WO2019062831A1 - Il-5抗体、其抗原结合片段及医药用途 - Google Patents

Il-5抗体、其抗原结合片段及医药用途 Download PDF

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WO2019062831A1
WO2019062831A1 PCT/CN2018/108240 CN2018108240W WO2019062831A1 WO 2019062831 A1 WO2019062831 A1 WO 2019062831A1 CN 2018108240 W CN2018108240 W CN 2018108240W WO 2019062831 A1 WO2019062831 A1 WO 2019062831A1
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seq
variable region
chain variable
antibody
amino acid
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PCT/CN2018/108240
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English (en)
French (fr)
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应华
石金平
王义芳
胡齐悦
葛虎
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to BR112020005766-5A priority Critical patent/BR112020005766A2/pt
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to KR1020207010252A priority patent/KR102709785B1/ko
Priority to AU2018340557A priority patent/AU2018340557A1/en
Priority to US16/651,639 priority patent/US11365247B2/en
Priority to MX2020003034A priority patent/MX2020003034A/es
Priority to CA3076941A priority patent/CA3076941A1/en
Priority to JP2020517497A priority patent/JP7307720B2/ja
Priority to EP18860941.6A priority patent/EP3689906A4/en
Priority to RU2020112984A priority patent/RU2772716C2/ru
Priority to CN201880058448.5A priority patent/CN111065651B/zh
Publication of WO2019062831A1 publication Critical patent/WO2019062831A1/zh
Priority to ZA2020/01807A priority patent/ZA202001807B/en
Priority to US17/747,627 priority patent/US20220356239A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54—F(ab')2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
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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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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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
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion

Definitions

  • the present disclosure relates to IL-5 antibodies and antigen-binding fragments thereof. Further, the present disclosure also relates to chimeric antibodies, humanized antibodies comprising the CDR regions of the IL-5 antibodies, and the present disclosure also relates to the inclusion of the IL-5 antibodies And pharmaceutical compositions thereof and antigen-binding fragments thereof, and their use as diagnostic and therapeutic agents for IL-5 related diseases.
  • Interleukin-5 is one of the important members of the interleukin family, also known as T cell replacement factor (TRF), B cell growth factor-II (BC cell growth factor-II BCGF). -II), IgA-enhancing factor IgA-EF, and eosinophil differentiation factor (EDF), a homodimeric glycoprotein secreted mainly by helper T cell 2 (Th2) .
  • TRF T cell replacement factor
  • B cell growth factor-II BC cell growth factor-II BCGF
  • IgA-enhancing factor IgA-EF IgA-enhancing factor
  • EDF eosinophil differentiation factor
  • Th2 helper T cell 2
  • Human IL-5 consists of 134 amino acid residues, including a signal peptide consisting of 22 amino acids and two glycosylation sites, and human and murine IL-5 have 70% homology at the amino acid level.
  • the active IL-5 is in the form of an oligodimer, the two peptide chains are linked by disulfide bonds and exist in an antiparallel configuration, while the monomers of IL-5 are not biologically active (Adv Immunol. 1994; 57:145-90).
  • Eosinophil is associated with a variety of inflammatory diseases in the lungs, including allergic reactions, which are chronic respiratory inflammatory diseases, affecting approximately 300 million patients worldwide. 10%. Its pathogenesis is related to a variety of cytokines, and IL-5 and its receptor IL-5R play an important role in the pathogenesis of asthma. There is a large amount of inflammatory cell infiltration in the bronchopulmonary tissues of asthma patients, among which eosinophilia is the most significant. Many studies have shown that eosinophils are one of the major cells leading to airway inflammation in asthma (Curr Opin Pulm Med. 2005 Jan; 11(1): 1-6).
  • IL-5 plays an extremely important role in the differentiation, maturation, adhesion, invasion and apoptosis of EOS.
  • a large number of animal studies and clinical studies have shown that IL-5 can activate EOS progenitor cells in the bone marrow and cause aggregation of EOS in peripheral blood and airways, leading to chronic inflammation and hyperresponsiveness of the airways (J Immunol.2014 Oct 15 ;193(8):4043-52).
  • IL-5 can prolong EOS survival, enhance its degranulation response to specific stimulating factors (such as IgA or IgG), and mediate eosinophil chemotaxis (J Asthma Allergy.2015 Nov 3;8 :125-34).
  • IL-5 Increased expression of IL-5 was detected in both asthmatic and human bronchial antigen-inducing models (Greenfeder et al, Respiratory Research, 2: 71-79, 2001); inhalation of recombinant human IL-5 protein in asthmatic patients resulted in eosinophilicity Increased numbers of granulocytes, bronchial hyperresponsiveness, and release of eosinophilic granules indicate that IL-5 is a key factor in the pathogenesis of asthma.
  • the most effective method for the treatment of asthma is to inhibit the expression of some key mediators (including IL-5) in asthma by nasal or oral administration of sterols to reduce lung inflammation.
  • IL-5 some key mediators
  • long-term use of sterols has many side effects, so it is necessary to find new drug targets for the treatment of asthma.
  • IL-5 antibodies are only mepolizumab from GSK and reslizumab from Teva Pharma. Other antibodies against the IL-5 target are in preclinical research.
  • the related patents are WO2017033121, WO2016040007, WO2015095539, WO2012083370, WO2012158954, WO2006046689.
  • WO9621000, WO9535375, etc. but the above drugs still have room for improvement in IL-5-induced elimination of eosinophils and improvement of lung function. Therefore, it is necessary to continue to develop antibodies with high selectivity, high affinity and good efficacy to provide more and better anti-IL-5 treatment regimens for the treatment of asthma.
  • the present disclosure provides monoclonal antibodies or antigen-binding fragments (also referred to as IL-5 binding molecules) that specifically bind to the amino acid sequence or three-dimensional structure of IL-5.
  • the disclosure provides a monoclonal antibody or antigen-binding fragment thereof, which binds to human IL-5, comprising a heavy chain variable region and a light chain variable region ,among them:
  • the heavy chain variable region comprises an HCDR1, HCDR2 and HCDR3 region selected from the group consisting of the amino acid sequences of SEQ ID NOs: 16-18 or having the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 16-18, respectively a 2 or 1 amino acid difference HCDR variant comprising a panel selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions set forth in the amino acid sequence of SEQ ID NO: 19-21 or SEQ ID NO: 19-21 LCDR variants with 3, 2 or 1 amino acid differences in the LCDR1, LCDR2 and LCDR3 regions, respectively; or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22-24 or the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 22-24, respectively.
  • a 2 or 1 amino acid difference HCDR variant comprising a panel selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions set forth in the amino acid sequence of SEQ ID NOs: 25-27 or SEQ ID NOS: 25-27 LCDR variants with 3, 2 or 1 amino acid differences in the LCDR1, LCDR2 and LCDR3 regions, respectively; or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions selected from the amino acid sequences set forth in SEQ ID NOs: 28-30 or the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 28-30, respectively.
  • a 2 or 1 amino acid difference HCDR variant comprising a panel selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions set forth in the amino acid sequences of SEQ ID NOs: 31-33 or SEQ ID NOs: 31-33 LCDR variants with 3, 2 or 1 amino acid differences in the LCDR1, LCDR2 and LCDR3 regions, respectively; or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions selected from the amino acid sequences set forth in SEQ ID NOs: 34-36 or the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 34-36, respectively.
  • a 2 or 1 amino acid difference HCDR variant comprising a panel selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions set forth in the amino acid sequence of SEQ ID NOs: 37-39 or SEQ ID NOs: 37-39 LCDR variants with 3, 2 or 1 amino acid differences in the LCDR1, LCDR2 and LCDR3 regions, respectively; or
  • the heavy chain variable region comprises an HCDR1, HCDR2 and HCDR3 region selected from the group consisting of the amino acid sequences of SEQ ID NOs: 40-42 or having the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NOs: 40-42, respectively a 2 or 1 amino acid difference HCDR variant comprising a light chain variable region comprising an LCDR1, LCDR2 and LCDR3 region selected from the group consisting of the amino acid sequences of SEQ ID NOs: 43-45 or SEQ ID NO: 43-45 LCDR variants with 3, 2 or 1 amino acid differences in the LCDR1, LCDR2 and LCDR3 regions, respectively; or
  • the heavy chain variable region comprises an HCDR1, HCDR2 and HCDR3 region selected from the group consisting of the amino acid sequences of SEQ ID NOs: 34-36 or the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 34, 82, 36, respectively
  • An HCDR variant having a 3, 2 or 1 amino acid difference the light chain variable region comprising an LCDR1, LCDR2 and LCDR3 region selected from the group consisting of the amino acid sequences of SEQ ID NOs: 37-39 or with SEQ ID NO: 37-39
  • the LCDR1, LCDR2 and LCDR3 regions shown have LCDR variants with 3, 2 or 1 amino acid differences, respectively.
  • the CDRs of the monoclonal antibody or antigen-binding fragment including 3 heavy chain CDRs and 3 light chain CDRs) having 3, 2 or 1 amino acid differences are screened by affinity maturation methods CDR variants with 3, 2 or 1 amino acid differences were obtained.
  • the monoclonal antibody or antigen-binding fragment has an affinity (KD) to IL-5 of less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, or less than 10 -11 M.
  • the monoclonal antibody or antigen-binding fragment specifically binds to human IL-5, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 16-18, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 19-21 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 22-24, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 25-27 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 28-30, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 31-33 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 34-36, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 37-39 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions as set forth in SEQ ID NOs: 40-42, and the light chain variable region comprises the LCDR1, LCDR2 and LCDR3 regions as set forth in SEQ ID NOs: 43-45 ;or
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NOs: 34, 82, 36, and the light chain variable region comprises the LCDR1, LCDR2 and SEQ ID NOs: 37-39 LCDR3 area.
  • the monoclonal antibody is a recombinant antibody.
  • the monoclonal antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a recombinant antibody to a humanized antibody, or an antigen-binding fragment thereof.
  • the light and heavy chain FR region sequences on the humanized antibody light and heavy chain variable regions are derived from human germline light and heavy chains, or mutated sequences thereof, respectively.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the humanized antibody comprises the heavy chain variable region of SEQ ID NO: 49, 57, 63, 69 or 75 Variant; the variant has a 1-10 amino acid mutation at the heavy chain variable region sequence set forth in SEQ ID NO: 49, 57, 63, 69 or 75.
  • the monoclonal antibody or antigen-binding fragment thereof, wherein the variant is the FR region of the heavy chain variable region set forth in SEQ ID NO: 49, 57, 63, 69 or 75 a back mutation having 1-10 amino acids; preferably, the back mutation is an amino acid back mutation selected from the group consisting of S49T, V93T and K98S or a combination thereof on the heavy chain variable region of SEQ ID NO: 49, or The back mutation is an amino acid back mutation selected from S49T, V93T and K98T or a combination thereof on the heavy chain variable region of SEQ ID NO: 57, or the back mutation is represented by SEQ ID NO: 63 An amino acid back mutation selected from the group consisting of R38K, M48I, R67K, V68A, M70L, R72V, T74K and L83F or a combination thereof, or the back mutation is a heavy chain variable as shown in SEQ ID NO: 69 An amino acid back mutation selected from the group consisting of F29I
  • the monoclonal antibody or antigen-binding fragment thereof wherein the humanized antibody comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 50 or 51, or comprises a selected from the group consisting of SEQ ID NO: a heavy chain variable region of 58 or 59, or comprising a heavy chain variable region selected from any one of SEQ ID NOS: 64, 65 and 66, or comprising SEQ ID NO: 70 or 71
  • the heavy chain variable region is shown, or comprises a heavy chain variable region selected from any one of SEQ ID NOs: 76 to 79.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the humanized antibody comprises the light chain variable region of SEQ ID NO: 46, 54, 60, 67 or 72 or Variant; the variant is a sequence having 1-10 amino acid changes on the light chain variable region set forth in SEQ ID NO: 46, 54, 60, 67 or 72.
  • the monoclonal antibody or antigen-binding fragment thereof wherein the variant is the FR region of the light chain variable region set forth in SEQ ID NO: 46, 54, 60, 67 or 72 a back mutation having 1-10 amino acids; preferably, the back mutation is selected from A43S, L47V, G66R, T69S, F71Y and Y87F or a combination thereof on the light chain variable region shown by SEQ ID NO:46 Amino acid back mutation; or amino acid back mutation of A43S, L47M, F71Y and Y87F or a combination thereof on the light chain variable region of SEQ ID NO: 54; or the light chain of SEQ ID NO: 60 Amino acid back mutation of E1D, I2T, I57V, V84T and Y86F or a combination thereof on the variable region; or amino acid of M4L, A42S, L45P and L46W or a combination thereof on the light chain variable region of SEQ ID NO: 67 Back mutation; or amino acid
  • the monoclonal antibody or antigen-binding fragment thereof wherein the humanized antibody comprises a light chain variable region selected from the group consisting of SEQ ID NO: 47 or 48; or NO: a light chain variable region of 55 or 56; or a light chain variable region selected from the group consisting of SEQ ID NO: 61 or 62; or a light chain variable region of SEQ ID NO: 68
  • the humanized antibody comprises a light chain variable region selected from the group consisting of SEQ ID NO: 47 or 48; or NO: a light chain variable region of 55 or 56; or a light chain variable region selected from the group consisting of SEQ ID NO: 61 or 62; or a light chain variable region of SEQ ID NO: 68
  • the monoclonal antibody or antigen-binding fragment thereof, wherein the humanized antibody comprises a selected from the group consisting of:
  • the monoclonal antibody or antigen-binding fragment thereof wherein the antibody is a full-length antibody, further comprising a human antibody constant region, wherein the heavy chain constant region is preferably human IgG1, IgG2, IgG3, and IgG4 antibody heavy
  • the chain constant region more preferably comprises the human antibody heavy chain constant region set forth in SEQ ID NO: 52 and the human light chain constant region set forth in SEQ ID NO:53.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F (ab') 2, single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized An antigen-binding fragment of the V region (dsFv) and a peptide comprising a CDR.
  • the present disclosure also provides an isolated monoclonal antibody or antigen-binding fragment thereof that competes for binding to human IL-5 with the monoclonal antibody or antigen-binding fragment thereof described above.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the present disclosure, and one or more pharmaceutically acceptable carriers, diluents, buffers or forms Agent.
  • the amount of the monoclonal antibody or antigen-binding fragment thereof contained in the unit dose of the pharmaceutical composition is preferably from 0.1 to 2000 mg, more preferably from 1 to 1000 mg.
  • the present disclosure also provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof according to the present disclosure.
  • the present disclosure also provides a recombinant vector comprising the nucleic acid molecule described above.
  • the present disclosure also provides a host cell transformed with a recombinant vector according to the present disclosure, the host cell being selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
  • the present disclosure also provides a method for producing a monoclonal antibody or antigen-binding fragment thereof according to the present disclosure, the method comprising culturing the above host cell in culture to form and accumulate the above monoclonal antibody or antigen-binding fragment thereof, And recovering the monoclonal antibody or antigen-binding fragment thereof from the culture.
  • the present disclosure also provides methods for detecting or determining human IL-5, the methods comprising using the monoclonal antibodies described above or antigen-binding fragments thereof.
  • the present disclosure also provides an agent for detecting or measuring human IL-5, which comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of the above.
  • the present disclosure also provides a diagnostic agent for a disease associated with human IL-5, the diagnostic agent comprising a monoclonal antibody or antigen-binding fragment thereof according to the above.
  • the present disclosure also provides methods for diagnosing a disease associated with human IL-5, the method comprising detecting or determining human IL-5 or IL-5 positive cells using the monoclonal antibodies or antigen-binding fragments thereof described above.
  • the present disclosure also provides the use of the above monoclonal antibody or antigen-binding fragment thereof for the preparation of a diagnostic agent for a disease associated with human IL-5.
  • the present disclosure also provides a medicament for treating a disease associated with human IL-5, comprising the above monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, or a nucleic acid molecule as described above.
  • the present disclosure also provides a method of treating a disease associated with human IL-5, the method comprising administering to a subject a pharmaceutically effective amount of the above monoclonal antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the same, or the above Nucleic acid molecules to prevent or treat human IL-5 related diseases.
  • the present disclosure also provides the above monoclonal antibody or antigen-binding fragment thereof, or the use of the above pharmaceutical composition, or a therapeutic agent of the above nucleic acid molecule for preparing a disease associated with human IL-5.
  • the above diseases or conditions are preferably asthma, malignant asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent Angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, helminth infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic hyperplasia Inflammation, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis dermatitis, endometriosis, steroid dependence Eosinophilic bronchitis.
  • the IL-5 monoclonal antibody or antigen-binding fragment of the present disclosure has high specificity and high affinity with IL-5, and the immunogenicity of the humanized antibody is greatly reduced, and the specificity of the mouse antibody is completely retained. High affinity and excellent in vitro and in vivo activity.
  • the IL-5 monoclonal antibodies or antigen-binding fragments of the present disclosure have good selectivity for specifically recognizing only IL5.
  • the IL-5 monoclonal antibody or antigen-binding fragment of the present disclosure has good metabolic kinetics in rats, exhibits a long half-life, and high bioavailability.
  • the IL-5 humanized antibody molecule of the present disclosure has good long-term stability, no obvious abnormal chemical modification, no obvious aggregation at high concentration, and high purity and thermal stability.
  • the IL-5 monoclonal antibody or antigen-binding fragment of the present disclosure has a good property of improving lung function.
  • Figure 1 FACS experiment of IL-5 antibody blocking IL-5 binding to IL-5 receptor
  • Figure 2 Binding specificity detection of IL-5 antibody and Th2 cytokine
  • Figure 3 IL-5 antibody enhances respiratory intermittent value (Penh) levels.
  • G1 normal control group (PBS);
  • G2 model group (IgG);
  • G3 h1705-008 antibody 10mpk group;
  • G4 h1705-008 antibody 2mpk group;
  • G5 h1706-009 antibody 10mpk group;
  • G6 h1706-009 antibody 2mpk group;
  • G7 Hu39D10 10mpk group; wherein, *p ⁇ 0.05, ** ⁇ 0.01 (compared by ANOVA/Bonferroni and G2 group);
  • FIG. 4A BALF eosinophil levels in the lungs of asthmatic mice;
  • Figure 4B Tracheal mucosal thickness scores in asthmatic mice.
  • G1 normal control group;
  • G2 model group;
  • G4 h1705-008 antibody 2mpk group;
  • G6 h1706-009 antibody 2mpk group;
  • G7 Hu39D10 10mpk group;
  • Figure 4C Percentage of BALF eosinophils in the lungs of asthmatic mice;
  • Figure 5A and Figure 5B shows the ability of IL5 mAb to reduce eosinophil levels in BALF.
  • an “antibody” as used in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain of the present disclosure may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in number and position.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • murine antibody in this disclosure is a monoclonal antibody to human IL-5 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the IL-5 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine IL-5 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, a lambda chain or a variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or variants thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is cloned.
  • the human constant region gene is ligated into a chimeric gene, inserted into an expression vector, and finally expressed in a eukaryotic or prokaryotic system.
  • the antibody light chain of the IL-5 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the IL-5 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or comprises An IgGl, IgG2 or IgG4 heavy chain constant region variant of an amino acid mutation (such as a YTE mutation or a back mutation).
  • humanized antibody refers to an antibody produced by grafting a murine CDR sequence into a human antibody variable region framework, ie, a different type of human germline antibody framework sequence. It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of the mouse protein component.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database (Internet www.mrccpe.com.ac.uk/vbase ), as well as at Kabat, EA et al. , 1991 Sequences of Proteins of Immunological Interest, found in the 5th edition.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured by phage display.
  • the CDR sequence of the mouse in the IL-5 humanized antibody is selected from the group consisting of SEQ ID NOs: 16-21, 22-27, 28-33, 34-39 or 40-45
  • the human antibody variable region framework is designed to be selected, wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from a human germline heavy chain sequence, and a human germline light chain sequence.
  • the human antibody variable region can be subjected to minimal reverse mutation (reversion mutation, that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues) to maintain activity.
  • minimal reverse mutation that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues
  • the CDR graft can attenuate the affinity of the IL-5 antibody or antigen-binding fragment thereof to the antigen due to changes in the framework residues that are contacted with the antigen. Such interactions can be the result of high mutations in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues involved in antigen binding from a non-human IL-5 antibody or antigen-binding fragment thereof can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be related.
  • the sequence can be compared to a subtype consensus sequence or a consensus sequence of a murine sequence with a high percent similarity.
  • Rare framework residues are thought to be the result of high somatic mutations and thus play an important role in binding.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, IL-5). It has been shown that fragments of full length antibodies can be utilized to achieve antigen binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of an antibody examples include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab') 2 fragment, including a divalent fragment of two Fab fragments joined by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of a single arm VH and VL domain of the antibody (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) A combination of two or more separate CDRs, optionally joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using a recombinant method such that they are capable of producing a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chains referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Antigen-binding fragments of the present disclosure include Fab, F(ab')2, Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv), inclusion Peptides of CDRs, etc.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity in a fragment obtained by treating an IgG antibody molecule with a protease papain (cleaving an amino acid residue at position 224 of the H chain), wherein the N-terminal side of the H chain About half of the entire L chain is bound by a disulfide bond.
  • the Fab of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure which specifically recognizes human IL-5 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with papain. Furthermore, the Fab can be produced by inserting a DNA encoding a Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express a Fab.
  • F(ab')2 is an antibody obtained by digesting the lower portion of two disulfide bonds in the IgG hinge region with an enzyme pepsin, having an molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions linked at the hinge position. Fragment.
  • the F(ab')2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure which specifically recognizes human IL-5 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with pepsin. Further, the F(ab') 2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above F(ab')2.
  • the Fab' of the present disclosure can be produced by treating F(ab')2 of the present disclosure which specifically recognizes IL-5 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting a DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.
  • single-chain antibody single-chain Fv
  • scFv single-chain Fv
  • scFv antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variant thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996). , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present disclosure can be produced by obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human IL-5 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the coding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express an scFv.
  • a diabody is an antibody fragment in which an scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity.
  • the two antigens may be the same or different.
  • the diabody of the present disclosure can be produced by obtaining the coding cDNA of VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human IL-5 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the coding.
  • the DNA of the scFv is such that the amino acid sequence length of the peptide linker is 8 residues or less, the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or true
  • the diabody is expressed in nuclear organisms.
  • dsFv is obtained by linking a polypeptide in which one of amino acid residues in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues.
  • the amino acid residue substituted with a cysteine residue can be selected based on a three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).
  • the dsFv of the present disclosure can be produced by obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human IL-5 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the coding dsFv
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express dsFv.
  • a peptide comprising a CDR is constructed by one or more regions of a CDR comprising a VH or VL. Peptides comprising a plurality of CDRs can be joined directly or via a suitable peptide linker.
  • the CDR-containing peptide of the present disclosure can be produced by constructing the encoding of the CDRs of the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human IL-5 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure. DNA, the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express the peptide.
  • the CDR-containing peptide can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
  • antibody framework refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain that does not have a CDR.
  • amino acid difference refers to the difference between a polypeptide and its variant, at a certain amino acid position on a polypeptide fragment, wherein the variant may be replaced, inserted or Deletion of amino acids was obtained.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on the IL-5 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 -8 M, such as less than about 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD affinity
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • the antibodies of the present disclosure bind to IL-5 with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10 -8 M, 10 -9 M, or 10-10 M or less, for example, as used.
  • SPR Surface plasmon resonance
  • the term “competition” is used in the context of an antigen binding protein that competes for the same epitope (eg, neutralizing an antigen binding protein or a neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay:
  • the antigen binding protein to be detected eg, an antibody or an immunologically functional fragment thereof
  • prevents or inhibits eg, reduces) the specificity of a reference antigen binding protein (eg, a ligand or reference antibody) and a common antigen (eg, an IL-5 antigen or fragment thereof) Sexual union.
  • a reference antigen binding protein eg, a ligand or reference antibody
  • a common antigen eg, an IL-5 antigen or fragment thereof
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al, 1983, Methods in Enzymology 9: 242-253
  • solid phase direct biotin-avidin EIA see, eg, Kirkland et al, 1986, J. Immunol.
  • solid Direct labeling assay solid phase direct label sandwich assay (see, eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling with I-125 label RIA (see, eg, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, eg, Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82).
  • the assay involves the use of a purified antigen (which is on a solid surface or cell surface) that binds to a reference antigen binding protein with an unlabeled detection antigen binding protein and label.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein to be tested.
  • the antigen binding protein to be tested is present in excess.
  • An antigen binding protein identified by a competitive assay includes: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and a table adjacent to an epitope that is sufficiently close to the binding of the reference antigen binding protein A binding antigen binding protein occurs where the two epitopes interfere with each other spatially.
  • a competing antigen binding protein when present in excess, it will inhibit (eg, reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70. -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to a DNA molecule and an RNA molecule.
  • the nucleic acid molecule may be single stranded or double stranded, but is preferably a double stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to the coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the vector is a "plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (for example, a bacterial vector having an origin of replication of bacteria and an episomal mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby The host genome is replicated together (eg, a non-episomal mammalian vector).
  • a mouse can be immunized with human IL-5 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be produced by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include microorganisms (eg, bacteria), plant or animal cells.
  • Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human IL-5. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • administering when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to an exogenous drug, therapeutic, diagnostic, or combination with an animal, Contact with a human, subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administering “administering,” and “treating” also means treating the cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure or a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, the patient having one or A variety of disease symptoms are known, and the therapeutic agents are known to have a therapeutic effect on these symptoms.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any clinically measured extent.
  • the amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by doctors or other health care professionals to assess the severity or progression of the condition.
  • any statistical test methods known in the art such as Student's t test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared x 100.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the present disclosure includes an agent for treating an IL-5-related disease, the agent comprising the monoclonal antibody of the present disclosure or an antibody fragment thereof as an active ingredient.
  • the IL-5-related disease there is no limitation on the IL-5-related disease as long as it is an IL-5-related disease, for example, the molecular-induced therapeutic response using the present disclosure can be followed by binding to human IL-5 and then repressing or inhibiting eosinophil stimulation. Produced by the role.
  • the molecules of the present disclosure are very useful for individuals who are suffering from allergic and/or atopic reactions or reactions associated with eosinophils when in preparations and formulations suitable for therapeutic applications.
  • asthma Such as, but not limited to, asthma, asthma exacerbation, asthmatic malignant episodes, chronic pneumonia, allergic rhinitis, perennial allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopy Dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, helminth infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria , eosinophilic excessive bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis, Endometriosis, steroid-dependent eosinophilic bronchitis, etc.
  • such treatment inhibits or reduces lung tissue infiltration of eosinophils.
  • the number of times the antibody or fragment thereof is administered can be from three times a day to once every six months, and the route of administration can be intravenous, subcutaneous, intramuscular, parenteral or topical.
  • the present disclosure relates to methods for immunodetection or assay of IL-5, reagents for immunodetection or assay of IL-5, methods for immunodetection or assay of cells expressing IL-5, and for diagnosis and IL-
  • a diagnostic agent for a related disease comprising the monoclonal antibody or antibody fragment of the present disclosure which specifically recognizes human IL-5 and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof as an active ingredient.
  • the method for detecting or measuring the amount of IL-5 may be any known method.
  • it includes immunodetection or assay methods.
  • the immunodetection or assay method is a method of detecting or measuring the amount of antibody or the amount of antigen using a labeled antigen or antibody.
  • immunoassay or assay methods include radioactive substance labeling immunological antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods Wait.
  • the above IL-5-related diseases can be diagnosed by detecting or measuring IL-5-expressing cells with the monoclonal antibodies or antibody fragments of the present disclosure.
  • a known immunodetection method can be used, and immunoprecipitation, fluorescent cell staining, immunohistochemical staining, or the like is preferably used. Further, a fluorescent antibody staining method or the like using the FMAT8100HTS system (Applied Biosystem) can be used.
  • a living sample for detecting or measuring IL-5 is not particularly limited as long as it has a possibility of containing cells expressing IL-5, such as tissue cells, blood, plasma, serum, pancreatic juice, urine. Liquid, feces, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody of the present disclosure or an antibody fragment thereof may further contain an agent for performing an antigen-antibody reaction or an agent for detecting a reaction, depending on a desired diagnostic method.
  • Agents for performing antigen-antibody reactions include buffers, salts, and the like.
  • the reagents for detection include reagents commonly used in immunoassays or assay methods, such as labeled secondary antibodies that recognize the monoclonal antibodies, antibody fragments or conjugates thereof, substrates corresponding to the labels, and the like.
  • Human IL-5 encoding His-tagged IL-5, rhesus IL-5, mouse IL-5, rat IL-5, human IL-5R ⁇ receptor extracellular domain fusion protein sequence containing human IgG1-Fc fragment
  • an expression plasmid was constructed and then transfected into HEK293.
  • the cell supernatant was collected by centrifugation at 4500 rpm for 10 min, and the supernatant containing recombinant IL-5 and IL-5 ⁇ receptor protein was purified using a nickel column.
  • the recombinant human IL-5-Fc fusion protein utilizes Protein.
  • a affinity chromatography column was used for purification.
  • the purified protein can be used in the experiments of the following examples.
  • the protein sequence of a specific antigen is as follows:
  • the italic part is the His6-tag tag
  • the italic part is the His6-tag tag.
  • the italic part is the His6-tag tag.
  • the italic part is the His6-tag tag.
  • the italic part is the human Fc tag.
  • Example 2 Construction and identification of recombinant IL-5 ⁇ receptor and IL-5 ⁇ / ⁇ receptor cell lines
  • the present disclosure constructs a CHO-S/IL-5 ⁇ cell line expressing IL-5 ⁇ , and a CHO-S/IL-5 ⁇ /IL-5 ⁇ cell line expressing both IL-5 ⁇ and IL-5 ⁇ .
  • the human IL-5 ⁇ full-length gene (Q01344) was cloned into a mammalian cell expression vector pTargeT, and the linearized plasmid was electrotransfected into CHO-S cells, screened by G418 for 2 weeks, and subjected to two limiting dilutions.
  • the IL-5 ⁇ gene on the cell surface was detected by FACS, and the CHO-S/IL-5 ⁇ cell line with high expression of IL-5 ⁇ was selected, and the linearized pcDNA3.1-IL-5 ⁇ was electroporated on the basis of G418.
  • IL-5 ⁇ and IL-5 ⁇ genes on the cell surface were detected by FACS, and CHO-S/IL-5 ⁇ /IL with high expression of IL-5 ⁇ and IL-5 ⁇ was selected.
  • -5 ⁇ cell line After screening with zeocin for 2 weeks, two limiting dilutions were performed, and the IL-5 ⁇ and IL-5 ⁇ genes on the cell surface were detected by FACS, and CHO-S/IL-5 ⁇ /IL with high expression of IL-5 ⁇ and IL-5 ⁇ was selected. -5 ⁇ cell line.
  • the recombinant protein rhIL-5-his and Freund's adjuvant CFA (Sigma, Lot#SLBQ1109V), IFA (Sigma, Lot#SLBJ2845V) were immunized with two doses of 100g/50g/50g and 25g/12.5g/12.5g, respectively.
  • the specific immune response of IL-5 was determined by ELISA, ligand receptor blocking assay and TF-1 proliferation inhibition assay for detecting serum titer. Mice with a better specific immune response were selected, and after sacrifice, spleen cells were taken and fused with myeloma cells.
  • the purified antibodies were subjected to SEC-HPLC, endotoxin content detection, biacore assay for various IL-5 affinities, FACS-based receptor blockade assay for IL-5, and TF-1 proliferation inhibition assay.
  • the sequence of cloning from a positive hybridoma is as follows. Hybridoma cells in logarithmic growth phase were collected, using Trizol (Invitrogen, Cat No.15596-018) according to kit instructions an RNA extraction step, with PrimeScript TM Reverse Transcriptase reverse transcription kit (Takara, Cat No.2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced.
  • amino acid sequences corresponding to the heavy and light chain variable region DNA sequences of mAb 1705 and mAb 1706, mAb 1780, mAb 1773, mAb 1779 were obtained (the amino acid residues of the VH/VL CDRs were determined and annotated by the Kabat numbering system):
  • the results show that the murine antibody of the present disclosure has a high affinity for the antigen.
  • Example 4 Purification of IL-5-related recombinant protein, and purification of hybridoma antibody and recombinant antibody
  • the sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume.
  • the NI-NTA affinity column (QIAGEN, Cat No. 30721) was equilibrated with PBS and washed 2-5 column volumes. The supernatant cells were subjected to supernatant analysis and the supernatant samples were applied to the column. Rinse the column with PBS until the A280 reading drops to baseline. Rinse the column with PBS, rinse the heteroprotein, and collect. The protein of interest was eluted sequentially with washing buffer (imidazole 20 mM) and elution buffer (imidazole 300 mM), and the eluted peak was collected.
  • the collected eluate was further purified by ion exchange (Hiload 16/600 superdex 200 column).
  • the PBS was equilibrated to about 2 column volumes to ensure pH 7.4.
  • the elution buffer containing the target protein was identified and concentrated, and the samples were collected, and identified by SDS-PAGE and LC-MS as correct and sub-equipment.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, and the hybridoma expression supernatant was purified using a Protein G column, and the Fc fusion protein expression supernatant was purified using a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline.
  • the protein of interest was eluted with 100 mM acetic acid pH 3.0 and neutralized with 1 M Tris-HCl, pH 8.0.
  • the eluted sample was appropriately concentrated and further purified by PBS-balanced gel chromatography Superdex 200 (GE). The peak of the depolymerized product was collected and used.
  • Example 5 Humanized design of anti-human IL-5 monoclonal antibody
  • Humanization of murine anti-human IL-5 monoclonal antibodies is carried out as disclosed in many literatures in the art. Briefly, the constant region of the murine antibody is replaced with a human constant region, and the CDRs of the murine antibody are grafted onto the highest human template of FR homology, and the antibody conformation and the key amino acids affecting the binding of the antibody to the antigen are maintained for the FR region. Perform a back mutation to achieve.
  • Heavy and light chain variable region lines with high amino acid sequence identity to mAb-1705, mAb-1706, mAb1780, mAb1773 and mAb1779 were selected by aligning the IMGT human antibody heavy light chain variable region germline gene database.
  • the gene is used as a template, and the CDRs of the murine antibody are separately grafted into the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • the homologous sequence of the light chain variable region (VL) and the heavy chain variable region (VH) was searched from the human germline database, according to the same FR
  • the source is ranked from high to low, and the germline with the highest FR homology is selected as the main template.
  • the CDR region of the mouse antibody is transplanted into the human template, and then embedded by software based on the three-dimensional structure of the mouse antibody. Residues, residues that interact directly with the CDR regions, and residues that have a significant effect on the conformation of VL and VH undergo back mutations and are optimized for chemically labile amino acid residues to yield the final humanized molecule.
  • VH of h1705 selects IGHV3-23*04 as a template
  • VL selects IGKV1-12*01 as a template
  • the CDR of mAb1705 is transplanted into the human template, and the embedded residues are found by software and have direct interaction with the CDR regions.
  • the residues were back-mutated and the light and heavy chain variable regions of the humanized antibodies were designed as shown in Table 3.
  • Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • A43S indicates that the 43rd position A of the grafted is mutated back to S according to the natural sequence numbering of the amino acid sequence.
  • This table represents the sequence obtained by various combinations of mutations.
  • the humanized mouse antibody h1705-007 contains two mutants, light chain h1705_VL.1A and heavy chain h1705_VH.1A. Other analogies.
  • variable region of humanized antibody h1705 is as follows:
  • the above light chain variable region is combined with the light chain constant region sequence as set forth in SEQ ID NO: 53 to form the final intact heavy light chain sequence.
  • Each heavy chain variable region is combined with a heavy chain constant region as set forth in SEQ ID NO: 52 to form the final heavy chain sequence.
  • Grafted represents the murine antibody CDRs implanted into the human germline FR region.
  • A43S indicates that the 43rd position A of the grafted is mutated back to S according to the natural sequence number of the amino acid sequence.
  • the designed humanized molecules were combined into different molecules as shown in Table 6, as shown in Table 6.
  • H1706-010 h1706_VH.1 h1706_VH.1A h1706_VH.1B h1706_VL.1 H1706-002 H1706-003 H1706-004 h1706_VL.1A H1706-005 H1706-006 H1706-007 h1706_VL.1B H1706-008 H1706-009 H1706-010
  • variable region of humanized antibody h1706 is as follows:
  • the above light chain variable region is combined with the light chain constant region sequence as set forth in SEQ ID NO: 53 to form the final intact heavy light chain sequence.
  • Each heavy chain variable region is combined with a heavy chain constant region as set forth in SEQ ID NO: 52 to form the final heavy chain sequence.
  • VH of h1780 selected IGHV1-2*02 as a template VL selected IGKV3-11*01 as a template, and the CDR region of the murine antibody mAb1780 was transplanted to the selected humanized template, and the embedded defect was found by software.
  • Grafted represents the murine antibody CDRs implanted into the human germline FR region.
  • E1D indicates that the first position E of the grafted is mutated back to D according to the natural sequence number of the amino acid sequence.
  • the designed humanized molecules were combined into different molecules as shown in Table 8, as shown in Table 8.
  • variable region of humanized antibody h1780 is as follows:
  • the above light chain variable region is combined with the light chain constant region sequence as set forth in SEQ ID NO: 53 to form the final intact heavy light chain sequence.
  • Each heavy chain variable region is combined with a heavy chain constant region as set forth in SEQ ID NO: 52 to form the final heavy chain sequence.
  • N in the HCDR2 (RIDPANGDTK HGPKFQG) of h1773 was mutated to V (ie, N55V) to form an HCDR2 change (the HCDR2 sequence after site mutation is SEQ ID NO: 82: RIDPAVGDTKHGPKFQG Heavy chain variable regions and antibodies.
  • Grafted represents the murine antibody CDRs implanted into the human germline FR region.
  • M4L indicates that the grafted position M is mutated back to L according to the natural sequence number of the amino acid sequence.
  • the designed humanized molecules were combined into different molecules as shown in Table 10, as shown in Table 10.
  • variable region of humanized antibody h1773 is as follows:
  • the above light chain variable region is combined with the light chain constant region sequence as set forth in SEQ ID NO: 53 to form the final intact heavy light chain sequence.
  • Each heavy chain variable region is combined with a heavy chain constant region as set forth in SEQ ID NO: 52 to form the final heavy chain sequence.
  • VH of h1779 selected IGHV1-2*02 as a template VL selected IGKV1-33*01 as a template, and the CDR region of mouse antibody h1779 was transplanted to the selected humanized template, and the embedded defect was found by software.
  • Grafted represents the murine antibody CDRs implanted into the human germline FR region.
  • A43S indicates that the 43rd position A of the grafted is mutated back to S according to the natural sequence number of the amino acid sequence.
  • the designed humanized molecules were combined into different molecules as shown in Table 12, as shown in Table 12.
  • variable region of humanized antibody h1779 is as follows:
  • the above light chain variable region is combined with the light chain constant region sequence as set forth in SEQ ID NO: 53 to form the final intact heavy light chain sequence.
  • Each heavy chain variable region is combined with a heavy chain constant region as set forth in SEQ ID NO: 52 to form the final heavy chain sequence.
  • the present disclosure uses the antibody Hu39D10 against IL5 in WO2012083370A1 as a positive control, and the heavy and light chain sequences thereof are shown in SEQ ID NO: 80 and SEQ ID NO: 81.
  • the positive antibody molecule obtained by hybridoma screening is sequenced to obtain a variable region coding gene sequence.
  • the primers were designed to sequence the primers, and the sequencing gene was used as a template to construct the VH/VK gene fragment of each antibody, and then the expression vector pHr (with signal peptide and hIgG1/hkappa constant region gene (CH1-Fc/CL) fragment).
  • Homologous recombination was carried out to construct a recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr/VL-CL-pHr to form five chimeric antibodies of Ch1705, Ch1706, Ch1780, Ch1773 and Ch1779.
  • the antibody sequence after humanization design is codon-optimized to generate the coding sequence of the human codon-preferred gene.
  • the primers are designed to construct the VH/VK gene fragment of each antibody, and then the expression vector pHr (with signal peptide and hIgG1/hkappa constant)
  • the region gene (CH1-Fc/CL) fragment was subjected to homologous recombination to construct a humanized antibody full-length expression plasmid VH-CH1-Fc-pHr/VL-CL-pHr.
  • the plasmids expressing the light and heavy heavy chains of the antibodies were transfected into HEK293E cells at a ratio of 1:1.2. After 6 days, the expression supernatants were collected, centrifuged at high speed to remove impurities, and purified by a protein A column. Rinse the column with PBS until the A280 reading drops to baseline. The protein of interest was eluted with an acidic eluent of pH 3.0 - pH 3.5 and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentrated, and further purified by PBS-balanced gel chromatography Superdex 200 (GE) to remove the aggregate, collect the monomer peak, and equilibrate the device.
  • PBS-balanced gel chromatography Superdex 200 GE
  • Test Example 1 Biacore assays binding of murine IL-5 antibodies to different species of IL-5
  • the affinity of the murine IL-5 antibody to be tested and human IL-5 was determined using a Biacore T200 (GE) instrument.
  • the molecule to be tested was affinity-trapped with a Protein A biosensor chip, and then the antigen was passed on the surface of the chip (recombinant human, monkey, mouse IL5 prepared in Example 1), and the reaction signal was detected in real time using a Biacore T200 instrument to obtain binding and dissociation curves. . After completion of the dissociation of each experimental cycle, the biosensor chip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5). The data was fitted with the (1:1) Langmuir model using BIAevaluation version 4.1, GE software, and the affinity values were obtained as shown in Table 13.
  • Test Example 2 Biacore measures the affinity of IL-5 humanized antibodies to different species of IL-5
  • the affinity of the humanized IL-5 antibody to be tested and human IL-5 was determined using a Biacore T200 (GE) instrument.
  • the molecule to be tested was affinity-trapped with a Protein A biosensor chip, and then passed through the antigen on the surface of the chip (prepared in Example 1), and the reaction signal was detected in real time using a Biacore T200 instrument to obtain a binding and dissociation curve. After completion of the dissociation of each experimental cycle, the biosensor chip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5). The data was fitted to the (1:1) Langmuir model using BIAevaluation version 4.1, GE software, and the affinity values were obtained as shown in Table 14.
  • humanized IL-5 antibody still has a high affinity with human IL-5 (except for the humanized mutation of h1705 in vitro, humanized mutants of other murine antibodies only provide exemplary data) .
  • Test Example 3 ELISA-based murine IL-5 antibody blocks IL-5 binding to IL-5 ⁇ receptor assay
  • IL-5 5 ⁇ g/ml in PBS
  • IL-5 5 ⁇ g/ml in PBS
  • 5% skim milk blocking solution diluted with PBS was added at 200 ⁇ l/well, and incubated at 37 ° C for 2.5 hours in an incubator. Closed.
  • the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20), and then 25 ⁇ l of 10 ⁇ g/labeled with the biotin labeling kit (Dongren Chemical, LK03) was added.
  • Ml of IL-5R ⁇ (in 1% BSA) was further added with 25 ⁇ l of a gradient-diluted antibody, and the antibody competed with IL-5R ⁇ for binding to IL-5 and incubated for 1 hour at 37 °C.
  • Test Example 4 FACS-based IL-5 antibody blocks IL-5 binding to IL-5 receptor assay
  • CHO-S-IL-5R ⁇ and ⁇ were cultured with CD-CHO containing 100 ng/ml G418 and 25 ng/ml zeozin. During the cell culture, the concentration should not exceed 3 ⁇ 10 6 cell/ml.
  • the IL-5R ⁇ / ⁇ -CHOS cells in good condition were centrifuged (1000 rpm, 5 min), washed once with 10% FBS in PBS, and counted, the cell concentration was adjusted to 4 ⁇ 10 6 cells/ml, and 25 ⁇ l was added to the circle. In the bottom of the 96-well plate.
  • the antibody to be tested was diluted with a PBS solution containing 10% FBS at an initial concentration of 200 ⁇ g/ml, and 8 gradients were diluted 1:10.
  • Test Example 5 IL-5 antibody inhibits IL-5-induced proliferation of TF1 cells
  • IL-5 can induce proliferation of TF-1 cells, and IL-5 antibody can prevent IL-5 from stimulating proliferation of TF-1 cells.
  • TF-1 cells (ATCC, CRL-2003) were cultured in 10% FBS and 2 ng/mL rhGM-CSF (Lianke Bio, Catalog No. 96-AF-300-03-20) RPMI1640, placed with 37 In a °C, 5% CO 2 incubator, the cell density does not exceed 1 ⁇ 10 6 /ml.
  • the cells in the logarithmic growth phase were washed with PBS three times at 800 rpm for 5 min, and the cell density was adjusted to 6000 cells/well/90 ⁇ l with RPMI1640 (FBS: 2%, recombinant human IL-5: 10 ng/ml). After 10 ⁇ l of the antibody to be tested was diluted to 96-well culture for 3 days, 30 ⁇ l of cell titer was added and mixed, and the IC50 was calculated based on the reading. The test results are shown in Table 17 below.
  • Test Example 6 IL5 antibody inhibits IL5-induced eosinophil adhesion assay
  • IL5 can induce the differentiation, maturation, migration and activation of eosinophils, causing inflammation of the respiratory tract and leading to asthma.
  • This experiment utilizes the principle that IL-5 cytokines can promote and activate eosinophils, and collect and purify eosinophils derived from human peripheral blood to test IL-5-specific antibodies against IL-5. Blocking of the pathway, in vitro detection of IL-5 antibodies blocking the adhesion of IL5-mediated eosinophils.
  • the isolated eosinophil count was added to a 96-well cell culture plate previously coated with IgG antibody, about 1 ⁇ 10 4 per well, and human IL-5 (20 ng/ml) was added, and different concentrations of IL- 5 antibody molecules (starting at 10 ⁇ g/ml, 3-fold dilution, 10 concentration points); the cell culture plate was incubated at 37 ° C, incubated in a 5% CO 2 incubator for 1 h, the plate was removed, 0.3% CTAB was added to lyse the cells, and finally added.
  • the oxidation reaction substrate TMB color reaction was carried out, and the OD450 absorption value was read by a microplate reader.
  • Test Example 7 Specificity evaluation of humanized IL-5 antibody Th2 cytokine
  • IL-5 is one of Th2 cytokines.
  • 12 Th2 and related cytokines, including IL2 were detected with Fortebio. , 202-IL-010/CF), IL4 (R&D, 204-IL-050/CF), IL-5 (R&D, 205-IL-025/CF), IFNgamma, IL6 (R&D, 7270-IL-025/ CF), IL9 (R&D, 209-IL-010/CF), IL10 (R&D, 217-IL-025/CF), IL13 (R&D, 213-ILB-025/CF), IL25 (R&D, 8134-IL- 025/CF), IL31 (R&D, 2824-IL-010/CF), and IL3 (203-IL-050/CF) and GMCSF (R&D, 215-GM-010/CF) sharing ⁇ receptor
  • IL-5 humanized antibodies h1705-008 and h1706-009 specifically bind to IL-5 only, and do not cross-react with other Th2 cytokines.
  • Test Example 8 Evaluation of pharmacokinetics of humanized IL-5 antibody in rats
  • mice (provided by Sipple-Beikai Experimental Animal Co., Ltd.) 18 males, 3 rats in each group, divided into 6 groups on average; Hu39D10, h1705-008 and h1706-009 were administered intravenously and subcutaneously; Another 9 SD rats were administered intravenously only h1773-007, h1779-014, h1780-017.
  • intravenous administration group 0.2 ml of whole blood was collected before administration and 5 min, 8 h, 1 d, 2 d, 4 d, 7 d, 10 d, 14 d, 21 d, 28 d after administration, without anticoagulation, and after taking blood, it was placed at 4 ° C for 30 min.
  • the supernatant (serum) was placed in an EP tube and stored at -80 ° C; the subcutaneous injection group was administered before and 1 h, 2 h, 4 h, 8 h, 1 d, 2 d, 4 d after administration.
  • Whole blood was collected at 7d, 10d, 14d, 21d, and 28d, and the antibody concentration in the serum was measured by Elisa.
  • Test Example 9 Efficacy evaluation of IL-5 antibody in OVA-induced mouse asthma model
  • This test is based on airway inflammatory response and airway remodeling to assess the efficacy of IL-5 antibodies in the ovalbumin (OVA) aerosol-induced BALB/c mouse asthma model.
  • OVA ovalbumin
  • mice were randomly divided into 7 groups according to body weight, 10 mice in each group: normal control group (G1); model group (G2); 2 test antibodies h1705-008 (G3 and G4) and h1706-009 (G5 and G6) , 2 treatment groups (10 mpk and 2 mpk) of each test antibody; and a positive antibody Hu39D10 control group (G7, 10 mpk).
  • G1 normal control group
  • G2 model group
  • h1705-008 G3 and G4
  • h1706-009 G5 and G6
  • 2 treatment groups (10 mpk and 2 mpk
  • Hu39D10 control group G7, 10 mpk
  • mice were intraperitoneally injected with different doses of different antibodies once a day for three consecutive days.
  • Fresh test antibodies were prepared prior to each injection and were completed within half an hour of dosing.
  • the mice of the first group were challenged with PBS for 30 minutes as a normal control group, and phosphate buffer was intraperitoneally injected 2 hours before the challenge, once a day for three consecutive days.
  • mice were tested for airway hyperresponsiveness using the WBP system. All animals were inhaled with 1.5625, 3.125, 6.25, 12.5, 25, 50 mg/mL doubling concentrations of methacholine to determine the enhanced expiratory intermittent value at the corresponding concentration.
  • a 1.2 mm diameter tracheal cannula was inserted into the trachea and fixed, and the lungs were lavaged twice, each time 0.8 ml of phosphate buffer containing 1% BSA and 0.6 mM EDTA. Record the recovery volume of the lavage fluid.
  • the BALF was centrifuged at 300 g for 4 minutes at 4 degrees Celsius and the supernatant was retained for cytokine analysis. After centrifugation, the cells were resuspended in 1.5 ml of PBS (containing 1% BSA and 0.6 mM EDTA) for cell counting. The total number of cells in BALF was counted by a hemocytometer and trypan blue staining experiments. After the cells were smeared, the Wright staining solution was stained for one minute and then stained with Giemsa for 7 minutes to distinguish between eosinophils, neutrophils, macrophages, and lymphocytes. Count under an optical microscope.
  • Test Example 10 Evaluation of in vivo efficacy of IL5 antibody by exogenous human IL5-induced guinea pig acute asthma model
  • Guinea pigs were divided into 9 groups, 8-10 in each group: normal control group, model group, hu39D10 (1mg/kg) group, h1705-008 (1mg/kg) group, h1706-009 (1mg/kg) group, h1780 -017 (1 mg/kg) group, h1773-007 (1 mg/kg) group and h1779-014 (1 mg/kg) group.
  • the guinea pigs in the model group and the drug-administered group were injected with 100 ⁇ l of human IL5 (containing 5 ⁇ g of IL5 antigen) in the first weather tube, and the normal control group was intratracheally injected with PBS.
  • Test Example 11 Stability of humanized anti-IL-5 antibody
  • Deamidation modification is a common chemical modification in antibodies that may affect late stability.
  • partial amino acids in the CDR regions are highly deamidated, oxidized or isomerized. Generally, selection is avoided or reduced. 100 ⁇ g samples taken at different time points were dissolved in 100 ⁇ l of 0.2 M His-HCl, 8 M Gua-HCl, pH 6.0 solution, 3 ⁇ l of 0.1 g/mL DTT, 50 ° C water bath for 1 hour, and then 0.02 M His-HCl, pH.
  • the solution of 6.0 was ultrafiltered twice, 3 ⁇ l of 0.25 mg/mL trypsin was added, and the solution was hydrolyzed overnight at 37 ° C in a water bath, and analyzed by LC-MS with Agilent 6530 Q-TOF.
  • the results showed that the chemical stability of the humanized antibody of the present disclosure was good, and the antibody was not abnormally modified after one month of acceleration at 40 ° C in the 529 buffer system.
  • the stability of the antibody to be tested was evaluated at a high concentration and different buffer systems and different temperature conditions for one month.
  • the concentration was 50 mg/ml.
  • 40 °C, 25 °C, 4 °C and -80 were investigated.
  • a Waters e2695 chromatograph was used with a column of Waters Xbridge BEH 200A SEC, a mobile phase of PBS (pH adjusted to 6.8 with dilute hydrochloric acid), 50 ⁇ g of protein was injected, and isocratic elution at a flow rate of 0.5 mL/min. It was investigated that IL-5 humanized antibody did not significantly aggregate all antibodies under high concentration conditions. After 4 weeks of acceleration at 40 ° C, the antibody monomer purity was greater than 95% in all three systems.
  • the stability of the antibody to be tested was evaluated at a high concentration and different buffer systems and different temperature conditions at a concentration of 50 mg/ml.
  • the stability of 40 ° C was examined in the His system, and the purity was monitored by CE-SDS.
  • the sample was replaced with a corresponding buffer (PBS buffer), and the sample concentration was controlled at about 1 mg/ml, and 9 ⁇ l was applied.
  • Parameter setting starting temperature 20 ° C; incubation 0 s; heating rate 0.3 ° C / min; Plate Hold 5 s; termination temperature 95 ° C.
  • the Tm value of the antibody was detected, and the antibody had a high Tm value, showing good thermal stability.

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Abstract

IL-5抗体、其抗原结合片段及医药用途。包含IL-5抗体CDR区的鼠源抗体、嵌合抗体、人源化抗体，以及包含IL-5抗体及其抗原结合片段的药物组合物，以及其作为药物的用途。

Description

IL-5抗体、其抗原结合片段及医药用途 技术领域
本公开涉及IL-5抗体以及其抗原结合片段，进一步地，本公开还涉及包含所述IL-5抗体CDR区的嵌合抗体、人源化抗体，本公开还涉及包含所述IL-5抗体及其抗原结合片段的药物组合物，以及其作为IL-5相关疾病诊断剂和治疗药物的用途。
背景技术
白细胞介素-5(IL-5)是白细胞介素家族的重要成员之一，又称为T细胞替代因子(T cell replacing factor TRF)、B细胞生长因子-II(B cell growth factor-II BCGF-II)、IgA增强因子(IgA-enhancing factor IgA-EF)、酸性粒细胞分化因子(eosinophil differentiation factor EDF)，是一种主要由辅助性T细胞2(Th2)分泌的同二聚体糖蛋白。人的IL-5由134个氨基酸残基组成，包括由22个氨基酸组成的信号肽和2个糖基化位点，人和鼠IL-5在氨基酸水平同源性为70％。具有活性的IL-5呈现寡二聚体形式，两条肽链以二硫键连接，以反向平行的构型存在，而IL-5的单体则无生物学活性(Adv Immunol.1994；57:145-90)。
嗜酸性粒细胞(eosinophil，EOS)与肺部多种炎性疾病包括与过敏性反应有关的变应性病症有关，其中哮喘是一种慢性呼吸道炎症疾病，全球约有3亿患者，发病率为10％。其发病机制与多种细胞因子有关，IL-5及其受体IL-5R在哮喘的发病过程中扮演着重要的角色。哮喘患者支气管肺组织内有大量的炎性细胞浸润，其中以嗜酸性粒细胞增多最为显著。许多研究都表明嗜酸性粒细胞是导致哮喘气道炎症的主要细胞之一(Curr Opin Pulm Med.2005Jan；11(1):1-6)。IL-5在EOS的分化、成熟、黏附、浸润和凋亡过程中发挥着极其重要的作用。大量的动物试验和临床研究均表明，IL-5可活化骨髓中的EOS祖细胞，并引起外周血和气道内EOS的聚集，从而导致气道的慢性炎症和高反应性(J Immunol.2014 Oct 15；193(8):4043-52)。此外，IL-5还能延长EOS的存活时间、增强其对特殊刺激因子(如IgA或IgG)的脱颗粒反应，介导嗜酸性粒细胞的趋化反应(J Asthma Allergy.2015 Nov 3；8:125-34)。在哮喘患者和人支气管抗原诱发模型中均检测到IL-5表达水平的增加(Greenfeder et al,Respiratory Research,2:71-79,2001)；哮喘患者吸入重组人IL-5蛋白会导致嗜酸性粒细胞数目增多，支气管高反应性及嗜酸性粒细胞毒性颗粒的释放，这些都表明IL-5是哮喘发病中的一个关键因素。
目前对哮喘治疗最有效的方法是通过鼻腔或口腔给予固醇类药物来抑制哮喘中一些关键介质(包括IL-5)的表达，以减轻肺部炎症。然而长期使用固醇类药物具有很多副作用，因此，有必要寻找新的治疗哮喘的药物靶点。已有研究证明，使用IL-5的抗体抑制IL-5与其受体结合，可显著降低嗜酸性粒细胞在肺内的聚集， 降低血液、组织、痰液中的嗜酸性粒细胞水平，并可减少嗜酸性粒细胞所介导的炎症反应，提高肺功能，对严重的嗜酸性粒细胞哮喘和复发性哮喘均具有很好的疗效(Drugs.2017May；77(7):777-784)。现有上市的IL-5抗体只有GSK公司的mepolizumab和Teva Pharma公司的reslizumab，其他针对IL-5靶点的抗体均处于临床前研究阶段，相关专利有WO2017033121、WO2016040007、WO2015095539、WO2012083370、WO2012158954、WO2006046689、WO9621000、WO9535375等，但是上述药物在IL-5诱导的嗜酸性粒细胞的消除和肺功能的改善方面仍有可提高的空间。因此，有必要继续开发具有高选择性、高亲和力和良好药效的抗体，为治疗哮喘提供更多和更优的抗IL-5治疗方案。
发明内容
本公开提供与IL-5的氨基酸序列或三维结构特异性结合的单克隆抗体或抗原结合片段(也可称IL-5结合分子)。
一方面，本公开提供一种单克隆抗体或其抗原结合片段，所述单克隆抗体或其抗原结合片段结合人IL-5，所述单克隆抗体包含重链可变区和轻链可变区，其中：
(i)重链可变区包含选自如SEQ ID NO：16-18氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：16-18所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：19-21氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：19-21所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
(ii)重链可变区包含选自如SEQ ID NO：22-24氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：22-24所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：25-27氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：25-27所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
(iii)重链可变区包含选自如SEQ ID NO：28-30氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：28-30所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：31-33氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：31-33所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
(iv)重链可变区包含选自如SEQ ID NO：34-36氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：34-36所示的HCDR1、HCDR2和HCDR3区分别 具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：37-39氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：37-39所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
(v)重链可变区包含选自如SEQ ID NO：40-42氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：40-42所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：43-45氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：43-45所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
(vi)重链可变区包含选自如SEQ ID NO：34-36氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：34、82、36所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：37-39氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：37-39所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体。
在一些实施方式中，所述单克隆抗体或抗原结合片段的CDR(包括3个重链CDR和3个轻链CDR)具有3，2或1个氨基酸差异的CDR变体是经亲和力成熟方法筛选获得的具有3，2或1个氨基酸差异的CDR变体。
在一些实施方式中，所述单克隆抗体或抗原结合片段与IL-5的亲和力(KD)小于10 -8M、小于10 -9M、小于10 -10M或小于10 -11M。
在一些实施方式中，所述单克隆抗体或抗原结合片段特异性结合人IL-5，所述单克隆抗体包含重链可变区和轻链可变区，其中：
(vii)重链可变区包含如SEQ ID NO：16-18所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：19-21所示的LCDR1、LCDR2和LCDR3区；或
(viii)重链可变区包含如SEQ ID NO：22-24所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：25-27所示的LCDR1、LCDR2和LCDR3区；或
(ix)重链可变区包含如SEQ ID NO：28-30所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：31-33所示的LCDR1、LCDR2和LCDR3区；或
(x)重链可变区包含如SEQ ID NO：34-36所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：37-39所示的LCDR1、LCDR2和LCDR3区；或
(xi)重链可变区包含如SEQ ID NO：40-42所示的HCDR1、HCDR2和HCDR3 区，轻链可变区包含如SEQ ID NO：43-45所示的LCDR1、LCDR2和LCDR3区；或
(xii)重链可变区包含如SEQ ID NO：34、82、36所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含如SEQ ID NO：37-39所示的LCDR1、LCDR2和LCDR3区。
在一些实施方式中，所述单克隆抗体是重组抗体。
在一些实施方式中，所述单克隆抗体选自鼠源抗体、嵌合抗体、人源化抗体的重组抗体或其抗原结合片段。
在一些实施方式中，所述的人源化抗体轻链和重链可变区上的轻链和重链FR区序列分别来源于人种系轻链和重链或其突变序列。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:49、57、63、69或75所示的重链可变区或其变体；所述变体是在SEQ ID NO:49、57、63、69或75所示的重链可变区序列上具有1-10个氨基酸突变。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述变体是在SEQ ID NO:49、57、63、69或75所示的重链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变是在SEQ ID NO:49所示的重链可变区上选自S49T，V93T和K98S或其组合的氨基酸回复突变，或所述回复突变是在SEQ ID NO:57所示的重链可变区上选自S49T,V93T和K98T或其组合的氨基酸回复突变，或所述回复突变是在SEQ ID NO:63所示的重链可变区上选自R38K，M48I，R67K，V68A，M70L，R72V，T74K和L83F或其组合的氨基酸回复突变，或所述回复突变是在SEQ ID NO:69所示的重链可变区上选自F29I，R38K，V48I，R72A，T97F和N55V或其组合的氨基酸回复突变，或所述回复突变是在SEQ ID NO:75所示的重链可变区上选自R38K，M48I，R67K，V68A，R72A，T74K，M81L，L83F和D89E或其组合的氨基酸回复突变。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:50或51所示的重链可变区，或包含选自SEQ ID NO:58或59所示的重链可变区，或包含选自SEQ ID NO:64，65和66中任一个所示的重链可变区，或包含选自SEQ ID NO:70或71所示的重链可变区，或包含选自SEQ ID NO:76至79中任一个所示的重链可变区。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:46、54、60、67或72所示的轻链可变区或其变体；所述变体是在SEQ ID NO:46、54、60、67或72所示的轻链可变区上具有1-10个氨基酸变化的序列。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述变体是在SEQ ID NO:46、54、60、67或72所示的轻链可变区的FR区位置上具有1-10 个氨基酸的回复突变；优选的，所述回复突变选自在SEQ ID NO:46所示的轻链可变区上的A43S，L47V，G66R，T69S，F71Y和Y87F或其组合的氨基酸回复突变；或在SEQ ID NO:54所示的轻链可变区上的A43S，L47M，F71Y和Y87F或其组合的氨基酸回复突变；或在SEQ ID NO:60所示的轻链可变区上的E1D，I2T，I57V，V84T和Y86F或其组合的氨基酸回复突变；或在SEQ ID NO:67所示的轻链可变区上的M4L，A42S，L45P和L46W或其组合的氨基酸回复突变；或在SEQ ID NO:72所示的轻链可变区上的A43S，I48V和F71Y或其组合的氨基酸回复突变。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:47或48所示的轻链可变区；或包含选自SEQ ID NO:55或56所示的轻链可变区；或包含选自SEQ ID NO:61或62中所示的轻链可变区；或包含SEQ ID NO:68所示的轻链可变区；或包含选自SEQ ID NO:73或74所示的轻链可变区。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自：
选自SEQ ID NO:49-51中任一个所示的重链可变区和选自SEQ ID NO:46-48中任一个所示的轻链可变区；或
选自SEQ ID NO:57-59中任一个所示的重链可变区和选自SEQ ID NO:54-56中任一个所示的轻链可变区；或
选自SEQ ID NO:63-66中任一个所示的重链可变区和选自SEQ ID NO:60-62中任一个所示的轻链可变区；或
选自SEQ ID NO:69-71中任一个所示的重链可变区和选自SEQ ID NO:67-68中任一个所示的轻链可变区；或
选自SEQ ID NO:75-79中任一个所示的重链可变区和选自SEQ ID NO:72-74中任一个所示的轻链可变区。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括人抗体恒定区，其中重链恒定区优选人IgG1、IgG2、IgG3和IgG4抗体重链恒定区，更优选包含SEQ ID NO:52所示的人抗体重链恒定区和SEQ ID NO:53所示的人轻链恒定区。
在一些实施方式中，所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
本公开还提供一种分离的单克隆抗体或其抗原结合片段，其与上述单克隆抗体或其抗原结合片段竞争结合人IL-5。
本公开还提供一种药物组合物，其含有治疗有效量的根据本公开的单克隆抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。药物组合物单位剂量中含单克隆抗体或其抗原结合片段的量优选为 0.1-2000mg，更优选为1-1000mg。
本公开还提供一种分离的核酸分子，其编码根据本公开的单克隆抗体或其抗原结合片段。
本公开还提供一种重组载体，其包含上述的核酸分子。
本公开还提供一种用根据本公开的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
本公开还提供用于生产根据本公开的单克隆抗体或其抗原结合片段的方法，所述方法包括将上述宿主细胞在培养物中进行培养以形成并积累上述单克隆抗体或其抗原结合片段，以及从培养物回收所述单克隆抗体或其抗原结合片段。
本公开还提供用于检测或测定人IL-5的方法，所述方法包括使用上述单克隆抗体或其抗原结合片段。
本公开还提供用于检测或测定人IL-5的试剂，所述试剂包含上述任一项所述的单克隆抗体或其抗原结合片段。
本公开还提供用于与人IL-5相关的疾病的诊断剂，所述诊断剂包含根据上述的单克隆抗体或其抗原结合片段。
本公开还提供用于诊断与人IL-5相关的疾病的方法，所述方法包括使用上述单克隆抗体或其抗原结合片段检测或测定人IL-5或IL-5阳性细胞。
本公开还提供上述单克隆抗体或其抗原结合片段在制备与人IL-5相关的疾病的诊断剂中的应用。
本公开还提供用于治疗与人IL-5相关的疾病的药物，所述治疗剂包含上述单克隆抗体或其抗原结合片段，或包含上述的药物组合物，或包含上述核酸分子。
本公开还提供治疗与人IL-5相关的疾病的方法，所述方法包括向受试者施用药物有效量的上述单克隆抗体或其抗原结合片段，或包含上述的药物组合物，或上述的核酸分子，以预防或治疗人IL-5相关的疾病。
本公开还提供上述单克隆抗体或其抗原结合片段，或包含上述药物组合物，或上述核酸分子在制备与人IL-5相关的疾病的治疗剂中的应用。
上述疾病或病症优选哮喘、哮喘恶性发作、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位、类固醇依赖性嗜酸细胞性支气管炎。
本公开IL-5单克隆抗体或抗原结合片段具有很高的特异性、与IL-5的高亲和力，人源化抗体的免疫原性大大降低，同时完全保留了鼠源抗体的特异性，较高的亲和力和优异的体内外活性。
本公开IL-5单克隆抗体或抗原结合片段具有只特异性识别IL5的良好选择性。
本公开IL-5单克隆抗体或抗原结合片段具有良好的大鼠上的代谢动力学特性，显示出很长的半衰期，很高的生物利用度。
本公开IL-5人源化抗体分子具有良好的长期稳定性，无明显异常化学修饰，高浓度下无明显聚集，有较高的纯度和热稳定性。
本公开IL-5单克隆抗体或抗原结合片段除具有降低嗜酸性粒细胞的增殖作用外，还具有良好的改善肺功能的特性。
附图说明
图1：IL-5抗体阻断IL-5结合IL-5受体的FACS实验；
图2：IL-5抗体与Th2细胞因子的结合特异性检测；
图3：IL-5抗体增强呼吸间歇值(Penh)水平。G1：正常对照组(PBS)；G2：模型组(IgG)；G3：h1705-008抗体10mpk组；G4：h1705-008抗体2mpk组；G5：h1706-009抗体10mpk组；G6：h1706-009抗体2mpk组；G7：Hu39D10 10mpk组；其中，*p<0.05,**<0.01(通过ANOVA/Bonferroni与G2组进行比较)；
图4A哮喘小鼠肺部BALF嗜酸性粒细胞水平；图4B哮喘小鼠气管粘膜厚度评分。G1：正常对照组；G2：模型组；G3：h1705-008抗体10mpk组；G4：h1705-008抗体2mpk组；G5：h1706-009抗体10mpk组；G6：h1706-009抗体2mpk组；G7：Hu39D10 10mpk组；图4C哮喘小鼠肺部BALF嗜酸性粒细胞百分比；
图5A和图5B：显示IL5单抗降低BALF中嗜酸性粒细胞水平的能力。
具体实施方式
一.术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本公开所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
在本公开中，本公开所述的抗体轻链可进一步包含轻链恒定区，所述的轻链 恒定区包含人源或鼠源的κ、λ链或其变体。
在本公开中，本公开所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人IL-5的单克隆抗体。制备时用IL-5抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源IL-5抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个具体的实施方案中，所述的IL-5嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的IL-5嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者包含氨基酸突变(如YTE突变或回复突变)的IgG1、IgG2或IgG4重链恒定区变体。
术语“人源化抗体(humanized antibody)”，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(因特网 www.mrccpe.com.ac.uk/vbase)中获得，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突 变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本公开一个优选的实施方案中，所述的IL-5人源化抗体中鼠的CDR序列选自SEQ ID NO:16-21、22-27、28-33、34-39或40-45；人的抗体可变区框架经过设计选择，其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链序列，和人种系轻链序列。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变(回复突变，即将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基)，以保持活性。
CDR的移植可由于与抗原接触的构架残基的变化而导致产生的IL-5抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人IL-5抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查鼠单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，IL-5)的能力的一个或多个片段。已显示可利用全长抗体的片段来实现抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science 242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
本公开的抗原结合片段包括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)、包含CDR的肽等。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
本公开的Fab可以通过用木瓜蛋白酶处理本公开的特异性识别人IL-5并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
本公开的F(ab')2可以通过用胃蛋白酶处理本公开的特异性识别人IL-5并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的特异性识别IL-5并与胞外区的氨基酸序列或其三维结构结合的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本公开的scFv可以通过以下步骤来生产：获得本公开的特异性识别人IL-5并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
双抗体是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本公开的双抗体可以通过以下步骤来生产：获得本公开的特异性识别人IL-5并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA 插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的dsFv可以通过以下步骤来生产：获得本公开的特异性识别人IL-5并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
包含CDR的肽是通过包含VH或VL的CDR中的一个或多个区域而构成的。包含多个CDR的肽可以被直接相连或经由适合的肽接头相连。
本公开的包含CDR的肽可以通过以下步骤来生产：构建本公开的特异性识别人IL-5并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的CDR的编码DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。
本文中使用的术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“氨基酸差异”是指多肽与其变体之间，在多肽片段上某个或某些氨基酸位点之间的差异，其中变体可以由多肽上某个或某些位点经替换、插入或缺失氨基酸获得。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，IL-5分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语"KD"是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10-7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合IL-5，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或 中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如 IL-5抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化抗原(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与充分接近参考抗原结合蛋白结合的表位所邻近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人IL-5或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样 可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括微生物(例如细菌)、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人IL-5特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“给予”、“施用”和“处理”当应用于动物、人、受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种抗体或其抗原结合片段的组合物或编码抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床有测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫 生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可 以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
此外，本公开包括用于治疗与IL-5相关的疾病的药剂，所述药剂包含本公开的单克隆抗体或其抗体片段作为活性成分。
对与IL-5相关的疾病没有限制，只要它是与IL-5相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类IL-5然后阻遏或抑制嗜酸性粒细胞刺激作用而产生。因此，当处于适于治疗应用的制备物和制剂中时，本公开的分子对这样一些人是非常有用的，他们患有变态和/或特应性反应，或与嗜酸性粒细胞有关的反应，例如但不限于，哮喘、哮喘恶化、哮喘恶性发作、慢性肺炎、过敏性鼻炎、常年过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位、类固醇依赖性嗜酸细胞性支气管炎等。在优选的实施方案中，这种治疗能抑制或减轻嗜酸性细胞的肺组织浸润。抗体或其片段给予的次数可以从每日三次到每6个月一次，给予的途径可以是静脉、皮下、肌肉、胃肠外或局部途径。
此外，本公开涉及用于免疫检测或测定IL-5的方法、用于免疫检测或测定IL-5的试剂、用于免疫检测或测定表达IL-5的细胞的方法和用于诊断与IL-5相关的疾病的诊断剂，其包含本公开的特异性识别人IL-5并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定IL-5的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的 方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与IL-5相关的疾病可以通过用本公开的单克隆抗体或抗体片段检测或测定表达IL-5的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定IL-5的活体样品没有特别限制，只要它具有包含表达IL-5的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
二.实施例与测试例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1、IL-5抗原及检测用蛋白的制备
IL-5抗原设计及表达
编码带His标签的人IL-5，恒河猴IL-5，小鼠IL-5，大鼠IL-5，含人IgG1-Fc片段的人IL-5Rα受体胞外区融合蛋白序列装到phr载体上，构建成表达质粒，然后转染HEK293。待转染第6天，取样4500rpm离心10min收集细胞上清，将含重组的IL-5和IL-5α受体蛋白上清利用镍柱进行纯化，重组的人IL-5-Fc融合蛋白利用Protein A亲和层析柱进行纯化。纯化的蛋白可用于下述各实施例实验中。具体抗原的蛋白序列如下：
1、带his标签的人IL-5氨基酸序列(rhIL-5-his)
Figure PCTCN2018108240-appb-000001
注释：斜体部分为His6-tag标记
                                            SEQ ID NO：1
2、带his标签的食蟹猴IL-5氨基酸序列
Figure PCTCN2018108240-appb-000002
注释：斜体部分为His6-tag标记。
3、带his标签的小鼠IL-5氨基酸序列
Figure PCTCN2018108240-appb-000003
注释：斜体部分为His6-tag标记。
4、带his标签的大鼠IL-5氨基酸序列
Figure PCTCN2018108240-appb-000004
注释：斜体部分为His6-tag标记。
5、融合人Fc片段的人IL-5α受体氨基酸序列
Figure PCTCN2018108240-appb-000005
注释：斜体部分为人Fc标签。
实施例2：重组IL-5α受体和IL-5α/β受体细胞系的构建和鉴定
为筛选有功能的抗体，本公开构建了表达IL-5α的CHO-S/IL-5α细胞株，以及同时表达IL-5α和IL-5β的CHO-S/IL-5α/IL-5β细胞株
具体为将人IL-5α全长基因(Q01344)克隆到哺乳动物细胞表达载体pTargeT上，将线性化质粒电转染至CHO-S细胞中，经G418筛选2周，再进行2次有限稀释。通过FACS检测细胞表面的IL-5α基因，挑选出IL-5α表达量高的CHO-S/IL-5α细胞株，在此基础上电转染线性化的pcDNA3.1-IL-5β，经G418和zeocin筛选2周，再进行2次有限稀释，通过FACS检测细胞表面的IL-5α和IL-5β基因，挑选出IL-5α和IL-5β表达量高的CHO-S/IL-5α/IL-5β细胞株。
实施例3：抗人IL-5鼠源单克隆抗体的制备
用重组蛋白rhIL-5-his和弗氏佐剂CFA(Sigma，Lot#SLBQ1109V)、IFA(Sigma，Lot#SLBJ2845V)分高低两个剂量100g/50g/50g和25g/12.5g/12.5g分别免疫两组Balb/c(5只/组)小鼠，四组SJL(5只/组)小鼠。IL-5的特异性免疫反应由检测血清效价的ELISA，配体受体阻断实验和TF-1增殖抑制实验来测定。选取有较好特异性免疫反应的小鼠，处死后，取脾细胞，与骨髓瘤细胞融合。
初次筛选用针对人IL-5的ELISA结合实验进行。当将杂交瘤细胞转移到24孔板后，用针对人、食蟹猴、小鼠IL-5的ELISA结合实验，基于ELISA的针对IL-5的受体阻断实验和TF-1增殖抑制实验，对其上清进行复筛。筛选出的阳性克隆经过两轮亚克隆后，得到杂交瘤克隆，用于抗体生产，并通过亲和层析方法纯化。
纯化后的抗体分别进行了SEC-HPLC，内毒素含量检测，biacore测定对各种属IL-5的亲和力，基于FACS的针对IL-5的受体阻断实验，和TF-1增殖抑制实验，嗜酸性粒细胞的粘附实验，以及小鼠哮喘模型和豚鼠体内中和模型上的药效评价，筛选出体内外活性好的单克隆杂交瘤细胞株mAb1705、mAb1706、mAb1780、mAb1773和mAb1779。
从阳性杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TM Reverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后测序。得到mAb1705和mAb1706，mAb1780，mAb1773，mAb1779的重链和轻链可变区DNA序列对应的氨基酸序列(VH/VL CDR的氨基酸残基由Kabat编号系统确定并注释)：
mAb1705鼠源重链可变区序列
Figure PCTCN2018108240-appb-000006
mAb1705鼠源轻链可变区序列
Figure PCTCN2018108240-appb-000007
Figure PCTCN2018108240-appb-000008
mAb1706鼠源重链可变区序列
Figure PCTCN2018108240-appb-000009
mAb1706鼠源轻链可变区序列
Figure PCTCN2018108240-appb-000010
mAb1780鼠源重链可变区序列
Figure PCTCN2018108240-appb-000011
mAb1780鼠源轻链可变区序列
Figure PCTCN2018108240-appb-000012
mAb1773鼠源重链可变区序列
Figure PCTCN2018108240-appb-000013
mAb1773鼠源轻链可变区序列
Figure PCTCN2018108240-appb-000014
mAb1779鼠源重链可变区序列
Figure PCTCN2018108240-appb-000015
mAb1779鼠源轻链可变区序列
Figure PCTCN2018108240-appb-000016
其中各抗体轻重链中CDR序列如表1所示。
表1各抗体重链及轻链CDR区序列
Figure PCTCN2018108240-appb-000017
Figure PCTCN2018108240-appb-000018
其Biacore活性检测结果见表2。
表2 IL-5鼠源抗体的体外活性
Figure PCTCN2018108240-appb-000019
结果显示，本公开中的鼠源抗体与抗原亲和力高。
实施例4：IL-5相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
4.1 IL-5-Flag-His重组蛋白的纯化步骤：
将样品高速离心去除杂质，并浓缩至适当体积。利用PBS平衡NI-NTA亲和柱(QIAGEN，Cat No.30721)，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品上柱。用PBS冲洗柱子，至A280读数降至基线。用PBS冲洗柱子，冲洗杂蛋白，并收集。依次用洗涤缓冲液(咪唑20mM)和洗脱缓冲液(咪唑300mM)洗脱目的蛋白，并收集洗脱峰。
收集的洗脱液用离子交换(Hiload 16/600 superdex 200柱)进一步纯化。用PBS平衡2个柱体积左右，保证pH7.4。将鉴定含目的蛋白的洗脱缓冲液浓缩后上样，收集样品，SDS-PAGE和LC-MS鉴定为正确后分装备用。
4.2杂交瘤表达抗体，Fc融合蛋白的纯化
将细胞表达上清样品高速离心去除杂质，杂交瘤表达上清用Protein G柱，Fc融合蛋白表达上清用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH3.0洗脱目的蛋白，用1M Tris-HCl,pH8.0中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，去聚体的峰收集好后分装备用。
实施例5：抗人IL-5单克隆抗体的人源化设计
鼠源抗人IL-5单克隆抗体人源化如本领域许多文献公示的方法进行。简言之， 鼠源抗体的恒定区替换为人恒定区，且将鼠源抗体的CDR移植到FR同源性最高人源模板上，并对FR区维持抗体构象和影响抗体与抗原结合的关键氨基酸进行回复突变来实现。
通过比对IMGT人类抗体重轻链可变区种系基因数据库，分别挑选与mAb-1705，mAb-1706，mAb1780，mAb1773和mAb1779抗体氨基酸序列同一性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号系统确定并注释。
人源FR区的选择和关键氨基酸的回复突变
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，从人源germline数据库中搜索轻链可变区(VL)和重链可变区(VH)的同源序列，按FR的同源性由高到低排序，选取FR同源性最高的germline作为主要模板，将鼠源抗体的CDR区移植到人源模板上，然后以鼠源抗体的三维结构为基础，通过软件对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并对化学不稳定氨基酸残基优化，得到最终的人源化分子。
5.1杂交瘤克隆mAb1705号人源化构架选择
h1705的VH选取IGHV3-23*04作为模板，VL选取IGKV1-12*01作为模板，将mAb1705的CDR移植到人源模板上，并通过软件找出包埋残基、与CDR区有直接相互作用的残基并回复突变，设计出人源化抗体不同的轻链和重链可变区，如表3所示。
表3 h1705的模板选择和回复突变设计
Figure PCTCN2018108240-appb-000020
注：Grafted代表鼠抗体CDR植入人种系FR区序列。如A43S表示依照氨基酸序列自然顺序编号，将grafted的第43位A突变回S。
表4 h1705人源化抗体重轻链可变区相互组合表
  h1705_VH.1 h1705_VH.1A h1705_VH.1B
h1705_VL.1 h1705-003 h1705-004 h1705-005
h1705_VL.1A h1705-006 h1705-007 h1705-008
h1705_VL.1B h1705-009 h1705-010 h1705-011
注：该表表示各种突变组合所得的序列。如h1705-007表示，人源化的鼠抗体h1705-007含轻链h1705_VL.1A、重链h1705_VH.1A两种突变体。其它类推。
h1705号人源化抗体可变区具体序列如下：
>h1705_VL.1(SEQ ID NO：46)
Figure PCTCN2018108240-appb-000021
>h1705_VL.1A(SEQ ID NO：47)
Figure PCTCN2018108240-appb-000022
>h1705_VL.1B(SEQ ID NO：48)
Figure PCTCN2018108240-appb-000023
>h1705_VH.1(SEQ ID NO：49)
Figure PCTCN2018108240-appb-000024
>h1705_VH.1A(SEQ ID NO：50)
Figure PCTCN2018108240-appb-000025
>h1705_VH.1B(SEQ ID NO：51)
Figure PCTCN2018108240-appb-000026
将上述轻链可变区与如SEQ ID NO:53所示的轻链恒定区序列组合形成最终的完整重轻链序列。将各重链可变区与如SEQ ID NO:52所示的重链恒定区组合形成最终的各重链序列。
人源化抗体恒定区序列
>重链IgG1恒定区：
Figure PCTCN2018108240-appb-000027
注：下划线为设计的M252Y、S254T、T256E突变
>轻链kappa恒定区：
Figure PCTCN2018108240-appb-000028
5.2杂交瘤克隆mAb1706号人源化构架选择
h1706的VH选取了IGHV3-23*04作为模板，VL选取了IGKV1-12*01作为模板，将鼠源抗体mab1706的CDR区移植到选择的人源化模板上，并通过软件找出包埋残基、与CDR区有直接相互作用的残基并回复突变，设计出人源化抗体不同的轻链和重链可变区，如表5所示。
表5 h1706的模板选择和回复突变设计
Figure PCTCN2018108240-appb-000029
注：Grafted代表鼠抗体CDR植入人种系FR区。如A43S表示依照氨基酸序列的自然顺序编号，将grafted的第43位A突变回S。
将设计的人源化分子按下表组合成不同的分子，如表6所示。
表6 h1706人源化抗体重轻链可变区相互组合表
  h1706_VH.1 h1706_VH.1A h1706_VH.1B
h1706_VL.1 h1706-002 h1706-003 h1706-004
h1706_VL.1A h1706-005 h1706-006 h1706-007
h1706_VL.1B h1706-008 h1706-009 h1706-010
h1706号人源化抗体可变区具体序列如下：
>h1706_VL.1(SEQ ID NO：54)
Figure PCTCN2018108240-appb-000030
>h1706_VL.1A(SEQ ID NO：55)
Figure PCTCN2018108240-appb-000031
>h1706_VL.1B(SEQ ID NO：56)
Figure PCTCN2018108240-appb-000032
>h1706_VH.1(SEQ ID NO：57)
Figure PCTCN2018108240-appb-000033
Figure PCTCN2018108240-appb-000034
>h1706_VH.1A(SEQ ID NO：58)
Figure PCTCN2018108240-appb-000035
>h1706_VH.1B(SEQ ID NO：59)
Figure PCTCN2018108240-appb-000036
将上述轻链可变区与如SEQ ID NO:53所示的轻链恒定区序列组合形成最终的完整重轻链序列。将各重链可变区与如SEQ ID NO:52所示的重链恒定区组合形成最终的各重链序列。
5.3杂交瘤克隆mAb 1780号人源化构架选择
h1780的VH选取了IGHV1-2*02作为模板，VL选取了IGKV3-11*01作为模板，将鼠源抗体mAb1780的CDR区移植到选择的人源化模板上，并通过软件找出包埋残基、与CDR区有直接相互作用的残基并回复突变，设计出人源化抗体不同的轻链和重链可变区，如表7所示。
表7 h1780的模板选择和回复突变设计
Figure PCTCN2018108240-appb-000037
注：Grafted代表鼠抗体CDR植入人种系FR区。如E1D表示依照氨基酸序列的自然顺序编号，将grafted的第1位E突变回D。
将设计的人源化分子按下表组合成不同的分子，如表8所示。
表8 h1780人源化抗体重轻链可变区相互组合表
  h1780_VH.1 h1780_VH.1A h1780_VH.1B h1780_VH.1C
h1780_VL.1 h1780-007 h1780-008 h1780-009 h1780-010
h1780_VL.1A h1780-011 h1780-012 h1780-013 h1780-014
h1780_VL.1B h1780-015 h1780-016 h1780-017 h1780-018
h1780号人源化抗体可变区具体序列如下：
>h1780_VL.1(SEQ ID NO：60)
Figure PCTCN2018108240-appb-000038
>h1780_VL.1A(SEQ ID NO：61)
Figure PCTCN2018108240-appb-000039
>h1780_VL.1B(SEQ ID NO：62)
Figure PCTCN2018108240-appb-000040
>h1780_VH.1(SEQ ID NO：63)
Figure PCTCN2018108240-appb-000041
>h1780_VH.1A(SEQ ID NO：64)
Figure PCTCN2018108240-appb-000042
>h1780_VH.1B(SEQ ID NO：65)
Figure PCTCN2018108240-appb-000043
>h1780_VH.1C(SEQ ID NO：66)
Figure PCTCN2018108240-appb-000044
将上述轻链可变区与如SEQ ID NO:53所示的轻链恒定区序列组合形成最终的完整重轻链序列。将各重链可变区与如SEQ ID NO:52所示的重链恒定区组合形成最终的各重链序列。
5.4杂交瘤克隆mAb1773号人源化构架选择
h1773的VH选取了IGHV3-73*01作为模板，VL选取了IGKV1-39*01作为模板，将鼠源抗体mab1773的CDR区移植到选择的人源化模板上，并通过软件找出包埋残基、与CDR区有直接相互作用的残基并回复突变，设计出人源化抗体不同的轻链和重链可变区，如表9所示。同时，为消除CDR区的异构化位点，将h1773的HCDR2(RIDPANGDTK HGPKFQG)中N突变为V(即N55V)，形成HCDR2变化(位点 突变后的HCDR2序列为SEQ ID NO:82：RIDPAVGDTKHGPKFQG)的重链可变区和抗体。
表9 h1773的模板选择和回复突变设计
Figure PCTCN2018108240-appb-000045
注：Grafted代表鼠抗体CDR植入人种系FR区。如M4L表示依照氨基酸序列的自然顺序编号，将grafted的第4位M突变回L。
将设计的人源化分子按下表组合成不同的分子，如表10所示。
表10 h1773人源化抗体重轻链可变区相互组合表
  h1773_VH.1 h1773_VH.1A h1773_VH.1B
h1773_VL.1 h1773-002 h1773-003 h1773-004
h1773_VL.1A h1773-005 h1773-006 h1773-007
h1773号人源化抗体可变区具体序列如下：
>h1773_VL.1(SEQ ID NO：67)
Figure PCTCN2018108240-appb-000046
>h1773_VL.1A(SEQ ID NO：68)
Figure PCTCN2018108240-appb-000047
>h1773_VH.1(SEQ ID NO：69)
Figure PCTCN2018108240-appb-000048
>h1773_VH.1A(SEQ ID NO：70)
Figure PCTCN2018108240-appb-000049
>h1773_VH.1B(SEQ ID NO：71)
Figure PCTCN2018108240-appb-000050
Figure PCTCN2018108240-appb-000051
将上述轻链可变区与如SEQ ID NO:53所示的轻链恒定区序列组合形成最终的完整重轻链序列。将各重链可变区与如SEQ ID NO:52所示的重链恒定区组合形成最终的各重链序列。
5.5杂交瘤克隆mAb1779号人源化构架选择
h1779的VH选取了IGHV1-2*02作为模板，VL选取了IGKV1-33*01作为模板，将鼠源抗体h1779的CDR区移植到选择的人源化模板上，并通过软件找出包埋残基、与CDR区有直接相互作用的残基并回复突变，设计出人源化抗体不同的轻链和重链可变区，如表11所示。
表11 h1779的模板选择和回复突变设计
Figure PCTCN2018108240-appb-000052
注：Grafted代表鼠抗体CDR植入人种系FR区。如A43S表示依照氨基酸序列的自然顺序编号，将grafted的第43位A突变回S。
将设计的人源化分子按下表组合成不同的分子，如表12所示。
表12 h1779人源化抗体重轻链可变区相互组合表
  h1779_VH.1 h1779_VH.1A h1779_VH.1B h1779_VH.1C h1779_VH.1D
h1779_VL.1 h1779-005 h1779-006 h1779-007 h1779-008 h1779-009
h1779_VL.1A h1779-010 h1779-011 h1779-012 h1779-013 h1779-014
h1779_VL.1B h1779-015 h1779-016 h1779-017 h1779-018 h1779-019
h1779号人源化抗体可变区具体序列如下：
>h1779_VL.1(SEQ ID NO：72)
Figure PCTCN2018108240-appb-000053
Figure PCTCN2018108240-appb-000054
>h1779_VL.1A(SEQ ID NO：73)
Figure PCTCN2018108240-appb-000055
>h1779_VL.1B(SEQ ID NO：74)
Figure PCTCN2018108240-appb-000056
>h1779_VH.1(SEQ ID NO：75)
Figure PCTCN2018108240-appb-000057
>h1779_VH.1A(SEQ ID NO：76)
Figure PCTCN2018108240-appb-000058
>h1779_VH.1B(SEQ ID NO：77)
Figure PCTCN2018108240-appb-000059
>h1779_VH.1C(SEQ ID NO：78)
Figure PCTCN2018108240-appb-000060
>h1779_VH.1D(SEQ ID NO：79)
Figure PCTCN2018108240-appb-000061
将上述轻链可变区与如SEQ ID NO:53所示的轻链恒定区序列组合形成最终的完整重轻链序列。将各重链可变区与如SEQ ID NO:52所示的重链恒定区组合形成最终的各重链序列。
同时，本公开将WO2012083370A1中针对IL5的抗体Hu39D10作为阳性对照，其重链和轻链序列如SEQ ID NO:80和SEQ ID NO:81所示。
Hu39D10的重链序列
Figure PCTCN2018108240-appb-000062
Figure PCTCN2018108240-appb-000063
Hu39D10的轻链序列
Figure PCTCN2018108240-appb-000064
实施例6.重组嵌合抗体以及人源化抗体的制备
1.重组嵌合抗体的分子克隆
杂交瘤筛选所获得的阳性抗体分子经过测序后，得到可变区编码基因序列。以测序所得序列设计首尾引物，以测序基因为模板，经过PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG1/hkappa恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建重组嵌合抗体全长表达质粒VH-CH1-Fc-pHr/VL-CL-pHr，形成Ch1705、Ch1706、Ch1780、Ch1773和Ch1779五个嵌合抗体。
2.人源化抗体的分子克隆
人源化设计之后的抗体序列，经过密码子优化后产生人密码子偏好的编码基因序列，设计引物PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG1/hkappa恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建人源化抗体全长表达质粒VH-CH1-Fc-pHr/VL-CL-pHr。
3.重组嵌合抗体以及人源化抗体的表达与纯化
分别表达抗体轻重链的质粒以1：1.2的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl,pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
以下用测试方法验证本公开抗体性能及有益效果。
体外活性生物学评价
测试例1：Biacore测定鼠源IL-5抗体与不同种属IL-5的结合
用Biacore T200(GE)仪器测定待测鼠源IL-5抗体和人IL-5的亲和力。
用Protein A生物传感芯片亲和捕获待测分子，然后于芯片表面流经抗原(实施例1制备所得的重组人，猴，鼠IL5)，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用甘氨酸-盐酸再生溶液(pH1.5)将生物传感芯片洗净再生。用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，如表13所示。
表13 IL-5鼠源抗体与不同种属IL-5亲和力的BIAcore检测结果
Figure PCTCN2018108240-appb-000065
本实施例证明，本公开的抗体mAb1705、mAb1706、mAb1780、mAb1773和mAb1779与不同种属(人、猴)的IL-5均有较高的亲和力。
测试例2：Biacore测定IL-5人源化抗体与不同种属IL-5的亲和力
用Biacore T200(GE)仪器测定待测人源化IL-5抗体和人IL-5的亲和力。
用Protein A生物传感芯片亲和捕获待测分子，然后于芯片表面流经抗原(实施例1制备所得)，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5)将生物传感芯片洗净再生。用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，如表14所示。
表14 IL-5人源化抗体与人IL-5亲和力的BIAcore检测结果
抗体 KD(M) 抗体 KD(M)
h1705-003 3.35E-09 h1706-003 1.89E-11
h1705-006 4.11E-09 h1706-006 1.73E-11
h1705-009 4.55E-09 h1706-009 5.45E-11
h1705-004 2.14E-11 h1780-017 7.78E-11
h1705-007 2.21E-11 h1773-007 2.07E-10
h1705-010 2.05E-11 h1779-014 4.12E-10
h1705-005 2.16E-11    
h1705-008 3.42E-11    
h1705-011 2.30E-11    
结果显示，人源化IL-5抗体与人IL-5仍均有较高的亲和力(除h1705的各人源化突变体外，其他鼠源抗体的人源化改造突变体仅提供示例性数据)。
测试例3：基于ELISA的鼠源IL-5抗体阻断IL-5结合IL-5α受体实验
为鉴定IL-5抗体阻断IL-5结合到重组表达的IL-5α受体蛋白胞外区的能力。将IL-5(5μg/ml in PBS)包被ELISA板，37℃孵育1小时，弃去液体后，加入用PBS稀释的5％脱脂牛奶封闭液200μl/孔，37℃孵育箱孵育2.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％Tween-20)洗板5次后，先加入25μl用生物素标记试剂盒(东仁化学，LK03)标记的10μg/ml的IL-5Rα(in 1％BSA)，再加入25μl梯度稀释的抗体，抗体和IL-5Rα竞争结合IL-5，37℃孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次后，加入50μl/孔用样品稀释液以1:600浓度稀释的Streptavidin-Peroxidase Polymer(Sigma，S2438-250UG)于37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-10min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算IL-5抗体阻断IL-5与IL-5Rα结合的IC50值，结果如表15所示，本公开两个抗体均可有效抑制IL-5与其受体的结合。
表15鼠源IL-5抗体阻断IL-5结合IL-5α受体的ELISA检测结果
  mAb1705 mAb1706
IC50(μg/ml) 0.42 0.40
测试例4：基于FACS的IL-5抗体阻断IL-5结合IL-5受体实验
为了鉴定筛选到的IL-5抗体可以阻断细胞表面的IL-5受体，我们构建了高表达IL-5Rα/β的两种受体的CHOS重组细胞系。本实验鉴定IL-5抗体可以分别阻断IL-5结合到CHOS细胞系表面的重组IL-5α/β受体。
具体方法：用含100ng/ml G418,25ng/ml zeozin的CD-CHO来培养CHO-S-IL-5Rα和β，细胞培养过程中，浓度不要超过3×10 6cell/ml。将状态良好的IL-5Rα/β-CHOS细胞离心(1000rpm，5min)，用10％FBS的PBS洗一遍，并且计数，将细胞浓度调整浓度至4×10 6cell/ml,取25μl加入到圆底的96孔板中。用含10％FBS的PBS溶液稀释待测抗体，初始浓度为200μg/ml，按1:10倍比稀释8个梯度。将25μl 100ng/ml的用生物素标记试剂盒(东仁化学，LK03)标记的IL-5和50μl稀释的各浓度抗体混合均匀，加到已加入细胞的96孔板中，在4℃孵育1小时。孵育结束后，4℃离心(400g，5min)，弃掉上清，用200μl的预冷PBS离心洗涤，重复两次后加入1:1333稀释的PE-Avidin二抗，4℃避光孵育40min后，4℃离心(400g，5min，弃掉上清，加入200μl的预冷PBS，吹起细胞，4℃离心洗涤，重复三次，加入100μl PBS，上机读板，根据荧光信号值计算IL-5抗体阻断IL-5与IL-5Rα/β结合的IC50值。结果如表16和图1所示。
表16 IL-5抗体阻断IL-5与IL-5Rα/β结合的检测结果
Figure PCTCN2018108240-appb-000066
结果显示，抗体h1705-008，h1706-009、h1780-017、h1773-007、h1779-014显示出较强的阻断IL-5结合到细胞表面IL-5受体的能力。
测试例5：IL-5抗体抑制IL-5诱导的TF1细胞增殖实验
IL-5可以诱导TF-1细胞增殖，IL-5抗体可以阻止IL-5对TF-1细胞的刺激增殖作用。
具体为：TF-1细胞(ATCC,CRL-2003)培养于10％FBS以及2ng/mL rhGM-CSF(联科生物,Catalog No.96-AF-300-03-20)的RPMI1640，放置与37℃，5％CO 2培养箱中，细胞密度不超过1×10 6个/ml。检测抗体时，取对数生长期的细胞用PBS洗三遍800rpm离心5min，用RPMI1640(FBS:2％，重组人IL-5:10ng/ml)调整细胞密度6000个/孔/90μl，并加入10μl梯度稀释的待测抗体到96孔培养3d后，加入30μl cell titer混匀后进行检测，根据读值计算IC50。检测结果如下表17所示。
表17 IL-5人源化抗体抑制IL-5诱导的TF1细胞增殖试验结果
抗体 IC50(nM) 抗体 IC50(nM)
Hu39D10 0.30 h1706-003 0.31
h1705-004 0.30 h1706-004 0.30
h1705-005 0.30 h1706-006 0.34
h1705-007 0.25 h1706-007 0.28
h1705-008 0.20 h1706-009 0.25
h1705-010 0.30 h1773-007 0.38
h1705-011 0.28 h1780-017 0.16
    h1779-014 0.20
测试例6：IL5抗体抑制IL5诱导的嗜酸性粒细胞粘附实验
IL5可以诱导嗜酸性粒细胞的分化，成熟，迁移和活化，引起呼吸道炎症而导致哮喘。本实验利用IL-5细胞因子能够促进嗜酸性粒细胞粘附并使之激活的原理，收集和纯化来源于人外周全血的嗜酸性粒细胞来测试IL-5特异性抗体对IL-5该通路的阻断作用，在体外检测IL-5抗体对IL5介导的嗜酸性粒细胞数粘附效果的阻断。
具体为：人外周全血用含2mM EDTA的PBS稀释5倍，采用Percoll TM(密度梯度为1.088)分离单核细胞和粒细胞，小心吸取含有粒细胞的红细胞层，利用红细胞裂解液去除红细胞；将剩余的细胞计数，按比例加人带有CD16抗体的分离磁珠(美天旎，Catalog No.130-045-701)，孵育30min后过磁珠柱，负选收集直接流出细胞亚群即主要为嗜酸性粒细胞。分离得到的嗜酸性粒细胞计数，加入预先以IgG抗体包被的96孔细胞培养板内，每孔约1×10 4个，加人IL-5(20ng/ml)，以 及不同浓度的IL-5抗体分子(10μg/ml起始，3倍稀释，10个浓度点)；细胞培养板置于37℃，5％CO2培养箱孵育1h，取出培养板，加入0.3％CTAB裂解细胞，最后加入过氧化物酶反应底物TMB显色反应，酶标仪读取OD450吸收值。最大吸附值为只加IL-5孔读值，不加IL-5及抗体药物孔为背景对照，计算各浓度下抗体药物相对于最大吸附孔的抑制值＝(最大吸附值-[抗体药物])/(最大吸附值-背景对照值)×100％，计算IC50。结果如下表18：
表18 IL-5抗体阻断IL-5诱导的嗜酸性粒细胞粘附
Figure PCTCN2018108240-appb-000067
结果表明，本公开的人源化抗体显示较强的抑制IL5介导的嗜酸性粒细胞粘附的能力。
测试例7：人源化IL-5抗体Th2细胞因子的特异性评价
IL-5是Th2细胞因子中的一种，为了验证IL-5抗体只特异性针对IL-5而与其他细胞因子没有交叉反应，用Fortebio检测了12种Th2以及相关细胞因子，包括IL2(R&D,202-IL-010/CF)，IL4(R&D,204-IL-050/CF)，IL-5(R&D,205-IL-025/CF)，IFNgamma，IL6(R&D,7270-IL-025/CF)，IL9(R&D,209-IL-010/CF)，IL10(R&D,217-IL-025/CF)，IL13(R&D,213-ILB-025/CF)，IL25(R&D,8134-IL-025/CF)，IL31(R&D,2824-IL-010/CF)，以及与IL-5共用α受体的IL3(203-IL-050/CF)和GMCSF(R&D,215-GM-010/CF)。
具体为：用Protein A Biosensor(PALL Fortebio,18-5010)来捕获抗体，记录捕获信号，然后再进样40nM各细胞因子，记录新的结合信号。最后将与IL-5的结合信号定义为100％，看其他细胞因子与抗体的的结合信号，结果如图2所示。
结果显示，12种相关细胞因子中，IL-5人源化抗体h1705-008和h1706-009只特异性结合IL-5，与其他Th2细胞因子无交叉反应。
药代动力学评价
测试例8：人源化IL-5抗体大鼠药代动力学评价
实验用SD大鼠(由西普尔-必凯实验动物有限公司提供)18只，雄性，每组3只，平均分成6组；静脉注射及皮下注射给药Hu39D10,h1705-008及h1706-009；另外9只SD大鼠仅静脉注射给药h1773-007,h1779-014,h1780-017。静脉给药组于给药前及给药后5min,8h,1d,2d,4d,7d,10d,14d,21d,28d采集全血0.2ml，不加抗凝，取血后在4℃放置30min，1000g离心15min，取上清(血清)置于EP管中，于-80℃保存；皮下注射给药组于给药前及给药后1h,2h,4h,8h,1d,2d,4d,7d,10d,14d,21d,28d采集全血，采用Elisa测定血清中的抗体浓度。
结果表明，本公开人源化抗体分子在大鼠体内半衰期长，生物利用度高。
表19抗体大鼠药代动力学评价表
Figure PCTCN2018108240-appb-000068
体内活性生物学评价
测试例9：IL-5抗体在OVA诱导的小鼠哮喘模型中的药效评价
本测试是基于气道炎性反应以及气道重塑来评估IL-5抗体在卵清蛋白(OVA)气雾诱导的BALB/c小鼠哮喘模型中的药效。
小鼠根据体重随机分成7组，每组10只小鼠：正常对照组(G1)；模型组(G2)；2个测试抗体h1705-008(G3和G4)和h1706-009(G5和G6)，每个测试抗体2个剂量(10mpk和2mpk)的治疗组；以及阳性抗体Hu39D10对照组(G7，10mpk)。在第1天和第14天，对所有小鼠腹腔注射致敏溶液进行致敏。在第28、29、30天，用OVA攻击溶液对第一组外的其他六组小鼠进行气雾攻击30分钟。攻击前2小时，第二组(G2)腹腔注射磷酸缓冲液，第三组至第七组(G3-G7)小鼠腹腔注射不同剂量的不同抗体，一天一次，连续三天。每次注射前配制新鲜的测试抗体，配制完成至给药半小时内完成。第一组的小鼠作为正常对照组用PBS进行气雾攻击30分钟，攻击前2小时腹腔注射磷酸缓冲液，一天一次，连续三天。
在第31天，使用WBP系统对动物进行气道高反应性的检测。所有动物均用雾化吸入1.5625、3.125、6.25、12.5、25、50mg/mL倍增浓度的乙酰甲胆碱，测定相应浓度下的增强呼气间歇值。
在第31天，WBP系统检测气道反应1小时后，直径1.2毫米的气管插管插入气管内并且固定，肺将被灌洗两次，每次0.8毫升的磷酸缓冲液含1％的BSA和0.6mM的EDTA。记录灌洗液的回收体积。
将BALF在4摄氏度300g下离心5分钟，上清液保留用于细胞因子分析。离心后，细胞重悬在1.5毫升的PBS里(含1％的BSA和0.6mM的EDTA)进行细胞计数。通过血球计数器和台盼蓝染色实验对BALF中的总细胞数进行计数。细胞涂片后，莱特染色液染色一分钟，接着用吉姆沙染色7分钟，从而区分出嗜酸细胞、中性白细胞、巨噬细胞和淋巴细胞。在光学显微镜下计数。
灌洗后，收集肺组织并用10％的中性甲醛溶液浸染，然后固定在10％的中性甲醛溶液中。固定的组织进行石蜡包埋，切边，H&E染色，评分。测试结果如图 3和图4A、图4B所示。
结果显示，本公开中抗体分子h1705-008和h1706-009可明显提高肺功能，且作用方式呈剂量依赖模式，而高剂量(10mpk)的阳性化合物却不能改善肺功能(参见图3)。同时，两个抗体显著降低了嗜酸性粒细胞水平和粘膜厚度，在同样剂量下(10mpk)显示出比阳性抗体更强的降低嗜酸性粒细胞的能力(参见图4A和4B)。而在同样类型小鼠哮喘模型中，重复实验也同样验证了1mg/ml的h1705-008，h1706-009和h1780-017均比阳性抗体更显著地降低BalF中的嗜酸性粒细胞水平(参见图4C)。
测试例10：外源性人IL5诱导的豚鼠急性哮喘模型评价IL5抗体的体内药效
本实验选用雄性豚鼠建立人IL5诱导的急性哮喘模型，以hu39D10为阳性抗体，评估本公开5个IL-5人源化单抗对人IL5诱导的豚鼠肺支气管灌流液(BALF)中的嗜酸性粒细胞增加是否有抑制作用。豚鼠共分为9组，每组8-10只：正常对照组、模型组、hu39D10(1mg/kg)组、h1705-008(1mg/kg)组、h1706-009(1mg/kg)组、h1780-017(1mg/kg)组、h1773-007(1mg/kg)组和h1779-014(1mg/kg)组。其中模型组和给药组豚鼠分别于第1天气管注射100μl human IL5(含IL5抗原5μg)激发，正常对照组气管注射PBS。给药组激发前2小时腹腔注射分别给予1mg/kg上述IL5单抗，给药体积为5ml/kg，模型组给予相应的IgG抗体，正常对照组腹腔给予PBS溶剂。气管注射后24h后麻醉豚鼠抽取肺支气管灌流液，待细胞浓度调整至5^10 6/ml时取15μl滴在载玻片上，晾干固定后进行HE染色，400倍镜下计数细胞总数和嗜酸性粒细胞数并计算嗜酸性粒细胞百分比。结果如图5A和图5B所示，表明本公开5个人源化抗体均可以显著降低BALF中嗜酸性粒细胞水平。
稳定性评价
测试例11：人源化抗IL-5抗体的稳定性
1.抗体的化学稳定性
脱酰胺修饰是抗体中可能影响后期稳定性的一种常见的化学修饰，尤其是CDR区域的部分氨基酸高度脱酰胺、氧化或者异构化修饰一般选择尽量避免或者突变降低。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl，8M Gua-HCl，pH 6.0溶液中，加3μl 0.1g/mL DTT，50℃水浴1小时，后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μl 0.25mg/mL的trypsin，37℃水浴酶解过夜，Agilent 6530 Q-TOF进行LC-MS检测分析。结果显示，本公开人源化抗体的化学稳定性良好，在40℃529缓冲液体系加速一个月后，抗体未发生异常修饰。
2.高浓度条件下抗体聚集程度考察
对待测抗体在高浓度及不同缓冲体系及不同温度条件下进行一个月稳定性评价，浓度在50mg/ml，在PB9、His和529三种体系中考察40℃、25℃、4℃及-80℃反复冻融的稳定性，以SEC-HPLC进行聚集程度监控。使用Waters e2695色谱仪， 色谱柱为Waters Xbridge BEH 200A SEC，流动相为PBS(用稀盐酸调节pH至6.8)，进样50μg蛋白，等度洗脱，流速为0.5mL/min。经考察，IL-5人源化抗体在高浓度条件下所有抗体均未发生明显聚集。40℃加速4周后，，在三种体系中抗体单体纯度均大于95％。
3.高浓度条件下抗体纯度考察
对待测抗体在高浓度及不同缓冲体系及不同温度条件下进行一个月稳定性评价，浓度在50mg/ml，在His体系中考察40℃的稳定性，以CE-SDS进行纯度监控。取100μg蛋白，加入sample buffer至95μl，若采用还原模式分析，加入5μl二巯基乙醇；若采用非还原模式分析，加入5μl IAA，70℃水浴反应10min，离心后取上清进样。使用Beckman PA800plus电泳仪采集数据并分析结果。经考察，抗体蛋白在高浓度条件下纯度稳定性良好,在40℃加速28天后，CE-SDS分析显示抗体主峰仅降低2％。
4.利用UNIT检测不同抗体的热稳定性
将样品置换到对应缓冲液(PBS buffer)中，控制样品浓度在1mg/ml左右，上样9μl。参数设置：起始温度20℃；孵育0s；升温速度0.3℃/min；Plate Hold 5s；终止温度95℃。检测抗体的Tm值，抗体有较高的Tm值，显示出良好的热稳定性。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本公开的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (21)

  1. 一种单克隆抗体或其抗原结合片段，所述单克隆抗体或其抗原结合片段特异性结合人IL-5，所述单克隆抗体包含重链可变区和轻链可变区，其中：
    (i)重链可变区包含选自如SEQ ID NO：16-18氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：16-18所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：19-21氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：19-21所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
    (ii)重链可变区包含选自如SEQ ID NO：22-24氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：22-24所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：25-27氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：25-27所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
    (iii)重链可变区包含选自如SEQ ID NO：28-30氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：28-30所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：31-33氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：31-33所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
    (iv)重链可变区包含选自如SEQ ID NO：34-36氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：34-36所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：37-39氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：37-39所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
    (v)重链可变区包含选自如SEQ ID NO：40-42氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：40-42所示的HCDR1、HCDR2和HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：43-45氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：43-45所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体；或
    (vi)重链可变区包含选自如SEQ ID NO：34-36氨基酸序列所示的HCDR1、HCDR2和HCDR3区或与SEQ ID NO：34、82、36所示的HCDR1、HCDR2和 HCDR3区分别具有3，2或1个氨基酸差异的HCDR变体，轻链可变区包含选自如SEQ ID NO：37-39氨基酸序列所示的LCDR1、LCDR2和LCDR3区或与SEQ ID NO：37-39所示的LCDR1、LCDR2和LCDR3区分别具有3，2或1个氨基酸差异的LCDR变体。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体是重组抗体，优选选自鼠源抗体、嵌合抗体、人源化抗体。
  3. 根据权利要求2所述的单克隆抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:49、57、63、69或75所示的重链可变区或其变体；所述变体是在SEQ ID NO:49、57、63、69或75所示的重链可变区序列上具有1-10个氨基酸突变。
  4. 根据权利要求3所述的单克隆抗体或其抗原结合片段，其中所述变体是在SEQ ID NO:49、57、63、69或75所示的重链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变是在SEQ ID NO:49所示的重链可变区上选自S49T，V93T和K98S或其组合的氨基酸回复突变，或所述回复突变是在SEQ ID NO:57所示的重链可变区上选自S49T,V93T和K98T或其组合的氨基酸回复突变，或所述回复突变是在SEQ ID NO:63所示的重链可变区上选自R38K，M48I，R67K，V68A，M70L，R72V，T74K和L83F或其组合的氨基酸回复突变，或所述回复突变是在SEQ ID NO:69所示的重链可变区上选自F29I，R38K，V48I，R72A，T97F的氨基酸回复突变和CDR中N55V的突变或其组合，或所述回复突变是在SEQ ID NO:75所示的重链可变区上选自R38K，M48I，R67K，V68A，R72A，T74K，M81L，L83F和D89E或其组合的氨基酸回复突变。
  5. 根据权利要求4所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:50或51所示的重链可变区，或包含选自SEQ ID NO:58或59所示的重链可变区，或包含选自SEQ ID NO:64，65和66中任一个所示的重链可变区，或包含选自SEQ ID NO:70或71所示的重链可变区，或包含选自SEQ ID NO:76至79中任一个所示的重链可变区。
  6. 根据权利要求2所述的单克隆抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:46、54、60、67或72所示的轻链可变区或其变体；所述变体是在SEQ ID NO:46、54、60、67或72所示的轻链可变区上具有1-10个氨基酸变化的序列。
  7. 根据权利要求6所述的单克隆抗体或其抗原结合片段，其中所述变体是在 SEQ ID NO:46、54、60、67或72所示的轻链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自在SEQ ID NO:46所示的轻链可变区上的A43S，L47V，G66R，T69S，F71Y和Y87F或其组合的氨基酸回复突变；或在SEQ ID NO:54所示的轻链可变区上的A43S，L47M，F71Y和Y87F或其组合的氨基酸回复突变；或在SEQ ID NO:60所示的轻链可变区上的E1D，I2T，I57V，V84T和Y86F或其组合的氨基酸回复突变；或在SEQ ID NO:67所示的轻链可变区上的M4L，A42S，L45P和L46W或其组合的氨基酸回复突变；或在SEQ ID NO:72所示的轻链可变区上的A43S，I48V和F71Y或其组合的氨基酸回复突变。
  8. 根据权利要求7所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:47或48所示的轻链可变区；或包含选自SEQ ID NO:55或56所示的轻链可变区；或包含选自SEQ ID NO:61或62中所示的轻链可变区；或包含SEQ ID NO:68所示的轻链可变区；或包含选自SEQ ID NO:73或74所示的轻链可变区。
  9. 根据权利要求1至8中任一项所述的单克隆抗体或其抗原结合片段，其中所述人源化抗体包含选自：
    选自SEQ ID NO:49-51中任一个所示的重链可变区和选自SEQ ID NO:46-48中任一个所示的轻链可变区；或
    选自SEQ ID NO:57-59中任一个所示的重链可变区和选自SEQ ID NO:54-56中任一个所示的轻链可变区；或
    选自SEQ ID NO:63-66中任一个所示的重链可变区和选自SEQ ID NO:60-62中任一个所示的轻链可变区；或
    选自SEQ ID NO:69-71中任一个所示的重链可变区和选自SEQ ID NO:67-68中任一个所示的轻链可变区；或
    选自SEQ ID NO:75-79中任一个所示的重链可变区和选自SEQ ID NO:72-74中任一个所示的轻链可变区。
  10. 根据权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括人抗体恒定区，优选包含SEQ ID NO:52所示的人抗体重链恒定区和SEQ ID NO:53所示的人轻链恒定区。
  11. 权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段，其中所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
  12. 一种分离的单克隆抗体或其抗原结合片段，其特征在于与权利要求1至11 中任一项所述的单克隆抗体或其抗原结合片段竞争结合人IL-5。
  13. 一种药物组合物，其含有治疗有效量的根据权利要求1至12中任一项所述的单克隆抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  14. 一种分离的核酸分子，其编码权利要求1至12中任一项的单克隆抗体或其抗原结合片段。
  15. 一种重组载体，其包含权利要求14所述的分离的核酸分子。
  16. 一种用根据权利要求15所述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
  17. 用于生产权利要求1至12中任一项所述的单克隆抗体或其抗原结合片段的方法，所述方法包括将权利要求16的宿主细胞在培养基中进行培养以形成并积累权利要求1至12任一项的单克隆抗体或其抗原结合片段，以及从培养物回收所述单克隆抗体或其抗原结合片段。
  18. 用于检测或测定人IL-5的方法，所述方法包括使用权利要求1至12中任一项的单克隆抗体或其抗原结合片段。
  19. 用于检测或测定人IL-5的试剂，所述试剂包含权利要求1至12中任一项所述的单克隆抗体或其抗原结合片段。
  20. 一种治疗与人IL-5相关的疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1至12中任一项的单克隆抗体或其抗原结合片段，或包含权利要求13所述的药物组合物，或权利要求14所述的分离的核酸分子，以治疗人IL-5相关的疾病，所述疾病优选哮喘、哮喘恶性发作、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位、类固醇依赖性嗜酸细胞性支气管炎。
  21. 如权利要求1至12中任一项的单克隆抗体或其抗原结合片段，或包含权利要求13所述的药物组合物，或权利要求14所述的分离的核酸分子或其组合在制备治疗或预防疾病或病症的药物中的用途，其中所述疾病或病症是人IL-5相关疾病，所述疾病或病症优选哮喘、哮喘恶性发作、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位、类固醇依赖性嗜酸细胞性支气管炎。
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