WO2020048312A1 - 结合人il-4r的抗体、其制备方法和用途 - Google Patents

结合人il-4r的抗体、其制备方法和用途 Download PDF

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WO2020048312A1
WO2020048312A1 PCT/CN2019/101628 CN2019101628W WO2020048312A1 WO 2020048312 A1 WO2020048312 A1 WO 2020048312A1 CN 2019101628 W CN2019101628 W CN 2019101628W WO 2020048312 A1 WO2020048312 A1 WO 2020048312A1
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antibody
antigen
human
binding fragment
seq
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PCT/CN2019/101628
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English (en)
French (fr)
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赵杰
蒋良丰
陈臣
吴惠玲
黄玉平
黄浩旻
朱祯平
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三生国健药业（上海）股份有限公司
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Priority to CN201980023589.8A priority Critical patent/CN112041347B/zh
Priority to EP19857454.3A priority patent/EP3848391A4/en
Priority to JP2020557997A priority patent/JP7047128B2/ja
Priority to US17/045,285 priority patent/US11667717B2/en
Publication of WO2020048312A1 publication Critical patent/WO2020048312A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies. More specifically, the present invention discloses an antibody that binds to human IL-4R, its preparation method and use.
  • the interleukin-4 receptor is a heterodimer composed of an ⁇ chain and a ⁇ c chain; wherein the ⁇ chain (IL-4R ⁇ ) can specifically bind to interleukin-4 (IL-R) 4), and then form a ternary complex with the ⁇ c chain to transduce signals that IL-4 promotes proliferation and gene transcription activation.
  • Interleukin-13 receptor is a heterodimer composed of IL-13R ⁇ 1 chain and IL-4R ⁇ chain; IL-13R ⁇ 1 can specifically bind to interleukin-13, and then Forms a ternary complex with the IL-4R ⁇ chain and transduces signals that IL-13 promotes proliferation and gene transcription activation.
  • IL-4 and IL-13 play important roles in the pathogenesis of allergic diseases such as allergic dermatitis and asthma.
  • Dupilumab injection is a monoclonal antibody drug targeting IL-4R ⁇ chain that is currently on the market. It was jointly developed by Regeneron Pharmaceuticals (Regeneron) and Sanofi-aventis (Sanofi).
  • Dupilumab is a fully human IgG4 type monoclonal antibody with a molecular weight of about 147kDa. It can specifically bind to the IL-4R ⁇ subunit shared by the IL-4 and IL-13 receptor complexes, thereby inhibiting IL-4 and IL- 13 cytokine-mediated inflammatory response.
  • Dupilumab was approved by the US FDA, European EMA, and Japanese PMDA on March 28, 2017, September 27, 2017, and January 19, 2018. Approved indications are the treatment of adult patients with moderate to severe atopic dermatitis with uncontrolled or inappropriately treated topical therapies, which can be used with or without topical corticosteroids.
  • dupilumab has been approved by the FDA for the treatment of atopic dermatitis, and may be approved for the treatment of moderate to severe asthma in the future.
  • IL-4R ⁇ there is still an urgent need to develop new, specific and efficient drugs targeting IL-4R ⁇ to fill the gap in the domestic similar drug market, so as to improve the quality of life of people with allergic autoimmune diseases in China and benefit domestic patients.
  • the inventors of the present invention carried out a large number of experiments, from antigen immunization, hybridoma screening, antibody expression purification to biological activity identification, and obtained a mouse-derived number 4-2 that specifically binds human IL-4R.
  • Antibodies and on this basis, further construction of the chimeric antibody 4-2-Chimeric-IgG1 and humanized antibodies 4-2-Humanized-IgG1 and 4-2-Humanized-IgG4.
  • the experimental results show that the chimeric antibody 4-2-Chimeric-IgG1 and the humanized antibodies 4-2-Humanized-IgG1 and 4-2-Humanized-IgG4 can effectively block the interaction between IL-4 and IL-4R And, it has the activity of inhibiting IL-4 induced TF-1 cell proliferation and human peripheral blood mononuclear cells (PBMC) expression of CD23 and secretion of IgE. Therefore, the antibody developed by the present invention that binds human IL-4R can be used to prepare a therapeutic drug for patients with IL-4R overexpression diseases.
  • PBMC peripheral blood mononuclear cells
  • the antibody of the present invention specifically binds to the following amino acid residues on the extracellular domain of IL-4R ⁇ : L39, F41, L42, L43, D72, and Y74.
  • a first object of the present invention is to provide an antibody or an antigen-binding fragment thereof that binds human IL-4R.
  • a second object of the present invention is to provide an isolated nucleotide encoding the human IL-4R-binding antibody or an antigen-binding fragment thereof.
  • a third object of the present invention is to provide an expression vector containing the nucleotide.
  • a fourth object of the present invention is to provide a host cell containing the expression vector.
  • a fifth object of the present invention is to provide a method for preparing the human IL-4R-binding antibody or an antigen-binding fragment thereof.
  • a sixth object of the present invention is to provide a pharmaceutical composition containing the human IL-4R-binding antibody or an antigen-binding fragment thereof.
  • a seventh object of the present invention is to provide the antibody or an antigen-binding fragment thereof that binds human IL-4R or the use of the pharmaceutical composition.
  • An eighth object of the present invention is to provide a method for improving the activity of the human IL-4R-binding antibody or antigen-binding fragment thereof.
  • a first aspect of the present invention provides an antibody or antigen-binding fragment thereof that binds human IL-4R, including:
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 7, and the HCDR3 is shown in FIG.
  • the amino acid sequence is shown in SEQ ID NO: 8, and
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a murine antibody, a chimeric antibody or a humanized antibody.
  • the antibody is an IgG1 antibody or an IgG4 antibody.
  • the antigen-binding fragment includes a Fab fragment, an F (ab ') 2 fragment, an Fv fragment, a single-chain antibody (scFv), a single-domain antibody (sdAb), and the like.
  • the human IL-4R-binding antibody or antigen-binding fragment thereof can block the interaction between IL-4 and IL-4R.
  • the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds human IL-4R is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region As shown in SEQ ID NO: 5; according to another preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment thereof that binds human IL-4R is shown in SEQ ID NO: 13 The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 15.
  • the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof that binds human IL-4R is shown in SEQ ID NO: 16, and the amino acid sequence of the light chain is shown in SEQ ID NO: 18
  • the amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof that binds human IL-4R is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is shown in SEQ.
  • amino acid sequence of the heavy chain of the antibody or antigen-binding fragment thereof that binds human IL-4R is shown in SEQ ID NO: 19, light chain
  • amino acid sequence is shown in SEQ ID NO: 18.
  • the antibody or antigen-binding fragment thereof that binds human IL-4R includes an Fc segment, and the Fc segment includes the following mutation sites: S267E and L328F.
  • a second aspect of the present invention provides an isolated nucleotide, which encodes the antibody or antigen-binding fragment thereof that binds human IL-4R as described in any one of the above.
  • the nucleotide has a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain encoding as shown in SEQ ID NO: 4 may Nucleotide sequence of a variable region; in another preferred embodiment of the present invention, the nucleotide has a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO: 12, and as SEQ ID NO: 14 shows a nucleotide sequence encoding a light chain variable region.
  • an expression vector containing the nucleotide according to any one of the above is provided.
  • a fourth aspect of the present invention provides a host cell containing the expression vector as described above.
  • a fifth aspect of the present invention provides a method for preparing a human IL-4R-binding antibody or an antigen-binding fragment thereof as described above, which method includes the following steps:
  • a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof that binds human IL-4R according to any one of the preceding claims, and a pharmaceutically acceptable carrier.
  • the human IL-4R-binding antibody or antigen-binding fragment thereof or the pharmaceutical composition as described above is provided for preparing a medicament for treating a disease related to IL-4R overexpression according to any one of the foregoing. Use.
  • the present invention also provides a method for treating a disease related to IL-4R overexpression, which method uses the antibody or an antigen-binding fragment thereof that binds to human IL-4R according to the present invention, or an antibody containing the antibody that binds to human IL-4R, or A pharmaceutical composition of the antigen-binding fragment is administered to a patient.
  • the present invention also provides an antibody or an antigen-binding fragment thereof for treating a disease associated with IL-4R overexpression or a pharmaceutical composition containing the antibody or an antigen-binding fragment thereof that binds human IL-4R.
  • the diseases related to IL-4R overexpression include atopic dermatitis, asthma, allergic reactions, eosinophilic esophagitis, skin infections, nasal polyposis and the like.
  • An eighth object of the present invention is to provide a method for improving the activity of a human IL-4R-binding antibody or an antigen-binding fragment thereof according to any one of the foregoing, the human IL-4R-binding antibody or an antigen-binding fragment thereof
  • the fragment includes an Fc segment, wherein the Fc segment includes the following mutation sites: S267E and L328F.
  • the invention prepares a series of antibodies that bind human IL-4R. They have the same variable region and different constant regions. The variable regions can specifically bind to human IL-4R. The constant region affects the entire antibody through a unique mechanism. Of activity.
  • the above-mentioned series of antibodies can be used to prepare drugs for treating diseases such as atopic dermatitis, asthma, etc., which have good clinical application prospects.
  • FIG. 1 shows the results of using TF-1 cells to detect the inhibitory effects of IL-4R-ECD-hFc and IL-4R-ECD-FLAG on IL-4-induced proliferation of TF-1 cells.
  • FIG. 2 is a result of detecting the relative affinity of the purified mouse-derived anti-human IL-4R monoclonal antibody to the IL-4R antigen by ELISA.
  • FIG. 3 shows the results of using TF-1 cells to detect the inhibitory effect of purified mouse-derived anti-human IL-4R monoclonal antibody on IL-4-induced proliferation of TF-1 cells.
  • FIG. 4 is a result of detecting the relative affinity of 4-2-Chimeric-IgG1 and 4-2-Humanized-IgG1 to the IL-4R antigen by ELISA.
  • FIG. 5 shows the results of using TF-1 cells to detect the inhibitory effect of 4-2-Chimeric-IgG1 and 4-2-Humanized-IgG1 on IL-4-induced proliferation of TF-1 cells.
  • FIG. 6 shows the results of using TF-1 cells to detect the inhibitory effects of 4-2-Chimeric-IgG1 and 4-2-Humanized-IgG1 on IL-13-induced proliferation of TF-1 cells.
  • Figure 7-1 to Figure 7-3 show the results of using TF-1 cells to detect the inhibitory effect of 4-2-Humanized-IgG1 and 4-2-Humanized-IgG4 on IL-4-induced proliferation of TF-1 cells.
  • FIG. 8 shows the results of using ELISA to detect the blocking activity of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on the interaction between IL-4 and IL-4R.
  • Figure 9 shows the results of using TF-1 cells to detect the inhibitory effect of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on IL-4-induced proliferation of TF-1 cells. .
  • Figure 10-1- Figure 10-3 shows the inhibitory effect of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on IL-4 induced CD23 expression in PBMC using PBMC. result.
  • Figure 11-1- Figure 11-2 shows the inhibitory effect of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on IL-4 induced IgE secretion by PBMC using PBMC. result.
  • Figure 12 shows the results of the pharmacokinetic study of 4-2-Humanized-IgG4.
  • the terms "antibody (Ab)” and “immunoglobulin G (IgG)” are isotetrasaccharide proteins of about 150,000 daltons having the same structural characteristics, which are composed of two identical light chains (L) And two identical heavy chains (H). Each light chain is connected to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds differs between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain .
  • the antibodies of the present invention include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) formed from at least two antibodies, antigen-binding fragments of antibodies, and the like.
  • the antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, and the like.
  • the term "monoclonal antibody (MAb)” refers to antibodies obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized through hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal” indicates the characteristics of an antibody and is obtained from a substantially homogeneous antibody population, which should not be interpreted as requiring any special method to produce the antibody.
  • mouse-derived antibody refers to an antibody derived from a rat or a mouse, preferably a mouse.
  • the mouse-derived antibody of the present invention is obtained by immunizing mice using the extracellular domain of human IL-4R as an antigen and screening hybridoma cells.
  • the mouse-derived antibody of the present invention is an antibody No. 4-2.
  • the term "chimeric antibody” refers to an antibody comprising heavy and light chain variable region sequences derived from one species and constant region sequences derived from another species, such as a mouse Antibodies to the light chain variable region.
  • the chimeric antibody of the present invention is obtained by splicing a heavy chain variable region sequence and a light chain variable region sequence of a mouse-derived antibody No. 4-2 to a human constant region.
  • the heavy chain of the chimeric antibody of the present invention is obtained by splicing the heavy chain variable region sequence of the mouse-derived antibody No. 4-2 with the human IgG1 or IgG4 (S228P) constant region.
  • the light chain variable region sequence of antibody -2 was obtained by splicing the human kappa chain.
  • the chimeric antibody of the present invention is 4-2-Chimeric-IgG1.
  • the term "humanized antibody” refers to a CDR derived from an antibody of a non-human species (preferably a mouse), and a residual portion (including a framework region and a constant region) of the antibody molecule is derived from a human antibody.
  • framework region residues can be altered to maintain binding affinity.
  • the humanized antibody of the present invention is recombined with the CDR region of the mouse-derived antibody No. 4-2 and the non-CDR region derived from the human antibody, and embeds residues and residues that directly interact with the CDR regions. , And the residues that have an important effect on the conformation of VL and VH of antibody 4-2 were obtained by back mutation.
  • the humanized antibodies of the present invention include 4-2-Humanized-IgG1 and 4-2-Humanized-IgG4.
  • binding refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed.
  • antibodies bind at an equilibrium dissociation constant (KD) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • KD equilibrium dissociation constant
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • antibodies bind antigen with KD less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less (for example, IL- 4R protein), for example, measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • the term "antigen-binding fragment” refers to a fragment of an antibody capable of specifically binding to a human IL-4R epitope.
  • the antigen-binding fragment of the present invention include a Fab fragment, an F (ab ') 2 fragment, an Fv fragment, a single-chain antibody (scFv), a single-domain antibody (sdAb), and the like.
  • Fab fragments are fragments produced by digestion of antibodies with papain.
  • the F (ab ') 2 fragment is a fragment produced by digesting the antibody with pepsin.
  • the Fv fragment is composed of a dimer in which the heavy chain variable region and the light chain variable region of the antibody are closely non-covalently linked.
  • a single-chain antibody is an antibody in which a variable region of an antibody heavy chain and a variable region of a light chain are linked by a short peptide of 15 to 20 amino acids (linker).
  • Single-domain antibody is also called nanobody or heavy chain antibody, which is only composed of heavy chain, and its antigen-binding region is only a single domain connected to the Fc region through the hinge region.
  • variable means that some parts of the variable regions of an antibody differ in sequence, and it forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the antibody variable region. It focuses on three fragments in the light and heavy chain variable regions called complementarity determining regions (CDRs) or hypervariable regions.
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy and light chains each contain four FR regions, which are generally in a ⁇ -sheet configuration, connected by three CDRs that form a linking loop, and in some cases may form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely brought together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC).
  • Fc segment means that papain can cleave an antibody into two identical Fab segments and one Fc segment.
  • the Fc segment can be a crystallizable fragment (Fc). Domain composition.
  • the Fc segment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.
  • the term "antibody that binds to human IL-4R” or "anti-human IL-4R antibody” refers to an antibody that specifically binds to human IL-4R.
  • the human IL-4R is the extracellular domain of human IL-4R. More preferably, the extracellular domain of human IL-4R has an amino acid sequence as shown in SEQ ID NO: 1.
  • the human IL-4R-binding antibody or antigen-binding fragment thereof of the present invention is capable of blocking the interaction between IL-4 and IL-4R.
  • expression vector may be pTT5, pSECtag series, pCGS3 series, pCDNA series vector, etc., as well as other vectors used in mammalian expression systems, etc.
  • the expression vector includes an appropriate transcription and translation regulatory sequence linked thereto Fusion DNA sequence.
  • the term "host cell” refers to a cell suitable for expressing the above-mentioned expression vector, and may be a eukaryotic cell.
  • mammalian or insect host cell culture systems can be used for the expression of the fusion protein of the present invention.
  • Ovary, Chinese (Hamster, Chinese), HEK293, COS, BHK, etc., and derived cells of the above-mentioned cells can all be used in the present invention.
  • the term "pharmaceutical composition” means that the antibody or an antigen-binding fragment thereof of the present invention that binds human IL-4R can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable therapeutic effect.
  • a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable therapeutic effect.
  • These preparations It can ensure the conformational integrity of the amino acid core sequence of the human IL-4R-binding antibody or its antigen-binding fragment disclosed in the present invention, and also protect the polyfunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation) .
  • the term "disease associated with IL-4R overexpression” means that the expression level of IL-4R in cells in an abnormal disease state is higher than the expression level of IL-4R in normal cells of the same tissue type.
  • the diseases related to IL-4R overexpression of the present invention include, but are not limited to, atopic dermatitis, asthma, allergic reactions, eosinophilic esophagitis, skin infections, and nasal polyposis.
  • the human IL-4R ⁇ extracellular domain (IL-4R-ECD) sequence as an antigen is from http://www.uniprot.org, and the amino acid sequence is shown in SEQ ID NO: 1. After the amino acid sequence is codon optimized, the whole gene is synthesized and ligated into a prokaryotic cloning vector. Recombinant PCR was used to add a hFc (IgG1) or FLAG tag to the end of the coding region of IL-4R-ECD, and the gene was reconstituted into a pTT5 transient transfection vector (purchased from NRC Biotechnology Research Institute).
  • hFc IgG1
  • FLAG tag FLAG tag
  • the above vector was transfected into HEK293 cells (purchased from NRC Biotechnology Research) according to standard operating procedures, cultured in Freestyle 293 Expression Medium (purchased from Gibco) medium, and the expressed proteins were purified from the cell culture supernatant after 5 days.
  • IL-4R-ECD-hFc and IL-4R-ECD-FLAG antigens were purified using protein A and anti-FLAG affinity chromatography columns, respectively.
  • the sequences of the heavy and light chains of the positive control antibody Dupilumab are from WHO Drug Information (2013,27 (3): 284-285). After the genes of the heavy chain variable region and light chain variable region of the antibody were synthesized, they were The heavy chain IgG4 (S228P) and light chain Kappa constant regions were recombined and constructed into pTT5 transient expression vectors, expressed using the HEK293E system, and purified with Protein A affinity chromatography.
  • TF-1 cells in the logarithmic growth phase were washed with RPMI1640 complete medium (RPMI1640 basal medium (purchased from Gibco) containing 10% fetal bovine serum) at 37 ° C, washed twice, 1000 rpm, and centrifuged 5min; count TF-1 cells, suspend in RPMI1640 complete medium to appropriate density, inoculate into a 96-well plate, 10,000 / 150 ⁇ l / well; add IL-4 (purchased from R & D systems) to RPMI1640 complete medium to make Its concentration reaches 80 ng / ml; then, the human IL-4R antigen (IL-4R-ECD-hFc and IL-4R-ECD-FLAG) and the positive control antibody Dupilumab are diluted to the appropriate concentration with IL-4 medium, Then dilute 9 gradients at appropriate magnifications; add the diluted antigen and antibody to a 96-well cell culture plate, 50 ⁇ l
  • IL-4R-ECD-hFc, IL-4R-ECD-FLAG, and Dupilumab can effectively inhibit IL-4-induced proliferation of TF-1 cells, with IC50 of 280.2ng / ml and 315.1ng / ml, respectively. And 502.8ng / ml.
  • the IL-4R-ECD-hFc antigen prepared in Example 1.1 was diluted to a suitable concentration with physiological saline, mixed with an equal volume of Freund's complete adjuvant, and 4-5 weeks old Balb was completely emulsified.
  • / c mice purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018) were injected subcutaneously at multiple points, each mouse was injected with 50 ⁇ g antigen / 100 ⁇ l. Three weeks later, an equal amount of protein was mixed with an equal volume of Freund's incomplete adjuvant.
  • mice were immunized subcutaneously at multiple points, and the immunization step was repeated again two weeks later. All mice took a drop of blood on the seventh day after the third immunization, separated the serum, and measured the serum titer by ELISA. For mice with a serum antibody titer> 100,000, a shock immunization was performed one week after the titer measurement: the tail vein was injected with 10 ⁇ g antigen protein / 100 ⁇ l physiological saline / rat.
  • PBST phosphate buffer containing 0.05% Tween-20
  • BSA bovine serum albumin
  • splenocytes were taken for fusion.
  • Myeloma sp2 / 0 cells (from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences) in good growth state were cultured in a 37 ° C, 5% CO 2 incubator, and the solution was changed the day before fusion.
  • a hybridoma strain stably expressing the antibody of interest was obtained by the aforementioned method, and these clones were expanded and the cells were frozen.
  • the aforementioned hybridoma strain was cultured in a serum-free medium HybridGRO SF (purchased from Corning) for 7 days, and then a mouse-derived anti-human IL-4R monoclonal antibody was purified from the culture supernatant using a Protein A / G affinity chromatography column.
  • Example 1.2 For the ELISA test method, see Example 1.2. The difference is that IL-4R-ECD-hFc is replaced with IL-4R-ECD-FLAG.
  • the relative affinity of the purified mouse-derived anti-human IL-4R monoclonal antibody to the IL-4R antigen is detected by an ELISA method.
  • Example 1.2 The experimental method is described in Example 1.2. The difference is that IL-4R-ECD-hFc is diluted to 100 ng / ml with sodium carbonate buffer, wherein the negative control used is a mouse-derived antibody that does not bind human IL-4R.
  • TF-1 cells are used to detect the inhibitory effect of the purified mouse-derived anti-human IL-4R monoclonal antibody on IL-4-induced proliferation of TF-1 cells.
  • clones 21, 31, 40, and 4-2 were finally selected as lead antibodies.
  • Trizol purchased from Lifetechnologies
  • Trizol was used to extract total RNA from four monoclonal antibody corresponding hybridoma monoclonal cell lines, and the mRNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Takara), and combined primers reported in the literature ("Antibody Engineering” Volume 1, Edited by Roland Kontermann and Stefan Dübel, the sequence of the combined primers is from page 323) PCR amplification of the light chain variable region and heavy chain of mouse anti-human IL-4R monoclonal antibody The variable region gene was then cloned into the pMD18-T vector, and the variable region gene sequence was sequenced and analyzed.
  • the sequence information is as follows: the heavy chain variable region gene sequence is 360bp in length and encodes 120 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 2 and the amino acid sequence is shown in SEQ ID NO: 3;
  • the variable region gene sequence is 318 bp in length and encodes 106 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 4 and the amino acid sequence is shown in SEQ ID NO: 5.
  • the amino acid sequences of the light chain variable region and the heavy chain variable region were analyzed, and three antigen complementarity determining regions (CDR) and four framework regions (FR) of antibody 4-2 were determined according to the Kabat rule.
  • the amino acid sequence of the heavy chain complementarity determining region is HCDR1: DDYIN (SEQ ID NO: 6), HCDR2: WIFPGNGNSYYNEKFKD (SEQ ID NO: 7), and HCDR3: GLVRYRALFDY (SEQ ID ID: 8).
  • the amino acid sequences are LCDR1: RASSSINYMH (SEQ ID NO: 9), LCDR2: AASNLAS (SEQ ID NO: 10), and LCDR3: QQWSSYPIT (SEQ ID NO: 11).
  • IGHV1-3 * 01 was selected as the template for heavy chain CDR transplantation, and the heavy chain CDR of mouse antibody 4-2 was transplanted into IGHV1-3. * 01 backbone region, constructed as a heavy chain CDR-grafted antibody.
  • IGKV1-16 * 01 was selected as the light chain CDR transplantation template after comparing with human IgG germline sequence homology, and the light chain CDRs of mouse-derived antibody No. 4-2 were transplanted into the backbone region of IGKV1-16 * 01.
  • a light chain CDR-grafted antibody was constructed, and the resulting antibody was defined as 4-2-Grafted.
  • the amino acid sequence was Kabat coded, and the position of the site was indicated by Kabat code.
  • the 48th position of Kabat code is returned as a rat-derived V
  • the 67th position is returned as A
  • the 69th position is returned as L
  • the 71st position is returned as R Is V
  • T at the 73rd position returns K
  • Y at the 91st position returns F.
  • variable region sequence F at position 36 of Kabat code is returned as Y, S at position 46 is returned as P, and L at position 47 is returned as W.
  • the above-mentioned variable region gene sequence was codon optimized and synthesized by Suzhou Jinweizhi Company according to the codon usage preference of Cricetulus griseus.
  • the synthetic humanized heavy chain variable region sequences were linked to human IgG1 and IgG4 (S228P) constant regions, respectively, and the resulting genes were named 4-2-Humanized-IgG1-HC and 4-2-Humanized-IgG4-HC, respectively. ;
  • the humanized light chain variable region is linked to the human Kappa chain constant region, and the resulting gene is named 4-2-Humanized-LC.
  • murine heavy chain variable region sequences were linked to the human IgG1 and IgG4 (S228P) constant regions, respectively, and the resulting genes were named 4-2-Chimeric-IgG1-HC and 4-2-Chimeric-IgG4-HC, respectively;
  • the variable region of the mouse light chain is connected to the constant region of the human Kappa chain, and the resulting gene is named 4-2-Chimeric-LC.
  • the humanized heavy chain variable region gene sequence of antibody No. 4-2 was 360 bp in length and encoded 120 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 12 and the amino acid sequence is shown in SEQ ID NO: 13
  • the humanized light chain variable region gene sequence is 318 bp in length and encodes 106 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 14 and the amino acid sequence is shown in SEQ ID NO: 15.
  • the light and heavy chain genes of the above antibodies were constructed into a pTT5 expression vector, and the light and heavy chain expression vectors were combined to perform transient transfection using the HEK293E system and express the antibody.
  • HEK293 cells were cultured in Free Style 293 Expression Medium medium, the plasmid was transferred into the cells by PEI transfection for 5 days, and the cell supernatant was collected. Purified antibodies were obtained using Protein A affinity chromatography.
  • the antibodies obtained after 4-2-Humanized-IgG1-HC and 4-2-Humanized-IgG4-HC were expressed in combination with 4-2-Humanized-LC were defined as 4-2-Humanized-IgG1 and 4-2-Humanized, respectively. -IgG4.
  • the antibody obtained after the combined expression of 4-2-Chimeric-IgG1-HC and 4-2-Chimeric-LC was defined as 4-2-Chimeric-IgG1.
  • the relative affinity of 4-2-Chimeric-IgG1 and 4-2-Humanized-IgG1 to the IL-4R antigen is detected using an ELISA method.
  • Example 1.2 The experimental method is described in Example 1.2. The difference is that IL-4R-ECD-hFc is replaced with IL-4R-ECD-FLAG (IL-4R-ECD-FLAG is diluted to 100ng / ml with sodium carbonate buffer), and HRP-labeled goat anti-mouse secondary antibody Replaced with HRP-labeled goat anti-human secondary antibody.
  • the negative control antibody used was a humanized antibody that did not bind human IL-4R.
  • 4-2-Humanized-IgG1, 4-2-Chimeric-IgG1 and Dupilumab can effectively bind the antigen IL-4R-ECD-FLAG, with EC50 of 12.39ng / ml, 11.81ng / ml and 12.52 respectively. ng / ml.
  • TF-1 cells are used to detect the inhibitory effect of 4-2-Chimeric-IgG1 and 4-2-Humanized-IgG1 on IL-4-induced proliferation of TF-1 cells.
  • Example 1.1 The experimental method is described in Example 1.1. Among them, the negative control used does not contain IL-4 but contains a control antibody that does not bind human IL-4R; the positive control contains IL-4 and a control antibody that does not bind human IL-4R.
  • Dupilumab, 4-2-Chimeric-IgG1, and 4-2-Humanized-IgG1 can effectively inhibit the proliferation of TF-1 cells induced by IL-4, with IC50 of 289.4ng / ml, 225.2ng / ml, and 207.4ng / ml.
  • TF-1 cells are used to detect the inhibitory effect of 4-2-Chimeric-IgG1 and 4-2-Humanized-IgG1 on IL-13-induced proliferation of TF-1 cells.
  • Example 1.1 The experimental method is described in Example 1.1. The difference is that IL-4 is replaced with IL-13, where the negative control used does not contain IL-13 but contains a control antibody that does not bind human IL-4R; the positive control contains IL-13 and a control that does not bind human IL-4R antibody.
  • Dupilumab, 4-2-Chimeric-IgG1, and 4-2-Humanized-IgG1 can effectively inhibit IL-13-induced proliferation of TF-1 cells, with IC50 of 73.8ng / ml, 81.6ng / ml, and 34.3ng / ml.
  • Example 4.1 Inhibitory effect of IL-4 induced proliferation of TF-1 cells
  • TF-1 cells are used to detect the inhibitory effect of humanized antibodies 4-2-Humanized-IgG1 and 4-2-Humanized-IgG4 on IL-4-induced proliferation of TF-1 cells.
  • Example 1.1 The experimental method is described in Example 1.1. Among them, the negative control used does not contain IL-4 but contains a control antibody that does not bind human IL-4R; the positive control contains IL-4 and a control antibody that does not bind human IL-4R.
  • the results of the first experiment are shown in Figure 7-1.
  • the IC50s of Dupilumab, 4-2-Humanized-IgG1, and 4-2-Humanized-IgG4 inhibiting IL-4-induced proliferation of TF-1 cells were 841.6ng / ml and 560.6, respectively. ng / ml and 1291 ng / ml.
  • the results of the second repeated experiment are shown in Figure 7-2.
  • the IC50s of Dupilumab, 4-2-Humanized-IgG1, and 4-2-Humanized-IgG4 in inhibiting IL-4-induced proliferation of TF-1 cells were 616.1ng / ml, 426.9ng / ml and 1113ng / ml.
  • the results of the third repeated experiment are shown in Figure 7-3.
  • the IC50s of Dupilumab, 4-2-Humanized-IgG1, and 4-2-Humanized-IgG4 inhibiting IL-4-induced proliferation of TF-1 cells were 687.1ng / ml, 520.4ng / ml and 1145ng / ml.
  • the order of functional activity of three independent repeated experiments is as follows: 4-2-Humanized-IgG1> Dupilumab> 4-2-Humanized-IgG4.
  • the capture molecule Protein A / G was covalently coupled to the chip surface using the activation reagent EDC / NHS and blocking reagent Ethanolamine in the amino coupling kit (purchased from GE Healthcare); on BiacoreT200, a protein coupled with ProteinA / G was used.
  • the CM5 chip captures the IL-4R antibody, and then injects the IL-4R-ECD-FLAG to obtain the binding-dissociation curve. After elution with 6M guanidine hydrochloride regeneration buffer, the next cycle is repeated; the data is analyzed using Biacore T200 Evaluation Software . The results are shown in Table 1.
  • Example 4.1 The experimental results in Example 4.1 show that compared with 4-2-Humanized-IgG1, 4-2-Humanized-IgG4 is slightly weaker in inhibiting the functional activity of IL-4-induced proliferation of TF-1 cells; Biacore affinity in Example 4.2
  • the test results showed that the affinity of 4-2-Humanized-IgG1 and 4-2-Humanized-IgG4 for IL-4R was basically the same, indicating that this difference was not caused by the change of affinity.
  • the above results suggest that the difference in activity between 4-2-Humanized-IgG1 and 4-2-Humanized-IgG4 in Example 4.1 may be caused by the difference between the Fc segments of the antibodies.
  • a S267E + L328F mutation is introduced into the Fc segment coding region of 4-2-Humanized-IgG1-HC, and the mutated antibody heavy chain gene is named 4-2-Humanized-IgG1-HC-SELF.
  • the heavy chain gene is related to 4 After the combination of -2-Humanized-LC, expression was performed in HEK293E cells. The specific expression and purification methods were as described in Examples 1.1 and 2.2. The resulting mutant antibodies were defined as 4-2-Humanized-IgG1-SELF.
  • the amino acid position of the antibody sequence in this example uses the Eu coding method.
  • This example uses ELISA to detect the blocking activity of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on the interaction between IL-4 and IL-4R.
  • the human IL-4 sequence is from http://www.uniprot.org, and the amino acid sequence is shown in SEQ ID NO: 20. After the amino acid sequence is codon optimized, the whole gene is synthesized and ligated into a prokaryotic cloning vector. A hFc (IgG1) tag was added to the end of the coding region of human IL-4 by recombinant PCR method, and the gene was recombined into a pTT5 transient transfection vector. The above vector was transfected into HEK293 cells according to standard operating procedures, and cultured in Freestyle 293 Expression Medium medium. After 5 days, the expressed IL-4-hFc was purified from the cell culture supernatant.
  • a hFc (IgG1) tag was added to the end of the coding region of human IL-4 by recombinant PCR method, and the gene was recombined into a pTT5 transient transfection vector.
  • the above vector was transfected into
  • IL-4-hFc served as a ligand to block detection.
  • IL-4R-ECD-hFc after dialysis and concentration measurement, was biotinylated with Biotin N-hydroxysuccinimide ester (Sigma / Cat. No. H1759-100MG).
  • IL-4-hFc Dilute IL-4-hFc to 2000ng / ml with sodium carbonate buffer, and then add 100 ⁇ l per well to the ELISA plate; incubate at room temperature for 4 hours; wash the plate with phosphate buffered saline (PBST) containing 0.05% Tween-20; each The wells were blocked by adding PBST containing 1% bovine serum albumin; washing the plate with PBST; diluting Biotinized IL-4R-ECD-hFc into PBST to a final concentration of 100ng / ml, and using this solution to test Dilute the antibody appropriately, and then perform a gradient dilution in a 96-well plate; transfer the diluted antibody to the above-mentioned ELISA plate coated with IL-4-hFc, and incubate for 1 hour at room temperature; wash the plate with PBST; Streptavidin-HRP diluted with 1% BSA in PBST at a dilution ratio
  • Dupilumab, 4-2-Humanized-IgG4, 4-2-Humanized-IgG1, and 4-2-Humanized-IgG1-SELF can effectively block the interaction between IL-4 and IL-4R, IC50 137.9ng / ml, 140.3ng / ml, 138.3ng / ml, and 144.1ng / ml, respectively.
  • the above results show that the ability of Dupilumab, 4-2-Humanized-IgG4, 4-2-Humanized-IgG1, and 4-2-Humanized-IgG1-SELF to block the interaction between IL-4 and IL-4R is basically the same without significant differences. .
  • TF-1 cells are used to detect the inhibitory effect of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on IL-4-induced proliferation of TF-1 cells.
  • Example 1.1 The experimental method is described in Example 1.1. Among them, the negative control used does not contain IL-4 but contains a control antibody that does not bind human IL-4R; the positive control contains IL-4 and a control antibody that does not bind human IL-4R.
  • the IC50s of Dupilumab, 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF inhibiting IL-4-induced proliferation of TF-1 cells were 1008ng / ml, respectively. , 667.7ng / ml, 2782ng / ml, and 291.9ng / ml.
  • the order of functional activity is as follows: 4-2-Humanized-IgG1-SELF> 4-2-Humanized-IgG1>
  • IL-4 can induce the expression of CD23 molecule in human peripheral blood mononuclear cells (PBMC), which is a low-affinity IgE receptor associated with allergic reactions.
  • PBMC peripheral blood mononuclear cells
  • PBMC is used to detect the inhibitory effect of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on IL-4-induced CD23 expression in PBMCs.
  • Isolate PBMC from human whole blood with Histopaque after counting the cells, inoculate RMPI-1640 complete medium (containing 10% fetal bovine serum) into a 96-well round-bottom cell culture plate, 2E5 cells / 150 ⁇ l per well; IL- 4 Dilute to 80ng / ml with RMPI-1640, use this medium to dilute the anti-IL-4R antibody gradient; add 50 ⁇ l of the mixed solution of anti-IL-4R antibody and IL-4 diluted RPMI-1640 gradient to the above 96-well plate Place the 96-well plate in a 37 ° C incubator for 2 days.
  • RMPI-1640 complete medium containing 10% fetal bovine serum
  • the results of the first experiment are shown in Figure 10-1.
  • Dupilumab, 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF can effectively inhibit IL-4-induced PBMC expression.
  • CD23 and IC50 were 90.91ng / ml, 29.38ng / ml, 232.1ng / ml and 7.404ng / ml, respectively.
  • the results of the second repeated experiment are shown in Figure 10-2.
  • Dupilumab, 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF inhibit IL-4-induced CD23 expression in PBMCs.
  • IC50 were 20.65ng / ml, 3.56ng / ml, 34.00ng / ml and 0.49ng / ml.
  • the order of functional activity of two independent replicate experiments is as follows: 4-2-Humanized-IgG1-SELF> 4-2-Humanized-IgG1> Dupilumab> 4-2-Humanized-IgG4. Of these, the blood used in the two experiments came from different donors.
  • Histopaque was used to isolate PBMCs from 6 blood from different donors; after counting the cells, RMPI-1640 complete medium was used to inoculate 96-well round bottom cell culture plates with 2E5 cells per well; then Add anti-IL-4R antibody (final concentration 25ng / ml) and IL-4 (final concentration 20ng / ml); incubate 96-well plates in a 37 ° C incubator for 2 days; 2 days later, use flow cytometry according to the above experimental method Analyze the expression of CD23.
  • the negative control used does not contain IL-4 but contains a control antibody that does not bind human IL-4R;
  • the positive control contains IL-4 and a control antibody that does not bind human IL-4R.
  • the experimental results are shown in Figure 10-3.
  • the functional activity of the 4-2 antibody is ranked as follows: 4-2-Humanized-IgG1-SELF> 4-2-Humanized-IgG1> 4-2-Humanized-IgG4, and has Statistically significant difference (statistical method is T-test, P ⁇ 0.05 is considered to have statistically significant difference).
  • IL-4 can induce human peripheral blood mononuclear cells (PBMCs) to secrete IgE molecules.
  • IgE molecules are highly related to allergic reactions.
  • PBMC is used to detect the inhibitory effect of 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF on IL-4-induced PBMC secretion of IgE.
  • Isolate PBMC from human whole blood with Histopaque after counting the cells, inoculate RMPI-1640 complete medium (containing 10% fetal bovine serum) into a 96-well round-bottom cell culture plate, 2E5 cells / 150 ⁇ l per well; IL- 4 Dilute to 80ng / ml with RMPI-1640, and add dexamethasone to a final concentration of 400ng / ml at the same time, use this medium to gradiently dilute the IL-4R antibody; add 50 ⁇ l of the above-mentioned 96-well plate with RPMI-1640 gradient dilution Mixed solution of anti-IL-4R antibody, IL-4 and dexamethasone; 96-well plate was incubated in a 37 ° C incubator for 14 days; the cell culture supernatant was taken after 14 days, and Anti-HumanIgEantibody was used as a capture antibody, Biotin-anti -HumanIgE antibody was used as the detection antibody, and the
  • the results of the first experiment are shown in Figure 11-1.
  • Dupilumab, 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF can effectively inhibit IL-4 induced PBMC secretion.
  • IgE and IC50 were 995.7ng / ml, 448.0ng / ml, 1953ng / ml and 50.85ng / ml, respectively.
  • the results of the second repeated experiment are shown in Figure 11-2.
  • Dupilumab, 4-2-Humanized-IgG1, 4-2-Humanized-IgG4, and 4-2-Humanized-IgG1-SELF inhibit IL-4-induced IgE secretion in PBMCs.
  • IC50 were 189.4ng / ml, 61.34ng / ml, 412.9ng / ml and 26.61ng / ml, respectively.
  • the order of functional activity of two independent replicate experiments is as follows: 4-2-Humanized-IgG1-SELF> 4-2-Humanized-IgG1> Dupilumab> 4-2-Humanized-IgG4. Of these, the blood used in the two experiments came from different donors.
  • intravenous injection Intravenous Injection, I.V.
  • rats Intravenous Injection, I.V.
  • IV. Intravenous Injection
  • ELISA plate is coated with IL-4R-ECD-FLAG at a concentration of 100ng / ml, 100 ⁇ l / well; after coating, it is blocked with PBST + 1% BSA; Rat serum; after a period of incubation, wash the plate and finally add HRP-labeled goat anti-human secondary antibody (purchased from Sigma; this antibody has been subjected to species cross-adsorption treatment and does not recognize rat endogenous antibodies); After incubation for a period of time, wash the plate and add a chromogenic solution to develop the color, and then detect the OD450 after stopping the chromogenic reaction with the stop solution; use the standard curve to convert the OD450 to the antibody concentration; use GraphPad
  • the amino acid residues located on the extracellular domain of IL-4R ⁇ are as follows: F13, M14, S15, L39, F41, L42, L43, D66, D67, V68, D69, S70, D72, N73, Y74, K91, P92, S93, E94, D125, N126, Y127, L128, Y129, and Y183.
  • a method of site-directed mutagenesis is used to mutate the above amino acid residues to alanine, respectively, to construct a series of IL-4R ⁇ mutants.
  • the IL-4R ⁇ mutants were expressed in HEK293E cells, and the corresponding mutants were purified by nickel affinity chromatography.
  • the purified IL-4R ⁇ mutant was used to coat the microtiter plate (10ng / well), and then 4-2-Humanized-IgG4 (4-2-Hu-IgG4) and Dupilumab were used to detect mutations of each IL-4R ⁇ . Body bonding strength.
  • the experimental method is described in Example 1.2.
  • the binding effect of the -4R ⁇ mutant did not change significantly, indicating that the three amino acid residues F41, L42 and L43 are necessary for the effective binding of 4-2-Hu-IgG4 to IL-4R ⁇ and the effective binding of Dupilumab to IL-4R ⁇ . necessary.
  • the above results indicate that 4-2-Hu-IgG4 and Dupilumab are different.

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Abstract

结合人IL-4R的抗体，它们具有相同的可变区和不同的恒定区，其可变区能够特异性与人IL-4R结合，恒定区通过氨基酸位点突变影响整个抗体的活性。上述抗体可应用于治疗与IL-4R过表达相关的疾病，例如特应性皮炎、哮喘等，具有良好的临床应用前景。

Description

结合人IL-4R的抗体、其制备方法和用途 技术领域
本发明涉及抗体领域，更具体地，本发明公开了一种结合人IL-4R的抗体、其制备方法和用途。
背景技术
白细胞介素-4受体(interleukin-4 receptor，IL-4R)是由α链及γc链组成的异二聚体；其中α链(IL-4Rα)能够特异结合白细胞介素-4(IL-4)，然后再与γc链形成三元复合体，转导IL-4促进增殖及基因转录激活的信号。白细胞介素-13受体(interleukin-13 receptor，IL-13R)是由IL-13Rα1链及IL-4Rα链组成的异二聚体；其中IL-13Rα1能够特异结合白细胞介素-13，然后再与IL-4Rα链形成三元复合体，转导IL-13促进增殖及基因转录激活的信号。IL-4和IL-13在过敏性皮炎和哮喘等过敏性疾病的病理机制中起重要作用。
Figure PCTCN2019101628-appb-000001
(Dupilumab)注射液是目前已上市的以IL-4Rα链为靶点的单克隆抗体药物，由Regeneron Pharmaceuticals(再生元公司)和Sanofi-aventis(赛诺菲公司)共同开发。Dupilumab是一种全人IgG4型单克隆抗体，分子量约147kDa，能够特异性地结合至被IL-4和IL-13受体复合物共享的IL-4Rα亚单位，从而抑制IL-4和IL-13细胞因子介导的炎症反应。Dupilumab分别于2017年3月28日、2017年9月27日和2018年1月19日获得美国FDA、欧洲EMA和日本PMDA批准上市。
Figure PCTCN2019101628-appb-000002
批准的适应症为患有局部治疗不可控或治疗不当的中度至重度特应性皮炎的成年患者的治疗，可以与或不与局部皮质类固醇共同使用。
最近的两项三期试验(Castro M,Corren J,Pavord I D,et al.Dupilumab efficacy and safety in moderate-to-severe uncontrolled asthma[J].New England Journal of Medicine,2018；378:2486-2496,DOI:10.1056/NEJMoa1804092)显示，接受dupilumab治疗的患者比接受安慰剂的对照组患者的严重哮喘恶化率显著降低，同时也有更好的肺功能和哮喘控制；在嗜酸性粒细胞水平较高的患者中，获益更加明显。基于上述临床试验结果，美国食品和药物管理局(FDA)已经接受了dupilumab作为对某些患有中度至重度哮喘的成人和青少年(12岁及以上)的附加维持治疗的补充生物许可申请(sBLA)。
目前，dupilumab已获FDA批准上市，用于治疗特应性皮炎，后续还可能被批准用于治疗中度至重度哮喘。然而，仍亟待开发新型、特异、高效的以IL-4Rα为靶点的药物填补国内同类药物市场空白，从而能够改善我国患有过敏性自身免疫疾病人群的生活质量，造福国内患者。
发明内容
为了解决上述技术问题，本发明的发明人进行了大量试验，从抗原免疫、杂交瘤筛选、抗体表达纯化到生物活性鉴定，筛选获得了一个特异性结合人IL-4R的鼠源4-2号抗体，并 在此基础上，进一步构建获得其嵌合抗体4-2-Chimeric-IgG1以及人源化抗体4-2-Humanized-IgG1和4-2-Humanized-IgG4。实验结果表明嵌合抗体4-2-Chimeric-IgG1以及人源化抗体4-2-Humanized-IgG1和4-2-Humanized-IgG4能够有效地阻断IL-4与IL-4R之间的相互作用，且具有抑制IL-4诱导的TF-1细胞增殖和人外周血单个核细胞(PBMC)表达CD23和分泌IgE的活性。因此，本发明开发的结合人IL-4R的抗体可用于制备IL-4R过表达疾病患者的治疗药物。
抗体表位分析表明，本发明的抗体与IL-4Rα胞外域上的下述氨基酸残基特异结合：L39、F41、L42、L43、D72和Y74。
因此，本发明的第一个目的在于提供一种结合人IL-4R的抗体或其抗原结合片段。
本发明的第二个目的在于提供编码所述的结合人IL-4R的抗体或其抗原结合片段的分离的核苷酸。
本发明的第三个目的在于提供含有所述核苷酸的表达载体。
本发明的第四个目的在于提供含有所述表达载体的宿主细胞。
本发明的第五个目的在于提供所述的结合人IL-4R的抗体或其抗原结合片段的制备方法。
本发明的第六个目的在于提供含有所述的结合人IL-4R的抗体或其抗原结合片段的药物组合物。
本发明的第七个目的在于提供所述的结合人IL-4R的抗体或其抗原结合片段或所述的药物组合物的用途。
本发明的第八个目的在于提供用于提高所述的结合人IL-4R的抗体或其抗原结合片段的活性的方法。
为了实现上述目的，本发明采用了如下技术方案：
本发明的第一个方面提供了一种结合人IL-4R的抗体或其抗原结合片段，包括：
(a)重链互补决定区HCDR1、HCDR2、HCDR3，所述的HCDR1的氨基酸序列如SEQ ID NO：6所示，所述的HCDR2的氨基酸序列如SEQ ID NO：7所示，所述的HCDR3的氨基酸序列如SEQ ID NO：8所示，和
(b)轻链互补决定区LCDR1、LCDR2、LCDR3，所述的LCDR1的氨基酸序列如SEQ ID NO：9所示，所述的LCDR2的氨基酸序列如SEQ ID NO：10所示，所述的LCDR3的氨基酸序列如SEQ ID NO：11所示。
根据本发明，所述的抗体为单克隆抗体或多克隆抗体。优选的，所述的抗体为单克隆抗体。
根据本发明，所述的抗体为鼠源抗体、嵌合抗体或人源化抗体等。
根据本发明，所述的抗体为IgG1型抗体或IgG4型抗体。
根据本发明，所述的抗原结合片段包括Fab片段、F(ab’)2片段、Fv片段、单链抗体(scFv)及单域抗体(sdAb)等。
根据本发明，所述的结合人IL-4R的抗体或其抗原结合片段能够阻断IL-4与IL-4R之间 的相互作用。
根据本发明的一个优选实施方式，所述的结合人IL-4R的抗体或其抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：3所示，轻链可变区的氨基酸序列如SEQ ID NO：5所示；根据本发明的另一个优选实施方式，所述的结合人IL-4R的抗体或其抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：13所示，轻链可变区的氨基酸序列如SEQ ID NO：15所示。
根据本发明的优选实施方式，所述的结合人IL-4R的抗体或其抗原结合片段的重链的氨基酸序列如SEQ ID NO：16所示，轻链的氨基酸序列如SEQ ID NO：18所示；在本发明的另一优选实施方式中，所述的结合人IL-4R的抗体或其抗原结合片段的重链的氨基酸序列如SEQ ID NO：17所示，轻链的氨基酸序列如SEQ ID NO：18所示；在本发明的另一优选实施方式中，所述的结合人IL-4R的抗体或其抗原结合片段的重链的氨基酸序列如SEQ ID NO：19所示，轻链的氨基酸序列如SEQ ID NO：18所示。
根据本发明的优选实施方式，所述的结合人IL-4R的抗体或其抗原结合片段包括Fc段，所述的Fc段包括如下突变位点：S267E和L328F。
本发明的第二个方面提供了一种分离的核苷酸，所述的核苷酸编码如上任一项所述的结合人IL-4R的抗体或其抗原结合片段。
根据本发明的优选实施方式，所述的核苷酸具有如SEQ ID NO：2所示的编码重链可变区的核苷酸序列，和如SEQ ID NO：4所示的编码轻链可变区的核苷酸序列；在本发明的另一种优选实施方式中，所述的核苷酸具有如SEQ ID NO：12所示的编码重链可变区的核苷酸序列，和如SEQ ID NO：14所示的编码轻链可变区的核苷酸序列。
本发明的第三个方面提供了一种表达载体，所述的表达载体含有如上任一项所述的核苷酸。
本发明的第四个方面提供了一种宿主细胞，所述的宿主细胞含有如上所述的表达载体。
本发明的第五个方面提供了如上所述的结合人IL-4R的抗体或其抗原结合片段的制备方法，所述方法包括以下步骤：
(a)在表达条件下，培养如上所述的宿主细胞，从而表达所述的结合人IL-4R的抗体或其抗原结合片段；
(b)分离并纯化(a)所述的结合人IL-4R的抗体或其抗原结合片段。
本发明的第六个方面提供了一种药物组合物，所述药物组合物含有如上任一项所述的结合人IL-4R的抗体或其抗原结合片段和药学上可接受的载体。
本发明的第七个方面提供了如上任一项所述的结合人IL-4R的抗体或其抗原结合片段或如上所述的药物组合物在制备治疗与IL-4R过表达相关的疾病的药物中的用途。
本发明也提供治疗与IL-4R过表达相关的疾病的方法，所述方法将本发明所述的结合人IL-4R的抗体或其抗原结合片段或含有所述结合人IL-4R的抗体或其抗原结合片段的药物组合物给药于患者。
本发明也提供一种用于治疗与IL-4R过表达相关的疾病的抗体或其抗原结合片段或含有所述结合人IL-4R的抗体或其抗原结合片段的药物组合物。
根据本发明的优选实施方式，所述的与IL-4R过表达相关的疾病包括特应性皮炎、哮喘、过敏反应、嗜酸性食道炎、皮肤感染、鼻息肉症等。
本发明的第八个目的在于提供用于提高如上任一项所述的结合人IL-4R的抗体或其抗原结合片段的活性的方法，所述的结合人IL-4R的抗体或其抗原结合片段包括Fc段，其特征在于，所述的Fc段包括如下突变位点：S267E和L328F。
有益效果：
本发明制备了一系列结合人IL-4R的抗体，它们具有相同的可变区和不同的恒定区，其可变区能够特异性与人IL-4R结合，恒定区通过独特的机制影响整个抗体的活性。上述一系列抗体可应用于制备治疗IL-4R过表达的疾病(例如特应性皮炎、哮喘等)的药物，具有良好的临床应用前景。
附图说明
图1为采用TF-1细胞检测IL-4R-ECD-hFc和IL-4R-ECD-FLAG对IL-4诱导的TF-1细胞增殖的抑制作用的结果。
图2为采用ELISA检测纯化的鼠源抗人IL-4R单克隆抗体对IL-4R抗原的相对亲和力的结果。
图3为采用TF-1细胞检测纯化的鼠源抗人IL-4R单克隆抗体对IL-4诱导的TF-1细胞增殖的抑制作用的结果。
图4为采用ELISA检测4-2-Chimeric-IgG1和4-2-Humanized-IgG1对IL-4R抗原的相对亲和力的结果。
图5为采用TF-1细胞检测4-2-Chimeric-IgG1和4-2-Humanized-IgG1对IL-4诱导的TF-1细胞增殖的抑制作用的结果。
图6为采用TF-1细胞检测4-2-Chimeric-IgG1和4-2-Humanized-IgG1对IL-13诱导的TF-1细胞增殖的抑制作用的结果。
[根据细则91更正 14.10.2019]　
图7-1-图7-3为采用TF-1细胞检测4-2-Humanized-IgG1和4-2-Humanized-IgG4对IL-4诱导的TF-1细胞增殖的抑制作用的结果。
图8为采用ELISA检测4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF对IL-4和IL-4R相互作用的阻断活性的结果。
图9为采用TF-1细胞检测4-2-Humanized-IgG1、4-2-Humanized-IgG4、4-2-Humanized-IgG1-SELF对IL-4诱导的TF-1细胞增殖的抑制作用的结果。
[根据细则91更正 14.10.2019]　
图10-1-图10-3为采用PBMC检测4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF对IL-4诱导PBMC表达CD23的抑制作用的结果。
[根据细则91更正 14.10.2019]　
图11-1-图11-2为采用PBMC检测4-2-Humanized-IgG1、4-2-Humanized-IgG4和 4-2-Humanized-IgG1-SELF对IL-4诱导PBMC分泌IgE的抑制作用的结果。
图12为4-2-Humanized-IgG4的药代动力学研究的结果。
具体实施方式
本发明中，术语“抗体(Ab)”和“免疫球蛋白G(IgG)”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。本发明的抗体包括单克隆抗体、多克隆抗体、由至少两种抗体形成的多特异性抗体(例如双特异性抗体)、抗体的抗原结合片段等。本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体等。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明中，术语“鼠源抗体”是指来源于大鼠或小鼠的抗体，优选小鼠。本发明的鼠源抗体为使用人IL-4R的胞外域为抗原免疫小鼠并进行杂交瘤细胞筛选获得。优选的，本发明的鼠源抗体为4-2号抗体。
本发明中，术语“嵌合抗体”是指包含来源于一个物种的重和轻链可变区序列以及来源于另一个物种的恒定区序列的抗体，例如具有与人恒定区连接的鼠重和轻链可变区的抗体。优选的，本发明的嵌合抗体是由鼠源抗体4-2号抗体的重链可变区序列和轻链可变区序列与人的恒定区拼接获得。更优选的，本发明的嵌合抗体的重链由鼠源抗体4-2号抗体的重链可变区序列与人的IgG1或IgG4(S228P)恒定区拼接获得，轻链由鼠源抗体4-2号抗体的轻链可变区序列与人的kappa链拼接获得。最优选的，本发明的嵌合抗体为4-2-Chimeric-IgG1。
本发明中，术语“人源化抗体”是指其CDR来源于非人物种(优选小鼠)抗体，抗体分子中残余的部分(包括框架区和恒定区)来源于人抗体。此外，框架区残基可被改变以维持结合亲和性。优选的，本发明的人源化抗体由鼠源抗体4-2号抗体的CDR区和来源自人抗体的非CDR区重组，并对包埋残基、与CDR区有直接相互作用的残基，以及对4-2号抗体的VL和VH的构象有重要影响的残基进行回复突变获得。更优选的，本发明的人源化抗体包括4-2-Humanized-IgG1和4-2-Humanized-IgG4。
本发明中，术语“结合”、“特异结合”、“特异性结合”是指两分子间的非随机的结合反 应，如抗体和其所针对的抗原之间的反应。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的平衡解离常数(KD)结合该抗原。
本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的KD结合抗原(例如，IL-4R蛋白)，例如，使用表面等离子体共振术(SPR)在BIACORE仪中测定。
本发明中，术语“抗原结合片段”是指能够与人IL-4R表位特异性结合的抗体的片段。本发明的抗原结合片段的例子包括Fab片段、F(ab’)2片段、Fv片段、单链抗体(scFv)、单域抗体(sdAb)等。Fab片段是用木瓜蛋白酶消化抗体产生的片段。F(ab’)2片段是用胃蛋白酶消化抗体产生的片段。Fv片段是由抗体的重链可变区和轻链可变区紧密非共价关联的二聚物组成。单链抗体(scFv)，是由抗体重链可变区和轻链可变区通过15～20个氨基酸的短肽(linker)连接而成的抗体。单域抗体(sdAb)又称为纳米抗体(nanobody)或重链抗体，仅由重链构成，其抗原结合区仅是一个通过铰链区与Fc区连接的单结构域。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。
本发明中，术语“Fc段”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab段和一个Fc段，Fc段即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc段无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。
本发明中，术语“结合人IL-4R的抗体”或“抗人IL-4R抗体”是指与人IL-4R特异性结合的抗体。优选的，所述的人IL-4R为人IL-4R的胞外域。更优选的，所述的人IL-4R的胞外域具有如SEQ ID NO：1所示的氨基酸序列。优选的，本发明的结合人IL-4R的抗体或其抗原结合片段能够阻断IL-4和IL-4R之间的相互作用。
本发明中，术语“表达载体”可以为pTT5，pSECtag系列，pCGS3系列，pCDNA系列载体等，以及其它用于哺乳动物表达系统的载体等，表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
本发明中，术语“宿主细胞”是指适用于表达上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养系统均可用于本发明的融合蛋白的表达，CHO(中国仓鼠卵 巢，Chinese Hamster Ovary)，HEK293，COS、BHK等及上述细胞的衍生细胞均可适用于本发明。
本发明中，术语“药物组合物”是指本发明的结合人IL-4R的抗体或其抗原结合片段可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的结合人IL-4R的抗体或其抗原结合片段的氨基酸核心序列的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
本发明中，术语“与IL-4R过表达相关的疾病”是指处于异常疾病状态的细胞中IL-4R的表达水平高于相同组织类型的正常细胞中的IL-4R表达水平。本发明的与IL-4R过表达相关的疾病包括但不限于：特应性皮炎、哮喘、过敏反应、嗜酸性食道炎、皮肤感染、鼻息肉症等。
以下实施例、实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例1鼠源抗人IL-4R单克隆抗体的筛选和制备
实施例1.1 IL-4R抗原以及阳性对照抗体Dupilumab的制备
作为抗原的人IL-4Rα胞外域(IL-4R-ECD)序列来自于http://www.uniprot.org，氨基酸序列如SEQ ID NO：1所示。该氨基酸序列经过密码子优化后，全基因合成并连接至原核克隆载体。用重组PCR法在IL-4R-ECD编码区的末端添加一个hFc(IgG1)或FLAG标签，将基因重组之后构建到pTT5瞬时转染载体(购自NRC biotechnology Research Institute)中。将上述载体按照标准操作流程转染到HEK293细胞(购自NRC biotechnology Research Institute)中，在Freestyle 293 Expression Medium(购自Gibco)培养基中进行培养，5天之后从细胞培养上清中纯化表达的IL-4R-ECD-hFc和IL-4R-ECD-FLAG抗原。上述抗原分别用protein A和anti-FLAG亲和层析柱进行纯化。
阳性对照抗体Dupilumab的重链和轻链的序列来自WHO Drug Information(2013,27(3):284-285)，该抗体的重链可变区和轻链可变区基因合成之后，分别与人重链IgG4(S228P)和轻链Kappa恒定区重组，并分别构建到pTT5瞬转表达载体中，用HEK293E系统表达，用Protein A亲和层析纯化。
上述蛋白纯化完毕后，采用TF-1细胞检测IL-4R-ECD-hFc、IL-4R-ECD-FLAG和Dupilumab对IL-4诱导TF-1细胞增殖的抑制作用的能力。处于对数生长期的TF-1细胞(购买自ATCC)用37℃预热的RPMI1640完全培养基(RPMI1640基础培养基(购自Gibco)中含有10％胎牛血清) 洗2遍，1000rpm，离心5min；TF-1细胞计数，用含RPMI1640完全培养基悬浮到适当密度，接种到96孔板中，10000个/150μl/孔；在RPMI1640完全培养基中加入IL-4(购自R&D systems)使其浓度达到80ng/ml；然后用加有IL-4的培养基将人IL-4R抗原(IL-4R-ECD-hFc和IL-4R-ECD-FLAG)和阳性对照抗体Dupilumab稀释到适当浓度，然后按照适当倍率稀释9个梯度；将稀释好的抗原和抗体加入96孔细胞培养板中，50μl/孔；96孔板周围用200μl/孔的蒸馏水补齐；在37℃、5％CO 2条件的孵箱中孵育培养4天；4天后在96孔板中每孔加入20μl CCK-8(购自Dojindo)溶液，在37℃孵箱中继续培养8h；震荡混匀后用酶标仪(SpectraMax 190，Molecular Devices)读取OD450值；GraphPad Prism6进行数据分析，作图并计算IC50。
结果如图1所示，IL-4R-ECD-hFc、IL-4R-ECD-FLAG和Dupilumab均能有效抑制IL-4诱导TF-1细胞增殖，IC50分别是280.2ng/ml、315.1ng/ml和502.8ng/ml。
实施例1.2 抗原免疫小鼠
将实施例1.1中制备的IL-4R-ECD-hFc抗原用生理盐水稀释成到合适的浓度，与等体积的弗氏完全佐剂混合，并经超声乳化完全后对4-5周龄的Balb/c小鼠(购自上海灵畅生物科技有限公司，动物生产许可证号：SCXK(沪)2013-0018)进行皮下多点注射，每只小鼠注射50μg抗原/100μl。三周后，等量蛋白与等体积弗氏不完全佐剂混合，超声乳化完全后对小鼠进行皮下多点免疫，两周后再次重复此免疫步骤。所有小鼠在第三次免疫后的第七天取一滴血，分离血清，利用ELISA进行血清滴度的检测。对于血清抗体效价>100000的小鼠，在滴度测定后一周进行冲击免疫：尾静脉注射10μg抗原蛋白/100μl生理盐水/鼠。
其中，利用ELISA进行血清滴度检测的方法说明如下：用碳酸钠缓冲液(1.59g Na 2CO 3和2.93g NaHCO 3溶于1L纯水中)将IL-4R-ECD-hFc稀释至1000ng/ml，然后100μl每孔加入ELISA板中；室温孵育4小时；用含0.05％Tween-20的磷酸盐缓冲液(简写为PBST：KH 2PO 40.2g，Na 2HPO 4·12H 2O2.9g，NaCl8.0g，KCl0.2g，Tween-200.5ml，加纯水至1000ml)洗板；每孔加入含有1％牛血清白蛋白(BSA)的PBST进行封闭；用PBST洗板，加入梯度稀释的小鼠血清，孵育适当时间；用PBST洗板，加入适当稀释的HRP标记的羊抗鼠二抗，孵育适当时间；洗板后加入显色液(底物显色A液：醋酸钠·三水13.6g，柠檬酸·一水1.6g，30％双氧水0.3ml，纯水500ml；底物显色B液：乙二胺四乙酸二钠0.2g，柠檬酸·一水0.95g，甘油50ml，TMB：0.15g溶于3ml DMSO中，纯水500ml；使用前A和B液等体积混匀)进行显色，用终止液(2M硫酸溶液)终止显色反应；用酶标仪读取OD450；用GraphPad Prism6进行数据分析，作图并计算血清滴度。
实施例1.3 杂交瘤的制备和筛选
小鼠冲击免疫三天后取脾细胞进行融合。生长状态良好的骨髓瘤sp2/0细胞(来源自中国科学院典型培养物保藏委员会细胞库)在37℃、5％CO 2孵箱中培养，融合前一天换液。融合与筛选过程如下：取小鼠脾脏，研磨洗涤后计数。按照脾细胞:sp2/0细胞＝2:1混合两种 细胞，1500rpm离心7分钟。洗去上清液。以1000rpm离心5min的条件，加入20ml细胞融合缓冲液(购自BTX)洗涤细胞三次。细胞沉淀以1×10 7个/ml的密度悬浮于细胞融合缓冲液中。加2ml细胞悬液于融合池中，置于电融合仪ECM2001上，30秒内按一定条件(AC60V，30S；DC1700V，40μS，3X；POST AC60V，3S)进行电融合。电融合后轻轻地将融合细胞转移至37度预热的含有10％血清(购自Gibco)的RPMI1640培养基中，室温继续放置60min。100μl/孔，将细胞按10 4个/孔接种入96孔板。次日每孔补加100μl的含有2×HAT(购自Gibco)和10％血清的RPMI1640培养基。于融合后第四天用新鲜的含有1×HAT和RPMI1640培养基置换一半旧培养液。于融合后第七天用新鲜的含有1×HAT和RPMI1640培养基置换大部分旧培养液。于融合后第九天取样进行ELISA检测。挑选出阳性杂交瘤克隆于24孔板中扩大培养并通过有限稀释法进行亚克隆。通过前述方法获得稳定表达目的抗体的杂交瘤株，对这些克隆进行扩增和细胞冻存。前述杂交瘤株用无血清培养基HybriGRO SF(购自Corning)培养7天，然后用Protein A/G亲和层析柱从培养上清中纯化鼠源抗人IL-4R单克隆抗体。
ELISA检测实验方法参见实施例1.2。区别在于将IL-4R-ECD-hFc更换为IL-4R-ECD-FLAG。
实施例1.4 ELISA检测
本实施例用ELISA方法检测纯化的鼠源抗人IL-4R单克隆抗体对IL-4R抗原的相对亲和力。
实验方法参见实施例1.2。区别在于用碳酸钠缓冲液将IL-4R-ECD-hFc稀释至100ng/ml，其中，使用的阴性对照是不结合人IL-4R的鼠源抗体。
结果如图2所示，根据EC50数据，选择具有较高相对亲和力的抗体，推进到下一个筛选环节。
实施例1.5 IL-4诱导的TF-1细胞增殖的抑制作用
本实施例采用TF-1细胞检测纯化的鼠源抗人IL-4R单克隆抗体对IL-4诱导的TF-1细胞增殖的抑制作用。
实验方法参见实施例1.1。
结果如图3所示，根据IC50数据，选择抑制作用较强的单克隆抗体，推进到下一步实验。
实施例2鼠源抗人IL-4R单克隆抗体的人源化
实施例2.1 鼠源抗人IL-4R单克隆抗体可变区序列的确定
根据实施例1.4和1.5中ELISA和细胞水平的功能实验筛选结果，最终挑取了21号、31号、40号和4-2号克隆作为先导抗体。使用Trizol(购自Life technologies)从四个单抗对应的杂交瘤单克隆细胞株中提取总RNA，用逆转录试剂盒(购自Takara公司)将mRNA逆转录成cDNA，通过文献报道的组合引物(《Antibody Engineering》Volume 1，Edited by Roland  Kontermann and Stefan Dübel，组合引物的序列来自第323页)用PCR扩增鼠源的抗人IL-4R单克隆抗体的轻链可变区和重链可变区基因，然后将PCR产物克隆入pMD18-T载体，测序并分析可变区基因序列。对各个克隆的可变区序列进行对比分析后发现，4-2号抗体的序列比较适合进行人源化，因此选择4-2号克隆作为最终的候选抗体。其序列信息如下：重链可变区基因序列全长360bp，编码120个氨基酸残基，核苷酸序列如SEQ ID NO：2所示，氨基酸序列如SEQ ID NO：3所示；轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO：4所示，氨基酸序列如SEQ ID NO：5所示。
实施例2.2 鼠源抗人IL-4R单克隆抗体的人源化
对上述轻链可变区和重链可变区的氨基酸序列进行分析，依据Kabat规则确定4-2号抗体的3个抗原互补决定区(CDR)和4个框架区(FR)。其中，重链互补决定区的氨基酸序列为HCDR1：DDYIN(SEQ ID NO：6)、HCDR2：WIFPGNGNSYYNEKFKD(SEQ ID NO：7)和HCDR3：GLVRYRALFDY(SEQ ID NO：8)，轻链互补决定区的氨基酸序列为LCDR1：RASSSINYMH(SEQ ID NO：9)、LCDR2：AASNLAS(SEQ ID NO：10)和LCDR3：QQWSSYPIT(SEQ ID NO：11)。
通过在NCBI IgBlast与人IgG胚系序列(Germline)进行同源性比较，选择IGHV1-3*01为重链CDR移植模板，将鼠源的4-2号抗体的重链CDR移植入IGHV1-3*01骨架区，构建成重链CDR移植抗体。同样地，经过与人IgG胚系序列同源性比较，选择IGKV1-16*01为轻链CDR移植模板，将鼠源4-2号抗体的轻链CDR移植入IGKV1-16*01的骨架区，构建成轻链的CDR移植抗体，所得的抗体定义为4-2-Grafted。同时，在此基础上，对一些框架区的氨基酸位点进行了回复突变，回复突变就是将人源框架区的某些氨基酸突变成鼠源框架区同一位置的氨基酸。在进行回复突变时，将氨基酸序列进行了Kabat编码，位点的位置由Kabat码指示。优选的，对于重链可变区序列，将Kabat编码第48位的M回复为鼠源的V，第67位的V回复为A，第69位的I回复为L，第71位的R回复为V，第73位的T回复为K，第91位的Y回复为F。对于轻链可变区序列，将Kabat编码第36位的F回复为Y，第46位的S回复为P，第47位的L回复为W。上述可变区基因序列由苏州金唯智公司按照Cricetulus griseus的密码子使用偏好进行密码子优化并合成。将合成的人源化重链可变区序列分别与人IgG1和IgG4(S228P)恒定区相连，得到的基因分别命名为4-2-Humanized-IgG1-HC和4-2-Humanized-IgG4-HC；人源化轻链可变区与人Kappa链恒定区相连，得到的基因命名为4-2-Humanized-LC。另外，将鼠源重链可变区序列分别与人IgG1和IgG4(S228P)恒定区相连，得到的基因分别命名为4-2-Chimeric-IgG1-HC和4-2-Chimeric-IgG4-HC；鼠源轻链可变区与人Kappa链恒定区相连，得到的基因命名为4-2-Chimeric-LC。
最终，4-2号抗体人源化重链可变区基因序列全长360bp，编码120个氨基酸残基，核苷酸序列如SEQ ID NO：12所示，氨基酸序列如SEQ ID NO：13所示；人源化轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO：14所示，氨基酸序 列如SEQ ID NO：15所示。人源化重链可变区与人IgG1恒定区相连后，最终得到447个氨基酸的4-2-Humanized-IgG1-HC重链(序列如SEQ ID NO：16所示)；人源化重链可变区与人IgG4(S228P)恒定区相连后，最终得到447个氨基酸的4-2-Humanized-IgG4-HC重链(序列如SEQ ID NO：17所示)；轻链可变区与人Kappa恒定区相连后，得到213个氨基酸的4-2-Humanized-LC轻链(序列如SEQ ID NO：18所示)。
上述抗体的轻重链基因构建到pTT5表达载体中，将上述轻重链表达载体组合后利用HEK293E系统进行瞬时转染并表达抗体。HEK293细胞在Free Style 293Expression Medium培养基中培养，利用PEI转染法将质粒转入细胞5天后收取细胞上清，利用Protein A亲和层析后获得纯化抗体。4-2-Humanized-IgG1-HC和4-2-Humanized-IgG4-HC分别与4-2-Humanized-LC组合表达后获得的抗体分别定义为4-2-Humanized-IgG1和4-2-Humanized-IgG4。另外，4-2-Chimeric-IgG1-HC与4-2-Chimeric-LC组合表达后获得的抗体定义为4-2-Chimeric-IgG1。
实施例3嵌合抗体4-2-Chimeric-IgG1和人源化抗体4-2-Humanized-IgG1的功能活性的检测
实施例3.1 ELISA检测
本实施例采用ELISA方法检测4-2-Chimeric-IgG1和4-2-Humanized-IgG1对IL-4R抗原的相对亲和力。
实验方法参见实施例1.2。区别在于将IL-4R-ECD-hFc更换为IL-4R-ECD-FLAG(用碳酸钠缓冲液将IL-4R-ECD-FLAG稀释至100ng/ml)，并将HRP标记的羊抗鼠二抗更换为HRP标记的羊抗人二抗。其中，使用的阴性对照抗体是不结合人IL-4R的人源化抗体。
如图4所示，4-2-Humanized-IgG1、4-2-Chimeric-IgG1和Dupilumab均能有效结合抗原IL-4R-ECD-FLAG，EC50分别是12.39ng/ml、11.81ng/ml和12.52ng/ml。
实施例3.2 IL-4诱导的TF-1细胞增殖的抑制作用
本实施例采用TF-1细胞检测4-2-Chimeric-IgG1和4-2-Humanized-IgG1对IL-4诱导的TF-1细胞增殖的抑制作用。
实验方法参见实施例1.1。其中，使用的阴性对照不含IL-4但含有不结合人IL-4R的对照抗体；阳性对照含有IL-4和不结合人IL-4R的对照抗体。
如图5所示，Dupilumab、4-2-Chimeric-IgG1和4-2-Humanized-IgG1均能有效抑制IL-4诱导TF-1细胞增殖，IC50分别是289.4ng/ml、225.2ng/ml和207.4ng/ml。
实施例3.3 IL-13诱导的TF-1细胞增殖的抑制作用
本实施例采用TF-1细胞检测4-2-Chimeric-IgG1和4-2-Humanized-IgG1对IL-13诱导的TF-1细胞增殖的抑制作用。
实验方法参见实施例1.1。区别在于将IL-4替换为IL-13，其中，使用的阴性对照不含IL-13但含有不结合人IL-4R的对照抗体；阳性对照含有IL-13和不结合人IL-4R的对照抗体。
如图6所示，Dupilumab、4-2-Chimeric-IgG1和4-2-Humanized-IgG1均能有效抑制IL-13诱导TF-1细胞增殖，IC50分别是73.8ng/ml、81.6ng/ml和34.3ng/ml。
实施例4人源化抗体4-2-Humanized-IgG1和4-2-Humanized-IgG4的功能活性的检测
实施例4.1 IL-4诱导的TF-1细胞增殖的抑制作用
本实施例采用TF-1细胞检测人源化抗体4-2-Humanized-IgG1和4-2-Humanized-IgG4对IL-4诱导的TF-1细胞增殖的抑制作用。
实验方法参见实施例1.1。其中，使用的阴性对照不含IL-4但含有不结合人IL-4R的对照抗体；阳性对照含有IL-4和不结合人IL-4R的对照抗体。
第一次实验结果如图7-1所示，Dupilumab、4-2-Humanized-IgG1和4-2-Humanized-IgG4抑制IL-4诱导TF-1细胞增殖的IC50分别是841.6ng/ml、560.6ng/ml和1291ng/ml。第二次重复实验结果如图7-2所示，Dupilumab、4-2-Humanized-IgG1和4-2-Humanized-IgG4抑制IL-4诱导TF-1细胞增殖的IC50分别是616.1ng/ml、426.9ng/ml和1113ng/ml。第三次重复实验结果如图7-3所示，Dupilumab、4-2-Humanized-IgG1和4-2-Humanized-IgG4抑制IL-4诱导TF-1细胞增殖的IC50分别是687.1ng/ml、520.4ng/ml和1145ng/ml。三次独立重复实验的功能活性强弱排序一致如下：4-2-Humanized-IgG1>Dupilumab>4-2-Humanized-IgG4。对三次实验结果进行统计分析，发现4-2-Humanized-IgG1和Dupilumab之间存在显著性差异(P＝0.026<0.05；统计方法为T-test，P<0.05认为具有统计学上的显著性差异)，说明4-2-Humanized-IgG1功能活性强于Dupilumab。
实施例4.2 Biacore检测
本实施例通过Biacore T200(GE healthcare)检测4-2-Humanized-IgG1和4-2-Humanized-IgG4与IL-4R之间的亲和力。
利用氨基偶联试剂盒(购自GE healthcare)中的活化试剂EDC/NHS和封闭试剂Ethanolamine将捕获分子Protein A/G共价偶联至芯片表面；在BiacoreT200上，使用偶联有ProteinA/G的CM5芯片捕获IL-4R抗体，再将IL-4R-ECD-FLAG进样，得到结合-解离曲线，用6M盐酸胍再生缓冲液洗脱后重复下一个循环；利用Biacore T200Evaluation Software对数据进行分析。结果如表1所示。
表1. 4-2-Humanized-IgG1/4对人IL-4R的亲和力检测
Figure PCTCN2019101628-appb-000003
实验结果显示，4-2-Humanized-IgG1和4-2-Humanized-IgG4对IL-4R的亲和力与Dupilumab相当，分别是7.17E-10、6.92E-10和6.71E-10，4-2-Humanized-IgG1和4-2-Humanized-IgG4能够比Dupilumab更加快速地结合IL-4R。
实施例5抗人IL-4R人源化抗体SELF突变体的构建
实施例4.1中实验结果显示，与4-2-Humanized-IgG1相比，4-2-Humanized-IgG4在抑制IL-4诱导TF-1细胞增殖的功能活性方面稍弱；实施例4.2中Biacore亲和力检测结果显示，4-2-Humanized-IgG1和4-2-Humanized-IgG4对IL-4R的亲和力基本一致，表明这种差异不是亲和力变化导致的。上述结果提示了实施例4.1中4-2-Humanized-IgG1和4-2-Humanized-IgG4之间的活性差异，可能由抗体Fc段之间的差异导致。
Xencor公司公开的专利US20090136485A1中报道，在抗体的Fc段恒定区引入S267E+L328F突变(同时将第267位的S突变成E，第328位的L突变成F)能够增强Fc段与CD32分子(一种Fc受体分子)之间的相互作用大约400倍(Chu S Y,Vostiar I,Karki S,et al.Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19and FcγRIIb with Fc-engineered antibodies[J].Molecular immunology,2008,45(15):3926-3933.)。本发明将S267E+L328F突变引入4-2-Humanized-IgG1-HC的Fc段编码区，突变后的抗体重链基因命名为4-2-Humanized-IgG1-HC-SELF，该重链基因与4-2-Humanized-LC组合后，在HEK293E细胞中进行表达，具体表达和纯化方法如实施例1.1和2.2中所述。最终获得的突变抗体分别定义为4-2-Humanized-IgG1-SELF。本实施例中抗体序列的氨基酸位置采用Eu编码法，具体方案参见链接http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html。其中，4-2-Humanized-IgG1-SELF抗体重链的氨基酸序列如SEQ ID NO：19所示。
实施例6抗人IL-4R人源化抗体的SELF突变体和非SELF突变体的功能活性的检测
实施例6.1 IL-4和IL-4R相互作用的阻断活性
本实施例采用ELISA检测4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF对IL-4和IL-4R相互作用的阻断活性。
人IL-4序列来自于http://www.uniprot.org，氨基酸序列如SEQ ID NO：20所示。该氨基酸序列经过密码子优化后，全基因合成并连接至原核克隆载体。用重组PCR法在人IL-4编码区的末端添加一个hFc(IgG1)标签，将基因重组之后构建到pTT5瞬时转染载体中。将上述载体按照标准操作流程转染到HEK293细胞中，在Freestyle 293Expression Medium培养基中进行培养，5天之后从细胞培养上清中纯化表达的IL-4-hFc。上述抗原分别用protein A亲和层析柱进行纯化。纯化的IL-4-hFc作为阻断检测的配体。IL-4R-ECD-hFc，经过透析和浓度测定后，用Biotin N-hydroxysuccinimide ester(Sigma/货号：H1759-100MG)进行生物素化标记。
用碳酸钠缓冲液将IL-4-hFc稀释至2000ng/ml，然后100μl每孔加入ELISA板中；室温孵育4小时；用含0.05％Tween-20的磷酸盐缓冲液(PBST)洗板；每孔加入含有1％牛血清白蛋白的PBST进行封闭；用PBST洗板；将Biotin化的IL-4R-ECD-hFc稀释到PBST中，使其终浓度达到100ng/ml，并用此溶液将待测抗体适当稀释，然后在96孔板中进行梯度稀释；将稀释好的抗体转移到上述包被有IL-4-hFc的ELISA酶标板中，室温孵育1小时；用PBST洗板；加入用含1％BSA的PBST稀释的Streptavidin-HRP，稀释比例为1:1000，100μl/孔，37℃孵育30min；加显色液(TMB底物溶液)，100μl/孔，室温孵育1～5min；加终止液终止显色反应，50μl/孔；用酶标仪读OD450值；用GraphPad Prism6进行数据整理分析和作图，计算IC50。其中，使用的阴性对照抗体是不结合人IL-4R的人源化抗体。
如图8所示，Dupilumab、4-2-Humanized-IgG4、4-2-Humanized-IgG1和4-2-Humanized-IgG1-SELF均能有效阻断IL-4和IL-4R的相互作用，IC50分别为137.9ng/ml、140.3ng/ml、138.3ng/ml和144.1ng/ml。以上结果表明，Dupilumab、4-2-Humanized-IgG4、4-2-Humanized-IgG1和4-2-Humanized-IgG1-SELF阻断IL-4和IL-4R相互作用的能力基本一致，无明显差别。
实施例6.2 IL-4诱导TF-1细胞增殖的抑制作用
本实施例采用TF-1细胞检测4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF对IL-4诱导的TF-1细胞增殖的抑制作用。
实验方法参见实施例1.1。其中，使用的阴性对照不含IL-4但含有不结合人IL-4R的对照抗体；阳性对照含有IL-4和不结合人IL-4R的对照抗体。
如图9所示，Dupilumab、4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF抑制IL-4诱导TF-1细胞增殖的IC50分别是1008ng/ml、667.7ng/ml、2782ng/ml和291.9ng/ml。功能活性强弱排序如下：4-2-Humanized-IgG1-SELF>4-2-Humanized-IgG1>Dupilumab>4-2-Humanized-IgG4。
实施例6.3 IL-4诱导PBMC表达CD23的抑制作用
IL-4能够诱导人外周血单个核细胞(PBMC)表达CD23分子，CD23分子是一种与过敏反应相关的低亲和力IgE受体。本实施例采用PBMC检测4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF对IL-4诱导PBMC表达CD23的抑制作用。
用Histopaque从人全血中分离PBMC；细胞计数后用RMPI-1640完全培养基(含有10％胎牛血清)接种至96孔圆底细胞培养板中，每孔2E5个细胞/150μl；将IL-4用RMPI-1640稀释至80ng/ml，用此培养基梯度稀释抗IL-4R抗体；在上述96孔板中加入50μl用RPMI-1640梯度稀释的抗IL-4R抗体和IL-4的混合液；将96孔板置于37℃培养箱中孵育2天；2天后用PE Mouse Anti-Human CD23荧光标记抗体将上述细胞染色，染色后加入4％的多聚甲醛固定；300g离心，洗涤后在流式细胞仪上(CytoFLEX Cytometer System，购自Beckman Coulter) 检测PE通道的荧光强度，用FCS/SSC或CD14荧光标记强度确定单核细胞群体；用流式细胞仪自带软件处理实验数据并计算平均荧光强度；用GraphPad Prism6进行数据整理分析和作图，计算IC50。
第一次实验结果如图10-1所示，Dupilumab、4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF均能有效抑制IL-4诱导PBMC表达CD23，IC50分别为90.91ng/ml、29.38ng/ml、232.1ng/ml和7.404ng/ml。第二次重复实验结果如图10-2所示，Dupilumab、4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF抑制IL-4诱导PBMC表达CD23的IC50分别为20.65ng/ml、3.56ng/ml、34.00ng/ml和0.49ng/ml。两次独立重复实验的功能活性强弱排序一致如下：4-2-Humanized-IgG1-SELF>4-2-Humanized-IgG1>Dupilumab>4-2-Humanized-IgG4。其中，两次实验使用的血液来自不同捐献者。
为了进一步验证上述实验结果，用Histopaque从6份来自不同捐献者的血液中分离PBMC；细胞计数后用RMPI-1640完全培养基接种至96孔圆底细胞培养板中，每孔2E5个细胞；然后加入抗IL-4R抗体(终浓度25ng/ml)和IL-4(终浓度20ng/ml)；将96孔板置于37℃培养箱中孵育2天；2天后按照上述实验方法用流式细胞仪分析CD23的表达。其中，使用的阴性对照不含IL-4但含有不结合人IL-4R的对照抗体；阳性对照含有IL-4和不结合人IL-4R的对照抗体。实验结果如图10-3所示，4-2抗体的功能活性强弱排序如下：4-2-Humanized-IgG1-SELF>4-2-Humanized-IgG1>4-2-Humanized-IgG4，并且具有统计学上的显著性差异(统计方法为T-test，P<0.05认为具有统计学上的显著性差异)。
实施例6.4 IL-4诱导PBMC分泌IgE的抑制作用
IL-4能够诱导人外周血单个核细胞(PBMC)分泌IgE分子，IgE分子与过敏反应相关高度相关。本实施例采用PBMC检测4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF对IL-4诱导PBMC分泌IgE的抑制作用。
用Histopaque从人全血中分离PBMC；细胞计数后用RMPI-1640完全培养基(含有10％胎牛血清)接种至96孔圆底细胞培养板中，每孔2E5个细胞/150μl；将IL-4用RMPI-1640稀释至80ng/ml，并同时补加地塞米松至终浓度400ng/ml，用此培养基梯度稀释IL-4R抗体；在上述96孔板中加入50μl用RPMI-1640梯度稀释的抗IL-4R抗体、IL-4和地塞米松的混合溶液；将96孔板置于37℃培养箱中孵育14天；14天后取细胞培养上清，Anti-HumanIgEantibody作为捕获抗体，Biotin-anti-HumanIgE antibody作为检测抗体，利用双抗夹心法检测细胞培养上清中IgE的分泌量；用GraphPad Prism6进行数据整理分析和作图，计算IC50。
第一次实验结果如图11-1所示，Dupilumab、4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF均能有效抑制IL-4诱导PBMC分泌IgE，IC50分别为995.7ng/ml、448.0ng/ml、1953ng/ml和50.85ng/ml。第二次重复实验结果如图11-2所示，Dupilumab、 4-2-Humanized-IgG1、4-2-Humanized-IgG4和4-2-Humanized-IgG1-SELF抑制IL-4诱导PBMC分泌IgE的IC50分别是189.4ng/ml、61.34ng/ml、412.9ng/ml和26.61ng/ml。两次独立重复实验的功能活性强弱排序一致如下：4-2-Humanized-IgG1-SELF>4-2-Humanized-IgG1>Dupilumab>4-2-Humanized-IgG4。其中，两次实验使用的血液来自不同捐献者。
实施例7抗人IL-4R人源化抗体的药代动力学研究
本实施例采用大鼠进行静脉注射(IntravenousInjection，I.V.)检测4-2-Humanized-IgG4的药代动力学。
每组四只大鼠，体重200g左右，每只大鼠通过静脉注射剂量为1mg的抗IL-4R抗体；分别在给药后的特定时间眼眶取血，血液自然凝固后离心取血清。血清中抗体药物浓度的测量方法如下：用IL-4R-ECD-FLAG包被ELISA板，浓度为100ng/ml，100μl/孔；包被完毕后用PBST+1％BSA封闭；然后加入适当稀释的大鼠血清；经过一段时间的孵育之后，洗板并最终加入HRP标记的羊抗人二抗(购自Sigma；该抗体经过种属交叉吸附处理，不识别大鼠内源抗体)；再经过一段时间的孵育之后，洗板并加入显色液进行显色，用终止液终止显色反应后检测OD450；用标准曲线将OD450换算成抗体浓度；用GraphPad Prism6进行数据整理分析和作图；用Phoenix软件计算抗体药物在大鼠体内的半衰期。
如图12所示，静脉注射药代动力学结果显示，4-2-Humanized-IgG4抗体在大鼠体内的半衰期与阳性对照抗体Dupilumab相当，分别是282.4小时和281.6小时。
实施例8抗人IL-4R人源化抗体的表位分析
根据文献报道(Zhang J L,Simeonowa I,Wang Y,et al.The high-affinity interaction of human IL-4 and the receptor α chain is constituted by two independent binding clusters[J].Journal of molecular biology,2002,315(3):399-407.)，位于IL-4Rα胞外域(氨基酸序列如SEQ ID NO：1所示)上的对结合IL-4比较重要的氨基酸残基如下：F13、M14、S15、L39、F41、L42、L43、D66、D67、V68、D69、S70、D72、N73、Y74、K91、P92、S93、E94、D125、N126、Y127、L128、Y129和Y183。本实施例用定点诱变的方法，将上述氨基酸残基分别突变成丙氨酸，构建一系列的IL-4Rα突变体。在HEK293E细胞中分别表达上述IL-4Rα突变体，用镍亲和层析法纯化相应突变体。用纯化所得的IL-4Rα突变体包被酶标板(10ng/孔)，然后用ELISA法检测4-2-Humanized-IgG4(4-2-Hu-IgG4)和Dupilumab对各个IL-4Rα的突变体的结合强度。实验方法参见实施例1.2。区别在于将IL-4R-ECD-hFc更换为上述IL-4Rα突变体，并将HRP标记的羊抗鼠二抗更换为HRP标记的羊抗人二抗。结果如表2所示。
表2. 4-2-Hu-IgG4和Dupilumab对IL-4Rα突变体的结合作用
Figure PCTCN2019101628-appb-000004
Figure PCTCN2019101628-appb-000005
注：“+”表示抗体对突变体的结合明显变弱(与野生型IL-4Rα相比，EC50升高超过三倍)；“-”表示抗体对突变体的结合无明显变化(与野生型IL-4Rα相比，EC50升高不超过三倍)。
实验结果显示，4-2-Hu-IgG4和Dupilumab对L39A、D72A和Y74A这三个IL-4Rα突变体的结合作用明显变弱，表明L39、D72和Y74这三个氨基酸残基对4-2-Hu-IgG4和Dupilumab有效结合IL-4Rα是必要的；4-2-Hu-IgG4对F41A、L42A和L43A这三个IL-4Rα突变体的结合作用明显变弱，但Dupilumab对这三个IL-4Rα突变体的结合作用没有明显变化，表明F41、L42和L43这三个氨基酸残基对4-2-Hu-IgG4有效结合IL-4Rα是必要的，而对Dupilumab有效结合IL-4Rα是不必要的。上述结果说明，4-2-Hu-IgG4和Dupilumab表位不同。

Claims (20)

  1. 结合人IL-4R的抗体或其抗原结合片段，其特征在于，包括：
    (a)重链互补决定区HCDR1、HCDR2、HCDR3，所述的HCDR1的氨基酸序列如SEQ ID NO：6所示，所述的HCDR2的氨基酸序列如SEQ ID NO：7所示，所述的HCDR3的氨基酸序列如SEQ ID NO：8所示，和
    (b)轻链互补决定区LCDR1、LCDR2、LCDR3，所述的LCDR1的氨基酸序列如SEQ ID NO：9所示，所述的LCDR2的氨基酸序列如SEQ ID NO：10所示，所述的LCDR3的氨基酸序列如SEQ ID NO：11所示。
  2. 如权利要求1所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的抗体为单克隆抗体或多克隆抗体。
  3. 如权利要求2所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的抗体为单克隆抗体。
  4. 如权利要求1所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的抗体为鼠源抗体、嵌合抗体或人源化抗体。
  5. 如权利要求1所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的抗体为IgG1型抗体或IgG4型抗体。
  6. 如权利要求1所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的抗原结合片段包括Fab片段、F(ab’)2片段、Fv片段、单链抗体及单域抗体。
  7. 如权利要求1所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的结合人IL-4R的抗体或其抗原结合片段能够阻断IL-4与IL-4R之间的相互作用。
  8. 如权利要求1所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的结合人IL-4R的抗体或其抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：3所示，轻链可变区的氨基酸序列如SEQ ID NO：5所示；或所述的结合人IL-4R的抗体或其抗原结合片段的重链可变区的氨基酸序列如SEQ ID NO：13所示，轻链可变区的氨基酸序列如SEQ ID NO：15所示。
  9. 如权利要求8所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的结合人IL-4R的抗体或其抗原结合片段的重链的氨基酸序列如SEQ ID NO：16，轻链的氨基酸序列如SEQ ID NO：18所示；或所述的结合人IL-4R的抗体或其抗原结合片段的重链的氨基酸序列如SEQ ID NO：17所示,轻链的氨基酸序列如SEQ ID NO：18所示；或所述的结合人IL-4R的抗体或其抗原结合片段的重链的氨基酸序列如SEQ ID NO：19所示，轻链的氨基酸序列如SEQ ID NO：18所示。
  10. 如权利要求1-9中任一项所述的结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述的结合人IL-4R的抗体或其抗原结合片段包括Fc段，所述的Fc段包括如下突变位点：S267E和L328F。
  11. 分离的核苷酸，其特征在于，所述的核苷酸编码如1-10中任一项所述的结合人IL-4R 的抗体或其抗原结合片段。
  12. 如权利要求11所述的核苷酸，其特征在于，所述的核苷酸具有如SEQ ID NO：2所示的编码重链可变区的核苷酸序列，和如SEQ ID NO：4所示的编码轻链可变区的核苷酸序列；或所述的核苷酸具有如SEQ ID NO：12所示的编码重链可变区的核苷酸序列，和如SEQ ID NO：14所示的编码轻链可变区的核苷酸序列。
  13. 表达载体，其特征在于，所述的表达载体含有如权利要求11或12所述的核苷酸。
  14. 宿主细胞，其特征在于，所述的宿主细胞含有如权利要求13所述的表达载体。
  15. 如权利要求1-10中任一项所述的结合人IL-4R的抗体或其抗原结合片段的制备方法，所述方法包括以下步骤：
    (a)在表达条件下，培养如权利要求14所述的宿主细胞，从而表达所述的结合人IL-4R的抗体或其抗原结合片段；
    (b)分离并纯化(a)所述的结合人IL-4R的抗体或其抗原结合片段。
  16. 药物组合物，其特征在于，所述药物组合物含有如权利要求1-10中任一项所述的结合人IL-4R的抗体或其抗原结合片段和药学上可接受的载体。
  17. 如权利要求1-10中任一项所述的结合人IL-4R的抗体或其抗原结合片段或如权利要求16所述的药物组合物在制备治疗与IL-4R过表达相关的疾病的药物中的用途。
  18. 如权利要求17所述的用途，其特征在于，所述的与IL-4R过表达相关的疾病包括特应性皮炎、哮喘、过敏反应、嗜酸性食道炎、皮肤感染、鼻息肉症。
  19. 用于提高如权利要求1-10中任一项所述的结合人IL-4R的抗体或其抗原结合片段的活性的方法，所述的结合人IL-4R的抗体或其抗原结合片段包括Fc段，其特征在于，所述的Fc段包括如下突变位点：S267E和L328F。
  20. 一种结合人IL-4R的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段与IL-4Rα胞外域上的下述氨基酸残基特异结合：L39、F41、L42、L43、D72和Y74。
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