WO2021213329A1 - 与人IL-4Rα中特定表位结合的抗体及其应用 - Google Patents

与人IL-4Rα中特定表位结合的抗体及其应用 Download PDF

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WO2021213329A1
WO2021213329A1 PCT/CN2021/088139 CN2021088139W WO2021213329A1 WO 2021213329 A1 WO2021213329 A1 WO 2021213329A1 CN 2021088139 W CN2021088139 W CN 2021088139W WO 2021213329 A1 WO2021213329 A1 WO 2021213329A1
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amino acid
acid sequence
sequence shown
antibody
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French (fr)
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杨欣
张立民
蒋洁
夏静
潘武宾
郑伟
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苏州康乃德生物医药有限公司
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Priority to EP21793385.2A priority Critical patent/EP4141031A4/en
Priority to JP2022564230A priority patent/JP2023525665A/ja
Priority to AU2021258884A priority patent/AU2021258884A1/en
Priority to US17/997,025 priority patent/US20230167180A1/en
Priority to CN202180030276.2A priority patent/CN115515979A/zh
Priority to KR1020227039724A priority patent/KR20230006837A/ko
Publication of WO2021213329A1 publication Critical patent/WO2021213329A1/zh

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    • 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
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5406—IL-4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to antibodies capable of binding to specific epitopes in human IL-4R ⁇ and their applications.
  • IL-4R Human interleukin 4 receptor
  • IL-4R Human interleukin 4 receptor
  • IL-4R binds to ligands such as interleukin 4 (IL-4) and interleukin 13 (IL-13), it can exert a variety of immunomodulatory effects, such as promoting the differentiation of Th2 cells, regulating the secretion of IgE antibodies by B cells, and Stimulate alternative activation of macrophages, etc.
  • IL-4R is a heterodimer composed of two polypeptide chains, one of the ⁇ chain (IL-4R ⁇ ) has a high affinity for IL-4; and, the IL-4R ⁇ chain will interact with the cell surface of IL-13
  • the receptor alpha chain (IL-13R ⁇ ) forms another form of IL-4R heterodimer, which also has a high affinity for IL-13.
  • IL-4R ⁇ can produce a soluble form of protein (sIL-4R ⁇ ), this soluble form of protein can inhibit a series of inflammation-related signal transduction, such as IL-4 mediated cell proliferation and T cell mediated IL-5 up-regulation and so on. Therefore, blocking antibodies targeting this protein can help treat and alleviate allergic rhinitis, sinusitis, asthma, or eczema.
  • both IL-4 and IL-13 are cytokines with a very broad-spectrum biological activity, involving many kinds of inflammation-related reactions, most of which are activated T cells, monocytes, mast cells, basophils and Eosinophils are produced. These two kinds of interleukins have many commonalities in biological functions.
  • IL-4 and IL-13 play a very important role in mediating the immune response of autoimmune diseases, allergic diseases, tumors and other diseases, and they have always been one of the research hotspots of people's attention.
  • sIL-4R ⁇ plays a dominant role in IL-4 binding, and it also involves other cytokines, there are many studies on antibodies related to sIL-4R ⁇ , and human monoclonal antibodies against this target have been clinically proven. It can effectively alleviate and treat diseases such as asthma and atopic dermatitis. However, there are few studies on the epitope of sIL-4R ⁇ in the immune effect, and people still do not know its specific epitope.
  • the inventors of the present invention conducted research on the epitope of sIL-4R ⁇ , and screened human sIL-4R ⁇ based on the newly obtained antibodies and the species specificity of humans and monkeys when they interact with sIL-4R ⁇ . Mutations were carried out at some of the sites, and related experiments were carried out, and several key epitope binding amino acids were found. These epitope results are of great significance for the further study of sIL-4R ⁇ and related antibodies.
  • an object of the present invention is to provide an antibody or antigen-binding fragment thereof that binds to human interleukin 4 receptor (IL-4R). -4R ⁇ ) in combination with specific epitopes.
  • IL-4R human interleukin 4 receptor
  • another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding a key domain in the antibody or antigen-binding fragment thereof of the present invention; Nucleic acid molecule carrier; providing host cells containing the vector; providing a method for preparing the antibody or antigen-binding fragment thereof; providing a pharmaceutical composition containing the antibody or antigen-binding fragment thereof; providing the antibody or antigen-binding fragment thereof Pharmaceutical use of the fragment or the pharmaceutical composition.
  • the present invention Based on the specific epitope in the ⁇ chain of human IL-4R provided by the present invention, the present invention also provides the epitope in the preparation of a binding agent of human IL-4R or blocking of the human IL-4/IL-13 signal transduction pathway Use in an agent or use of the epitope in evaluating the efficacy of the binding agent or blocking agent.
  • the present invention provides an antibody or antigen-binding fragment thereof that binds to human interleukin 4 receptor (IL-4R), the epitope to which the antibody or antigen-binding fragment binds is located in the ⁇ chain of human IL-4R ( IL-4R ⁇ ), and the epitope includes one of the amino acid residues D92, V94, D97, L67, L68, A96, H156, C207, Q63 and L64 selected from the amino acid sequence shown in SEQ ID NO:1 or Multiple.
  • IL-4R human interleukin 4 receptor
  • the epitope includes one or more of the amino acid residues D92, V94 and D97 in the amino acid sequence shown in SEQ ID NO:1.
  • the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1:
  • the binding epitope of the antibody or antigen-binding fragment thereof provided by the present invention to human IL-4R ⁇ includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1:
  • the antibody or antigen-binding fragment thereof provided by the present invention does not have species cross-binding activity with monkey IL-4R; preferably, it does not bind monkey IL-4R; more preferably, it does not bind monkey IL-4R ⁇ ; further preferably , Does not bind to the amino acid sequence shown in SEQ ID NO: 2.
  • the present invention provides an antibody or antigen-binding fragment thereof that binds to human interleukin 4 receptor (IL-4R), and the epitope bound by the antibody or antigen-binding fragment is located in human IL- 4R ⁇ chain (IL-4R ⁇ ), and the epitope includes one or more of amino acid residues L67, L68 and A96 in the amino acid sequence shown in SEQ ID NO:1.
  • IL-4R human interleukin 4 receptor
  • IL-4R ⁇ human IL- 4R ⁇ chain
  • the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1:
  • the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1:
  • the epitope of the present invention includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1:
  • the binding of the antibody of the present invention to the antigen IL-4R ⁇ is detected, and it is found that the EC50 of the binding activity of the antibody of the present invention to sIL-4R ⁇ does not exceed 50ng/ml, 30ng/ml, 20ng/ml or 10ng/ml. And it was found that the antibody of the present invention did not show binding activity with 100ul ⁇ 0.5ug/ml coated sIL-4R ⁇ -97 under 100ul ⁇ 2500ng/ml, and the sIL-4R ⁇ -97 was the amino acid at position 97 of sIL-4R ⁇ Residue D is mutated to A.
  • the antibody of the present invention does not show binding activity to at least one mutant antigen selected from sIL-4R ⁇ -QSMDH and sIL-4R ⁇ -63/64 under 100ul ⁇ 2500ng/ml coated with 100ul ⁇ 0.5ug/ml Or the binding activity is significantly reduced.
  • the binding activity is measured by the operation procedure described in the section "(1) Binding Activity Detection Method" in the "Best Mode for Carrying Out the Invention" of the present invention.
  • the antibody of the present invention can block the binding of IL-4 or IL-13 to IL-4R, and therefore can be used as a blocking agent for the binding of IL-4 or IL-13 to IL-4R, or as IL-4 /IL-13 signal transduction pathway blocker.
  • the antibodies or antigen-binding fragments provided by the present invention comprise the following combinations of the heavy chain complementarity determining regions HCDR1, HCDR2, HCDR3 and the light chain complementarity determining regions LCDR1, LCDR2, LCDR3:
  • HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 35, 36, 37; and LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 53, 54, 55;
  • HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 52, 36, 37 in sequence
  • LCDR1, LCDR2, LCDR3 shown in SEQ ID NO: 53, 54, 55 in sequence.
  • the combination of light and heavy chain CDRs provided by the present invention is derived from the antibody or fragments thereof of the present invention. Based on the amino acid sequence of the variable region contained in a given antibody or antigen-binding fragment thereof, those skilled in the art can routinely determine the CDRs contained therein. For example, according to a specific embodiment of the present invention, the IMGT tool is used to divide the CDRs in the amino acid sequence of the variable region. The combination of light and heavy chain CDRs divided by methods known in the art is also encompassed within the scope of the present invention.
  • the heavy chain variable region comprises a sequence selected from the group consisting of: shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, The amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27 or have at least 75% identity with the amino acid sequence
  • SEQ ID NO: 3 SEQ ID NO: 7
  • SEQ ID NO: 9 amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27 or have at least 75% identity with the amino acid sequence
  • SEQ ID NO: 11 amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27 or have at least 75% identity with the amino acid sequence
  • SEQ ID NO: 11 amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID
  • the light chain variable region comprises a sequence selected from the group consisting of: shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ The amino acid sequence of ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 33, or SEQ ID NO: 34 or an amino acid sequence having at least 75% identity with the amino acid sequence.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 3; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 4;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 7; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 8;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 9; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 10;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 12;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 16;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 17; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 18;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 19; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 20;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 34;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 33;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 27; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 34;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23; and, the light chain may The variable region comprises the amino acid sequence shown in SEQ ID NO: 33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 33; or
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 23; and, the light chain may The variable region includes the amino acid sequence shown in SEQ ID NO: 28 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof of the present invention includes at least a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs and an interspaced framework region (FR), and the arrangement of each domain It is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 the arrangement of each domain It is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • at most 25% difference in the amino acid sequence caused by the "at least 75% identity" may exist in any framework region in the heavy chain variable region or the light chain variable region, or in the present invention.
  • the difference can be caused by amino acid deletion, addition or substitution at any position, wherein the substitution can be a conservative substitution or a non-
  • the antibody or antigen-binding fragment thereof provided by the present invention binds to human interleukin 4 receptor (IL-4R), preferably binds to the ⁇ chain of human IL-4R (IL-4R ⁇ ), and more preferably binds to human soluble IL -4R ⁇ (sIL-4R ⁇ ).
  • IL-4R human interleukin 4 receptor
  • IL-4R ⁇ ⁇ chain of human IL-4R
  • sIL-4R ⁇ human soluble IL -4R ⁇
  • the antibody provided by the present invention may be a monoclonal antibody or a single chain antibody.
  • the antibody is a murine antibody, a chimeric antibody or a fully or partially humanized antibody.
  • the antigen-binding fragment is a fragment that the antibody can specifically bind to a target.
  • the antigen-binding fragment is an antibody Fab fragment, Fab' fragment, F(ab')2 fragment, or Fv fragment (e.g., scFv).
  • the antibody or antigen-binding fragment thereof further comprises a constant region of human or murine origin, preferably a heavy chain constant region (CH) and/or light chain constant region (CL) of human or murine origin.
  • the antibody molecule or antigen-binding fragment thereof comprises a heavy chain and a light chain.
  • the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG or IgM, such as a human subtype of IgA, IgD, IgE, IgG or IgM, more preferably a human IgG1, IgG2, IgG3 or IgG4 subtype.
  • the antibody or antigen-binding fragment thereof provided by the present invention comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region, for example, the heavy chain constant region is an IgG (such as IgG1). Or IgG4) type, and the light chain constant region is ⁇ type.
  • the heavy chain constant region of the monoclonal antibody comprises an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71, or an amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 70 or SEQ ID NO.
  • the amino acid sequence encoded by the nucleic acid sequence of 71 has an amino acid sequence that is at least 75% identical; the light chain constant region of the monoclonal antibody contains the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 72 or the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 72.
  • the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 72 has an amino acid sequence with at least 75% identity.
  • At least 75% identity is the identity of any percentage number between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
  • the present invention also provides a nucleic acid molecule comprising a core encoding a heavy chain variable region, a light chain variable region, a heavy chain, and/or a light chain in the antibody or antigen-binding fragment thereof of the present invention. Nucleotide sequence.
  • the nucleic acid molecule of the present invention can be cloned into a vector, and then transformed or transfected into a host cell. Therefore, in another aspect, the present invention also provides a vector, which contains the nucleic acid molecule of the present invention.
  • the vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
  • the vector or nucleic acid molecule of the present invention can be used to transform or transfect host cells for preservation or antibody expression and other purposes. Therefore, in yet another aspect, the present invention provides a host cell that contains the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and/or vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
  • the antibodies or antigen-binding fragments thereof provided by the present invention can be obtained by any method known in the art.
  • the heavy chain variable region and/or light chain variable region of the antibody may be obtained first from the nucleic acid molecule provided by the present invention, or the heavy chain and/or light chain of the antibody may be obtained, and then combined with any of the antibody Select other domains to assemble into an antibody; or, in the host cell provided by the present invention to allow expression of the heavy chain variable region and/or light chain variable region of the antibody or the heavy chain and/or light chain of the antibody to assemble
  • the host cell is cultured.
  • the method further includes the step of recovering the produced antibody.
  • the antibodies or antigen-binding fragments thereof, nucleic acid molecules, vectors and/or host cells provided by the present invention can be included in the composition, more specifically included in a pharmaceutical composition, such as a pharmaceutical preparation, so as to be used for each according to actual needs.
  • a pharmaceutical composition such as a pharmaceutical preparation
  • the present invention also provides a composition, preferably a pharmaceutical composition, which comprises the antibody or fragment thereof, nucleic acid molecule, vector and/or host cell of the present invention, and optionally Pharmaceutically acceptable excipients.
  • the above-mentioned pharmaceutical composition can be made into a certain dosage form, preferably oral administration and parenteral administration (including subcutaneous, intramuscular and intravenous), including but not limited to solid dosage form, liquid dosage form, semi-liquid dosage form, aerosol or Suppositories, etc.
  • dosage forms suitable for oral administration include tablets, capsules, granules, powders, pills, powders, lozenges, syrups or suspensions; dosage forms suitable for parenteral administration include aqueous or non-aqueous solutions or Emulsion, such as subcutaneous injection.
  • the pharmaceutical composition according to the present invention can be administered via a parenteral or non-parenteral (enteric) route that is therapeutically effective for antibody drugs.
  • the pharmaceutical composition of the present invention is formed into a formulation containing pharmaceutically acceptable excipients (for example, a vehicle), and administered systemically or locally.
  • the present invention also provides the use of the antibody or its antigen-binding fragment, nucleic acid molecule, vector, host cell and/or composition in the preparation of medicine.
  • the drug is used to prevent, treat or ameliorate diseases or disorders related to human interleukin 4 receptor (IL-4R) or to human interleukin 4 (IL-4) or human interleukin 13 (IL-13) diseases or disorders related to signal transduction pathways.
  • IL-4R human interleukin 4 receptor
  • IL-4 human interleukin 4
  • IL-13 human interleukin 13
  • the present invention also provides a method for preventing, treating or ameliorating a disease or disorder, the method comprising administering (e.g., a therapeutically effective amount) of the antibody or antigen-binding fragment thereof of the present invention to a subject in need thereof , Nucleic acid molecules, vectors, host cells and/or compositions.
  • the disease or disorder is related to human interleukin 4 receptor (IL-4R) or is related to human interleukin 4 (IL-4) or human interleukin 13 (IL-13) signaling
  • IL-4R human interleukin 4 receptor
  • IL-4R human interleukin 4 receptor
  • IL-13 human interleukin 13
  • the diseases or disorders include autoimmune diseases, allergic diseases, tumors or cancers.
  • the disease or disorder includes an autoimmune disease or an allergic disease, such as dermatitis (e.g., atopic dermatitis), asthma (e.g., inflammatory asthma, such as type 2 inflammatory asthma), chronic esophagitis (e.g., eosinophilic asthma) Granulocyte esophagitis), eczema, rhinitis (such as allergic rhinitis), nasal polyps (such as chronic sinusitis with nasal polyps), conjunctivitis, inflammatory bowel disease (such as ulcerative colitis, Crohn's disease), inflammatory Neuropathy, arthritis (e.g. rheumatoid arthritis), multiple sclerosis, lupus erythematosus, psoriasis or insulin and non-insulin dependent diabetes and other immune or allergy related diseases.
  • dermatitis e.g., atopic dermatitis
  • asthma e.g., inflammatory asthma, such as type 2 inflammatory asthma
  • the disease or disorder includes tumors or cancers, such as bowel cancer, melanoma, skin cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, mesothelioma, prostate cancer, Bladder cancer, anal cancer, glioblastoma, astrocytoma, liver cancer, kidney cancer, esophageal cancer, stomach cancer, lung cancer, head and neck cancer, myeloma, bone cancer, AIDS-related Kaposi's sarcoma, Hodgkin’s or non-Hodgkin’s lymphoma, prostate cancer, prostate tumor, lymphoma and pancreatic cancer, etc.
  • tumors or cancers such as bowel cancer, melanoma, skin cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, testicular cancer, mesothelioma, prostate cancer, Bladder cancer, anal cancer, glioblastoma,
  • the medicine is used to prevent, treat or ameliorate one or more of the above-mentioned disease types.
  • the subject is a mammal, preferably a primate; more preferably, the subject is a human.
  • the present invention Based on the specific epitope in the ⁇ chain of human IL-4R (IL-4R ⁇ ) provided by the present invention, the present invention also provides the use of the specific epitope to screen human IL-4R binding agents or human IL-4/IL- 13 Use in a signal transduction pathway blocker or use the specific epitope to evaluate the efficacy of the binding agent or blocker.
  • the epitope includes one or more of amino acid residues D92, V94, D97, L67, L68, A96, H156, C207, Q63 and L64 selected from the amino acid sequence shown in SEQ ID NO: 1. .
  • the epitope includes one or more of the amino acid residues D92, V94 and D97 in the amino acid sequence shown in SEQ ID NO:1.
  • the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO: 1:
  • the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1: (i) Q63 and L64.
  • the epitope includes one or more of the amino acid residues L67, L68 and A96 in the amino acid sequence shown in SEQ ID NO:1.
  • the epitope includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1:
  • the epitope includes the following amino acid residues in the helium acid sequence shown in SEQ ID NO: 1:
  • the epitope of the present invention includes the following amino acid residues in the amino acid sequence shown in SEQ ID NO:1:
  • the present invention provides a binding agent that specifically binds to human interleukin 4 receptor (IL-4R) or specifically blocks human interleukin 4 (IL-4) or human interleukin 4 (IL-4) or human interleukin 4 (IL-4).
  • a screening method for a blocker of the IL-13 signal transduction pathway comprising the following steps: contacting the reagent to be screened with human IL-4R or a part thereof, and then detecting whether the reagent to be screened binds to the present The epitope described in the invention. When it is detected that the reagent to be screened binds to the epitope of the present invention, it is confirmed to be the binding agent or blocking agent.
  • the present invention also provides a method for evaluating the efficacy of a reagent, which includes the following steps: contacting the reagent to be screened with human interleukin 4 receptor (IL-4R) or a part thereof, and then detecting State whether the reagent to be screened binds to the epitope described in the present invention.
  • IL-4R human interleukin 4 receptor
  • the reagent to be screened binds to the epitope of the present invention, it is confirmed that it is a binding agent that specifically binds to human interleukin 4 receptor (IL-4R) or specifically blocks human interleukin 4 (IL- 4) Or a blocker of the human interleukin 13 (IL-13) signal transduction pathway, which in turn has the effect of preventing, treating or improving the disease or disorder of the present invention.
  • IL-4R human interleukin 4 receptor
  • IL- 4 human interleukin 4
  • IL-13 human interleukin 13
  • the reagent to be screened is an antibody, such as a polyclonal antibody or a monoclonal antibody. More preferably, the antibody is obtained by immunizing an animal with human IL-4R or a part thereof as an immunogen.
  • the detection of whether the reagent to be screened binds to the epitope of the present invention can be carried out as follows: by site-directed mutagenesis of human IL-4R or part thereof, Make one or more amino acid residues included in the epitope provided by the present invention mutate, and compare the binding activity or affinity of the reagent to be screened with the human IL-4R before and after the mutation or a part thereof, when compared with the mutated human IL-4R or When part of its binding activity or affinity decreases or even loses, it is determined that the reagent binds to the epitope of the present invention.
  • the screening method or pharmacodynamic evaluation method provided by the present invention may include the following steps:
  • binding activity or affinity has significantly decreased or lost. For example, when the binding activity or affinity of the reagent to be screened to the mutant of human IL-4R or part thereof is 1/10 of the binding activity or affinity before mutation, it can be determined that the binding activity or affinity is significantly decreased.
  • one or more amino acid residues included in the epitope are mutated to alanine or mutated to the amino acid residues at the corresponding positions of the monkey interleukin 4 receptor to obtain human Mutants of IL-4R or parts thereof.
  • the part of human IL-4R used in the method of the present invention is the ⁇ chain of human IL-4R, and further is human sIL-4R ⁇ . Therefore, it is preferable to use human IL-4R ⁇ and obtain a mutant of human IL-4R ⁇ to perform the method. Further, human sIL-4R ⁇ can be used for the method, and one or more of the human sIL-4R ⁇ muteins listed in Table 3 below are used as the mutants in step 1).
  • human IL-4R ⁇ refers to NCBI reference sequence NP_000409.1; the amino acid sequence of human sIL-4R ⁇ is shown in SEQ ID NO: 1; the amino acid sequence of monkey sIL-4R ⁇ is shown in SEQ ID NO: 2.
  • the binding epitope of the antibody or its antigen-binding fragment provided by the present invention to human interleukin 4 receptor (IL-4R) is located in the ⁇ chain of human IL-4R (IL-4R ⁇ ), and the epitope includes It is selected from one or more of amino acid residues D92, V94, D97, L67, L68, A96, H156, C207, Q63 and L64 in the amino acid sequence shown in SEQ ID NO:1.
  • the present invention firstly clarified the key site of anti-IL-4R antibody binding to IL-4R.
  • the present invention provides an anti-human IL-4R, particularly IL-4R ⁇ antibody with a novel IL-4R binding epitope, which can be used as a novel and effective antibody against human IL-4R, particularly IL-4R ⁇ .
  • the present invention provides another way to develop potential antibodies or drugs against IL-4R, especially human IL-4R ⁇ .
  • Figure 1 shows the sequence alignment results of human IL-4R ⁇ (sequence_1; SEQ ID NO: 1) and monkey IL-4R ⁇ (sequence_0; SEQ ID NO: 2).
  • Fig. 2 shows the stability of the antibody of the present invention, wherein Fig. 2A shows the result after storage at 40°C for 2 weeks, and Fig. 2B shows the result after storage at 40°C for 4 weeks.
  • epitope refers to the amino acid residues of the antigen corresponding to the binding site when the antibody or its fragment binds to the antigen and its corresponding position in the antigen sequence; the method of detecting the binding activity of "the operation of the process measured mutated amino acid residue of the epitope will result in a position of binding activity decreased more than 10 2 ng / ml or loss of binding activity.
  • Preparation of antigen Use PBS to formulate the antigen into a 100ug/ml solution, and store it in a refrigerator at -20°C.
  • Preparation of the antibody to be tested Use PBS containing 1% BSA to dilute the antibody to be tested to the required concentration: 0, 0.032, 0.16, 0.8, 4, 20, 100, 500, 2500 ng/ml.
  • Preparation of the working solution of the secondary antibody Dilute the mother solution of the secondary antibody (Goat Anti-Human IgG-Fc Secondary Antibody (HRP), sino biological, Cat Number: SSA001) 16000 times with PBS containing 1% BSA, which is the working solution. For example, add 1 ul of the secondary antibody mother solution to 15999 ul of 1% BSA in PBS, and mix up and down repeatedly. It can be scaled up in equal proportions according to the experimental needs to prepare the required volume of the secondary antibody working solution.
  • HRP Goat Anti-Human IgG-Fc Secondary Antibody
  • Preparation of human IL-4 (Invivogen, article number: rhIL-4): Use PBS to prepare a 100ug/ml solution of human IL-4, and store it in a refrigerator at -20°C after being divided into aliquots.
  • Preparation of human IL-13 (Invivogen, article number: rhIL-13): Use PBS to prepare a 100ug/ml solution of human IL-13, and store it in a refrigerator at -20°C after aliquoting.
  • Preparation of the antibody to be tested Dilute the antibody to the required concentration (1000, 200, 40, 8) with DMEM medium (HyClone, Cat No. SH30022.01) containing 10% FBS (Hyclone, Cat No. SV30184.02) , 1.6, 0.32, 0.064, 0.0128, 0.00256, 0ng/ml).
  • HEK Blue IL-4/IL13 (Invivogen, Cat No.hkb-il413) cells in liquid nitrogen, and shake them gently in a 37°C water bath to quickly dissolve them. Transfer the dissolved cell suspension to a 15ml centrifuge tube, add DMEM medium (containing 10% FBS, 10 ⁇ g/ml Blasticidin, 100 ⁇ g/ml Zeocin, 100 ⁇ g/ml Normocin) to 10ml, centrifuge at 800rpm for 5min, and aspirate the supernatant.
  • DMEM medium containing 10% FBS, 10 ⁇ g/ml Blasticidin, 100 ⁇ g/ml Zeocin, 100 ⁇ g/ml Normocin
  • mice were immunized with human sIL-4R ⁇ (SEQ ID NO: 1), and blood was collected to perform the above (1) binding activity and (2) cell functional activity detection. Combining the results of the two tests, the two mice with the best results were selected for fusion.
  • the multiple sets of cell supernatants obtained by the fusion were tested again for the above (1) binding activity and (2) cell functional activity.
  • the parent clone with the highest activity was selected for subcloning, and then (1) binding activity And (2) Cell function activity detection, and finally 14 strains of monoclonal murine antibodies were screened.
  • the 14 clones were sequenced, and they were 9 pairs of antibodies with new sequences.
  • the corresponding heavy chain variable regions and light chain variable regions are shown below; the CDRs divided by the IMGT tool are underlined.
  • Heavy chain variable region (SEQ ID NO: 3; CDR sequence is: SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37)
  • Light chain variable region (SEQ ID NO: 4; CDR sequence is: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)
  • Heavy chain variable region (SEQ ID NO: 5; CDR sequence is: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40)
  • Light chain variable region (SEQ ID NO: 6; CDR sequence is: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58)
  • Heavy chain variable region (SEQ ID NO: 7; CDR sequence is: SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43)
  • Light chain variable region (SEQ ID NO: 8; CDR sequence is: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61)
  • Heavy chain variable region (SEQ ID NO: 9; CDR sequence is: SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46)
  • Light chain variable region (SEQ ID NO: 10; CDR sequence is: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 62)
  • Heavy chain variable region (SEQ ID NO: 11; CDR sequence is: SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 46)
  • Light chain variable region (SEQ ID NO: 12; CDR sequence is: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65)
  • Heavy chain variable region (SEQ ID NO: 13; CDR sequence is: SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40)
  • Light chain variable region (SEQ ID NO: 14; CDR sequence is: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58)
  • Heavy chain variable region (SEQ ID NO: 15; CDR sequence is: SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 46)
  • Light chain variable region (SEQ ID NO: 16; CDR sequence is: SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68)
  • Heavy chain variable region (SEQ ID NO: 17; CDR sequence is: SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 51)
  • Light chain variable region (SEQ ID NO: 18; CDR sequence is: SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 69)
  • Heavy chain variable region (SEQ ID NO: 19; CDR sequence is: SEQ ID NO: 52, SEQ ID NO: 36, SEQ ID NO 37)
  • Light chain variable region (SEQ ID NO: 20; CDR sequence is: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55)
  • 9 chimeric antibodies were further constructed based on the above variable region sequences, and the sequence shown in SEQ ID NO: 71 was used as the coding sequence of the heavy chain constant region, and the sequence shown in SEQ ID NO: 72 was used as the coding sequence of the light chain constant region for expression and purification These 9 chimeric antibodies are named after the mouse antibody name plus Q.
  • the IC50 of the chimeric antibody is detected using the procedure described in "(2) Cell Function Activity Detection Method" above, and the chimeric antibody and human sIL are detected by Biacore -4R ⁇ affinity KD, the results are shown in Table 1 below.
  • the chimeric antibody of clone 14 (Y0188-14Q) was selected for humanization, and 7 humanized heavy chain variable regions and 7 humanized light chain variable regions were obtained.
  • the humanized heavy chain variable region and light chain variable region are shown below; the CDRs divided by the IMGT tool are underlined.
  • Human sIL-4R ⁇ protein and the different sIL-4R ⁇ mutant proteins shown in Table 3 are used as antigens, and 6 humanized high-activity antibodies are used as primary antibodies.
  • the antibodies are detected by the operation described in "(1) Binding Activity Detection Method" above Binding activity with antigen. The results are shown in Table 4.
  • human sIL-4R ⁇ protein and the different sIL-4R ⁇ mutant proteins shown in Table 3 were used as antigens, and 9 chimeric antibodies obtained from the screening were used as primary antibodies, and the binding activity of the antibodies to the antigen was also tested. The results are shown in Table 5.

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Abstract

本发明公开了一种人IL-4R抗体或其抗原结合片段，该抗体或其抗原结合片段特异性结合人IL-4Rα且与人IL-4Rα中特定表位相结合。并且，本发明还提供所述表位在筛选人IL-4R的结合剂或人IL-4/IL-13信号传导通路的阻断剂中的用途或者使用所述特定表位评价所述结合剂或阻断剂的药效中的用途。

Description

与人IL-4Rα中特定表位结合的抗体及其应用
相关申请的交叉引用
本专利申请要求于2020年4月24日提交的申请号为CN202010331685.3的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明涉及生物制药领域，具体而言，本发明涉及能够与人IL-4Rα中特定表位结合的抗体及其应用。
背景技术
人白细胞介素4受体(interleukin 4 receptor，IL-4R)是一种I型跨膜蛋白，其于全身各种组织器官中、尤其是免疫器官中广泛表达，例如扁桃体、阑尾、淋巴结、脾脏和骨髓等。IL-4R与白细胞介素4(IL-4)和白细胞介素13(IL-13)等配体结合后可以发挥多种免疫调节效应，例如促进Th2细胞的分化、调节B细胞分泌IgE抗体以及刺激巨噬细胞替代性激活等。
IL-4R是由两条多肽链组成的异二聚体，其中的一条α链(IL-4Rα)对IL-4有很高的亲和力；并且，IL-4Rα链会与IL-13的细胞表面受体α链(IL-13Rα)组成另一种形式的IL-4R异二聚体，由此对IL-13也有很高的亲和力。
IL-4Rα可产生一种可溶形式的蛋白(sIL-4Rα)，这种可溶形式的蛋白可以抑制一系列炎症相关的信号传导，例如IL-4介导的细胞增殖和T细胞介导的IL-5上调等。因此将该蛋白作为靶点的阻断抗体有助于治疗和缓解过敏性鼻炎、鼻窦炎、哮喘或湿疹等病症。并且，IL-4和IL-13都是具有非常广谱生物学活性的细胞因子，涉及很多种炎症相关的反应，大部分由活化的T细胞、单核细胞、肥大细胞、嗜碱粒细胞和嗜酸粒细胞产生。这两种白细胞介素在生物功能方面有许多共性，其主要的生物作用有刺激TH2细胞的分化增殖、刺激活化B细胞分泌IgE抗体等。研究表明，IL-4和IL-13在介导自身免疫性疾病、过敏性疾病、肿瘤等疾病的免疫反应中有非常重要的作用，一直是人们关注的研究热点之一。
由于sIL-4Rα对IL-4结合起主导所用，并且其还涉及其他细胞因子，因此目前对sIL-4Rα这个靶点相关抗体的研究很多，并且临床上已经证明针对 该靶点的人单克隆抗体可以有效缓解和治疗哮喘、特应性皮炎等疾病。但是，目前对sIL-4Rα在免疫效应中的表位研究较少，人们尚不清楚其具体的表位情况。
发明内容
针对上述技术问题，本发明的发明人对sIL-4Rα的表位进行了研究，根据新获得的抗体及其与sIL-4Rα作用时存在的人和猴的物种特异性，筛选人sIL-4Rα中的一些位点进行了突变，并进行相关实验，找到了若干关键的表位结合氨基酸。这些表位结果对sIL-4Rα以及相关抗体的进一步研究具有重大意义。
因此，本发明的一个目的是提供一种结合人白细胞介素4受体(IL-4R)的抗体或其抗原结合片段，所述抗体或其抗原结合片段与人IL-4R的α链(IL-4Rα)中特定表位相结合。
基于本发明提供的所述抗体或其抗原结合片段，本发明的其他目的是提供一种核酸分子，其包含编码本发明的抗体或其抗原结合片段中关键结构域的核苷酸序列；提供含有核酸分子的载体；提供含有所述载体的宿主细胞；提供所述抗体或其抗原结合片段的制备方法；提供含有所述抗体或其抗原结合片段的药物组合物；提供所述抗体或其抗原结合片段或所述药物组合物的制药用途。
基于本发明提供的人IL-4R的α链中的特定表位，本发明还提供所述表位在制备人IL-4R的结合剂或者人IL-4/IL-13信号传导通路的阻断剂中的用途或者所述表位在评价所述结合剂或者阻断剂的药效中的用途。
本发明的具体方案如下：
一方面，本发明提供一种结合人白细胞介素4受体(IL-4R)的抗体或其抗原结合片段，所述抗体或其抗原结合片段结合的表位位于人IL-4R的α链(IL-4Rα)，且所述表位包括选自SEQ ID NO：1所示氨基酸序列中的氨基酸残基D92、V94、D97、L67、L68、A96、H156、C207、Q63和L64中的一个或多个。
优选地，所述表位包括SEQ ID NO：1所示氨基酸序列中的氨基酸残基D92、V94和D97中的一个或多个。
根据本发明的具体实施方式，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
(1)D92；
(2)D97；
(3)D92、V94和D97；
(4)D92和V94；
(5)D92和D97；或
(6)V94和D97。
和/或，本发明提供的抗体或其抗原结合片段与人IL-4Rα的结合表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
(i)Q63和L64。
并且，本发明提供的抗体或其抗原结合片段不具有与猴IL-4R的种属交叉结合活性；优选地，不结合猴IL-4R；更优选地，不结合猴IL-4Rα；进一步优选地，不结合SEQ ID NO：2所示氨基酸序列。
就上述交叉结合活性而言，本发明提供一种结合人白细胞介素4受体(IL-4R)的抗体或其抗原结合片段，所述抗体或其抗原结合片段结合的表位位于人IL-4R的α链(IL-4Rα)，且所述表位包括SEQ ID NO：1所示氨基酸序列中的氨基酸残基L67、L68和A96中的一个或多个。
优选地，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
①L67和L68；
②L67、L68和A96；或
③A96。
根据本发明的具体实施方式，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
(A)L67、L68、A96、H156和C207；
(B)A96、H156和C207；
(C)L67、L68、H156和C207；
(D)L67、L68、A96和C207；或
(E)L67、L68、A96和H156。
特别优选地，本发明的表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
(P-1)D97、L67和L68；
(P-2)D97、L67、L68、A96和D92；
(P-3)D97、L67、L68和D92；
(P-4)D97、Q63和L64；
(P-5)D97、Q63、L64和D92；
(P-6)D97、Q63、L64、D92和A96；或
(P-7)D97、Q63、L64、L67和L68。
检测本发明的抗体与抗原IL-4Rα的结合，发现本发明的抗体与sIL-4Rα的结合活性EC50不超过50ng/ml、30ng/ml、20ng/ml或10ng/ml。并且发现，本发明的抗体在100ul×2500ng/ml下与包被的100ul×0.5ug/ml的sIL-4Rα-97不显示结合活性，所述sIL-4Rα-97为sIL-4Rα的97位氨基酸残基D被突变为A后所得。进一步还发现，本发明的抗体在100ul×2500ng/ml下与包被的100ul×0.5ug/ml的选自sIL-4Rα-QSMDH和sIL-4Rα-63/64中至少一个突变抗原不显示结合活性或结合活性显著下降。所述结合活性通过本发明的“实施发明的最佳方式”部分“(一)结合活性检测方法”所述操作过程测得。
此外，本发明的抗体可以阻断IL-4或IL-13与IL-4R的结合，因此可以用作IL-4或IL-13与IL-4R结合的阻断剂，或者用作IL-4/IL-13信号传导通路的阻断剂。
就所包含的结构域氨基酸序列而言，本发明提供的抗体或其抗原结合片段包含以下重链互补决定区HCDR1、HCDR2、HCDR3和轻链互补决定区LCDR1、LCDR2、LCDR3的组合：
(1)依次示于SEQ ID NO：35、36、37的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：53、54、55的LCDR1、LCDR2、LCDR3；
(2)依次示于SEQ ID NO：41、42、43的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：59、60、61的LCDR1、LCDR2、LCDR3；
(3)依次示于SEQ ID NO：44、45、46的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：56、57、62的LCDR1、LCDR2、LCDR3；
(4)依次示于SEQ ID NO：44、47、46的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：63、64、65的LCDR1、LCDR2、LCDR3；
(5)依次示于SEQ ID NO：48、49、46的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：66、67、68的LCDR1、LCDR2、LCDR3；
(6)依次示于SEQ ID NO：44、50、51的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：56、57、69的LCDR1、LCDR2、LCDR3；或
(7)依次示于SEQ ID NO：52、36、37的HCDR1、HCDR2、HCDR3； 和，依次示于SEQ ID NO：53、54、55的LCDR1、LCDR2、LCDR3。
本发明提供的轻重链CDR的组合来自本发明的抗体或其片段，基于给定抗体或其抗原结合片段包含的可变区氨基酸序列，本领域技术人员可以常规地确定其中包含的CDR。例如，根据本发明的具体实施方式，采用IMGT工具划分可变区氨基酸序列中的CDR。以本领域已知方法划分得到的轻重链CDR的组合也被涵盖在本发明的范围内。
在本发明提供的抗体或其抗原结合片段中，优选地，所述重链可变区包含选自以下的序列：示于SEQ ID NO：3、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：23或SEQ ID NO：27的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，
优选地，所述轻链可变区包含选自以下的序列：示于SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20、SEQ ID NO：28、SEQ ID NO：33或SEQ ID NO：34的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
根据本发明的具体实施方式，本发明提供的抗体或其抗原结合片段中：
(1)所述重链可变区包含示于SEQ ID NO：3的氨基酸序列或与示于SEQ ID NO：3的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：4的氨基酸序列或与示于SEQ ID NO：4的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)所述重链可变区包含示于SEQ ID NO：7的氨基酸序列或与示于SEQ ID NO：7的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：8的氨基酸序列或与示于SEQ ID NO：8的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)所述重链可变区包含示于SEQ ID NO：9的氨基酸序列或与示于SEQ ID NO：9的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：10的氨基酸序列或与示于SEQ ID NO：10的氨基酸序列具有至少75％同一性的氨基酸序列；
(4)所述重链可变区包含示于SEQ ID NO：11的氨基酸序列或与示于SEQ ID NO：11的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：12的氨基酸序列或与示于SEQ ID NO：12的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)所述重链可变区包含示于SEQ ID NO：15的氨基酸序列或与示于SEQ ID NO：15的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：16的氨基酸序列或与示于SEQ ID NO：16的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)所述重链可变区包含示于SEQ ID NO：17的氨基酸序列或与示于SEQ ID NO：17的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：18的氨基酸序列或与示于SEQ ID NO：18的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)所述重链可变区包含示于SEQ ID NO：19的氨基酸序列或与示于SEQ ID NO：19的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：20的氨基酸序列或与示于SEQ ID NO：20的氨基酸序列具有至少75％同一性的氨基酸序列；
(8)所述重链可变区包含示于SEQ ID NO：27的氨基酸序列或与示于SEQ ID NO：27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：34的氨基酸序列或与示于SEQ ID NO：34所示的氨基酸序列具有至少75％同一性的氨基酸序列；
(9)所述重链可变区包含示于SEQ ID NO：27的氨基酸序列或与示于SEQ ID NO：27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：33的氨基酸序列或与示于SEQ ID NO：33的氨基酸序列具有至少75％同一性的氨基酸序列；
(10)所述重链可变区包含示于SEQ ID NO：27的氨基酸序列或与示于SEQ ID NO：27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：28的氨基酸序列或与示于SEQ ID NO：28的氨基酸序列具有至少75％同一性的氨基酸序列；
(11)所述重链可变区包含示于SEQ ID NO：23的氨基酸序列或与示于SEQ ID NO：23的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：34的氨基酸序列或与示于SEQ ID NO：34的氨基酸序列具有至少75％同一性的氨基酸序列；
(12)所述重链可变区包含示于SEQ ID NO：23的氨基酸序列或与示于SEQ ID NO：23的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：33的氨基酸序列或与示于SEQ ID NO：33的氨基酸序列具有至少75％同一性的氨基酸序列；或
(13)所述重链可变区包含示于SEQ ID NO：23的氨基酸序列或与示于SEQ ID NO：23的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：28的氨基酸序列或与示于SEQ ID NO：28的氨基酸序列具有至少75％同一性的氨基酸序列。
特别地，本发明的抗体或其抗原结合片段至少包含重链可变区和轻链可变区，二者均包括上述CDR以及间隔的框架区(framework region，FR)，各个结构域的排列方式为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。进一步可选地，所述“至少75％同一性”导致的氨基酸序列的至多25％差异可存在于重链可变区或轻链可变区中的任意框架区中，或者存在于本发明的抗体或其抗原结合片段中重链可变区和轻链可变区以外的任意结构域或序列中。所述差异可以由任何位置的氨基酸缺失、添加或置换产生，其中置换可以是保守置换或非保守置换。
优选地，本发明提供的抗体或其抗原结合片段结合人白细胞介素4受体(IL-4R)，优选地结合人IL-4R的α链(IL-4Rα)，更优选地结合人可溶性IL-4Rα(sIL-4Rα)。
优选地，本发明提供的抗体可以为单克隆抗体或单链抗体。优选地，所述抗体为鼠源抗体、嵌合抗体或完全或部分人源化抗体。优选地，所述抗原结合片段为所述抗体能够特异性结合靶标的片段。例如，所述抗原结合片段为抗体的Fab片段、Fab’片段、F(ab’)2片段或Fv片段(如scFv)。
优选地，所述抗体或其抗原结合片段还包含人或鼠源的恒定区，优选包含人或鼠源的重链恒定区(CH)和/或轻链恒定区(CL)。进一步优选地，所述抗体分子或其抗原结合片段包含重链和轻链。例如，所述抗体为免疫球蛋白，具体为IgA、IgD、IgE、IgG或IgM，例如IgA、IgD、IgE、IgG或IgM的人亚型，更优选为人IgG1、IgG2、IgG3或IgG4亚型。
优选地，本发明提供的抗体或其抗原结合片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区，例如重链恒定区为IgG(如IgG1或IgG4)型，轻链恒定区为κ型。进一步优选地，所述单克隆抗体的重链恒定区包含由示于SEQ ID NO：70或SEQ ID NO：71的核酸序列编码的氨基酸序列或者与由示于SEQ ID NO：70或SEQ ID NO：71的核酸序列编码的氨基酸序列具有至少75％同一性的氨基酸序列；所述单克隆抗体的轻链恒定区包含由示于SEQ ID NO：72的核酸序列编码的氨基酸序列或者与由示于SEQ ID NO：72的核酸序列编码的氨基酸序列具有至少75％同一性的氨基酸 序列。
在本发明的上下文中，“至少75％同一性”为75％至100％之间的任何百分比数字的同一性，例如75％、80％、85％、90％，甚至91％、92％、93％、94％、95％、96％、97％、98％或99％同一性。
另一方面，本发明还提供一种核酸分子，所述核酸分子包含编码本发明抗体或其抗原结合片段中的重链可变区、轻链可变区、重链和/或轻链的核苷酸序列。
本发明的核酸分子可以被克隆到载体中，进而转化或转染宿主细胞。因此又一方面，本发明还提供一种载体，所述载体包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞，用于保存或抗体表达等目的。因此，还一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
本发明提供的抗体或其抗原结合片段可以利用本领域已知的任何方法获得。例如，可以先由本发明提供的核酸分子获得所述抗体的重链可变区和/或轻链可变区，或者获得所述抗体的重链和/或轻链，然后与所述抗体的任选其他结构域组装成抗体；或者，在允许本发明提供的宿主细胞表达所述抗体的重链可变区和/或轻链可变区或者所述抗体的重链和/或轻链以组装成所述抗体的情况下，培养所述宿主细胞。任选地，所述方法还包括回收产生的抗体的步骤。
本发明提供的抗体或其抗原结合片段、核酸分子、载体和/或宿主细胞可以被包含在组合物中，更特别地被包含在药物组合物、例如药物制剂中，从而根据实际需要用于各种目的。因此，在又一方面，本发明还提供一种组合物，优选药物组合物，所述组合物包含本发明所述的抗体或其片段、核酸分子、载体和/或宿主细胞，以及任选的药学上可接受的辅料。
上述药物组合物可制成一定的剂型，优选经口服给药和肠胃外给药(包括皮下、肌肉内和静脉内)，包括但不限于固体剂型、液体剂型、半液体剂型、气雾剂或栓剂等。例如，适合经口给药的剂型包括片剂、胶囊剂、颗粒剂、散剂、丸剂、粉剂、锭剂、糖浆剂或混悬剂；适合肠胃外给药的剂型包括水性或非水性的溶液或乳液，例如皮下注射剂。
优选地，可以经由对抗体药物治疗上有效的肠胃外或非肠胃外(肠)途径施用根据本发明的药物组合物。例如，将本发明的药物组合物形成为含有药学上可接受的辅料(例如溶媒)的制剂，全身或局部地施用。
再一方面，本发明还提供所述抗体或其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物在制备药物中的用途。优选地，所述药物用于预防、治疗或改善与人白细胞介素4受体(IL-4R)相关的疾病或障碍或者与人白细胞介素4(IL-4)或人白细胞介素13(IL-13)信号传导通路相关的疾病或障碍。
另一方面，本发明还提供一种预防、治疗或改善疾病或障碍的方法，所述方法包括给有此需要的受试者施用(例如治疗有效量的)本发明的抗体或其抗原结合片段、核酸分子、载体、宿主细胞和/或组合物。优选地，如上所述，所述疾病或障碍与人白细胞介素4受体(IL-4R)相关或者与人白细胞介素4(IL-4)或人白细胞介素13(IL-13)信号传导通路相关。
在本发明提供的上述用途或方法中，所述疾病或障碍包括自身免疫性疾病、过敏性疾病、肿瘤或癌症。
优选地，所述疾病或障碍包括自身免疫性疾病或过敏性疾病，例如皮炎(例如特应性皮炎)、哮喘(例如炎症性哮喘，诸如2型炎症性哮喘)、慢性食管炎(例如嗜酸性粒细胞食管炎)、湿疹、鼻炎(例如过敏性鼻炎)、鼻息肉(例如慢性鼻窦炎伴鼻息肉)、结膜炎、炎症性肠病(例如溃疡性结肠炎、克罗恩病)、炎性神经病、关节炎(例如类风湿性关节炎)、多发性硬化症、红斑狼疮、银屑病或胰岛素和非胰岛素依赖性糖尿病以及其它免疫或过敏相关疾病。
或者，所述疾病或障碍包括肿瘤或癌症，例如肠癌、黑色素瘤、皮肤癌、乳腺癌、子宫癌、子宫颈癌、子宫内膜癌、卵巢癌、睾丸癌、间皮瘤、前列腺癌、膀胱癌、肛门癌、胶质母细胞瘤、星形细胞瘤、肝癌、肾癌、食道癌、胃癌、肺癌、头部和颈部癌、骨髓瘤、骨癌、艾滋病相关卡波济氏肉瘤、何杰金氏或非何杰金氏淋巴瘤、前列腺癌、前列腺肿瘤、淋巴瘤和胰腺癌等。
优选地，所述药物用于预防、治疗或改善上述疾病类型中的一种或多种。
优选地，所述受试者为哺乳类动物，优选灵长类动物；更优选地，所述受试者为人。
基于本发明提供的人IL-4R的α链(IL-4Rα)中的特定表位，本发明还提供使用所述特定表位在筛选人IL-4R的结合剂或人IL-4/IL-13信号传导通 路的阻断剂中的用途或者使用所述特定表位评价所述结合剂或阻断剂的药效中的用途。如上所述，所述表位包括选自SEQ ID NO：1所示氨基酸序列中的氨基酸残基D92、V94、D97、L67、L68、A96、H156、C207、Q63和L64中的一个或多个。
优选地，所述表位包括SEQ ID NO：1所示氨基酸序列中的氨基酸残基D92、V94和D97中的一个或多个。
根据本发明的具体实施方式，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
(1)D92；
(2)D97；
(3)D92、V94和D97；
(4)D92和V94；
(5)D92和D97；或
(6)V94和D97。
和/或，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：(i)Q63和L64。
和/或，所述表位包括SEQ ID NO：1所示氨基酸序列中的氨基酸残基L67、L68和A96中的一个或多个。
优选地，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
①L67和L68；
②L67、L68和A96；或
③A96。
根据本发明的具体实施方式，所述表位包括SEQ ID NO：1所示氦基酸序列中的如下氨基酸残基：
(A)L67、L68、A96、H156和C207；
(B)A96、H156和C207；
(C)L67、L68、H156和C207；
(D)L67、L68、A96和C207；或
(E)L67、L68、A96和H156。
特别优选地，本发明的表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
(P-1)D97、L67和L68；
(P-2)D97、L67、L68、A96和D92；
(P-3)D97、L67、L68和D92；
(P-4)D97、Q63和L64；
(P-5)D97、Q63、L64和D92；
(P-6)D97、Q63、L64、D92和A96；或
(P-7)D97、Q63、L64、L67和L68。
就上述特定表位的应用，本发明提供一种特异性结合人白细胞介素4受体(IL-4R)的结合剂或者特异性阻断人白细胞介素4(IL-4)或人白细胞介素13(IL-13)信号传导通路的阻断剂的筛选方法，所述筛选方法包括以下步骤：使待筛选试剂与人IL-4R或其部分接触，然后检测所述待筛选试剂是否结合本发明所述的表位。在检测到待筛选试剂结合本发明所述的表位时，确认其为所述结合剂或阻断剂。
或者，本发明还提供一种试剂的药效评价方法，所述药效评价方法包括以下步骤：使待筛选试剂与人白细胞介素4受体(IL-4R)或其部分接触，然后检测所述待筛选试剂是否结合本发明所述的表位。在检测到待筛选试剂结合本发明所述的表位时，确认其为特异性结合人白细胞介素4受体(IL-4R)的结合剂或者特异性阻断人白细胞介素4(IL-4)或人白细胞介素13(IL-13)信号传导通路的阻断剂，进而具有预防、治疗或改善本发明所述的疾病或障碍的作用。
在本发明提供的筛选方法或药效评价方法中，优选地，所述待筛选试剂为抗体，例如多克隆抗体或单克隆抗体。更优选地，所述抗体是以人IL-4R或其部分作为免疫原免疫动物得到的。
在所述筛选方法或药效评价方法中，优选地，对所述待筛选试剂是否结合本发明所述的表位的检测可以通过如下进行：通过对人IL-4R或其部分进行定点突变，使本发明提供的表位所包括的一个或多个氨基酸残基突变，比较待筛选试剂与突变前后的人IL-4R或其部分的结合活性或亲和力，当与突变后的人IL-4R或其部分的结合活性或亲和力下降、甚至丧失时，确定所述试剂与本发明所述的表位结合。
具体地，本发明提供的筛选方法或药效评价方法可包括以下步骤：
1)通过对人白细胞介素4受体(IL-4R)或其部分进行定点突变，使本发明提供的表位所包括的一个或多个氨基酸残基突变，获得人IL-4R或其部 分的突变体；
2)使待筛选试剂分别与人IL-4R或其部分和人IL-4R或其部分的突变体接触，并分别检测与其的结合活性或亲和力；
3)相比于与人IL-4R或其部分的结合活性或亲和力，当所述待筛选试剂与人IL-4R或其部分的突变体的结合活性或亲和力显著下降或丧失时，确定所述试剂与本发明提供的表位结合。
本领域技术人员可以常规地判断该结合活性或亲和力是否有显著下降或丧失。例如，当所述待筛选试剂与人IL-4R或其部分的突变体的结合活性或亲和力为突变之前的结合活性或亲和力的1/10时，可以确定该结合活性或亲和力显著下降。
优选地，在步骤1)中，将表位所包括的一个或多个氨基酸残基突变为丙氨酸或突变为猴白细胞介素4受体在对应位点处的氨基酸残基，以获得人IL-4R或其部分的突变体。
优选地，在本发明的方法中采用的人IL-4R的部分为人IL-4R的α链，进一步为人sIL-4Rα。因此优选地，采用人IL-4Rα并获得人IL-4Rα的突变体进行所述方法。进一步地，可以采用人sIL-4Rα进行所述方法，并将下文表3中所列的人sIL-4Rα突变蛋白中的一个或多个作为步骤1)中的突变体。
本发明中，人IL-4Rα参见NCBI参考序列NP_000409.1；人sIL-4Rα的氨基酸序列示于SEQ ID NO：1；猴sIL-4Rα的氨基酸序列示于SEQ ID NO：2。
实验证明，本发明提供的抗体或其抗原结合片段对人白细胞介素4受体(IL-4R)的结合表位位于人IL-4R的α链(IL-4Rα)，并且所述表位包括选自SEQ ID NO：1所示氨基酸序列中的氨基酸残基D92、V94、D97、L67、L68、A96、H156、C207、Q63和L64中的一个或多个。在此之前，对sIL-4Rα在免疫效应中的表位研究较少，而本发明首次明确了抗IL-4R抗体与IL-4R结合的关键位点。
因此，本发明提供了具有全新的IL-4R结合表位的抗人IL-4R、特别是IL-4Rα的抗体，该抗体可以作为针对人IL-4R、特别是IL-4Rα的新型有效抗体。并且，通过本发明抗体与人IL-4Rα结合的关键位点的阐明，本发明为开发针对IL-4R、特别是人IL-4Rα潜在抗体或药物提供了另一途径。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出人IL-4Rα(序列_1；SEQ ID NO：1)和猴IL-4Rα(序列_0；SEQ ID NO：2)的序列比对结果。
图2示出本发明抗体的稳定性，其中图2A为40℃放置2周后的结果，图2B为40℃放置4周的结果。
实施发明的最佳方式
在本发明中，术语“表位”是指抗体或其片段与抗原结合时结合部位所对应的抗原氨基酸残基及其在抗原序列中的相应位置；并且，如通过本申请下文“(一)结合活性检测方法”所述操作过程测得的，突变所述表位位置的氨基酸残基将导致结合活性的下降大于10 2ng/ml或失去结合活性。
本发明中涉及以下实验操作或定义。应注意，本发明还可采用本领域其他常规技术进行实施，并不仅限于以下实验操作。
(一)结合活性检测方法
1、试剂的配制
抗原的的配制：使用PBS将抗原配制成100ug/ml溶液，分装后于-20℃冰箱中保存。
待测抗体的准备：使用含1％BSA的PBS将待测抗体稀释至所需浓度：0、0.032、0.16、0.8、4、20、100、500、2500ng/ml。
二抗工作溶液的配制：使用含1％BSA的PBS将二抗母液(Goat Anti-Human IgG-Fc Secondary Antibody(HRP)，sino biological，Cat Number：SSA001)稀释16000倍，即为工作溶液。例如取1ul二抗母液加入到15999ul的1％BSA的PBS中，反复上下颠倒混匀。可根据实验需求等比例放大，配制所需体积的二抗工作溶液。
2、ELISA检测
取50ul 100ug/ml抗原溶液，加入到9.95ml PBS中，上下颠倒混匀，即为0.5ug/ml抗原包被液。将配好的抗原包被液加入到加样槽中，使用12通道移液器(RAININ)加入96-well酶标板(Corning)中，每孔100ul。将96-well酶标板用保鲜膜包裹(或加盖)后，4℃冰箱中孵育过夜。第二天，将96-well酶标板取出，弃去其中溶液，并在洁净的纸巾上轻轻拍干。向96-well酶标板中逐排加入PBS，每孔300ul。室温静置3min，弃溶液，并在洁净的纸巾上轻轻拍干。重复洗涤3次。向96-well酶标板中逐排加入含2％BSA的PBS，每孔300ul。将酶标板用保鲜膜包裹(或加盖)后，于37℃恒温培养箱中孵 育2h。将96-well酶标板取出，弃去其中溶液，并在洁净的纸巾上轻轻拍干。向96-well酶标板中逐排加入含0.05％Tween-20(Beyotime，Cat Number：ST825)的PBS，每孔300ul。室温静置3min，弃溶液，并在洁净的纸巾上轻轻拍干。重复洗涤3次。
将稀释好的待测抗体分别依次加入到相应的孔中，每个样品做三个复孔，每孔100ul。将酶标板用保鲜膜包裹(或加盖)后，置于37℃恒温培养箱中孵育1h。将96-well酶标板取出，弃去其中溶液，并在洁净的纸巾上轻轻拍干。向96-well酶标板中逐排加入含0.05％Tween-20的PBS，每孔300ul。室温静置3min，弃溶液，并在洁净的纸巾上轻轻拍干。重复洗涤3次。向96-well酶标板中逐排加入二抗工作溶液，每孔100ul。将酶标板用保鲜膜包裹(或加盖)后，置于37℃恒温培养箱中孵育1h。将96-well酶标板取出，弃去其中溶液，并在洁净的纸巾上轻轻拍干。向96-well酶标板中逐排加入含0.05％Tween-20的PBS，每孔300ul。室温静置3min，弃溶液，并在洁净的纸巾上轻轻拍干。重复洗涤3次。向96-well酶标板中逐排加入TMB溶液(SurModics，Cat Number：TMDS-1000-01)，每孔100ul。置于37℃恒温培养箱中放置5分钟，立即向96-well酶标板中加入2M H2SO4溶液终止反应。将96-well酶标板置于flexstation 3(Molecular Devices)中，读取OD450的值，数据收集计算分析。
(二)细胞功能活性检测方法
1、试剂的配制
人IL-4(Invivogen，货号：rhIL-4)的配制：使用PBS将人IL-4配制成100ug/ml溶液，分装后于-20℃冰箱中保存。
人IL-13(Invivogen，货号：rhIL-13)的配制：使用PBS将人IL-13配制成100ug/ml溶液，分装后于-20℃冰箱中保存。
Quanti-blue(I)溶液的配制：将一包Quanti-blue粉末倒入一个无菌的250mL瓶子里，加入100mL无菌水，轻轻混匀，37℃温浴30min，完全溶解后，在4℃冰箱避光放置过夜，然后按10mL每支分装，于-20℃避光保存，可保存6个月。
待测抗体的准备：用含10％FBS(Hyclone，Cat No.SV30184.02)的DMEM培养基(HyClone，Cat No.SH30022.01)将抗体稀释至所需浓度(1000、200、40、8、1.6、0.32、0.064、0.0128、0.00256、0ng/ml)。
2、细胞培养
在液氮中取出冻存的HEK Blue IL-4/IL13(Invivogen，Cat No.hkb-il413)细胞，在37℃水浴中不停轻柔震荡，使其迅速溶解。将溶解后的细胞悬液移至15ml离心管内，加DMEM培养基(含10％FBS，10μg/ml Blasticidin，100μg/ml Zeocin，100μg/ml Normocin)至10ml，800rpm离心5min，吸去上清，保留细胞沉淀，重复洗涤一次，加入10ml DMEM培养基，调整细胞密度为1×10 5-1×10 6个/ml，移至T75细胞培养瓶(Nunc)中，置于37℃，5％CO2培养箱(Thermo)中静置培养。每隔2-3天，取细胞悬液，800rpm离心5min，用10ml培养基重悬细胞，计数细胞1×10 6个细胞移入新的T75细胞瓶中，同时补加培养基至10ml，连续传代2-3次，至细胞状态良好(细胞透亮，单一贴壁的梭状细胞)后可进行实验。
3、阻断实验
取T75细胞瓶中生长状态良好细胞，弃上清，用10ml PBS将贴壁细胞轻轻吹打下来，移入15ml离心管内，800rpm离心5min，取细胞沉淀，再次用10ml PBS重悬细胞，800rpm离心5min，弃上清，取细胞沉淀。用5ml含10％FBS的DMEM培养基重悬细胞，细胞计数后，补加培养基，调整细胞密度至6.6×10 5个/ml。将细胞悬液按每孔30ul体积加入384孔板内。将准备好的不同浓度的抗体10ul加入384孔的细胞内(3个重复孔)。用含10％FBS的DMEM培养基稀释人IL-4或人IL-13至2.5ng/ml。将2.5ng/ml的人IL-4或人IL-13按照10ul每孔的体积分别加入384孔板上对应的细胞孔内，使得每孔中最终的细胞数量为2×104个，人IL-4或人IL-13终浓度为0.5ng/ml，最终384孔板每孔体积为50ul。将384孔细胞板置于37℃，5％CO2培养箱中静置培养。
4、数据统计
384孔细胞板在5％CO2培养箱中静置培养22h后，在新的384孔板中每孔加入45ul的Quanti-blue试剂。将之前孵育了22h的板子里每孔吸取5ul上清加入到新的384孔板对应的孔中。37℃静置孵育60min。将384孔板置于flexstation III中，读取OD650的值，数据收集计算分析。
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。其中：
人sIL-4Rα：NP_000409.1，Met1-His232，见SEQ ID NO：1；
猴sIL-4Rα：EHH60265.1，Met1-Arg232，见SEQ ID NO：2。
实施例1鼠抗、嵌合抗体、人源化抗体的制备
使用人sIL-4Rα(SEQ ID NO：1)免疫十只小鼠，采血，进行如上(一)结合活性和(二)细胞功能活性检测。综合两种检测的结果，选择结果最好的两只小鼠分别进行融合。
将融合得到的多组细胞上清再次进行如上(一)结合活性和(二)细胞功能活性检测，综合两种检测的结果，筛选出活性最高的母克隆进行亚克隆，经(一)结合活性和(二)细胞功能活性检测，最终筛选得到14株单克隆鼠源抗体。对这14株单克隆进行测序，为9对全新序列的抗体，其对应的重链可变区和轻链可变区见下；采用IMGT工具划分的CDR以下划线示出。
Y0188-1
重链可变区(SEQ ID NO：3；CDR依次为：SEQ ID NO：35，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000001
轻链可变区(SEQ ID NO：4；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000002
Y0188-2
重链可变区(SEQ ID NO：5；CDR依次为：SEQ ID NO：38，SEQ ID NO：39，SEQ ID NO：40)
Figure PCTCN2021088139-appb-000003
轻链可变区(SEQ ID NO：6；CDR依次为：SEQ ID NO：56，SEQ ID NO：57，SEQ ID NO：58)
Figure PCTCN2021088139-appb-000004
Y0188-3
重链可变区(SEQ ID NO：7；CDR依次为：SEQ ID NO：41，SEQ ID NO：42，SEQ ID NO：43)
Figure PCTCN2021088139-appb-000005
轻链可变区(SEQ ID NO：8；CDR依次为：SEQ ID NO：59，SEQ ID NO：60，SEQ ID NO：61)
Figure PCTCN2021088139-appb-000006
Y0188-4
重链可变区(SEQ ID NO：9；CDR依次为：SEQ ID NO：44，SEQ ID NO：45，SEQ ID NO：46)
Figure PCTCN2021088139-appb-000007
轻链可变区(SEQ ID NO：10；CDR依次为：SEQ ID NO：56，SEQ ID NO：57，SEQ ID NO：62)
Figure PCTCN2021088139-appb-000008
Y0188-6
重链可变区(SEQ ID NO：11；CDR依次为：SEQ ID NO：44，SEQ ID NO：47，SEQ ID NO：46)
Figure PCTCN2021088139-appb-000009
轻链可变区(SEQ ID NO：12；CDR依次为：SEQ ID NO：63，SEQ ID NO：64，SEQ ID NO：65)
Figure PCTCN2021088139-appb-000010
Y0188-8
重链可变区(SEQ ID NO：13；CDR依次为：SEQ ID NO：38，SEQ ID NO：39，SEQ ID NO：40)
Figure PCTCN2021088139-appb-000011
轻链可变区(SEQ ID NO：14；CDR依次为：SEQ ID NO：56，SEQ ID NO：57，SEQ ID NO：58)
Figure PCTCN2021088139-appb-000012
Y0188-9
重链可变区(SEQ ID NO：15；CDR依次为：SEQ ID NO：48，SEQ ID NO：49，SEQ ID NO：46)
Figure PCTCN2021088139-appb-000013
轻链可变区(SEQ ID NO：16；CDR依次为：SEQ ID NO：66，SEQ ID NO：67，SEQ ID NO：68)
Figure PCTCN2021088139-appb-000014
Y0188-10
重链可变区(SEQ ID NO：17；CDR依次为：SEQ ID NO：44，SEQ ID NO：50，SEQ ID NO：51)
Figure PCTCN2021088139-appb-000015
轻链可变区(SEQ ID NO：18；CDR依次为：SEQ ID NO：56，SEQ ID NO：57，SEQ ID NO：69)
Figure PCTCN2021088139-appb-000016
Figure PCTCN2021088139-appb-000017
Y0188-14
重链可变区(SEQ ID NO：19；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO37)
Figure PCTCN2021088139-appb-000018
轻链可变区(SEQ ID NO：20；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000019
根据上述可变区序列进一步构建得到9个嵌合抗体，采用SEQ ID NO：71所示序列作为重链恒定区编码序列，SEQ ID NO：72所示序列作为轻链恒定区编码序列，表达纯化这9个嵌合抗体，以鼠抗名称加上Q进行命名，使用上文“(二)细胞功能活性检测方法”所述操作过程检测嵌合抗体的IC50，用Biacore检测嵌合抗体与人sIL-4Rα的亲和力KD，结果见下表1。
表1.嵌合抗体的细胞功能活性和亲和力检测结果
Figure PCTCN2021088139-appb-000020
选择14号克隆的嵌合抗体(Y0188-14Q)进行人源化，得到7条人源化的重链可变区和7条人源化的轻链可变区。人源化的重链可变区和轻链可变 区见下；采用IMGT工具划分的CDR以下划线示出。
重链可变区：
>HV3-15-14H(SEQ ID NO：21；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000021
>HV3-48-14H(SEQ ID NO：22；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000022
>HV3-73*2-14H(SEQ ID NO：23；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000023
>HV3-72-14H(SEQ ID NO：24；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000024
>Y01-14H(SEQ ID NO：25；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000025
>162-14H(SEQ ID NO：26；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000026
>VH73-14H(SEQ ID NO：27；CDR依次为：SEQ ID NO：52，SEQ ID NO：36，SEQ ID NO：37)
Figure PCTCN2021088139-appb-000027
轻链可变区：
>Y01-14L(SEQ ID NO：28；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000028
>164-14L(SEQ ID NO：29；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000029
>KV4-14L(SEQ ID NO：30；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000030
>KV1-27-14L(SEQ ID NO：31；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000031
>KV1-9-14L(SEQ ID NO：32；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000032
>KV1-NL1-14L(SEQ ID NO：33；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000033
>KV1D-43-14L(SEQ ID NO：34；CDR依次为：SEQ ID NO：53，SEQ ID NO：54，SEQ ID NO：55)
Figure PCTCN2021088139-appb-000034
将上述轻重链人源化序列两两相互搭配，并采用SEQ ID NO：71所示序列作为重链恒定区编码序列，SEQ ID NO：72所示序列作为轻链恒定区编码序列，进行表达，然后同样采用上文“(二)细胞功能活性检测方法”所述操作过程检测得到的49个组合抗体的IC50，并且进一步用Biacore验证细胞功能活性(KD)，最终优选出6个组合，如表2所示。
表2.人源化抗体的细胞功能活性检测结果
Figure PCTCN2021088139-appb-000035
实施例2结合表位研究
2.1抗体与人猴IL-4Rα结合的种属差异
以猴sIL-4Rα(EHH60265.1，Met1-Arg232；SEQ ID NO：2)为抗原，采用上文“(一)结合活性检测方法”所述操作检测抗体与抗原的结合活性。
2.2抗原序列中的突变位点选择
将人和猴的IL-4Rα进行序列比对，见图1。推测人猴的IL-4Rα序列存在差异的部分位点有可能是抗体与IL-4Rα结合的关键位点，使得其与人的差异序列结合，而不与猴的差异序列结合，于是对人的sIL-4Rα中与猴差异的位点进行单点突变或者组合突变成猴sIL-4Rα中对应位点上的氨基酸，并且对人猴sIL-4Rα差异位点附近的非丙氨酸进行选择性单点或者组合突变成丙氨酸，然后构建具有不同突变组合的人sIL-4Rα突变体(见下表3)，并应 用细胞功能活性实验加以验证。
表3.人sIL-4Rα突变蛋白
Figure PCTCN2021088139-appb-000036
2.3结合活性检测
以人sIL-4Rα蛋白和表3所示的不同sIL-4Rα突变蛋白为抗原，以6个人源化高活性抗体为一抗，采用上文“(一)结合活性检测方法”所述操作检测抗体与抗原的结合活性。结果见表4。
同样地，以人sIL-4Rα蛋白和表3所示的不同sIL-4Rα突变蛋白为抗原，以筛选得到的9个嵌合抗体为一抗，同样检测抗体与抗原的结合活性。结果见表5。
通过筛选，发现抗体与多个人猴差异位点及其附近位点的sIL-4Rα突变蛋白的结合活性发生变化甚至反转。
Figure PCTCN2021088139-appb-000037
Figure PCTCN2021088139-appb-000038
由表4中数据可知，就L67、L68、A96、H156和C207这五个位点而言，将任一个上的氨基酸残基突变成猴IL-4Rα中对应位点上的氨基酸后，当突变位点组合中包含L67和L68时，本发明人源化抗体的结合活性显著下降；而同时，当仅L67和L68单独突变时，则本发明人源化抗体保持了原有的结合活性。
就D92、V94和D97这三个位点而言，将任一个上的氨基酸残基突变成丙氨酸后，当突变位点组合中包含D97或仅突变D97时，本发明人源化抗体结合活性显著下降。
Figure PCTCN2021088139-appb-000039
Figure PCTCN2021088139-appb-000040
由表5中数据可知，就D92、V94和D97这三个位点而言，将任一个上的氨基酸残基突变成丙氨酸后，当突变位点组合中包含D97或仅突变D97时，除Y0188-2Q和Y0188-8Q外，其余嵌合抗体的结合活性显著下降。这一点与上文人源化抗体得到的结合结果一致。
而就L67、L68、A96、H156和C207这五个位点而言，将任一个上的氨基酸残基突变成猴IL-4Rα中对应位点上的氨基酸后，当突变位点组合中包含L67和L68或L67、L68和A96时，嵌合抗体Y0188-1Q、Y0188-3Q、Y0188-10Q结合活性显著下降；而同时，当仅L67和L68单独突变时，则除Y0188-2Q和Y0188-8Q外的嵌合抗体均保持了原有的结合活性。此外，就A96而言，当突变位点组合包含A96时，嵌合抗体Y0188-3Q结合活性均显著下降。
而就Q63和L64这三个位点而言，将二者同时突变成丙氨酸后，除Y0188-1Q、Y0188-2Q和Y0188-8Q外的嵌合抗体结合活性显著下降、甚至失去结合活性。
实施例3抗体稳定性
取抗体ES、B5，在10mM Histidine，150mM NaCl，pH 6中配制浓度分别为0.59mg/ml、0.54mg/ml、6.44mg/ml，分别在40℃放置2周和4周后，使用上文所述“(二)细胞功能活性检测方法”检测过程进行实验，得到抗体浓度与OD650的关系曲线图，见图2，然后由此计算IC50(ng/mL)，见下表6。
表6.抗体的稳定性结果
Figure PCTCN2021088139-appb-000041
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (22)

  1. 一种结合人白细胞介素4受体(IL-4R)的抗体或其抗原结合片段，所述抗体或其抗原结合片段结合的表位位于人IL-4R的α链(IL-4Rα)，且所述表位包括选自SEQ ID NO：1所示氨基酸序列中的氨基酸残基D92、V94、D97、L67、L68、A96、H156、C207、Q63和L64中的一个或多个。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述表位包括SEQ ID NO：1所示氨基酸序列中的氨基酸残基D92、V94和D97中的一个或多个；更优选地，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
    (1)D92；
    (2)D97；
    (3)D92、V94和D97；
    (4)D92和V94；
    (5)D92和D97；或
    (6)V94和D97；
    优选地，所述抗体或其抗原结合片段与人IL-4Rα的结合表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：(i)Q63和L64。
  3. 根据权利要求1或2所述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不具有与猴IL-4R的种属交叉结合活性；优选地，不结合猴IL-4R；更优选地，不结合猴IL-4Rα；进一步优选地，不结合SEQ ID NO：2所示氨基酸序列；
    优选地，所述表位包括SEQ ID NO：1所示氨基酸序列中的氨基酸残基L67、L68和A96中的一个或多个；优选地包括：
    ①L67和L68；
    ②L67、L68和A96；或
    ③A96；
    更优选地，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
    (A)L67、L68、A96、H156和C207；
    (B)A96、H156和C207；
    (C)L67、L68、H156和C207；
    (D)L67、L68、A96和C207；或
    (E)L67、L68、A96和H156。
  4. 根据权利要求1至3中任一项所述的抗体或其抗原结合片段，其特征在于，所述表位包括SEQ ID NO：1所示氨基酸序列中的如下氨基酸残基：
    (P-1)D97、L67和L68；
    (P-2)D97、L67、L68、A96和D92；
    (P-3)D97、L67、L68和D92；
    (P-4)D97、Q63和L64；
    (P-5)D97、Q63、L64和D92；
    (P-6)D97、Q63、L64、D92和A96；或
    (P-7)D97、Q63、L64、L67和L68。
  5. 根据权利要求1至4中任一项所述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在其重链可变区和轻链可变区中包含以下重链互补决定区HCDR1、HCDR2、HCDR3和轻链互补决定区LCDR1、LCDR2、LCDR3：
    (1)依次示于SEQ ID NO：35、36、37的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：53、54、55的LCDR1、LCDR2、LCDR3；
    (2)依次示于SEQ ID NO：41、42、43的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：59、60、61的LCDR1、LCDR2、LCDR3；
    (3)依次示于SEQ ID NO：44、45、46的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：56、57、62的LCDR1、LCDR2、LCDR3；
    (4)依次示于SEQ ID NO：44、47、46的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：63、64、65的LCDR1、LCDR2、LCDR3；
    (5)依次示于SEQ ID NO：48、49、46的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：66、67、68的LCDR1、LCDR2、LCDR3；
    (6)依次示于SEQ ID NO：44、50、51的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：56、57、69的LCDR1、LCDR2、LCDR3；或
    (7)依次示于SEQ ID NO：52、36、37的HCDR1、HCDR2、HCDR3；和，依次示于SEQ ID NO：53、54、55的LCDR1、LCDR2、LCDR3；
    优选地，所述重链可变区包含选自以下的序列：
    示于SEQ ID NO：3、SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：23或SEQ ID  NO：27的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，所述轻链可变区包含选自以下的序列：
    示于SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：10、SEQ ID NO：12、SEQ ID NO：16、SEQ ID NO：18、SEQ ID NO：20、SEQ ID NO：28、SEQ ID NO：33或SEQ ID NO：34的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
  6. 根据权利要求1至5中任一项所述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中：
    (1)所述重链可变区包含示于SEQ ID NO：3的氨基酸序列或与示于SEQ ID NO：3的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：4的氨基酸序列或与示于SEQ ID NO：4的氨基酸序列具有至少75％同一性的氨基酸序列；
    (2)所述重链可变区包含示于SEQ ID NO：7的氨基酸序列或与示于SEQ ID NO：7的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：8的氨基酸序列或与示于SEQ ID NO：8的氨基酸序列具有至少75％同一性的氨基酸序列；
    (3)所述重链可变区包含示于SEQ ID NO：9的氨基酸序列或与示于SEQ ID NO：9的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：10的氨基酸序列或与示于SEQ ID NO：10的氨基酸序列具有至少75％同一性的氨基酸序列；
    (4)所述重链可变区包含示于SEQ ID NO：11的氨基酸序列或与示于SEQ ID NO：11的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：12的氨基酸序列或与示于SEQ ID NO：12的氨基酸序列具有至少75％同一性的氨基酸序列；
    (5)所述重链可变区包含示于SEQ ID NO：15的氨基酸序列或与示于SEQ ID NO：15的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：16的氨基酸序列或与示于SEQ ID NO：16的氨基酸序列具有至少75％同一性的氨基酸序列；
    (6)所述重链可变区包含示于SEQ ID NO：17的氨基酸序列或与示于SEQ ID NO：17的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：18的氨基酸序列或与示于SEQ ID NO：18的氨基酸序列具有至少75％同一性的氨基酸序列；
    (7)所述重链可变区包含示于SEQ ID NO：19的氨基酸序列或与示于SEQ ID NO：19的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：20的氨基酸序列或与示于SEQ ID NO：20的氨基酸序列具有至少75％同一性的氨基酸序列；
    (8)所述重链可变区包含示于SEQ ID NO：27的氨基酸序列或与示于SEQ ID NO：27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：34的氨基酸序列或与示于SEQ ID NO：34所示的氨基酸序列具有至少75％同一性的氨基酸序列；
    (9)所述重链可变区包含示于SEQ ID NO：27的氨基酸序列或与示于SEQ ID NO：27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：33的氨基酸序列或与示于SEQ ID NO：33的氨基酸序列具有至少75％同一性的氨基酸序列；
    (10)所述重链可变区包含示于SEQ ID NO：27的氨基酸序列或与示于SEQ ID NO：27的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：28的氨基酸序列或与示于SEQ ID NO：28的氨基酸序列具有至少75％同一性的氨基酸序列；
    (11)所述重链可变区包含示于SEQ ID NO：23的氨基酸序列或与示于SEQ ID NO：23的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：34的氨基酸序列或与示于SEQ ID NO：34的氨基酸序列具有至少75％同一性的氨基酸序列；
    (12)所述重链可变区包含示于SEQ ID NO：23的氨基酸序列或与示于SEQ ID NO：23的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：33的氨基酸序列或与示于SEQ ID NO：33的氨基酸序列具有至少75％同一性的氨基酸序列；或
    (13)所述重链可变区包含示于SEQ ID NO：23的氨基酸序列或与示于SEQ ID NO：23的氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO：28的氨基酸序列或与示于SEQ ID NO：28的氨基酸序列具有至少75％同一性的氨基酸序列。
  7. 根据权利要求1至6中任一项所述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结合人白细胞介素4受体(IL-4R)，优选地结合人IL-4R的α链(IL-4Rα)，更优选地结合可溶性人IL-4Rα；
    优选地，所述抗体为单克隆抗体或单链抗体；
    优选地，所述抗体为鼠源抗体、嵌合抗体或完全或部分人源化抗体；优选地，所述抗原结合片段为所述抗体能够特异性结合IL-4R或IL-4Rα的片段。
  8. 根据权利要求1至7中任一项所述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还包含人或鼠源的恒定区，优选包含人或鼠源的重链恒定区(CH)和/或轻链恒定区(CL)；
    优选地，所述抗体分子或其抗原结合片段包含重链和轻链；
    优选地，所述抗体为免疫球蛋白，具体为IgA、IgD、IgE、IgG或IgM，例如IgA、IgD、IgE、IgG或IgM的人亚型，更优选为人IgG1、IgG2、IgG3或IgG4亚型；
    更优选地，所述抗体或其抗原结合片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区，例如重链恒定区为IgG(如IgG1或IgG4)型，轻链恒定区为κ型。
  9. 一种核酸分子，所述核酸分子包含编码根据权利要求1至8中任一项所述的抗体或其抗原结合片段中的VH、VL、CH和/或CL的核苷酸序列。
  10. 一种载体，所述载体包含权利要求9所述的核酸分子。
  11. 一种宿主细胞，所述宿主细胞包含权利要求9所述的核酸分子或权利要求10所述的载体，或者所述宿主细胞被权利要求9所述的核酸分子或权利要求10所述的载体转化或转染。
  12. 一种组合物，所述组合物包含权利要求1至8中任一项所述的抗体或其抗原结合片段、权利要求9所述的核酸分子、权利要求10所述的载体或权利要求11所述的宿主细胞。
  13. 根据权利要求12所述的组合物，其特征在于，所述组合物为药物组合物；优选地，所述药物组合物还包含药学上可接受的辅料；
    优选地，所述药物组合物制成经口服给药和肠胃外给药的剂型。
  14. 权利要求1至8中任一项所述的抗体或其抗原结合片段、权利要求9所述的核酸分子、权利要求10所述的载体、权利要求11所述的宿主细胞或者权利要求12或13所述的组合物在制备药物中的用途，所述药物用于预防、治疗或改善与人白细胞介素4受体(IL-4R)相关的疾病或障碍或者与人白细胞介素4(IL-4)或人白细胞介素13(IL-13)信号传导通路相关的疾病或障碍。
  15. 一种预防、治疗或改善疾病或障碍的方法，所述方法包括给有此需 要的受试者施用(例如治疗有效量的)权利要求1至8中任一项所述的抗体或其抗原结合片段、权利要求9所述的核酸分子、权利要求10所述的载体、权利要求11所述的宿主细胞或者权利要求12或13所述的组合物，所述疾病或障碍与人白细胞介素4受体(IL-4R)相关的疾病或障碍或者与人白细胞介素4(IL-4)或人白细胞介素13(IL-13)信号传导通路相关。
  16. 根据权利要求14所述的用途或权利要求15所述的方法，其特征在于，所述疾病或障碍包括自身免疫性疾病、过敏性疾病、肿瘤或癌症；
    优选地，所述疾病或障碍包括自身免疫性疾病或过敏性疾病，例如皮炎(例如特应性皮炎)、哮喘(例如炎症性哮喘，诸如2型炎症性哮喘)、慢性食管炎(例如嗜酸性粒细胞食管炎)、湿疹、鼻炎(例如过敏性鼻炎)、鼻息肉(例如慢性鼻窦炎伴鼻息肉)、结膜炎、炎症性肠病(例如溃疡性结肠炎、克罗恩病)、炎性神经病、关节炎(例如类风湿性关节炎)、多发性硬化症、红斑狼疮、银屑病或胰岛素和非胰岛素依赖性糖尿病以及其它免疫或过敏相关疾病；
    或者优选地，所述疾病或障碍包括肿瘤或癌症，例如肠癌、黑色素瘤、皮肤癌、乳腺癌、子宫癌、子宫颈癌、子宫内膜癌、卵巢癌、睾丸癌、间皮瘤、前列腺癌、膀胱癌、肛门癌、胶质母细胞瘤、星形细胞瘤、肝癌、肾癌、食道癌、胃癌、肺癌、头部和颈部癌、骨髓瘤、骨癌、艾滋病相关卡波济氏肉瘤、何杰金氏或非何杰金氏淋巴瘤、前列腺癌、前列腺肿瘤、淋巴瘤和胰腺癌。
  17. 根据权利要求14至16中任一项所述的用途或方法，其特征在于，所述药物用于预防、治疗或改善权利要求16中所限定的疾病或障碍中的一种或多种；
    优选地，所述受试者为哺乳类动物，优选灵长类动物；更优选地，所述受试者为人。
  18. 一种特异性结合人白细胞介素4受体(IL-4R)的结合剂或者特异性阻断人白细胞介素4(IL-4)或人白细胞介素13(IL-13)信号传导通路的阻断剂的筛选方法，所述筛选方法包括以下步骤：使待筛选试剂与人IL-4R或其部分接触，然后检测所述待筛选试剂是否结合权利要求1至3中任一项所限定的表位。
  19. 一种试剂的药效评价方法，所述药效评价方法包括以下步骤：使待筛选试剂与人白细胞介素4受体(IL-4R)或其部分)接触，然后检测所述 待筛选试剂是否结合权利要求1至3中任一项所限定的表位。
  20. 根据权利要求18所述的筛选方法或权利要求19所述的药效评价方法，其特征在于，所述待筛选试剂为抗体，例如多克隆抗体或单克隆抗体；
    优选地，所述抗体是以人IL-4R或其部分作为免疫原免疫动物得到的；
    更优选地，对所述待筛选试剂是否结合所述表位的检测可以通过如下进行：通过对人IL-4R或其部分进行定点突变，使所述表位所包括的一个或多个氨基酸残基突变，比较待筛选试剂与突变前后的人IL-4R或其部分的结合活性或亲和力，当与突变后的人IL-4R或其部分的结合活性或亲和力下降、甚至丧失时，确定所述试剂与所述表位结合。
  21. 根据权利要求18至20中任一项所述的筛选方法或药效评价方法，其特征在于，所述方法包括以下步骤：
    1)通过对人IL-4R或其部分进行定点突变，使所述表位所包括的一个或多个氨基酸残基突变，获得人IL-4R或其部分的突变体；
    2)使待筛选试剂分别与人IL-4R或其部分和人IL-4R或或其部分的突变体接触，并分别检测与其的结合活性或亲和力；
    3)相比于与人IL-4R或其部分的结合活性或亲和力，当所述待筛选试剂与人IL-4R或其部分的突变体的结合活性或亲和力显著下降或丧失时，确定所述试剂与所述表位结合。
  22. 根据权利要求18至21中任一项所述的筛选方法或药效评价方法，其特征在于，在步骤1)中，将所述表位所包括的一个或多个氨基酸残基突变为丙氨酸或突变为猴白细胞介素4受体在对应位点处的氨基酸残基，以获得人IL-4R或其部分的突变体；
    优选地，所述人IL-4R的部分为人IL-4R的α链，进一步为人sIL-4Rα；优选地，采用人IL-4Rα并获得人IL-4Rα的突变体进行所述方法；
    优选地，采用人sIL-4Rα进行所述方法，并将说明书表3中所列的人sIL-4Rα突变蛋白中的一个或多个作为步骤1)中的突变体。
PCT/CN2021/088139 2020-04-24 2021-04-19 与人IL-4Rα中特定表位结合的抗体及其应用 WO2021213329A1 (zh)

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