesthesia was maintained with 1.5%-5% isoflurane during immunization.
  • Body weight measurement The body weight of animals was measured one day before immunization, and body weight was measured once a week thereafter until the end of experiment.
  • Arthritis score score the degree of inflammation of the arthritis of the limbs of the monkey on days 0 and 21, and score once a week after 21 days until the end of the experiment (if there is early onset, the corresponding arthritis score will be evaluated once a week in advance) ,
  • the scoring criteria are shown in Table 2.
  • the following 15 joints of each paw palm were scored: 5 metacarpophalangeal joints (MCP), 4 proximal knuckles (PIP), 5 distal knuckles (DIP), 1 wrist or ankle .
  • MCP metacarpophalangeal joints
  • PIP proximal knuckles
  • DIP distal knuckles
  • the sum of the scores of the joints is the animal's arthritis score, and the maximum score is 192 (16 ⁇ 3 ⁇ 4).
  • Arthritis scoring criteria 0 points, normal; 1 point, mild arthritis, mild onset but clearly distinguishable; 2 points, moderate swelling; 3 points severe arthritis, severe swelling or obvious joint deformation.
  • the normal cynomolgus monkey (G1) has a stable body weight; after arthritis induction, the average weight of the cynomolgus monkey in the vehicle-treated group (G2) continues to decline.
  • Antibodies hu31 and hu59 both controlled this downward trend. Therefore, under this experimental condition, hu31 and hu59 have a certain improvement effect on weight loss caused by arthritis (**P ⁇ 0.01, ****P ⁇ 0.0001, compared with "G2: vehicle group”; One-way ANOVA /Dunnett).
  • the arthritis clinical score of the normal control animals remained at 0; the arthritis score of the model-vehicle group (G2) increased gradually, while the test antibodies hu31 and hu59 significantly inhibited Animal arthritis clinical scores are increasing. Therefore, the test antibodies hu31 and hu59 have the effect of inhibiting the progressive development of arthritis.
  • the test antibody hu31 significantly inhibited the increasing trend of the clinical score of cynomolgus monkey arthritis (***P ⁇ 0.001, #P ⁇ 0.05, and G2 : Comparison of vehicle group; One-way ANOVA/Dunnett).
  • Example 17 Effect of humanized antibody on mouse joint swelling induced by NIH3T3-IL17 cells
  • Cells NIH3T3 cells, NIH3T3 cells expressing human IL-17.
  • NIH3T3-IL-17+ control IgG antibody group (30mg/kg);
  • NIH3T3-IL-17+ test antibody high-dose administration group (antibody hu31, 3mg/kg);
  • NIH3T3-IL-17+ test antibody mid-dose administration group (antibody hu31, 10mg/kg);
  • NIH3T3-IL-17+ test antibody low-dose administration group (antibody hu31, 30mg/kg);
  • NIH3T3-IL-17+ positive drug administration group (cosentyx, 10mg/kg).
  • NIH3T3-IL-17 cells and NIH3T3 control cells were injected into the articular cavity of the right ankle joint of each group of mice.
  • Intraperitoneal injection of antibody hu31 (3, 10, 30mg/kg) and cosentyx (10mg/kg) were started for interventional administration 1 day before model selection, once every 3 days to investigate the effect of antibody hu31 injection on mouse arthritis model .
  • a vernier caliper measures the thickness of the ankle joint of the mouse and calculates the degree of swelling.
  • NIH3T3 cells stably transfected with hIL-17 were injected into the metaphyseal joint cavity of the mouse, and the next day, severe swelling of the metaphyseal joint of the mouse was observed.
  • inhibition rate (%) (NIH3T3-ILI7 group ankle joint thickness-administration group ankle joint thickness)/(NIH3-ILl7 fine ankle joint thickness- NIH3T3 group ankle thickness) ⁇ 100.
  • the results showed that on the second day after the administration, the inhibitory effect of each dose of the drug group on the swelling of the ankle joint of mice was observed until the end of the experiment, and the maximum inhibition efficiency was reached on the sixth day after the administration.
  • the inhibition rates of antibody hu31 (3, 10, 30 mg/kg) in each group were 49.4%, 65.9%, and 74.1%, respectively.
  • the swelling inhibition rate of cosentyx (10 mg/kg) was 67.1%. Calculate the average inhibition rate of different days of each group, the results show that the average inhibition rate of each group of 3, 10 and 30mg/kg is 45.2%, 57.0% and 73.9% respectively.
  • the swelling inhibition rate of cosentyx (10 mg/kg) was 60.4%.
  • the experimental results suggest that the antibody hu31 can dose-dependently inhibit the IL-17-induced swelling of joints and ankles in mice, and its effect of 10 mg/kg is comparable to the positive control drug cosentyx (10 mg/kg). The effect of 30mg/kg is better than the positive control drug cosentyx (10mg/kg).
  • Example 18 Effect of humanized antibody on mouse air bladder inflammation induced by NIH3T3-IL17 cells
  • Cells NIH3T3 cells, NIH3T3 cells expressing human IL-17.
  • NIH3T3-IL-17 cell group + test antibody high-dose administration group (antibody hu31, 30mg/kg);
  • NIH3T3-IL-17 cell group + test antibody mid-dose administration group (antibody hu31, 10mg/kg);
  • NIH3T3-IL-17 cell group + test antibody low-dose administration group (antibody hu31, 3mg/kg);
  • Route of administration intraperitoneal injection.
  • Air balloon 2.5 ml of air was injected into the back of the mice on days 0 and 3, respectively. On the 5th day, cells were injected into the air bladder. The number of cells injected per mouse was 2 ⁇ 10 5 cells/500 ⁇ l PBS. 8 mice per group.
  • Number of neutrophils total number of cells ⁇ Gr1 + cell ratio
  • NIH-3T3 cells stably transfected with hlL-17A were injected into the air sacs on the back of mice. From the total number of infiltrated cells, compared with the NIH3T3 cell group, the NIH3T3-IL-17 cell group air sacs The number of infiltrated leukocytes increased significantly, and the proportion and number of Grl + cells also increased significantly, and the model selection was successful.
  • the antibody hu31 was injected intraperitoneally on the day of modeling (the doses were 3, 10, and 30 mg/kg, respectively).
  • inhibition rate (%) (NIH3T3-ILI7-lgG group cell number-drug group cell number) / (NIH3T3-ILl7-IgG Number of cells in the group-NIH3T3 cells) ⁇ 100.
  • inhibition rate of the total number of infiltrating cells of antibody hu31 (3, 10, 30 mg/kg) in each group was 50.0%, 56.7%, and 78.3%, respectively.

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Abstract

一种以高亲和力与IL-17A特异性结合的抗体或其功能性片段。一种编码上述抗体或其功能性片段的核酸分子，用于表达抗体或其功能性片段的表达载体和宿主细胞，以及抗体或其功能性片段的生产方法。一种包含抗体或其功能性片段的药物组合物，以及使用抗体或其功能性片段治疗免疫功能失调性疾病的方法。

Description

抗IL-17A抗体及其应用 技术领域
本发明涉及特异性结合IL-17A的抗体及其抗原结合片段。本发明更具体地涉及抑制IL-17A介导的生物活性的抗体及其抗原结合片段，以及使用所述抗体或其抗原结合片段的组合物和治疗相关疾病的方法。本发明更具体地涉及应用抗IL-17A抗体或其抗原结合片段来治疗免疫性病理性疾病，所述疾病包括自身免疫性和炎性疾病，诸如类风湿性关节炎、银屑病、强制性脊柱炎、多发性硬化症、系统性红斑狼疮(SLE)、狼疮性肾炎或慢性阻塞性肺病、哮喘、感染性肉芽肿、囊性纤维化或癌症。
背景技术
白细胞介素17(Interleukin 17,IL-17)，也称为CTLA-8或IL-17A，在免疫系统中扮演着关键的协调作用。IL-17家族共六个成员，包括IL-17A、IL-17B、IL-17C、IL-17D、IL-17E和IL-17F，都含有至关重要的4个高度保守的半胱氨酸残基，但成员之间生物学效应差异较大，其中，IL-17A和IL-17F同源性和生物学功能最接近，目前研究也最深入。体内表达的IL-17A具有23个氨基酸的N-末端信号肽，其经切割产生成熟的IL-17A。成熟的IL-17A通过二硫键连接，一般以同源二聚体形式分泌与存在，有时也与IL-17F连接形成异源二聚体IL-17AF。一般IL-17A或IL-17指IL-17A同源二聚体蛋白，其主要由辅助性T细胞17(T helper 17,Thl7)产生，也可以由其它免疫细胞如γδT细胞、LTi(Lymphoid Tissue inducer cells)、ILCs(Innate Lymphoid Cells)和NKT(Natural Killer T)细胞合成分泌(Cua DJ,Tato CM.，Nature reviews.2010,10:479–489)。IL-17A的表达调控非常复杂，研究发现细胞因子IL-6、IL-1β和TGFβ等诱导初始CD4 +T细胞分化为Th17，但此时Th17细胞稳定性弱，分泌少量IL-17A，其组织损伤作用小；当IL-23存在时，其通过促进Th17细胞稳定性并持续分泌IL-17A、上调促炎因子(IL-22、CSF-2和IFN-γ)和下调抑炎因子(IL-2、IL-27和IL-12)表达等途径，引起炎性爆发和组织损伤(McGeachy MJ,et al.，Nature immunology.2009,10:314–324)。所以，当组织中IL-23异常表达时，IL-17A途径在组织损伤中发挥着关键性的作用。
IL-17一般定点分泌，在局部组织中与靶细胞表面的IL-17受体(IL-17recptor，IL-17R) 结合发挥效应。IL-17R家族包括IL-17RA、IL-17RB、IL-17RC、IL-17RD及IL-17RE五个成员，广泛表达于多种细胞膜上(Iwakura Y,et al.，Immunity.2011；34:149–162)。IL-17主要与非造血细胞来源的细胞(如上皮细胞、间质细胞)表面的IL-17RA/IL-17RC复合体结合发挥效应(Ishigame H,et al.，Immunity.2009；30:108–119)，促进细胞分泌细胞因子(诸如IL-6、G-CSF、GM-CSF、IL-10、TGF-β、TNF-α)、趋化因子(包括IL-8、CXCL1和MCP-1)和前列腺素(例如，PGE2)，诱导中性粒细胞和巨噬细胞聚集，释放活性氧类物质(ROS)而损伤组织(Stark MA,et al.，Immunity.2005；22:285–294)。
自身免疫性疾病严重威胁着人类的健康，如银屑病、类风湿性关节炎、强直性脊柱炎、克罗恩病、多发性硬化病等。研究发现，IL-17的分泌失调与该类疾病发生发展密切相关。靶向IL-17的抗体通过抑制IL-17-IL-17R信号通路，能有效地缓解自身免疫性疾病的症状(Sarah L,et al.，Nat Rev Immunol.2014,14(9):585–600)。诺华公司开发的Cosentyx(secukinumab)是全球首个IL-17单抗，获批的适应症包括中度至重度斑块型银屑病(plaque psoriasis)，为广大银屑病群体提供一个重要的一线生物治疗选择。但是，开发结构不同、疗效更优、适应症更广等不同特性的抗IL-17抗体，治疗银屑病、类风湿性关节、强直性脊柱炎等自身免疫相关病症以及IL-17相关的其它疾病具有迫切的需求和重要的意义。
发明概述
本发明提供一种特异性结合人IL-17A的抗体或其抗原结合片段，其包含至少一个选自SEQ ID NO:1-24、60-65的互补决定区(CDR)序列。
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含至少一个选自SEQ ID NO:1-3、4-6、7-9、10-12或60-62的重链CDR结构域和/或至少一个选自SEQ ID NO:13-15、16-18、19-21、22-24或63-65的轻链CDR结构域。
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含重链可变区(VH),其特征为所述的重链可变区包含选自SEQ ID NO:1、4、7、10或60的HCDR1，选自SEQ ID NO:2、5、8、11或61的HCDR2，和选自SEQ ID NO:3、6、9、12或62的HCDR3。
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含重链可变区(VH),其特征为所述的重链可变区包含的HCDR1、HCDR2和HCDR3氨基酸序列选自以下组A到组E中的任一组：
组号 HCDR1 HCDR2 HCDR3
A SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3
B SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6
C SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9
D SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12
E SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:62
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含轻链可变区(VL),其特征为所述的轻链可变区包含选自SEQ ID NO:13、16、19、22或63的LCDR1，选自SEQ ID NO:14、17、20、23或64的LCDR2，和选自SEQ ID NO:15、18、21、24或65的LCDR3。
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含轻链可变区(VL),其特征为所述的轻链可变区包含的LCDR1、LCDR2和LCDR3的氨基酸序列选自以下组F到组J中的任一组：
组号 LCDR1 LCDR2 LCDR3
F SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
G SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18
H SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21
I SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24
J SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其特征为所述可变区包含的6个CDR的氨基酸序列选自以下组I到组VI中的任一组：
组号 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3
I SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
II SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18
III SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21
IV SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24
V SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24
VI SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含重链可变区(VH)和/或轻链可变区(VL)，其特征为所述的重链可变区氨基酸序列选自SEQ ID NO:25、26、27、28、33、35、38或40，和/或所述的轻链可变区(LV)氨基酸序列选自SEQ ID NO:29、30、31、32、34、36、38、39或41。
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其特征为所述的可变区氨基酸序列选自以下组1到组7中的任一组：
组号 VH VL
1 SEQ ID NO:25 SEQ ID NO:29或30
2 SEQ ID NO:26 SEQ ID NO:31
3 SEQ ID NO:27或28 SEQ ID NO:32
4 SEQ ID NO:33或35 SEQ ID NO:34
5 SEQ ID NO:35 SEQ ID NO:36
6 SEQ ID NO:37 SEQ ID NO:38或39
7 SEQ ID NO:40 SEQ ID NO:41
在一个实施方式中，本发明所述的抗体或其抗原结合片段包含轻链(LC)和/或重链(HC)，其特征为所述的重链(HC)氨基酸序列选自序列编号SEQ ID NO:42、44、46或49，和/或所述的轻链(LC)氨基酸序列选自序列编号SEQ ID NO:43、45、47、48或50。
在一个实施方式中，本发明所述的抗体或其抗原结合片段，其包含轻链(LC)和重链(HC)，其特征为所述的轻链的氨基酸序列如SEQ ID NO:43所示，和重链的氨基酸序列如SEQ ID NO:42或44所示；或轻链的氨基酸序列如SEQ ID NO:45所示，和重链的氨基酸序列如SEQ ID NO:44所示；或轻链的氨基酸序列如SEQ ID NO:47或48所示，和重链的氨基酸序列如SEQ ID NO:46所示；或轻链的氨基酸序列如SEQ ID NO:50所示，和重链的氨基酸序列如SEQ ID NO:49所示。
在一个实施方式中，本发明所述的抗体为完全抗体，优选为IgG，更优选为IgG4型。
在一个具体的实施方式中，本发明所述的抗体为1F8、2B2、2F5、ch1、ch2、ch16、hu31、hu43、hu44、hu59、hu60或hu250。
在一个实施方式中，本发明所述的抗体或其抗原结合片段的特征为：a)特异性结合IL-17A同源二聚体和IL-17AF异源二聚体；b)阻断IL-17A与其受体之间的结合；和/或c)抑制IL-17A介导的生物活性。
在一个实施方式中，本发明所述的IL-17A、IL-17AF或IL-17F选自食蟹猴、小鼠 或人中的一种或多种。
在一个实施方案中，本发明所述的抗体或抗原结合片段不特异性结合a)人IL-17F同二聚体、IL-17B同二聚体、IL-17C同二聚体、IL-17D同二聚体、IL-17E同二聚体的任一种或多种，和/或b)食蟹猴IL-17F同二聚体、小鼠IL-17F同二聚体的任一种或多种。
在另一方面，本发明所述的抗体或其抗原结合片段具有抑制IL-17A与其受体之间的结合和/或降低由IL-17A介导的细胞信号转导和/或生物活性的功能。
在一个实施方式中，本发明所述的抗体或其抗原结合蛋白在体外活性评估时，能够抑制IL-17A诱导上皮细胞分泌CXCL1。
在一个实施方式中，本发明所述的抗体或其抗原结合片段在体内活性评估时，能够抑制IL-17A诱导小鼠体内分泌CXCL1。
在一个实施方式中，本发明所述的抗体或其抗原结合蛋白在体内活性评估时，能够在咪喹莫特诱导的银屑病实验模型中抵抗小鼠发病，小鼠银屑病发病的临床评分及耳部肿胀程度的显著降低。
在一个实施方式中，本发明所述的分离抗体或其抗原结合片段在体内评估时，能够在抗原诱导的关节炎实验模型诸如食蟹猴AIA-模型中抑制膝关节肿胀。
本发明还提供所述抗体或其抗原结合片段，优选hu31、hu43、hu44、hu59、hu60或hu250作为药物的用途，更优选作为用于治疗由IL-17A介导的病理性疾病和/或通过抑制IL-17A信号转导来治疗的病理性疾病的药物的用途。
在一个具体实施方式中，由IL-17A介导的病理性疾病是炎性病症或病况，诸如关节炎、类风湿性关节炎、银屑病、强直性脊柱炎、慢性阻塞性肺病、系统性红斑狼疮(SLE)、狼疮性肾炎、哮喘、多发性硬化或囊性纤维化等。
在本发明提供了治疗由IL-17A介导的病理性疾病的方法，所述方法包括施用有效量的本发明所述的分离的抗体或其抗原结合片段，优选为hu31、hu43、hu44、hu59、hu60或hu250抗体，以使所述病况被缓解。
本发明还涉及用于产生本发明的抗体或其抗原结合片段的方法。此类方法包括编码本发明的抗体或蛋白质的至少重链和/或轻链可变区的分离的核酸分子，或包含此类核酸的克隆性表达载体，特别是用于在宿主细胞中重组产生本发明所述的抗体或蛋白质例如hu31、hu43、hu44、hu59、hu60或hu250。
本发明还涉及包含一种或多种上述克隆性载体或表达载体的宿主细胞以及用于产生本发明的抗体或包含其抗原结合片段的蛋白质，具体地比如产生hu31、hu43、hu44、hu59、hu60或hu250抗体的方法，所述方法包括培养所述宿主细胞、纯化和回收所述抗 体或蛋白质。
在一个实施方式中，本发明所述的分离抗体或包含其抗原结合片段的蛋白质缀合于其它活性部分。
在一个实施方式中，本发明所述的抗体或其抗原结合片段可以是单克隆抗体或其抗原结合片段，优选嵌合抗体、人源化抗体或人抗体或其部分。
在本发明的一方面，提供了药物组合物，其包含与一种或多种可药用的赋形剂、稀释剂或载体组合的本发明任一实施方式所述的抗体或包含其抗原结合片段的蛋白质。
在一个实施方式中，所述药物组合物包含一种或多种额外的活性成分。
在一个具体实施方式中，所述药物组合物是冻干粉剂。在另一个具体实施方式中，所述药物组合物是包含治疗上可接受的量的本发明所述的抗体或分子的稳定的液体制剂。
在一个实施方式中，本发明涉及所述的抗体或其抗原结合片段用于制备用于治疗任何一种由IL-17A介导的病理性病症的药物中的用途。
本发明提供了编码上述的抗体或其抗原结合片段的分离的核酸分子，包含上述核酸分子的表达载体或重组载体，以及转化所述载体的宿主细胞。
本发明提供了一种药物组合物，其包含如上述的抗体或其抗原结合片段、上述核酸分子、载体或宿主细胞和药学上可接受的载体或赋形剂的组合物。
本发明提供了上述的抗体或其抗原结合片段、核酸分子、载体或宿主细胞或上述的药物组合物在制备用于治疗和/或预防IL-17A介导的疾病或病症的药物中的用途。
在一些方案中，上述药物是用于治疗关节炎、类风湿性关节炎、银屑病、强制性脊柱炎、慢性阻塞性肺疾病、系统性红斑狼疮(SLE)、狼疮性肾炎、哮喘、多发性硬化或囊性纤维化的药物。
附图简述
图1：人IL-17A-mFc重组蛋白的SDS-PAGE电泳图。
图2：FACS检测人IL-17A-mFc与293F细胞上的IL-17RA结合(MFI表示平均荧光强度,实验组v.s control:7695vs308)。
图3：ELISA检测杂交瘤抗体阻断IL-17A介导的体内生物活性作用。其中杂交瘤抗体明显抑制IL-17A诱导小鼠表达CXCL1。
图4：ELISA检测嵌合抗体与人IL-17A的结合作用。其中嵌合抗体ch1，ch2，ch4，ch7和ch16与人IL-17A结合具有较高的特异性，其EC 50分别为6.62ng/ml，5.17ng/ml，88.48ng/ml，39.96ng/mL和15.42ng/mL。
图5：FACS检测嵌合抗体阻断人IL-17A与293F细胞上的IL-17RA结合作用。其中嵌合抗体ch1，ch2，ch7和ch16具有高效的阻断作用，其IC 50分别为4.46ug/ml，3.145ug/ml，1.220ug/ml和1.445ug/ml。
图6：ELISA检测人源化抗体与人IL-17A的结合作用。其中抗体hu31，hu43，hu44，hu59，hu60和hu250与人IL-17A结合具有较高的特异性，其EC 50分别为8.13ng/mL，8.64ng/mL，6.764ng/ml，6.102ng/mL，5.776ng/mL和6.351ng/mL。
图7：FACS检测人源化抗体阻断人IL-17A与293F细胞上的IL-17RA结合作用。其中抗体hu31，hu43，hu44，hu59，hu60和hu250均具有高效的阻断作用，其IC 50分别为867.6ng/mL，780.8ng/mL，828.5ng/ml，467.4ng/mL，482.8ng/mL和577.8ng/mL。
图8：ELISA检测人源化抗体阻断IL-17A介导的上皮细胞分泌CXCL1的效应。其中抗体hu31，hu43，hu44，hu59，hu60和hu250均高效抑制IL-17A诱导上皮细胞表达CXCL1，并且比对照抗体的阻断作用更强。
图9：ELISA检测人源化抗体阻断IL-17A介导的体内生物活性作用。其中抗体hu31，hu43，hu44，hu60和hu250具有高效抑制IL-17A诱导小鼠表达CXCL1，并且比对照抗体的阻断作用更强。
图10-1：人源化抗体给药对咪喹莫特诱导的银屑病小鼠临床评分影响。而给予hu31和hu44可明显抑制咪喹莫特诱导的小鼠银屑病模型皮肤鳞屑、硬结、红肿等情况,即临床评分降低(*P<0.05vs KLH)。
图10-2：人源化抗体对咪喹莫特诱导的银屑病小鼠耳肿胀程度的影响。给予hu31和hu44可明显改善耳肿胀程度。(*P<0.05vs KLH,**P<0.01vs KLH)。
图11-1：人源化抗体对II型胶原蛋白诱导的雌性食蟹猴体重的影响。hu31和hu59对关节炎引起的体重下降具有一定改善作用(**P<0.01，****P<0.0001，和“G2:溶媒组”相比较；One-way ANOVA/Dunnett)。
图11-2：人源化抗体对II型胶原蛋白诱导的雌性食蟹猴关节炎评分的影响。hu31显著抑制了食蟹猴关节炎临床评分的增加趋势(***P<0.001，#P<0.05，和G2:溶媒组相比较；One-way ANOVA/Dunnett)。
图12：人源化抗体对NIH3T3-IL17细胞诱导的小鼠关节肿胀的作用。
图13：人源化抗体对NIH3T3-IL17细胞诱导的小鼠空气囊炎症的作用。
发明详述
本发明涉及特异性结合人IL-17A并阻断由IL-17A参与介导的生物活性的抗体。本 发明涉及在下文中进一步描述的全长IgG形式抗体及其抗原结合片段。
定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
IL-17或IL-17A是白介素-17。除非另有表述，所述的IL-17通常指人IL-17A。
体内表达的IL-17A是具有23个氨基酸的N-末端信号肽，NCBI登录号NP_443104.1，其经切割产生成熟的IL-17A。在一个具体的实施方式中，本公开内容中IL-17A指成熟的IL-17A，而不包含N-末端信号肽，其氨基酸序列为SEQ ID NO:66，核苷酸序列为SEQ ID NO:67。
体内表达的IL-17F是具有30个氨基酸的N-末端信号肽，NCBI登录号NP_443104.1，其经切割产生成熟的IL-17F。在一个具体的实施方式中，本文中IL-17F指成熟的IL-17F，而不包含N-末端信号肽，其氨基酸序列为SEQ ID NO:68。
本文中IL-17AF是IL-17A亚单位与IL-17F亚单位的异二聚体，如本领域普通技术人员所理解的。
“免疫应答”是指由例如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝产生可溶性大分子(包括抗体、细胞因子和补体)的作用，该作用导致从人体选择性损害、破坏或清除侵入的病原体、感染病原体的细胞或组织、癌细胞或者在自体免疫或病理性炎症的情况下的正常人细胞或组织。
“信号转导途径”或“信号转导活性”是指通常由蛋白质间相互作用诸如生长因子对受体的结合启动的生化因果关系，所述关系导致信号从细胞的一部分传递至细胞的另一部分。一般地，传递包括引起信号转导的系列反应中的一种或多种蛋白质上的一个或多个酪氨酸、丝氨酸或苏氨酸残基的特定磷酸化。倒数第二过程通常包括细胞核事件，从而导致基因表达的变化。
关于本发明的抗体或其抗原结合片段，术语“活性”或“生物活性”，或术语“生物性质” 或“生物特征”此处可互换使用，并包括但不局限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗IL-17A活性的能力、IC50、抗体的体内稳定性和抗体的免疫原性质。本领域公知的抗体的其它可鉴定的生物学性质或特征包括，例如，交叉反应性(即通常与靶定肽的非人同源物，或与其它蛋白质或组织的交叉反应性)，和保持哺乳动物细胞中蛋白质高表达水平的能力。使用本领域公知的技术观察、测定或评估前面提及的性质或特征，所述技术包括但不局限于ELISA、FACS或BIACORE等离子体共振分析、不受限制的体外或体内中和测定、受体结合、细胞因子或生长因子的产生和/或分泌、信号转导和不同来源(包括人类、灵长类或任何其它来源)的组织切片的免疫组织化学。
“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、完全人抗体、嵌合抗体和骆驼源化单结构域抗体。
“分离抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
“全长抗体”，天然存在时包含四条肽链的免疫球蛋白分子，两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为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区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)的结合。
抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗 体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab') 2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。术语“结合化合物”是指抗体及其结合片段两者。
“单链Fv”或“scFv”抗体是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单条多肽链中。Fv多肽一般还包含VH和VL结构域之间的多肽接头，其使scFv能够形成用于抗原结合的所需结构。
“结构域抗体”是只含有重链可变区或轻链可变区的免疫功能性免疫球蛋白片段。在某些情况下，两个或更多个VH区与肽接头共价连接形成二价结构域抗体。二价结构域抗体的2个VH区可靶向相同或不同的抗原。
“二价抗体”包含2个抗原结合部位。在某些情况下，2个结合部位具有相同的抗原特异性。然而，二价抗体可以是双特异性的。
“双抗体”是指具有两个抗原结合部位的小抗体片段，所述片段包含在同一多肽链(VH-VL或VL-VH)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用短得不允许在同一链的两个结构域之间配对的接头，迫使该结构域与另一链的互补结构域配对并产生两个抗原结合部位。
“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常可变结构域获自啮齿动物等实验动物的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
“人源化抗体”是指含有来自人和非人(例如鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
“完全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则完全人抗体可含有鼠糖链。同样，“小鼠 抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则完全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
“同种型”是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语包括具有与参照核酸相似的结合性质并且以与天然存在的核苷酸相似的方式被代谢的含有已知的天然核苷酸的类似物的核酸。(参见，属于Kariko等人的美国专利No.8,278,036,其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。可使用经修饰mRNA的方法，例如，属于Kariko等人的美国专利No.8,278,036和属于Moderna公司的专利申请W02013/090186A1中公开的那些方法。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、SNP和互补序列以及明确指出的序列。具体地，简并密码子取代可通过生成其中一个或多个选择的(或全部)密码子的第三位被混合碱基和/或脱氧肌苷残基取代的序列来实现(Batzer等人，Nucleic Acid Res.19:5081(1991)；Ohtsuka等人，J.Biol.Chem.260:2605-2608(1985)；和Rossolini等人，Mol.Cell.Probes 8:91-98(1994))。
“构建体”是指任何重组多核苷酸分子(诸如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体或线性或环状单链或双链DNA或RNA多核苷酸分子)，衍生自任何来源，能够与基因组整合或自主复制，构成如下多核苷酸分子，其中已经以功能操作的方式连接(即，可操作地连接)一或多个多核苷酸分子。重组构建体通常会包含可操作地连接至转录起始调节序列的本发明的多核苷酸，这些序列会导引多核苷酸在宿主细胞中的转录。可使用异源及非异源(即，内源)启动子两者导引本发明的核酸的表达。
“载体”是指任何重组多核苷酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。一种类型的载体为“质粒”，是指环状双链DNA环，可将额外DNA区段连接至该环中。另一类型的载体为病毒载体，其中可将额外DNA区段连接至病毒基因组中。某些载体能够在被引入到的宿主细胞中自主复制(例如，具有细菌复制起点的细菌载体及游离型哺乳动物载体)。在引入到宿主细胞中后，其他载体(例如，非游离型哺乳动物载体)整合至宿主细胞的基因组中，且因此与宿主基因组一起复制。此外，某些载体能够导引被操作性连接的基因的表达。本文将此类载体称为“表达载体”。
本文所用术语“表达载体”是指能够在转化、转染或转导至宿主细胞中时复制及表达目的基因的核酸分子。表达载体包含一或多个表型选择标记及复制起点，以确保维护载体及以在需要的情况下于宿主内提供扩增。
如本文中所用，术语“IL-17A拮抗剂”或“IL-17A阻断分子”意指抑制IL-17A通过IL-17R诱导的信号转导活性，从而降低或中和IL-17A活性的抗体或其抗原结合蛋白。这可在人细胞测定诸如人细胞的依赖IL-17A的CXCL1产生测定中得以显示。此类测定在下面实施例中进行了更详细描述。
用于细胞或受体的“活化”、“刺激”和“处理”可具有相同含义，例如细胞或受体用配体活化、刺激或处理，除非上下文另外或明确规定。“配体”包括天然和合成配体，例如细胞因子、细胞因子变体、类似物、突变蛋白和来源于抗体的结合化合物。“配体”还包括小分子，例如细胞因子的肽模拟物和抗体的肽模拟物。“活化”可指通过内部机制以及外部或环境因素调节的细胞活化。“应答/反应”，例如细胞、组织、器官或生物体的应答，包括生化或生理行为(例如生物区室内的浓度、密度、粘附或迁移、基因表达速率或分化状态)的改变，其中改变与活化、刺激或处理有关，或者与例如遗传编程等内部机制有关。
如本文中所用，术语任何疾病或病症的“治疗”或“医治”在一个实施方案中是指改善疾病或病症(即，减缓或阻止或减少疾病的进展或其临床症状的至少一个)。在另一个实施方案中，“治疗”或“医治”是指缓解或改善至少一个身体参数，包括可能不能被患者辨别出的那些物理参数。在另一个实施方案中，“治疗”或“医治”是指在身体上(例如，可辨别的症状的稳定)、生理上(例如，身体参数的稳定)或在这两方面调节疾病或病症。除非在本文中明确描述，否则用于评估疾病的治疗和/或预防的方法在本领域中通常是已知的。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的IL-17A抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活 性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％；通常至少20％；优选至少约30％；更优选至少40％，最优选至少50％。
抗体的产生
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的人IL-17A都可用作产生抗体的免疫原(抗原)。通过举例而非限制，任何人IL-17A同种型或其片段都可用作免疫原。实例包括但不限于本文所述的天然的成熟型人IL-17A(氨基酸序列为SEQ ID NO:66)。
在一个优选方案中，产生鼠源的单克隆抗人IL-17A抗体的杂交瘤细胞可通过本领域公知的方法产生。这些方法包括但不限于最初由Kohler等(1975)(Nature 256:495-497)研发的杂交瘤技术。优选根据标准方案，分离出小鼠脾细胞，用PEG或通过电融合与小鼠骨髓瘤细胞系融合。然后可筛选产生抗原特异性抗体的所得杂交瘤。例如，在50％PEG的情况下，可将来源于免疫小鼠的脾淋巴细胞的单细胞悬液与1/6数目的小鼠骨髓瘤细胞SP20(ATCC)融合。可将细胞以约2×10 5个细胞/mL接种在平底微量滴定板中，接着在含有20％胎牛血清的完全培养基和1×HAT(Sigma；融合后24小时加入HAT)的选择培养基中孵育2周。2周后，可将细胞在HAT用HT替换的培养基中培养。然后可通过ELISA针对抗人IL-17A单克隆IgG抗体筛选各孔。一旦出现大规模杂交瘤生长，通常可在10-14天后对培养基进行观察。分泌抗体的杂交瘤可再次接种、筛选，如果仍为人IgG阳性，则可通过有限稀释对抗人IL-17A单克隆抗体杂交瘤进行亚克隆至少两次。然后可体外培养稳定的亚克隆以在组织培养基中产生少量的抗体用于表征。
在一个优选实施方式中，本发明获得的单克隆杂交瘤细胞为1F8、2F5和2B2，其分泌的抗体与IL-17A高特异性结合、阻断IL-17A与IL-17RA结合并抑制IL-17A介导的生物活性，如抑制CXCL1分泌。
在一个优选实施方式中，本发明利用基于简并引物PCR的方法，测定由候选杂交瘤细胞1F8、2F5和2B2免疫球蛋白可变区的DNA序列。其中杂交瘤细胞1F8具有两种抗体轻链基因和一种抗体重链基因，2F5具有两种抗体重链基因和一种抗体轻链基因。所以1F8、2F5分泌的抗体可能均含有两种混合完全抗体，在本文中，1F8分泌的抗体称为1F8-1和1F8-2，2F5分泌的抗体称为2F5-1和2F5-2。
表1：杂交瘤抗体可变区的氨基酸序列
Figure PCTCN2019124486-appb-000001
Figure PCTCN2019124486-appb-000002
来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而最高则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84:3439-43)。然而，嵌合抗体中的完整啮齿动物可变区的保留仍可在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321:522；Verhoeyen等(1988)Science 239:1534)。
在一些实施方案中，本发明的嵌合或人源化抗体可基于所述制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方案中，本发明所述的嵌合抗体，可使用本领域已知的方法将杂交瘤来源的编码免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链。其中人IgG可选自任何亚型，如IgG1、IgG2、IgG3、IgG4，优选IgG4。
在一个具体的实施方式中，本发明的嵌合抗体可由一种嵌合型轻链与一种嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的IL-17A结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试，优选的ch1，ch2，ch4，ch7和ch16具有最优的结合和阻断活性，其可变区氨基酸序列如表2所示。
表2：嵌合抗体可变区氨基酸序列
嵌合抗体 重链可变区(VH) 轻链可变区(VL)
ch1 SEQ ID NO:25 SEQ ID NO:29
ch2 SEQ ID NO:25 SEQ ID NO:30
ch4 SEQ ID NO:25 SEQ ID NO:32
ch7 SEQ ID NO:26 SEQ ID NO:31
ch16 SEQ ID NO:28 SEQ ID NO:32
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案中的任何一种方案来确定，包括由Kabat等人(1991)，“Sequences of Proteins of Immunological Interest，第5版.Public Health Service，National Institutes of Health，Bethesda，MD(“Kabat”编号方案)描述的Kabat方案和Lefranc M.-P.等人描述的IMGT方案(1999Nucleic Acids Research,27,209-212)。在一些实施方式中，本发明优选的鼠源抗体可变区的CDR具体定义方案和氨基酸序列如表3所示。
表3：鼠源抗体可变区CDR定义
Figure PCTCN2019124486-appb-000003
在一些实施方案中，本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370。简言之，发明人藉由NCBI (http://www.ncbi.nlm.nih.gov/igblast/)网站中的人类免疫球蛋白基因数据库搜寻与鼠源抗体可变区的cDNA序列同源的人类种系IgG基因，原则上藉由选定的CDR嫁接实现人源化。然而，CDR环交换仍不能均匀产生具有与起始抗体相同的结合性质的抗体。在人源化抗体中，常常还需要构架残基(FR)(参与CDR环支持的残基)改变以保持抗原结合亲和力。Kabat等(1991)J.Immunol.147:1709。简言之，人源化改造过程涉及以下步骤：A、把各候选抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；B、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架序列；C、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析各候选的抗体基因序列中可与IL-17A作用以及维护空间构架的关键氨基酸个体，将其嫁接回已经选择的人胚胎系基因框架，并在此基础上标配出必须保留的框架区氨基酸位点，合成人源化抗体。
在一些实施方式中，本发明获得的优选人源化抗体为hu31，hu43，hu44，hu59，hu60和hu250。
人源化抗体hu31，hu43，hu44，hu59，hu60和hu250可变区及其对应的CDR氨基酸序列如表4所示。
表4：人源化抗体可变区和CDR序列
抗体 hu31 hu43 hu44 hu59 hu60 hu250
VH SEQ ID NO:33 SEQ ID NO:35 SEQ ID NO:35 SEQ ID NO:37 SEQ ID NO:37 SEQ ID NO:40
VL SEQ ID NO:34 SEQ ID NO:34 SEQ ID NO:36 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:41
HCDR1 SEQ ID NO:1 SEQ ID NO:1 SEQ ID NO:1 SEQ ID NO:10 SEQ ID NO:10 SEQ ID NO:60
HCDR2 SEQ ID NO:2 SEQ ID NO:2 SEQ ID NO:2 SEQ ID NO:11 SEQ ID NO:11 SEQ ID NO:61
HCDR3 SEQ ID NO:3 SEQ ID NO:3 SEQ ID NO:3 SEQ ID NO:12 SEQ ID NO:12 SEQ ID NO:62
LCDR1 SEQ ID NO:13 SEQ ID NO:13 SEQ ID NO:13 SEQ ID NO:22 SEQ ID NO:22 SEQ ID NO:63
LCDR2 SEQ ID NO:14 SEQ ID NO:14 SEQ ID NO:14 SEQ ID NO:23 SEQ ID NO:23 SEQ ID NO:64
LCDR3 SEQ ID NO:15 SEQ ID NO:15 SEQ ID NO:15 SEQ ID NO:24 SEQ ID NO:24 SEQ ID NO:65
人源化抗体hu31，hu43，hu44，hu59，hu60和hu250的轻/重链氨基酸和核苷酸序列如表5所示。
表5：人源化抗体氨基酸/核苷酸序列
Figure PCTCN2019124486-appb-000004
Figure PCTCN2019124486-appb-000005
本发明的其它抗体包括具有已通过氨基酸缺失、插入或取代突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方案中，本发明的抗体是hu31，hu43、hu44、hu59、hu60和hu250的任一个的突变体，其中所述突变体包括突变氨基酸序列，在所述突变氨基酸序列中，当与上述序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
编码本发明的抗体的其它核酸包括已通过核苷酸缺失、插入或取代突变的，但仍然与上文中所述的序列中描绘的CDR对应编码区具有至少60、70、80、90、95或100％同一性的核酸。
抗体表达
在一些实施方案中，本发明涉及包含一种或多种表达载体或表达载体的宿主细胞以及用于产生本发明的抗体或包含其抗原结合片段的方法，所述方法包括培养所述宿主细胞、纯化和回收所述抗体或抗原结合片段。
可使用例如本领域中公知的重组DNA技术与基因转染方法的组合(例如，Morrison,S.1985,Science 229:1202)在宿主表达细胞中产生本发明的抗体。例如，为了表达抗体或其抗体片段，编码部分或全长轻链和重链的DNA可利用标准分子生物学或生物化学技术(例如，DNA化学合成、PCR扩增或使用表达目标抗体的杂交瘤的cDNA克隆)来获得，并且可将所述DNA插入表达载体，以便所述基因被有效地连接于转录和翻译控制序列。在本说明书中，术语“有效连接的”意指将抗体基因连接进入载体，以使载体内的转录和翻译控制序列行使它们的调节抗体基因的转录和翻译的预定功能。选择可与所使用的表达宿主细胞相容的表达载体和表达调控序列。可将抗体轻链基因和抗体重链基因插入至不同的载体中，或者更通常地，将两个基因插入至同一表达载体中。通过标准方法将抗 体基因插入至表达载体中(例如，连接抗体基因片段和载体上互补的限制性位点，或者如果不存在限制性位点，则进行平端连接)。可以使用本文所述抗体的轻链和重链可变区来生成任何抗体同种型的全长抗体基因，其通过将它们插入已编码期望同种型的重链恒定区和轻链恒定区的表达载体中，从而使得VH区段与载体中的CH段有效连接，VL区段与载体中的CL区段有效连接。此外/或可选择地，重组表达载体可以编码信号肽(也称为前导序列)，其有利于抗体链从宿主细胞分泌。可将抗体链基因克隆进载体中以使信号肽与抗体链基因的氨基末端连接于同一读码框中。信号肽可以是免疫球蛋白信号肽或异源信号肽(即来自非免疫球蛋白的信号肽)。
用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。
在将编码重链或抗原结合片段或其片段、轻链和/或其抗原结合片段的重组表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中，来产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
医药用途
本发明的抗体或其抗原结合片段具有体外和体内诊断及治疗用途。优选地，人源化抗体hu31、hu43、hu44、hu59、hu60或hu250抗体可用于治疗与IL-17A相关疾病症。
一方面，本发明所述的分离抗体或其抗原结合片段在体内活性评估时，能够在咪喹莫特诱导的银屑病实验模型中抵抗小鼠发病，小鼠银屑病发病的临床评分及耳部肿胀程度显著降低。
在一个具体的实施方式中，在咪喹莫特诱导的银屑病实验模型中，人源化抗体hu31和hu44可明显抵抗小鼠的发病，小鼠银屑病发病的临床评分及耳部肿胀程度降低。
一方面，本发明所述的分离抗体或其抗原结合片段在体内评估时，能够在抗原诱导的关节炎实验模型诸如食蟹猴AIA-模型中抑制膝关节肿胀。
在一个具体的实施方式中，在食蟹猴AIA-模型中，人源化抗体hu31显著抑制食蟹猴关节炎临床评分的增加趋势。
本发明的一个方面，提供了治疗由IL-17A介导的病理性疾病的方法，所述方法包括施用有效量的根据本发明的分离的抗体或其抗原结合片段，特别地hu31、hu43、hu44、hu59、hu60或hu250抗体，以使所述病症被缓解。
在一个实施方式中，本发明所述的分离抗体或包含其抗原结合片段的蛋白质缀合于其它活性部分。
在一个实施方式中，本发明所述的分离抗体或包含其抗原结合片段的蛋白质可以是单克隆抗体或其抗原结合片段，优选嵌合抗体、人源化抗体或人抗体或其部分。
在本发明的方面，提供了药物组合物，其包含与一种或多种可药用的赋形剂、稀释剂或载体组合的本发明所述的实施方式的抗体或包含其抗原结合片段的蛋白质。
在实施方式中，所述药物组合物包含一种或多种额外的活性成分。
在一个具体实施方式中，所述药物组合物是冻干粉剂。在另一个具体实施方式中，所述药物组合物是包含治疗上可接受的量的本发明所述的抗体或分子的稳定的液体制剂。
具体地，本发明提供治疗与IL-17A相关病症和/或自身免疫性和炎性病症的方法。在某些实施方式中，所述方法包括向有此需要的受试者施用根据本发明的分离的抗体或其抗原结合片段的步骤。
本发明还提供了方法，所述方法通过将细胞与包含治疗有效剂量的本发明的抗体的组合物接触，来减弱或抑制IL-17A或IL-17AF在靶细胞或组织中诱导的信号转导应答。
在本发明中，术语“IL-17A介导的疾病”或“IL-17A相关病症”包括IL-17A或IL-17AF在疾病或医学病况中起作用(无论是直接还是间接地)的所有疾病和病况，包括疾病或病况的原因、发展、进展、持续或病理学。因此，这些术语包括与异常IL-17A或IL-17AF水平相关或以异常IL-17A或IL-17AF水平为特征的病况和/或可通过减弱或抑制IL-17A/AF在靶细胞或组织中诱导的活性(例如CXCL1)来治疗的疾病或病况。这类疾病或病况包括炎性病况和自身免疫性疾病，诸如关节炎、类风湿性关节炎、强直性脊柱炎、多发性硬化症或银屑病。这类疾病还包括过敏症和过敏性病况、超敏反应、慢性阻塞性肺疾病、囊性纤维化以及器官或组织移植排斥。
本发明所用“抑制”或“治疗”或“处理”包括病症相关症状的发展延迟和/或这类病症 症状的严重程度减轻。该术语还包括改善现有的不受控制的或有害的症状，预防其它症状和改善或预防这类症状的根本起因。因此，该术语表明已为患有病症、疾病或症状的脊椎动物受试者或者可能发生这类病症、疾病或症状的脊椎动物受试者提供有益结果。
本发明所用术语“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的IL-17结合化合物当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的结合化合物的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％；通常至少20％；优选至少约30％；更优选至少40％；最优选至少50％。
类风湿性关节炎(RA)
RA是以滑膜关节的炎症为特征的进行性全身性疾病，影响约0.5％的全球人口。参见Emery(2006)BMJ332:152-155。关节炎症可导致畸形、疼痛、僵硬和肿胀，最终导致关节不可逆退化。受影响关节包括膝、肘、颈和手足关节。常规治疗包括使用NSAID缓解症状，接着给予缓解疾病的抗风湿性药物(DMRD)，例如金、青霉胺、柳氮磺吡啶和甲氨蝶呤。最近进展包括用TNF-α抑制剂的治疗，包括英利昔单抗、阿达木单抗和戈利木单抗等单克隆抗体，以及受体融合蛋白，例如依那西普。用这些TNF-α抑制剂治疗显著降低因疾病所致的结构损害。
本发明的抗IL-17A抗体可用来治疗需要这种治疗的受试者的RA。本发明的抗IL-17A抗体还可与RA的其它治疗组合，例如甲氨蝶呤、硫唑嘌呤、环磷酰胺、霉酚酸吗乙酯、NSAID或TNF-α抑制剂。
银屑病
皮肤是机体内环境与外界之间的重要屏障，防止与潜在的有害抗原接触。在抗原/病原体侵入的情况下，皮肤接触部位的T细胞、多形核细胞和巨噬细胞等局部浸润并启动炎症反应以消除抗原。(参见例如Williams和Kupper，(1996)Life Sci.，58:1485-1507)。通常，由病原体引发的这种炎症反应处于严密严监控之下，并在病原体消除时停止。在某些情况下，这种炎症反应在没有外部刺激的和没有适当控制的情况下发生，导致皮肤炎症。本发明提供用于治疗和诊断皮肤炎症的方法。皮肤炎症(上述细胞浸润以及从这些细胞分泌的细胞因子的结果)包括若干炎性病症，例如疤痕性类天疱疮、硬皮病、化脓性汗腺炎、中毒性表皮坏死松解症、痤疮、骨炎、移植物抗宿主病(GVHD)、坏疽性 脓皮症(pyroderma gangrenosum)和贝切特综合征(Behcet’s Syndrome)(参见例如Williams和Griffiths，(2002)Clin.Exp.Dermatol.，27:585-590)。最常见的皮肤炎症形式是银屑病。
银屑病的特征在于T细胞介导的角质形成细胞过度增殖伴发炎症浸润。该病具某些明显重叠的临床表型，包括慢性斑块病变、皮疹和脓疱病变(参见例如Gudjonsson等，(2004)Clin Exp.Immunol.135:1-8)。大约10％银屑病患者发生关节炎。该疾病具有强而复杂的遗传素因，在单卵孪生中有60％一致性。
典型的银屑病病变是边界十分清楚的红斑，红斑被厚的银色鳞屑覆盖。与正常皮肤相比，银屑病组织的炎症和过度增殖与不同的组织学、抗原和细胞因子概况有关。与银屑病有关的细胞因子为：TNF-α、IL-19、IL-18、IL-15、IL-12、IL-7、IFN-γ、IL-17A和IL-23(参见Gudjonsson等，同上)。
单独或与其它作用剂组合的本发明的抗IL-17A抗体，也可用于预防、治疗、诊断和预测银屑病突发。
为了制备本发明的IL-17A抗体的药物组合物或无菌组合物，将抗体与药学上可接受的载体或赋形剂混合。参见例如Remington’s Pharmaceutical Sciences and U.S.Pharmacopeia：National Formulary,Mack Publishing Company,Easton,PA(1984)。
可通过与可接受的载体、赋形剂或稳定剂混合来制备以下形式的治疗剂和诊断剂的剂型：例如冻干粉、膏剂、水溶液剂或混悬剂。在一个实施方式中，将本发明的IL-17A抗体在乙酸钠溶液(pH 5-6)中稀释到适当浓度，加入NaCl或蔗糖以利渗透压。可加入其它物质(例如聚山梨醇酯20或聚山梨醇酯80)以提高稳定性。
给药方案取决于若干因素，包括治疗性抗体的血清或组织周转率、症状水平、治疗性抗体的免疫原性和生物基质中靶细胞的可及性。优选给药方案递送足够的治疗性抗体以实现靶疾病状态的改善，同时使不良副作用降到最低。因此，递送的生物剂(biologic)的量部分取决于具体的治疗性抗体和待治疗病况的严重程度。可获得有关选择适当剂量的治疗性抗体方面的指引。例如利用本领域已知或推测影响治疗的参数或因素，临床医生可确定适当剂量。总的来讲，剂量以稍小于最佳剂量的量开始，之后以少的增量增加直到达到相对于任何负面的副作用是所需的效果或最佳效果。重要的诊断方法包括例如炎症症状或所产生的炎性细胞因子水平的诊断方法。优选可使用的生物剂来源于与靶向治疗的动物相同的物种，从而使对试剂的炎症、自身免疫或增殖应答降到最低。例如，在人受试者的情况下，优选嵌合、人源化和完全人抗体。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性 的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
实施例
实施例1.重组蛋白人IL-17A-mFc
从义翘神州购买编码全长人IL-17A的cDNA序列(NCBI登录号:NP_002181.1)的质粒HG12047-G，通过常规PCR技术扩增人IL-17A成熟片段(NCBI登录号NP_002181.1的第24-155位氨基酸，氨基酸序列为SEQ ID NO:66，核苷酸序列为SEQ ID NO:67)。扩增片段经BSPQI酶切后，克隆到自主构建的真核表达质粒系统(MXT1-Fc，包含鼠源IgG重链的Fc域)，从而产生重组融合蛋白表达质粒IL-17A-mFc。利用常规技术，将鉴定正确的质粒转染表达细胞293F，表达并纯化获取人IL-17A-mFc重组蛋白。图1所示为人IL-17A-mFc重组蛋白的SDS-PAGE电泳图。
实施例2.构建表达人IL-17RA的293F稳转细胞株
从义翘神州购买含有编码人类全长IL-17RA的cDNA序列的质粒HG10895-G，通过常规PCR扩增编码全长人IL-17RA的DNA序列(SEQ ID NO:69)。利用常规的克隆技术，将扩增片段克隆到自主构建的真核表达质粒系统(HXP)中，其含嘌呤霉素筛选体系。将构建成功的IL-17RA重组表达质粒转染至293F(ATCC)细胞中。转染24h后，通过嘌呤霉素(2μg/ml)进行筛选，直至形成293F IL-17RA稳转细胞库。利用常规方法分离单个克隆，如通过有限稀释法，按每孔0.8个细胞，铺96孔板，15天后，挑选出IL-17RA-293F单克隆，并进行传代，形成293F IL-17RA稳转细胞株，藉由FACS等分析筛选所有克隆，及选择顶级表达克隆用于FACS结合检定以筛选杂交瘤单克隆抗体，或在功能性检定中使用。
实施例3.重组蛋白IL-17A-mFc与293F IL-17RA稳转细胞株的结合
FACS实验检测重组蛋白IL-17A-mFc与293F细胞上IL-17RA的结合特异性。简而言之，将细胞(293F IL-17RA稳转细胞株)制备成1×10 6/ml的细胞悬液，按每孔20ul加入96孔板中，实际每孔细胞数为2×10 4个，将重组蛋白IL-17A-mFc(3ug/ml，20ul/ 孔；实验组)或1％BSA(20ul/孔；阴性对照组)与细胞悬液混合，37摄氏度孵育30分钟后，FACS缓冲液洗脱3次后，加入抗小鼠IgG(1：200)在室温孵育30分钟。以FACS缓冲液洗脱3次后上细胞流式仪检测，比较各组平均荧光强度值(MFI)。如图2所示，重组蛋白IL-17A-mFc可以与293F细胞上的IL-17RA特异性的结合。
实施例4.杂交瘤抗体的制备与筛选
使用标准分子生物学技术产生杂交瘤抗体。简言之，将从HumanZyme购买的天然人IL-17A蛋白作为抗原与等量免疫佐剂混合，取5只6周大雌性FVB小鼠进行免疫。在初次免疫以后，每周进行一次加强免疫，共进行七次免疫。在最后一针加强免疫后，选择血清中具有高抗IL-17A抗体滴度的小鼠进行细胞融合实验。使用标准杂交瘤技术，使脾细胞分离及与鼠类骨髓瘤细胞系SP2/0细胞(ATCC)融合。将融合细胞重悬于含有HAT的RPMI-1640完全培养基，涂板在具有腹膜细胞饲养层的孔中。
基于初始所希望的抗体/抗原结合特征(诸如对于IL-17A的结合亲和力、阻断IL-17A对其受体的结合的能力、种属交叉反应以及在体外测定中阻断IL-17A介导的生物效应的能力)，鉴定单克隆杂交瘤分泌上清，其中杂交瘤1F8、2B2、2F5、2F2、2H1和2H5分泌的上清抗体用于进一步表征。
实施例5.杂交瘤抗体体内阻断IL-17A生物活性的作用
大量研究表明，IL-17A在体内促进细胞因子CXCL1表达与释放，所以可通过ELISA定量检测小鼠血清中CXCL1表达变化，判断杂交瘤抗体对小鼠体内IL-17A介导的生物活性的影响。简而言之，选择40只10周龄的雌性Balb/c小鼠，分为8组，每组5只。给药前4天，搜集血清，并检测CXCL1表达量作为基础值。给药当天，心内注射候选杂交瘤抗体、生理盐水(cntrol)或参照抗体mAb317(商业抗IL-17A抗体，购自R&D)药物，剂量为1mg/kg；给药后1小时，按150ug/kg给药量通过皮下注射天然型的人IL-17A(HumanZyme)；注射人IL-17A后2小时，搜集血清检测血液中CXCL1的浓度，并与基础值进行对比，计算各组在给药前后CXCL1的浓度变化倍数(均值±标准误(mean±SEM))。各抗体组与对照组的比较分析使用Student’s-t test检验，P<0.05认为有显著差异，*P<0.05，**P<0.01，***P<0.001。
如图3所示，实施例4获得的杂交瘤抗体同商业抗体mAb317都能显著抑制IL-17A诱导小鼠体内CXCL1表达。
实施例6.候选抗体可变区序列的获得
用基于简并引物PCR的方法，测定由杂交瘤1F8、2B2和2F5表达的抗体可变区的DNA序列。简言之，将杂交瘤细胞株1F8、2B2和2F5分别扩大培养，1000rpm离心收集细胞，并以Trizol提取总RNA。以此为模板，合成第一链cDNA后，以第一链cDNA为后续模板PCR扩增对应的可变区DNA序列,所用PCR引物基于Ig-引物组。回收纯化PCR产物并测序分析，获得候选杂交瘤抗体重链和轻链可变区序列。其中杂交瘤细胞1F8具有两种抗体轻链可变区基因序列和一种抗体重链可变区基因序列，2F5具有两种抗体重链可变区基因序列和一种抗体轻链可变区基因序列。所以1F8、2F5分泌的抗体可能均含有两种混合完全抗体，其中1F8分泌的抗体称为1F8-1和1F8-2，2F5分泌的抗体称为2F5-1和2F5-2。
1F8、2F5、2B2表达的抗体的重链可变区和轻链可变区的氨基酸序列如表1(见说明书详细描述)所示。
实施例7.构建嵌合抗体重组表达载体
从人血细胞(北京血液研究所)中克隆人IgG4重链恒定区Fc片段和轻链κ恒定区，联入pCDNA3.1质粒加以改造。上述重链和轻链可变区序列片段由Genscript公司合成，重链经Bspq I酶切，轻链经Bspq I酶切后，联入相对应改造的pCDNA3.1质粒中，经测序确定IgG4嵌合型的重链(ch-HC)或轻链(ch-LC)的表达质粒。将上述不同的嵌合型重链和轻链的表达质粒混合配对转染表达细胞，获得16种嵌合抗体，编号为ch1到ch16(见表6)。后续的实验材料均由此系列质粒转染表达细胞后提取获得。
表6：嵌合抗体轻/重链来源
LC/HC 1F8-ch-HC 2B2-ch-HC 2F5-ch-HC1 2F5-ch-HC2
1F8-ch-LC1 ch1 ch5 ch9 ch13
1F8-ch-LC2 ch2 ch6 ch10 ch14
2B2-ch-LC ch3 ch7 ch11 ch15
2F5-ch-LC ch4 ch8 ch12 ch16
实施例8.嵌合抗体与人IL-17A的结合特异性
利用常规ELISA检测方法检测嵌合抗体与人IL-17A结合特异性。即将0.5μg/ml的人IL-17A-mFc包被于96孔酶标板，37摄氏度恒温孵育60-90分钟。然后弃去孔内溶液，用洗涤缓冲液洗3次，加入含有2％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗3 次后加入不同浓度的嵌合抗体稀释液，37摄氏度孵育60分钟后用洗涤缓冲液冲洗3次，然后加入1：10000倍稀释的生物素-抗IgG4，37摄氏度孵育1小时，经洗涤缓冲液冲洗三次后，加入用洗涤缓冲液以1：10000倍稀释的HPR-Strep，室温孵育1小时，经洗涤缓冲液冲洗3次后，加入100μl TMB底物溶液显色，室温反应30分钟后，以100μl2M的盐酸溶液终止反应并在450nm处读出吸光度。
如图4所示，嵌合抗体ch1，ch2，ch4，ch7和ch16与人IL-17A结合具有较高的特异性，其EC 50分别为6.62ng/mL，5.17ng/mL，88.48ng/ml，39.96ng/mL和15.42ng/mL。
实施例9.嵌合抗体阻断人IL-17A与IL-17RA的结合作用
采用竞争性基于细胞的流式细胞(FACS)测定检测嵌合抗体阻断IL-17A与细胞上IL-17RA的结合作用。简而言之，将不同浓度的嵌合抗体稀释液(起始10ug/ml，3倍滴定)与预先生物素标记的实施例1获得的人IL-17A-mFC(3ug/ml)混合，室温孵育30分钟。然后将混合物与细胞悬液(实施例2获得的293F IL-17RA稳转细胞株，1.5×10 5细胞/孔)在37摄氏度孵育15分钟，以PBS洗脱3次后，加入5μg/ml的抗小鼠IgG并室温孵育30分钟。以PBS洗脱3次后，通过流式细胞仪检测嵌合抗体对IL-17A与293F细胞表面的IL-17RA结合的抑制作用。
如图5所示，嵌合抗体ch1，ch2，ch7和ch16均能显具抑制人IL-17与293F细胞表面的IL-17RA结合。综合实验结果，选择ch1和ch16继续进行人源化改造。
实施例10.抗体的人源化改造
对于抗体的人源化，首先在NCBI(http://www.ncbi.nlm.nih.gov/igblast/)网站中的人类免疫球蛋白基因数据库搜寻与鼠源抗体可变区的cDNA序列同源的人类种系IgG基因。再藉由Kabat编号系统或IMGT编号系统定义可变区CDR的氨基酸序列及其精确边界。原则上将与鼠源抗体可变区具有高同源性的人类IGVH及IGVk选为人源化模板，藉由CDR嫁接实施人源化。简言之，人源化改造过程涉及以下步骤：A、把各候选抗体的基因序列与人胚胎系抗体基因序列进行比对，找出同源性高的序列；B、分析考察HLA-DR亲和性，选出亲和力低的人胚胎系框架序列；C、利用计算机模拟技术，应用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式。通过计算静电力，范德华力，亲疏水性和熵值，分析各候选的抗体基因序列中可与IL-17A作用以及维护空间构架的关键氨基酸个体，将其嫁接回已经选择的人胚胎系基因框架，并在此基础上标配出必须保留的框架区氨基酸位点，合成人源化抗体。
鼠源抗体可变区CDR定义方案及其氨基酸序列见本公开内容详细说明部分的表3。
基于前述实验筛选的结果，选取嵌合抗体ch1和ch16继续人源化改造。在一系列抗体/抗原结合特征(诸如对于IL-17A的结合亲和力、阻断IL-17A对其受体的结合的能力)初级筛选后，选取人源化抗体hu31，hu43，hu44，hu59，hu60和hu250继续后续验证。
人源化抗体hu31，hu43，hu44，hu59，hu60和hu250可变区及其CDR氨基酸序列见本公开内容详细说明部分的表4。
实施例11.人源化抗体与人IL-17A的结合特异性
利用常规ELISA检测方法检测人源化抗体与人IL-17A结合特异性，实验方法与步骤参见实施例8。如图6所示，人源化抗体hu31，hu43，hu44，hu59，hu60和hu250均与IL-17A特异性结合。其EC 50分别为8.13ng/mL，8.64ng/mL，6.76ng/ml，6.10ng/mL，5.78ng/mL和6.35ng/mL。
实施例12.人源化抗体阻断人IL-17A与IL-17RA的结合作用
采用竞争性基于细胞的流式细胞测定(FACS)，检测嵌合抗体阻断IL-17A与细胞上IL-17RA的结合作用，实验方法与步骤参见实施例9。如图7所示，人源化抗体hu31，hu43，hu44，hu59，hu60和hu250均能显著地抑制IL-17A与细胞上的IL-17RA特异性结合。其IC 50分别为867.6ng/mL，780.8ng/mL，828.5ng/ml，467.4ng/mL，482.8ng/mL和577.8ng/mL。
实施例13.人源化抗体拮抗IL-17A诱导上皮细胞表达CXCL1
IL-17A可以刺激多种上皮细胞和其它细胞分泌细胞因子CXCL1表达与释放，可通过ELISA定量检测细胞上清中CXCL1表达量变化，判断人源化抗体对IL-17A在细胞中介导的生物活性的影响。
使用标准技术在组织培养物处理的烧瓶中的培养/测定培养基中维持HT-29细胞(人结肠直肠腺癌上皮细胞，ATCC)。HT-29在组织培养瓶中生长直至它们在测定当天达到50-80％汇合。在测定当天，用PBS冲洗细胞并用胰蛋白酶+EDTA从培养瓶中脱离细胞，并制成细胞悬液。取人源化抗体hu31、hu59、hu60、hu250或参照抗体(Secukinumab,诺华)稀释液(起始浓度为55ug/ml，3倍浓度梯度稀释)与人IL-17A(1ug/ml)混合，铺入96孔板中，孵育1h。向每孔中加入100ul(2×10 4个)HT-29细胞 (ATCC,人结肠直肠腺癌上皮细胞)悬液，37℃，7％CO 2培养48h。500Xg，5min离心，将培养上清转入新的96孔板中，利用ELISA试剂盒检测CXCL1的表达。
如图8所示，相比参照抗体Secukinumab，人源化抗体hu31、hu59、hu60和hu250对IL-17A刺激上皮细胞释放CXCL1具有更强得拮抗作用。
实施例14.人源化抗体拮抗IL-17A诱导小鼠表达CXCL1
同实施例5，通过检测小鼠血清CXCL1水平变化，判断人源化抗体对IL-17A在体内介导生物活性的影响。简言之，选择40只10周龄的雌性Balb/c小鼠，分为8组，每组5只。给药前4天，搜集血清，并检测CXCL1表达量作为基础值。给药当天，按1mg/kg，分别心内注射候选抗体(人源化抗体hu31，hu43，hu44，hu60和hu250)和IgG4同种型对照(hlgG)；给药后1小时，按150ug/kg给药量通过皮下注射人IL-17A；人IL-17A注射后2小时，搜集血清检测血液中CXCL1的浓度,并与基础值进行对比，计算各组在给药前后CXCL1的浓度变化倍数(均值±标准误(mean±SEM))。候选抗体与IgG4同种型对照的比较分析使用Student’s-t test检验，P<0.05认为有显著差异，*P<0.05，**P<0.01，***P<0.001。
如图9所示，相较于IgG4同种型对照，候选人源化抗体hu31，hu43，hu44，hu60和hu250对IL-17A刺激小鼠体内释放CXCL1具有更强的拮抗作用。
实施例15.人源化抗体改善咪喹莫特诱导的小鼠银屑病模型的疗效研究
在小鼠耳背皮肤涂抹咪喹莫特可诱导银屑病样病理学特征，即角质形成细胞过度增生、炎症细胞聚集和真皮乳头部血管增生等，构建银屑病小鼠模型。以临床评分、耳部肿胀度等为指标判断药物对银屑病小鼠的治疗作用。
15.1实验方法
取6-8周C57BL/6雌性小鼠(南京大学模式动物研究所购买，动物合格证号为201605578)48只，背部脱毛，除假手术组外，二天后致敏。致敏前两天随机分成5组(每组8只)：I组为假手术组；II组为PBS组，III组为KLH对照组(同型IgG)，给予KLH；IV组为hu31组；V组为hu43给药组；VI组为hu44给药组，各组给药剂量均为50mg/kg；上述各组于分组第0天及第3天腹腔注射药物1次。致敏当天(day 1)，II-VI组小鼠于右耳及背部皮肤上涂抹约62.5mg咪喹莫特乳膏(艾达乐，5％，3M Health Care Limited)，连续4天。
15.2评价方法
致敏当天起每天用螺旋测微仪测量小鼠右耳厚度，以day 1右耳厚度为对照,计算小鼠耳肿胀厚度数值。同时给小鼠每天称重，观察皮肤鳞屑、硬结、红斑情况，进行评分，采用4级评分法：0分，不发病；1分，轻微；2分，中度；3分，严重；4分，非常严重。结果以平均值±标准误(mean±SEM)表示，先使用单因素方差分析(ANOVA)，有差异后两组之间的比较使用Student’s-t test检验，P<0.05认为有显著差异。
如图10-1所示，给予本发明人源化抗体可明显抑制咪喹莫特诱导的小鼠银屑病模型皮肤鳞屑、硬结、红肿等情况，即评分值较小。
如图10-2所示，自致敏当天起于小鼠右耳涂抹咪喹莫特乳膏，其右耳会发生严重肿胀，耳厚度增加，而本发明人源化抗体均可明显改善耳肿胀程度。
在咪喹莫特诱导的小鼠银屑病模型中，本发明人源化抗体可明显抵抗小鼠的发病，表型为小鼠临床评分及耳部肿胀程度的降低。
实施例16.人源化抗体改善II型胶原蛋白诱导的雌性食蟹猴关节炎的疗效研究
II型胶原蛋白诱导的关节炎是广泛用于类风湿关节炎(RA)研究的动物模型，具有与人RA相同的组织病理学特征，其特征在于小关节的炎症和软骨及骨的进行性侵蚀。人/人源化的生物大分子包括抗体常常与食蟹猴体内抗原具有更优的交叉反应，所以食蟹猴关节炎模型是检测本发明中人源化抗体IL-17A抗风湿作用的一种有效体系。本实验即在食蟹猴类风关节炎模型上评价候选抗体药效。
16.1实验方法
将II型牛胶原(CII，四川大学)溶解在醋酸(货号10000218；国药；上海；中国)中放入4℃冰箱搅拌过夜，然后用等体积的完全弗氏佐剂(货号：F5881，Sigma-Aldrich，美国)乳化胶原，乳剂胶原终浓度2mg/ml。第0天，舒泰(1.5-5mg/kg，i.m)麻醉动物，于背部及尾根部进行多点的胶原乳剂免疫注射，根据需要，免疫时用1.5％-5％的异氟烷维持麻醉。3个星期后(第21天)重新注射一次胶原，方法同第一次。本实验分为4组，G1为正常动物组，不进行关节炎的诱导；G2为溶媒对照组；G3为抗体hu31给药组；G4为抗体hu59给药组。当某只动物的临床评分达到临床评分最大值的5％(192×5％≈10)时，按顺序分别分到各实验组，先达分值的先入组，如此循环直到所有符合条件的动物都依序分入各组中。入组后开始给药，每周一次，每次7.5mg/kg，由输液泵(infusion bump)30分钟持续泵入，持续5周。
16.2评价方法
体重测量：免疫前一天测量动物体重，以后每周测量一次体重直到实验结束。
关节炎评分：在第0天和第21天对猴子四肢关节炎发炎程度进行评分，并在21天后每周评分一次直至实验结束(如果出现提早发病，则相应提前每周一评的关节炎评分)，评分标准见表2。对每个爪掌的下述15个关节进行评分：5个掌指关节(MCP)，4个近端指关节(PIP)，5个远端指关节(DIP)，1个腕关节或踝关节。同时还需要评估四肢膝盖/肘关节的发病程度。各个关节评分的总和即为这只动物的关节炎评分，最大分值为192(16×3×4)。关节炎的评分标准：0分，正常；1分，轻微关节炎，发病轻微但可以明确分辨出；2分，中度肿胀；3分严重关节炎，严重肿胀或有明显的关节变形。
实验数据表示成均值±标准误(mean±S.E.M)。统计分析溶媒对照、参照药和测试药组各项参数的组间差异，p<0.05被认为具有统计学差异(One-way ANOVA/Dunnett)。
如图11-1所示，正常的食蟹猴(G1)体重稳定；关节炎诱导后的食蟹猴，溶媒处理组(G2)的食蟹猴平均体重持续下降，与之相比，受试抗体hu31和hu59均使这种下降趋势得到控制。故在本实验条件下，hu31和hu59对关节炎引起的体重下降具有一定改善作用(**P<0.01，****P<0.0001，和“G2:溶媒组”相比较；One-way ANOVA/Dunnett)。
如图11-2所示，动物入组后，正常对照动物关节炎临床评分保持为0；模型-溶媒组(G2)动物关节炎评分呈渐进性增加，而受试抗体hu31和hu59显著抑制了动物的关节炎临床评分的增加趋势。故受试抗体hu31和hu59有抑制关节炎病情渐进性发展的作用，受试抗体hu31显著抑制了食蟹猴关节炎临床评分的增加趋势(***P<0.001，#P<0.05，和G2:溶媒组相比较；One-way ANOVA/Dunnett)。
实施例17.人源化抗体对NIH3T3-IL17细胞诱导的小鼠关节肿胀的作用
1.动物：C57BL/6，雌性，6-8周，北京维通利华实验动物技术有限公司。
2.细胞：NIH3T3细胞，表达人IL-17的NIH3T3细胞。
3.分组及给药方案：
NIH3T3组；
NIH3T3-IL-17+对照IgG抗体组(30mg/kg)；
NIH3T3-IL-17+受试抗体高剂量给药组(抗体hu31,3mg/kg)；
NIH3T3-IL-17+受试抗体中剂量给药组(抗体hu31,10mg/kg)；
NIH3T3-IL-17+受试抗体低剂量给药组(抗体hu31,30mg/kg)；
NIH3T3-IL-17+阳性药给药组(cosentyx,10mg/kg)。
4.造模及给药：
将NIH3T3-IL-17细胞及NIH3T3对照细胞(2.5×10 5个/只，每只注射体积为25uL) 分别注射入各组小鼠的右踝关节的关节腔内。
选模前1天开始腹腔注射给予抗体hu31(3、10、30mg/kg)和cosentyx(10mg/kg)干预给药，每3天1次，考察抗体hu31注射液对于小鼠关节炎模型的影响。
5.检测：
游标卡尺测量小鼠踝关节厚度，计算肿胀度。
6.实验结果：
如图12所示，在小鼠跺关节腔内注射稳转hIL-17的NIH3T3细胞，第二天可观察到小鼠跺关节处严重肿胀。
依据小鼠踝关节的厚度进行肿胀抑制率的计算，计算公式为：抑制率(％)＝(NIH3T3-ILI7组踝关节厚度-给药组踝关节厚度)/(NIH3-ILl7细踝关节厚度-NIH3T3组踝关节厚度)×100。结果显示，给药后第2天，观察到各剂量药物组别对小鼠踝关节肿胀的抑制作用，直至试验结束，在给药后第6天达到最大抑制效率。第10天时，抗体hu31(3、10、30mg/kg)各组抑制率分别为49.4％、65.9％和74.1％。cosentyx(10mg/kg)肿胀抑制率为67.1％。计算各组不同天数的平均抑制率，结果显示，3、10和30mg/kg各组的平均抑制率分别为45.2％、57.0％和73.9％。cosentyx(10mg/kg)肿胀抑制率为60.4％。实验结果提示：抗体hu31能剂量依赖性的抑制IL-17诱导的小鼠关节踝肿胀，其10mg/kg的效果与阳性对照药物cosentyx(10mg/kg)相当。30mg/kg的效果优于阳性对照药物cosentyx(10mg/kg)。
实施例18.人源化抗体对NIH3T3-IL17细胞诱导的小鼠空气囊炎症的作用
1.动物：C57BL/6，雄性，6-8周，由北京维通利华实验动物技术有限公司购买)。
2.细胞：NIH3T3细胞，表达人IL-17的NIH3T3细胞。
3.试剂：Gr1-FITC抗体，Biolegend公司
4.分组及给药方案：
NIH3T3细胞组
NIH3T3-IL-17细胞组+对照IgG抗体组(30mg/kg)；
NIH3T3-IL-17细胞组+受试抗体高剂量给药组(抗体hu31,30mg/kg)；
NIH3T3-IL-17细胞组+受试抗体中剂量给药组(抗体hu31,10mg/kg)；
NIH3T3-IL-17细胞组+受试抗体低剂量给药组(抗体hu31,3mg/kg)；
给药途径：腹腔注射。
5.造模：
空气囊：分别在第0和3天在小鼠背部注射2.5ml空气。第5天开始将细胞注入空气囊内。每只小鼠注射的细胞数量为2×10 5个/500微升PBS。每组8只小鼠。
6.检测：
白细胞向空气囊的迁移，计算灌洗液的细胞总数，流式测定Gr1 +细胞比例，计算中性粒细胞数量。
中性粒细胞数量＝细胞总数×Gr1 +细胞比例
7.实验结果
如图13所示，在小鼠背部空气囊内注射稳转hlL-17A的NIH-3T3细胞，从浸润的细胞总数来看，相比于NIH3T3细胞组，NIH3T3-IL-17细胞组空气囊中浸润的白细胞数显著增加，Grl +细胞比例和数量也显著增加，选模成功。造模当天腹腔注射给予抗体hu31(剂量分别为3、l0、30mg/kg)干预给药。依据浸润的总细胞数和Grl +细胞数进行抑制率的计算，计算公式为：抑制率(％)＝(NIH3T3-ILI7-lgG组细胞数-给药组细胞数)/(NIH3T3-ILl7-IgG组细胞数－NIH3T3细胞数)×100。结果显示，抗体hu31(3、10、30mg/kg)各组浸润细胞总数抑制率分别为50.0％、56.7％和78.3％。计算Grl +细胞数的抑制率，结果显示：抗体hu31(3、10、30mg/kg)各组平均Grl +细胞抑制率分别为59.1％、54.5％和81.8％，抗体hu31能剂量依赖性的抑制IL-17诱导的小鼠总细胞及炎症细胞浸润。

Claims (18)

  1. 一种抗体或其抗原结合片段，其特异性结合IL-17A，其中所述抗体或其抗原结合片段包含至少一个选自SEQ ID NO:1-24、60-65的互补决定区(CDR)。
  2. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含至少一个选自SEQ ID NO:1-3、4-6、7-9、10-12或60-62的重链CDR结构域和/或至少一个选自SEQ ID NO:13-15、16-18、19-21、22-24或63-65的轻链CDR结构域。
  3. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区(VH)，且所述重链可变区的HCDR1选自SEQ ID NO:1、4、7、10和60中的一个，HCDR2选自SEQ ID NO:2、5、8、11和61中的一个，和HCDR3选自SEQ ID NO:3、6、9、12和62中的一个。
  4. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区(VH)，且所述重链可变区包含的HCDR1、HCDR2和HCDR3氨基酸序列选自以下组A到组E中的任一组：
    组号 HCDR1 HCDR2 HCDR3 A SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 B SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 C SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 D SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 E SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:62
  5. 如权利要求1或3所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含轻链可变区(VL)，且所述轻链可变区的LCDR1选自SEQ ID NO:13、16、19、22和63中的一个，LCDR2选自SEQ ID NO:14、17、20、23和64中的一个，和LCDR3选自SEQ ID NO:15、18、21、24和65的一个。
  6. 如权利要求1或4所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含轻链可变区(VL)，且所述轻链可变区包含的LCDR1、LCDR2和LCDR3的氨基酸序列选自以下组F到组J中的任一组：
    组号 LCDR1 LCDR2 LCDR3 F SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 G SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18
    H SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 I SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 J SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65
  7. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，所述可变区包含的6个CDR的氨基酸序列选自以下组I到组VI中的任一组：
    组号 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 I SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15 II SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18 III SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21 IV SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 V SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24 VI SEQ ID NO:60 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63 SEQ ID NO:64 SEQ ID NO:65
  8. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区(VH)和/或轻链可变区(VL)，所述重链可变区的氨基酸序列选自SEQ ID NO:25、26、27、28、33、35、37和40中的一个，和/或所述轻链可变区(VL)的氨基酸序列选自序列编号SEQ ID NO:29、30、31、32、34、36、38、39和41中的一个。
  9. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，所述可变区氨基酸序列选自以下组1到组7中的任一组：
    组号 VH VL 1 SEQ ID NO:25 SEQ ID NO:29或30 2 SEQ ID NO:26 SEQ ID NO:31 3 SEQ ID NO:27或28 SEQ ID NO:32 4 SEQ ID NO:33或35 SEQ ID NO:34 5 SEQ ID NO:35 SEQ ID NO:36
    6 SEQ ID NO:37 SEQ ID NO:38或39 7 SEQ ID NO:40 SEQ ID NO:41
  10. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体或全人抗体。
  11. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体为完全抗体，所述抗原结合片段选自单链抗体、Fab抗体、Fab’抗体、(Fab’)2抗体、和双(多)特异性抗体。
  12. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段为任何IgG亚型，如IgG1、IgG2、IgG3或IgG4。
  13. 如权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含轻链(LC)和/或重链(HC)，且所述重链(HC)的氨基酸序列选自SEQ ID NO:42、44、46或49，和/或所述轻链(LC)的氨基酸序列选自SEQ ID NO:43、45、47、48或50。
  14. 如权利要求13所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段轻链的氨基酸序列如SEQ ID NO:43所示，和重链的氨基酸序列如SEQ ID NO:42或44所示；或轻链的氨基酸序列如SEQ ID NO:45所示，和重链的氨基酸序列如SEQ ID NO:44所示；或轻链的氨基酸序列SEQ ID NO:47或48所示，和重链的氨基酸序列SEQ ID NO:46所示；或轻链的氨基酸序列如SEQ ID NO:50所示，和重链的氨基酸序列如SEQ ID NO:49所示。
  15. 编码如权利要求1-14任一项所述的抗体或其抗原结合片段的分离的核酸分子，包含所述核酸分子的表达载体或重组载体，以及转化所述载体的宿主细胞。
  16. 一种药物组合物，其包含如权利要求1-14任一项所述的抗体或其抗原结合片段、权利要求15所述核酸分子、载体或宿主细胞和药学上可接受的载体或赋形剂的组合物。
  17. 如权利要求1-14任一项所述的抗体或其抗原结合片段、权利要求15所述核酸分子、载体或宿主细胞或权利要求16所述的药物组合物在制备用于治疗和/或预防IL-17A介导的疾病或病症的药物中的用途。
  18. 如权利要求17所述的用途，其中，所述药物是用于治疗关节炎、类风湿性关节炎、银屑病、强制性脊柱炎、慢性阻塞性肺疾病、系统性红斑狼疮(SLE)、狼疮性肾炎、哮喘、多发性硬化或囊性纤维化的药物。
PCT/CN2019/124486 2018-12-12 2019-12-11 抗il-17a抗体及其应用 WO2020119707A1 (zh)

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MX2021007028A MX2021007028A (es) 2018-12-12 2019-12-11 Anticuerpo anti-il-17a y uso del mismo.
EP19897274.7A EP3896086A4 (en) 2018-12-12 2019-12-11 ANTI-IL-17A ANTIBODIES AND USE THEREOF
SG11202106128TA SG11202106128TA (en) 2018-12-12 2019-12-11 Anti-il-17a antibody and use thereof
US17/413,152 US20230159632A1 (en) 2018-12-12 2019-12-11 Anti-il-17a antibody and use thereof
JP2021534214A JP2022513224A (ja) 2018-12-12 2019-12-11 抗ｉｌ‐１７ａ抗体及びその適用
BR112021011257-0A BR112021011257A2 (pt) 2018-12-12 2019-12-11 Anticorpo anti-il-17a e uso do mesmo
CA3123124A CA3123124A1 (en) 2018-12-12 2019-12-11 Anti-il-17a antibody and use thereof
CN201980082789.0A CN113166239B (zh) 2018-12-12 2019-12-11 抗il-17a抗体及其应用
KR1020217021674A KR20210102946A (ko) 2018-12-12 2019-12-11 항il-17a 항체 및 이의 응용
AU2019397309A AU2019397309A1 (en) 2018-12-12 2019-12-11 Anti-IL-17A antibody and use thereof
IL283881A IL283881A (en) 2018-12-12 2021-06-10 Anti-il-17a antibody and its use
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