WO2019200787A1 - 抗il-4r抗体及其用途 - Google Patents

抗il-4r抗体及其用途 Download PDF

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Publication number
WO2019200787A1
WO2019200787A1 PCT/CN2018/100263 CN2018100263W WO2019200787A1 WO 2019200787 A1 WO2019200787 A1 WO 2019200787A1 CN 2018100263 W CN2018100263 W CN 2018100263W WO 2019200787 A1 WO2019200787 A1 WO 2019200787A1
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Prior art keywords
antibody
sequence
seq
amino acid
variable region
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PCT/CN2018/100263
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English (en)
French (fr)
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刘志刚
刘玉兰
郝小勃
蒋磊
郭晶晶
Original Assignee
北京智仁美博生物科技有限公司
智翔（上海）医药科技有限公司
重庆智翔金泰生物制药有限公司
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Application filed by 北京智仁美博生物科技有限公司, 智翔（上海）医药科技有限公司, 重庆智翔金泰生物制药有限公司 filed Critical 北京智仁美博生物科技有限公司
Priority to RU2020136302A priority Critical patent/RU2758091C1/ru
Priority to US17/049,288 priority patent/US11897960B2/en
Publication of WO2019200787A1 publication Critical patent/WO2019200787A1/zh
Priority to ZA2020/06904A priority patent/ZA202006904B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—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
    • 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
    • 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
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates generally to the field of genetic engineering and antibody drugs; in particular, to the field of anti-human interleukin-4 receptor (IL-4R) antibodies and uses thereof.
  • the present application develops novel anti-human IL-4R antibodies and provides the use of such antibodies in the treatment of IL-4R mediated diseases.
  • Interleukin-4 consists of 153 amino acids with a molecular weight of approximately 17 kDa. Initially, IL-4 was discovered to stimulate B cell proliferation and was named B cell stimulating factor-1 (BSF-1) [1] . Like IL-13, IL-4 belongs to the type I cytokine family and has a quaternary structure composed of a hydrophobic bundle core of 4 ⁇ helix [2] . IL-4 is secreted by TH2 cells and participates in TH2-mediated immune responses. It has a wide range of biological activities, including stimulation of T cells, mast cells, granulocytes, megakaryocytes and erythrocyte proliferation [3] .
  • BSF-1 B cell stimulating factor-1
  • IL-4 belongs to the type I cytokine family and has a quaternary structure composed of a hydrophobic bundle core of 4 ⁇ helix [2] .
  • IL-4 is secreted by TH2 cells and participates in TH2-mediated immune responses. It has a wide
  • IL-4 can also stimulate B cells to express major histocompatibility complex class 2 molecules.
  • IL-13 and IL-4 have approximately 30% amino acid sequence homology and a variety of similar functions [4] . Both IL-4 and IL-13 promote B cell proliferation, and combined with CD40/CD40L co-stimulation induces the conversion of IgM type to IgE [5] .
  • IL-4 promotes mast cell aggregation, up-regulates the expression of IgE low-affinity receptor CD23 (Fc ⁇ RII) on mast cell high-affinity IgE receptor and B cells, up-regulates vascular endothelial cell adhesion molecule (VCAM-1) expression, and promotes eosinophils , T lymphocytes, monocytes and basophils. Unlike IL-13, IL-4 promotes the differentiation of naive T cells into TH2 [6] .
  • IgE low-affinity receptor CD23 Fc ⁇ RII
  • VCAM-1 vascular endothelial cell adhesion molecule
  • IL-4 needs to bind to membrane receptors to exert biological functions.
  • the human interleukin receptor (IL-4R) is a heterodimer formed by two polypeptide chains, one of which has a high affinity for IL-4 due to the IL-4R alpha chain to IL in the IL-4R complex.
  • the combination of -4 plays a leading role, so IL-4R ⁇ is commonly used in many scientific studies and reports to replace IL-4R.
  • IL-4R on a variety of human B cells, mast cells, eosinophils, basophils, macrophages / monocytes, the DC cells, fibroblasts, smooth muscle and airway epithelial expression of both [ 7] .
  • IL-4R ⁇ can form two types of receptor complexes with other subunits, and mainly express type I receptors composed of IL-4R ⁇ and ⁇ c in hematopoietic stem cells [3] .
  • IL-4 acts primarily through type II receptors composed of IL-4R ⁇ and IL-13R ⁇ 1 [8,9] .
  • the type II receptor is a co-receptor of IL-4 and IL-13, and IL-13 functions in combination with IL-13R ⁇ 1. Both type I and type II receptors transduce signals through the Jak/STAT pathway.
  • IL-4R ⁇ , ⁇ c and IL-13R ⁇ 1 bind to Jak1, Jak3 and Tyk2, respectively, to activate downstream pathways.
  • IL-4 and IL-13 can also pass.
  • the insulin receptor substrate family (IRS) transduces the signal and ultimately activates PI3-K, NF- ⁇ B in the nucleus [10] .
  • Blocking IL-4R inhibits both IL-4 and the biological function of IL-13.
  • Atopic dermatitis also known as atopic dermatitis or atopic dermatitis
  • AD is a common dermatological disease, more common in children and adolescents, often associated with certain genetic allergic diseases such as allergic rhinitis, asthma, etc. [11 ] .
  • the study found that TH2 levels of IL-2, IL-5, IL-10, and IL-13 were elevated in AD patients [12] , and IgE levels were elevated [13] .
  • TH2 factors were also associated with AD disease progression, overexpression.
  • mice with TH2 factors such as IL-4, IL-13 and the like exhibited skin protection defects and AD-like conditions [14] [15] . Elevated levels of IL-4 and IL-13 in AD patients impede epidermal differentiation and production of antimicrobial peptides [16] . IL-4 deficient mice reduce the incidence of skin allergic inflammation [17] . These studies suggest that blocking IL-4R may be effective in treating AD. Foreign countries have already listed anti-IL-4R monoclonal antibodies, showing good therapeutic effects on AD [11] .
  • IL-13 and IL-4 also play important roles in asthma.
  • Asthma is a common pulmonary inflammatory disease characterized by airway hyperresponsiveness (AHR), excessive mucus secretion, fibrosis, and elevated IgE levels.
  • AHR airway hyperresponsiveness
  • Non-specific stimuli such as cold air often lead to increased airway hyperresponsiveness, and excessive secretion of AHR and mucus leads to airway obstruction, which is the leading cause of asthma death.
  • TH2 factor plays an important role in the progression of asthmatic diseases [18] , and bronchial and alveolar lavage fluids in asthma patients overexpress IL-4 and IL-13 [19] .
  • IL-13 and IL-4 have some functional similarities, some studies have shown that IL-13 plays a more important role in the progression of asthma than other Th2 cytokines [20] .
  • IL-13 can promote the differentiation and fibrosis of goblet cells. Injecting recombinant IL-13 into the airway of mice that have not been stimulated with allergens can cause airway inflammation, excessive mucus secretion and airway hyperresponsiveness [21,22] . Injection of soluble IL13R ⁇ 2 can prevent mouse AHR and mucus secretion. More and the occurrence of lung inflammation. Injection of IL-4R ⁇ antibody in an asthma model reduced eosinophils in AHR and alveolar lavage fluid. Studies have shown that blocking IL-4R ⁇ may be effective in treating asthma.
  • the application provides an antibody that binds to human IL-4R, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, wherein
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQSLLYSIGYNYLD
  • the LCDR2 sequence is LGSNRAS
  • the LCDR3 sequence is MQSFKAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSRNVIYGNGYNYLD
  • the LCDR2 sequence is LGNNNVA
  • the LCDR3 sequence is MQSLQAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQNVVYGNGYNYLD
  • the LCDR2 sequence is LGTNVAA
  • the LCDR3 sequence is MQSLQAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQNVVYGNGYNYLD
  • the LCDR2 sequence is LGNNNVA
  • the LCDR3 sequence is MQSLKAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSHNLLYSNGYNYLD
  • the LCDR2 sequence is LGSNRAY
  • the LCDR3 sequence is MQALQSPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQSLLYSNGYNYLD
  • the LCDR2 sequence is LGSNRAS
  • the LCDR3 sequence is MQALETPYA
  • HCDR and LCDR sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of an antibody is set forth in SEQ ID NO:18.
  • amino acid sequence of the light chain variable region of an antibody is set forth in SEQ ID NO: 26, 27, 28, 29, 30 or 31.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:26.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 28.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:29.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:30.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:31.
  • the application provides an antibody that binds to human IL-4R, wherein the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to SEQ ID NO: 18, and the light chain of the antibody The amino acid sequence of the variable region has at least 90% identity to any of SEQ ID NO: 26, 27, 28, 29, 30 or 31.
  • the antibody is directed against IL-4R ⁇ .
  • the antibody is capable of binding to recombinant human IL4R (SEQ ID NO: 1) and recombinant monkey IL4R (SEQ ID NO: 3), and has a low KD when combined with recombinant human IL4R At 1nM.
  • the antibody can be lower than the IC 50 values of inhibition of recombinant 100pM IL4 (SEQ ID NO: 4) activation of HEK-Blue IL4 / IL-13 cells .
  • the antibody can be lower than the IC 50 values of inhibition 50pM recombinant IL13 (SEQ ID NO: 32) activation of HEK-Blue IL-4 / IL13 cells .
  • the antibody can be lower than the IC 50 values of inhibition of recombinant 200pM IL4: proliferation (SEQ ID NO 4) induction of TF-1 cell.
  • the antibody is a whole antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or comprising a selected from the ⁇ subtype or the ⁇ subtype Light chain constant region.
  • the antibody is a monoclonal antibody.
  • the antibody is a neutralizing antibody.
  • the antibody binds to and neutralizes human IL4R, thereby blocking the IL4-IL4R and IL13-IL4R signaling pathways.
  • the application provides a nucleic acid molecule encoding the antibody of the first aspect to the second aspect, or an antigen binding portion thereof.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first aspect to the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is for treating an IL-4R mediated disease.
  • the present application provides the use of the antibody of the first aspect to the second aspect for the preparation of a medicament for preventing or treating an IL-4R mediated disease.
  • the present application provides a method of preventing or treating an IL-4R-mediated disease, comprising administering the antibody of the first aspect to the second aspect or the pharmaceutical composition of the fourth aspect to an individual in need thereof.
  • the IL-4R mediated disease is an autoimmune disease.
  • the autoimmune disease is selected from the group consisting of asthma or atopic dermatitis.
  • Figure 1 shows an exemplary anti-IL4R phage-scFv binding IL4R epitope analysis of the present application.
  • Figure 2 shows inhibition curves of exemplary anti-IL4R monoclonal antibodies of the present application against IL-4 induced expression of SEAP in HEK-Blue IL-4/IL-13 cells.
  • Figure 3 shows inhibition curves of exemplary anti-IL4R monoclonal antibodies of the present application against IL-13-induced expression of SEAP in HEK-Blue IL-4/IL-13 cells.
  • Figure 4 shows that an exemplary S1E6 light chain mutant of the present application inhibits IL4 binding to IL4R.
  • Figure 5 shows that an exemplary S1E6 light chain mutant of the present application inhibits IL4 binding to IL4R.
  • Figure 6 shows inhibition curves of exemplary S1E6 light chain mutants of the present application against IL-4 induced HEK-Blue IL-4/IL-13 cells expressing SEAP.
  • Figure 7 shows inhibition curves of exemplary S1E6 light chain mutants of the present application against IL-13-induced expression of SEAP in HEK-Blue IL-4/IL-13 cells.
  • Figure 8 shows inhibition curves of exemplary S1E6 light chain mutants of the present application against IL-4 induced HEK-Blue IL-4/IL-13 cells expressing SEAP.
  • Figure 9 shows inhibition curves of exemplary S1E6 light chain mutants of the present application against IL-13-induced expression of SEAP in HEK-Blue IL-4/IL-13 cells.
  • Figure 10 shows that an exemplary S1E6 light chain mutant of the present application inhibits IL-4 induced proliferation of TF-1 cells.
  • Figure 11 shows that an exemplary S1E6 light chain mutant of the present application inhibits IL-4 induced human PBMC CD23 expression.
  • SEQ ID NO: 1 shows the amino acid sequence of the human (homo sapiens) IL-4R extracellular domain (hIL-4R).
  • SEQ ID NO: 2 shows the amino acid sequence of mouse (mus musculus) IL-4R extracellular domain (mIL-4R).
  • SEQ ID NO: 3 shows the amino acid sequence of the extracellular domain of the macaca mulatta IL-4R (mmIL-4R).
  • SEQ ID NO: 4 shows the amino acid sequence of the human IL-4 extracellular domain (hIL-4).
  • SEQ ID NO: 5 shows the amino acid sequence of the His tag (His).
  • SEQ ID NO: 6 shows the amino acid sequence of the Fc fragment (Fc) of human antibody IgG1.
  • SEQ ID NO: 7 shows the amino acid sequence of the Fc fragment (mFc) of the murine antibody IgG2a.
  • SEQ ID NO: 8 shows the amino acid sequence of the human IgGl subtype heavy chain constant region.
  • SEQ ID NO: 9 shows the amino acid sequence of the human IgG2 subtype heavy chain constant region.
  • SEQ ID NO: 10 shows the amino acid sequence of the human IgG4 subtype heavy chain constant region.
  • SEQ ID NO: 11 shows the amino acid sequence of the heavy chain constant region of the murine IgGl subtype.
  • SEQ ID NO: 12 shows the amino acid sequence of the heavy chain constant region of the murine IgG2a subtype.
  • SEQ ID NO: 13 shows the amino acid sequence of the human kappa subtype light chain constant region.
  • SEQ ID NO: 14 shows the amino acid sequence of the human lambda subtype light chain constant region.
  • SEQ ID NO: 15 shows the amino acid sequence of the murine kappa subtype light chain constant region.
  • SEQ ID NO: 16 shows the amino acid sequence of the murine lambda subtype light chain constant region.
  • SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 show the full length amino acid sequence, VH amino acid sequence, and VL amino acid sequence of clone S1E6, respectively.
  • SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 show the full length amino acid sequence, VH amino acid sequence and VL amino acid sequence of clone S1H9, respectively.
  • SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 show the full length amino acid sequence, VH amino acid sequence and VL amino acid sequence of clone S2C2, respectively.
  • SEQ ID NO:26 is the amino acid sequence of the light chain mutant L18D7.
  • SEQ ID NO:27 is the amino acid sequence of the light chain mutant L28G5.
  • SEQ ID NO:28 is the amino acid sequence of the light chain mutant L28F8.
  • SEQ ID NO: 29 is the amino acid sequence of the light chain mutant L28C9.
  • SEQ ID NO: 30 is the amino acid sequence of light chain mutant L10B2.
  • SEQ ID NO: 31 is the amino acid sequence of the light chain mutant L10C2.
  • SEQ ID NO: 32 shows the amino acid sequence of human recombinant IL-13.
  • a novel anti-human IL-4R antibody or antigen-binding fragment thereof obtained novel anti-human IL-4R antibodies by antibody engineering techniques.
  • a novel anti-human IL-4R antibody or antigen-binding fragment thereof a polynucleotide encoding the antibody or antigen-binding fragment thereof, a vector comprising the polynucleotide, comprising the multinuclear Host cells for a glycoside or vector, methods of making and purifying the antibodies, and medical and biological applications of the antibodies or antigen-binding fragments thereof.
  • a full-length antibody molecule can be constructed as a medicament for the treatment of a clinically mediated IL-4R-mediated disease.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule.
  • Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like.
  • antibody includes not only intact (ie, full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') 2 , Fv), variants thereof, and antibody-containing portions thereof.
  • Fusion proteins humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (eg bispecific antibodies) and any other immunoglobulin containing an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • a full or full length antibody comprises two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variant region (VH) and first, second and third constant regions (CH1, CH2 and CH3).
  • Each light chain contains a light chain variant region (VL) and a constant region (CL).
  • the full length antibody can be any kind of antibody, such as IgD, IgE, IgG, IgA or IgM (or a subclass of the above), but the antibody does not need to belong to any particular class.
  • Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of the heavy chain.
  • immunoglobulins there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to different immunoglobulin classes are referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • antigen binding domain refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variant region (VH), a light chain variant region (VL), or both.
  • VH and VL typically contains three complementarity determining regions, CDR1, CDR2 and CDR3.
  • CDRs complementarity determining regions
  • VH or VL There are two common definitions for the CDR sequences of VH or VL, namely the kabat definition and the Chothia definition.
  • CDR sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In an embodiment of the present application, CDR sequences are defined using Kabat.
  • variable region sequences of a given antibody the CDR region sequences in the variable region sequences can be analyzed in a variety of ways, for example, using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab') 2 fragment, which may have two a bivalent fragment of a Fab' fragment joined by a disulfide bridge of the hinge region (ie, a dimer of Fab'); (3) an Fv fragment of the VL and VH domains of the one arm of the antibody; 4) a single-chain Fv (scFv), which may be a single multi-peptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) 2 , which may comprise two linked by a peptide A coupled VH domain and two VL domains are combined with the two VH domains via a disulfide bridge.
  • a Fab fragment which may be a monovalent fragment having a VL-CL chain and a VH-
  • the term "specifically binds" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population are identical except that there may be naturally occurring mutations in a small number of individuals.
  • Monoclonal antibodies as used herein specifically include “chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence derived from a particular species or antibody belonging to a particular antibody or subclass, but The remainder of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies as long as they exhibit desired Biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • R represents A or G
  • Y represents C or T
  • M represents A or C
  • K represents G or T
  • S represents C or G
  • W represents A or T
  • H represents A or C or T
  • B represents C or G or T
  • V represents A or C or G
  • D represents A or G or T
  • N represents A or C or G or T.
  • the application provides an antibody that binds to human IL-4R, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences, and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, wherein
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQSLLYSIGYNYLD
  • the LCDR2 sequence is LGSNRAS
  • the LCDR3 sequence is MQSFKAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSRNVIYGNGYNYLD
  • the LCDR2 sequence is LGNNNVA
  • the LCDR3 sequence is MQSLQAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQNVVYGNGYNYLD
  • the LCDR2 sequence is LGTNVAA
  • the LCDR3 sequence is MQSLQAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQNVVYGNGYNYLD
  • the LCDR2 sequence is LGNNNVA
  • the LCDR3 sequence is MQSLKAPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSHNLLYSNGYNYLD
  • the LCDR2 sequence is LGSNRAY
  • the LCDR3 sequence is MQALQSPYT;
  • the HCDR1 sequence is GFTFSSYAMS
  • the HCDR2 sequence is SITGGGGGIYYADSVKG
  • the HCDR3 sequence is DRISITIRPRYFGLDF
  • the LCDR1 sequence is RSSQSLLYSNGYNYLD
  • the LCDR2 sequence is LGSNRAS
  • the LCDR3 sequence is MQALETPYA
  • HCDR and LCDR sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of an antibody is set forth in SEQ ID NO:18.
  • amino acid sequence of the light chain variable region of an antibody is set forth in SEQ ID NO: 26, 27, 28, 29, 30 or 31.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:26.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:27.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 28.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:29.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:30.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:31.
  • the application provides an antibody that binds to human IL-4R, wherein the amino acid sequence of the heavy chain variable region of the antibody is at least 90% identical to SEQ ID NO: 18, and the light chain of the antibody The amino acid sequence of the variable region has at least 90% identity to any of SEQ ID NO: 26, 27, 28, 29, 30 or 31.
  • the antibody is directed against IL-4R ⁇ .
  • the antibody is capable of binding to recombinant human IL4R (SEQ ID NO: 1) and recombinant monkey IL4R (SEQ ID NO: 3), and has a low KD when combined with recombinant human IL4R At 1nM.
  • the antibody can be lower than the IC 50 values of inhibition of recombinant 100pM IL4 (SEQ ID NO: 4) activation of HEK-Blue IL4 / IL-13 cells .
  • the antibody can be lower than the IC 50 values of inhibition 50pM recombinant IL13 (SEQ ID NO: 32) activation of HEK-Blue IL-4 / IL13 cells .
  • the antibody can be lower than the IC 50 values of inhibition of recombinant 200pM IL4: proliferation (SEQ ID NO 4) induction of TF-1 cell.
  • the antibody is a whole antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or comprising a selected from the ⁇ subtype or the ⁇ subtype Light chain constant region.
  • the antibody is a monoclonal antibody.
  • the antibody is a neutralizing antibody.
  • the antibody binds to and neutralizes human IL4R, thereby blocking the IL4-IL4R and IL13-IL4R signaling pathways.
  • the application provides a nucleic acid molecule encoding the antibody of the first aspect to the second aspect, or an antigen binding portion thereof.
  • the nucleic acid molecule is operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first aspect to the second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition may further comprise one or more of the following: a lubricant such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier; a suspending agent; a preservative, Such as benzoic acid, sorbic acid and calcium propionate; sweeteners and / or flavoring agents.
  • a lubricant such as talc, magnesium stearate, and mineral oil
  • a wetting agent such as talc, magnesium stearate, and mineral oil
  • an emulsifier such as benzoic acid, sorbic acid and calcium propionate
  • a preservative such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and / or flavoring agents such as benzoic acid, sorbic acid and calcium propionate
  • the pharmaceutical compositions of the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical compositions of the present application can be delivered using any physiologically acceptable administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration. Drug, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
  • a pharmaceutical composition for therapeutic use may be formulated in a lyophilized formulation or as an aqueous solution by mixing an agent having the desired purity with a pharmaceutically acceptable carrier, excipient, or the like, as appropriate. storage.
  • the pharmaceutical composition is for treating an IL-4R mediated disease.
  • the present application provides the use of the antibody of the first aspect to the second aspect for the preparation of a medicament for preventing or treating an IL-4R mediated disease.
  • the present application provides a method of preventing or treating an IL-4R-mediated disease, comprising administering the antibody of the first aspect to the second aspect or the pharmaceutical composition of the fourth aspect to an individual in need thereof.
  • the IL-4R mediated disease is an autoimmune disease.
  • the autoimmune disease is selected from the group consisting of asthma or atopic dermatitis.
  • the application further provides a vector comprising an isolated nucleic acid molecule encoding an antibody of the invention or an antigen binding portion thereof, and a host cell comprising the nucleic acid molecule or vector.
  • the present application also provides methods of producing the antibodies of the present application.
  • a method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid.
  • the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.
  • IL-4R extracellular domain hIL-4R, SEQ ID NO: 1
  • mouse IL-4R extracellular domain mIL-4R, SEQ ID NO: 2
  • cynomolgus IL-4R extracellular domain mmIL-4R, SEQ ID NO: 3
  • human IL-4 extracellular domain hIL-4, SEQ ID NO: 4
  • human Recombinant IL-13 SEQ ID NO: 32
  • proteins all have post-translational modifications (eg, glycosylation or disulfide bonds, etc.), and thus the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of the recombinant protein.
  • His tag His, SEQ ID NO: 5
  • Fc human antibody IgG1
  • mFc murine antibody IgG2a
  • the antibody heavy chain constant region can be a human IgG1 subtype (SEQ ID NO: 8), a human IgG2 subtype (SEQ ID NO: 9), a human IgG4 subtype (SEQ ID NO: 10) or a murine IgG1 subtype (SEQ ID NO: 11), murine IgG2a subtype (SEQ ID NO: 12), the light chain constant region may be human kappa subtype (SEQ ID NO: 13), human lambda subtype (SEQ ID NO: 14) or murine kappa Type (SEQ ID NO: 15), murine lambda subtype (SEQ ID NO: 16).
  • the genes of the above various recombinant proteins were designed and synthesized based on the amino acid sequence of the recombinant protein for various purposes of the Uniprot database.
  • the various recombinant protein genes synthesized are cloned into a suitable eukaryotic expression vector (such as invitrogen pcDNA3.1, etc.) using conventional molecular biology techniques, and then using liposomes (such as 293fectin of Invitrogen, etc.) or other cations.
  • the recombinant protein expression plasmid prepared by transfection reagent (such as PEI) is transfected into HEK293 cells (such as HEK293F of Invitrogen) and cultured in serum-free suspension culture for 3-4 days. The culture supernatant is then harvested by centrifugation or the like.
  • the recombinant protein expressed by the His-tag fusion is subjected to one-step purification of the recombinant protein in the culture supernatant by a metal chelate affinity chromatography column (such as GE's HisTrap FF).
  • the recombinant protein expressed by fusion of Fc and mFc was subjected to one-step purification using a Protein A/G affinity chromatography column (e.g., Mabselect SURE, GE).
  • the recombinant protein storage buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (eg, Hitrap desaulting, GE, etc.). If necessary, the antibody samples can be sterilized by filtration and then stored separately at -20 °C.
  • Example 3 Screening of anti-human IL4R mAb using phage display antibody library technology
  • Example 2 The recombinant hIL4R-his prepared in Example 2 was used as an antigen, and the solid phase screening strategy was utilized (experimental program reference phage display: general experimental guide / (American) Clarkson (Clackson, T.), (United States) Lohman (Lowman, HB Edited by Ma Wei et al.
  • the recombinant phage (phage-scFv) was prepared by immobilizing the IL4R recombinant antigen, and the monoclonal antibodies S1E6, S1H9 and S2C2 were prepared, and a series of concentration gradients were performed on the recombinant protein IL4 by different titer of different monoclonal antibody phage-scFv.
  • the HRP-anti-M13 secondary antibody detects the ability of the IL4 recombinant protein to block the binding of three monoclonal antibody phage-scFvs to IL4R.
  • the results (Fig. 1) show that the recombinant protein IL4 can compete with the S1E6 phage-scFv for binding to IL4R, indicating that the S1E6 single chain antibody and IL4 have similar IL4R binding sites.
  • HEK-Blue TM IL-4 / IL-13 cells (InvivoGen, hkb-il413) based on a report to a company InvivoGen HEK293 cell development gene cell line.
  • the cell line is stably transfected into the human STAT6 gene and SEAP (base phosphatase) reporter gene.
  • SEAP base phosphatase reporter gene.
  • IL-4 interleukin-4
  • IL-13 interleukin-13
  • IL-4 human heavy chain constant region IgG4 subtypes
  • the antibody S1E6 mAb was subjected to in vitro affinity maturation using a strategy of light chain CDR (LCDR) mutation based on a dual vector phage display system (for example, see Example 5 of Chinese Patent No. 201510097117.0 filed by the applicant).
  • LCDR light chain CDR
  • a S1E6VK-CDR123 mutant library with a library capacity of more than 1.4 ⁇ 10E8 was constructed using the classical overlap extension PCR method.
  • the primers required for introduction of mutations in the three CDRs of the S1E6 light chain (S1E6VK) are shown in Table 5.
  • the light chain mutant library was then screened for three rounds using recombinant hIL4R-His as the antigen.
  • L18D7 amino acid sequence SEQ ID NO: 26
  • L28G5 amino acid sequence SEQ ID NO: 27
  • L28F8 amino acid sequence SEQ ID NO: 28
  • L28C9 L28C9
  • the light chain replacement strategy is utilized (for details, refer to Example 4.3 of the Chinese Patent No. 201510097117.0 filed by the present applicant) for the in vitro affinity of the antibody S1E6.
  • two high affinity light chain mutants L10B2 (amino acid sequence SEQ ID NO: 30) and L10C2 (amino acid sequence SEQ ID NO: 31) were obtained.
  • Recombinant human IgG4-kappa form fully human antibodies were prepared using conventional molecular biology methods using the high affinity of 4.1 obtained binding to the light chain mutant of IL4R.
  • a 96-well plate (3 ⁇ g/ml, 100 ⁇ l/well) was coated with the antigen IL4R-mFc and coated overnight at 4 °C.
  • Each anti-IL4R recombinant antibody was serially diluted with a fixed concentration of IL4-his, and 100 ⁇ l/well was added to a 96-well plate and incubated at 37 ° C for 1 h.
  • HRP-mouse-anti-his IgG (Kangwei Century, CW0285M) detects the binding of IL4-his and IL4R-mFc.
  • the results of the ELISA analysis (Fig. 4 and Fig. 5) showed that the six light chain mutants of S1E6 were able to effectively block the binding of IL4R and IL4, and the ability was superior to that of S1E6, and IC 50 is shown in Table 6 and Table 7.
  • each anti-IL4R IgG4 chimeric antibody was determined using Biacore X100.
  • Amino coupling kit, human antibody capture kit, CM5 chip and related reagents and consumables such as 10 ⁇ HBS-EP of pH 7.4 were purchased from GE healthcare.
  • the anti-human Fc-segment antibody was coupled to the surface of the CM5 chip using an amino coupling method according to the instructions in the kit, and then the antibody protein was diluted to an appropriate concentration to ensure that about 100 RU of the antibody was captured by the antibody against human Fc.
  • IL4R-his was subjected to a series of concentration gradients (100 nm, 33.3 nm, 11.1 nm, 3.7 nm, 1.23 nm) flowing through the surface of the stationary phase, and the surface of the chip was regenerated with 3M MgCl2, and the affinity of each monoclonal antibody was measured at 25 °C.
  • the biacore data was analyzed using Biacore X100 Evaluation software (version 2.0.1) and the results of the fitting are shown in Tables 8 and 9.
  • the anti-IL4R mAb was captured, and a series of concentration gradients (50 nm, 16.7 nm, 5.56 nm, 31.85 nm, 0.62 nm) were set for mmIL4R-mFc, and the affinity data of each anti-IL4R monoclonal antibody binding to mmIL4R was measured as shown in Table 10. Shown.
  • Example 3.3 The results (FIGS. 6 and 7) shows four light chain mutants S1E6 S1E6 relative biological activity significantly increased, and four kinds of light-chain mutant S1E6 inhibition IC 50 values of IL-4 or IL-13 is relatively close to (and Table 11 Table 12).
  • Results Figures 8 and 9 show two other S1E6 light chain mutants suppress IL-4 and IL-13 increased activity, L10B2 superior L10C2, IC 50 values in Table 13 show L10C2 inhibition the IC 50 of IL-4 is L10B2 1.6 times, the IC 50 values in table 14 show L10C2 inhibiting IL-13 IC 50 values of 2.4 times L10B2.
  • Table 11 S1E6 light chain mutants to inhibit 50 values of IC IL-4
  • Table 14 S1E6 light chain mutants to inhibit 50 values of IC IL-13
  • TF-1 human erythrocytic leukemia cell line
  • TF-1 cell growth is completely dependent on GM-CSF or IL-3, and erythropoietin (EPO) also maintains short-term growth of TF-1 cells, but does not induce differentiation of TF-1 cells.
  • EPO erythropoietin
  • cytokines have effects on TF-1, and cytokines such as IL-4 and IL-13 can stimulate the proliferation of TF-1 cells.
  • TF-1 cells were stimulated with a certain concentration of IL-4 (80 pM), and a series of concentration gradient anti-IL-4R monoclonal antibodies were added (concentration range 65.536 nM to 0.25 pM). , four times dilution), use The number of viable cells was determined by the luminescence cell viability assay kit (Promega, G7571). The results in Figure 10 show that the curves of the four S1E6 light chain mutants inhibiting TF-1 proliferation almost coincide, and the IC 50 values of Table 15 were not significantly different.
  • CD23 (Fc ⁇ RII) is a cell surface receptor with low affinity for IgE and is expressed on the surface of various inflammatory cells. Up-regulation of CD23 can increase the uptake and presentation of antigen by bronchial mucosa and lead to allergic reactions. IL-4 can up-regulate CD23 expression on the surface of monocytes, macrophages and B lymphocytes. PBMC were isolated from healthy human whole blood by Ficoll density gradient centrifugation, PBMC cells were stimulated with a certain concentration of IL-4 (100 pM), and a series of concentration gradient anti-IL-4R monoclonal antibodies were added (maximum concentration 16384 pM to 0.25 pM, four times dilution).
  • the cells were cultured at 37 °C 5% CO 2 environment at 48h, cells were harvested, with anti-CD23-PE (BD Pharmingen, 555711 ) staining, PBMC CD23 expression was measured by flow cytometry (BD Accuri TM C6).
  • the results of Figure 11 show the ability to suppress the expression of PBMC CD23 L18D7 stronger than L28C9, L28C9 IC 50 values of inhibition of IL-4 is 2.3 times the L18D7.

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Abstract

本申请公开了结合人IL-4R的抗体或其抗原结合部分，编码所述抗体或其抗原结合部分的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体或其抗原结合部分的应用。

Description

抗IL-4R抗体及其用途
相关申请的引用
本申请要求2018年4月20日提交的发明名称为“抗IL-4R抗体及其用途”的201810360234.5号中国专利申请的优先权，在此通过引用的方式将上述两篇专利申请的全部内容并入本文。
技术领域
本申请大体涉及基因工程和抗体药物领域；具体而言，涉及抗人白介素-4受体(IL-4R)抗体领域及其用途。本申请开发了新的抗人IL-4R抗体，并提供了该抗体在治疗IL-4R介导的疾病中的用途。
发明背景
白介素4(Interleukin-4，IL-4)由153个氨基酸组成，分子量约为17kDa。最初，因为IL-4能够刺激B细胞增殖而被发现，并被命名为B细胞刺激因子-1(BSF-1) [1]。IL-4与IL-13一样属于I型细胞因子家族，具有4α螺旋的疏水束核心所构成的四级结构 [2]。IL-4由TH2细胞分泌，参与TH2介导的免疫应答，具有广泛的生物学活性，包括刺激T细胞、肥大细胞、粒细胞、巨核细胞和红细胞增殖 [3]。此外，IL-4还可以刺激B细胞表达主要组织相容性复合物2类分子。IL-13与IL-4具有大约30％的氨基酸序列同源性和多种相似的功能 [4]。IL-4和IL-13都能促进B细胞增殖，并联合CD40/CD40L共刺激诱导IgM类型转变成IgE [5]。IL-4促进肥大细胞聚集，上调肥大细胞高亲和力IgE受体和B细胞上IgE低亲和力受体CD23(FcεRII)的表达，上调血管内皮细胞黏附分子(VCAM-1)表达，促进嗜酸性粒细胞、T淋巴细胞、单核细胞和嗜碱性粒细胞的转移。与IL-13不同的是，IL-4可以促进幼稚T细胞分化成TH2 [6]。
IL-4需要与膜受体结合而发挥生物学功能。人白介素受体(IL-4R)是由两条多肽链形成的异二聚体，其中一条α链对IL-4有很高的亲和力，由于在IL-4R复合物中IL-4Rα链对IL-4的结合起主导作用，因此很多科学研究和报道中常用IL-4Rα替代IL-4R。IL-4R在人B细胞、肥大细胞、嗜酸性粒细胞、嗜碱性粒细胞、巨噬细胞/单核细胞、DC细胞、纤维细胞、气道上皮和平滑肌等多种细胞上都有表达 [7]。IL-4Rα可以与其他亚基形成两类受体复合物，在造血干细胞中主要表达由IL-4Rα和γc组成的I型受体 [3]。在非造血干细胞中IL-4主要通过IL-4Rα和IL-13Rα1组成的II型受体发挥作用 [8,9]。II型受体是IL-4和IL-13的共同受体，IL-13与IL-13Rα1结合发挥功能。I型受体和II型受体都通过Jak/STAT通路转导信号，IL-4Rα、γc和IL-13Rα1分别与Jak1、Jak3和Tyk2结合激活下游通路，IL-4和IL-13还可以通过胰岛素受体底物家族(IRS)转导信号，最终激活核内的PI3-K、NF-κB [10]。阻断IL-4R既可以抑制IL-4也可以抑制IL-13的生物学功能。
多项研究表明IL-4和IL-13与TH2免疫应答相关的疾病有关。特应性皮炎(AD)，又称异位性皮炎或遗传过敏性皮炎，是皮肤科常见疾病，多见于儿童和青少年，常与某些遗传过敏性疾病如过敏性鼻炎、哮喘等并发 [11]。研究发现AD患者的TH2因子IL-4、IL-5、IL-10和IL-13水平上升 [12]，IgE水平升高 [13]，此外还发现，TH2因子与AD疾病进程相关，过表达IL-4、IL-13等TH2因子的小鼠表现出皮肤保护缺陷和类似AD病症 [14] [15]。AD患者IL-4和IL-13水平升高阻碍了表皮分化和抗菌肽的产生 [16]。IL-4缺陷小鼠降低皮肤过敏性炎症的发生 [17]。这些研究表明阻断IL-4R可能对治疗AD有效。国外已经有抗IL-4R的单抗上市，对AD表现出良好的治疗效果 [11]。
此外IL-13和IL-4在哮喘中也发挥重要作用。哮喘是一种常见的肺部炎症疾病，以 气道高反应性(AHR)、粘液分泌过多、纤维化和IgE水平升高为特征。非特异性刺激如冷空气等常常导致气道高反应性加剧，AHR和粘液分泌过多导致气道阻碍这是哮喘致死的主要原因。TH2因子在哮喘疾病进程中发挥重要作用 [18]，哮喘患者支气管和肺泡灌洗液过表达IL-4和IL-13 [19]。尽管IL-13和IL-4具有某些功能相似性，但是一些研究表明IL-13在哮喘的疾病进展中发挥了比其他Th2细胞因子更为重要的作用 [20]。IL-13可以促进杯状细胞的分化和纤维化。给未经过过敏原刺激的小鼠气道注射重组IL-13会导致气道炎症，粘液分泌过多和气道高反应性 [21,22]，注射可溶性的IL13Rα2可以阻止小鼠AHR、粘液分泌过多和肺部炎症的发生。在哮喘模型中注射IL-4Rα抗体可以降低AHR和肺泡灌洗液中的嗜酸性粒细胞。研究表明阻断IL-4Rα可能对治疗哮喘有效。
新的抗IL-4R抗体的开发和应用是本领域所需要的。
发明概述
第一方面，本申请提供了结合人IL-4R的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区和含LCDR1、LCDR2和LCDR3序列的轻链可变区，其中
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQSLLYSIGYNYLD，所述LCDR2序列为LGSNRAS，所述LCDR3序列为MQSFKAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSRNVIYGNGYNYLD，所述LCDR2序列为LGNNVAA，所述LCDR3序列为MQSLQAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQNVVYGNGYNYLD，所述LCDR2序列为LGTNVAA，所述LCDR3序列为MQSLQAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQNVVYGNGYNYLD，所述LCDR2序列为LGNNVAA，所述LCDR3序列为MQSLKAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSHNLLYSNGYNYLD，所述LCDR2序列为LGSNRAY，所述LCDR3序列为MQALQSPYT；
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQSLLYSNGYNYLD，所述LCDR2序列为LGSNRAS，所述LCDR3序列为MQALETPYA；
其中HCDR和LCDR序列根据Kabat定义。
在一些实施方案中，抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示。
在一些实施方案中，抗体的轻链可变区的氨基酸序列如SEQ ID NO:26、27、28、29、30或者31所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:26所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:27所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:28所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:29所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:30所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:31所示。
第二方面，本申请提供了结合人IL-4R的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:18具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:26、27、28、29、30或者31中任何一项具有至少90％的一致性。
在第一方面至第二方面的一些实施方案中，所述抗体针对IL-4Rα。
在第一方面至第二方面的一些实施方案中，所述抗体能够结合重组人IL4R(SEQ ID NO:1)和重组猴IL4R(SEQ ID NO:3)，且结合重组人IL4R时的KD低于1nM。
在第一方面至第二方面的一些实施方案中，所述抗体能够以低于100pM的IC 50值抑制重组IL4(SEQ ID NO:4)对HEK-Blue IL-4/IL-13细胞的激活。
在第一方面至第二方面的一些实施方案中，所述抗体能够以低于50pM的IC 50值抑制重组IL13(SEQ ID NO:32)对HEK-Blue IL-4/IL-13细胞的激活。
在第一方面至第二方面的一些实施方案中，所述抗体能够以低于200pM的IC 50值抑制重组IL4(SEQ ID NO:4)诱导的TF-1细胞的增殖。
在第一方面至第二方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面至第二方面的一些实施方案中，所述抗体为全人源抗体。
在第一方面至第二方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区和/或包含选自κ亚型或者λ亚型的轻链恒定区。
在第一方面至第二方面的一些实施方案中，所述抗体为单克隆抗体。
在第一方面至第二方面的一些实施方案中，所述抗体为中和抗体。
在第一方面至第二方面的一些实施方案中，所述抗体能结合并中和人IL4R，进而阻断IL4-IL4R和IL13-IL4R信号通路。
第三方面，本申请提供了核酸分子，其编码第一方面至第二方面所述的抗体或其抗原结合部分。
第四方面，本申请提供了药物组合物，其包含第一方面至第二方面所述的抗体和药学可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述药物组合物用于治疗IL-4R介导的疾病。
第五方面，本申请提供了第一方面至第二方面所述的抗体在制备用于预防或治疗IL-4R介导的疾病的药物中的用途。
第六方面，本申请提供了预防或治疗IL-4R介导的疾病的方法，包括向有需要的个体给予第一方面至第二方面所述的抗体或第四方面的药物组合物。
在第四方面、第五方面和第六方面的一些实施方案中，IL-4R介导的疾病为自身免疫性疾病。在一些具体实施方案中，自身免疫性疾病选自哮喘或过敏性皮炎。
附图说明
图1显示了本申请的示例性抗IL4R噬菌体-scFv结合IL4R表位分析。
图2显示了本申请的示例性抗IL4R单克隆抗体对IL-4诱导的HEK-Blue IL-4/IL-13细胞表达SEAP的抑制曲线。
图3显示了本申请的示例性抗IL4R单克隆抗体对IL-13诱导的HEK-Blue IL-4/IL-13细胞表达SEAP的抑制曲线。
图4显示了本申请的示例性S1E6轻链突变体抑制IL4结合IL4R。
图5显示了本申请的示例性S1E6轻链突变体抑制IL4结合IL4R。
图6显示了本申请的示例性S1E6轻链突变体对IL-4诱导的HEK-Blue IL-4/IL-13细胞表达SEAP的抑制曲线。
图7显示了本申请的示例性S1E6轻链突变体对IL-13诱导的HEK-Blue IL-4/IL-13细胞表达SEAP的抑制曲线。
图8显示了本申请的示例性S1E6轻链突变体对IL-4诱导的HEK-Blue IL-4/IL-13细胞表达SEAP的抑制曲线。
图9显示了本申请的示例性S1E6轻链突变体对IL-13诱导的HEK-Blue IL-4/IL-13细胞表达SEAP的抑制曲线。
图10显示了本申请的示例性S1E6轻链突变体抑制IL-4诱导的TF-1细胞的增殖。
图11显示了本申请的示例性S1E6轻链突变体抑制IL-4诱导的人PBMC CD23表达。
序列说明
SEQ ID NO:1显示人(homo sapiens)IL-4R胞外区(hIL-4R)的氨基酸序列。
SEQ ID NO:2显示小鼠(mus musculus)IL-4R胞外区(mIL-4R)的氨基酸序列。
SEQ ID NO:3显示猕猴(Macaca mulatta)IL-4R胞外区(mmIL-4R)的氨基酸序列。
SEQ ID NO:4显示人IL-4胞外区(hIL-4)的氨基酸序列。
SEQ ID NO:5显示His标签(His)的氨基酸序列。
SEQ ID NO:6显示人抗体IgG1的Fc段(Fc)的氨基酸序列。
SEQ ID NO:7显示鼠抗体IgG2a的Fc段(mFc)的氨基酸序列。
SEQ ID NO:8显示人IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:9显示人IgG2亚型重链恒定区的氨基酸序列。
SEQ ID NO:10显示人IgG4亚型重链恒定区的氨基酸序列。
SEQ ID NO:11显示鼠IgG1亚型重链恒定区的氨基酸序列。
SEQ ID NO:12显示鼠IgG2a亚型重链恒定区的氨基酸序列。
SEQ ID NO:13显示人κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:14显示人λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:15显示鼠κ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:16显示鼠λ亚型轻链恒定区的氨基酸序列。
SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19分别显示克隆S1E6的全长氨基酸序列、VH氨基酸序列和VL氨基酸序列。
SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22分别显示克隆S1H9的全长氨基酸序列、VH氨基酸序列和VL氨基酸序列。
SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25分别显示克隆S2C2的全长氨基酸序列、VH氨基酸序列和VL氨基酸序列。
SEQ ID NO:26为轻链突变体L18D7的氨基酸序列。
SEQ ID NO:27为轻链突变体L28G5的氨基酸序列。
SEQ ID NO:28为轻链突变体L28F8的氨基酸序列。
SEQ ID NO:29为轻链突变体L28C9的氨基酸序列。
SEQ ID NO:30为轻链突变体L10B2的氨基酸序列。
SEQ ID NO:31为轻链突变体L10C2的氨基酸序列。
SEQ ID NO:32显示人重组IL-13的氨基酸序列。
发明详细描述
本申请的发明人通过抗体工程技术得到了新的抗人IL-4R抗体。在本申请的多个方面，提供了新的抗人IL-4R抗体或其抗原结合片段，编码该抗体或其抗原结合片段的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该抗体的方法及所述抗体或其抗原结合片段的医学和生物学应用。根据本申请提供的抗体的可变区的序列，可构建全长的抗体分子作为药物用于治疗临床上由IL-4R介导的疾病。
除非另外指明，本发明的实施采用本领域常规的分子生物学、微生物学、细胞生物学、生物化学以及免疫学技术。
除非另外指明，本申请中所用的术语具有本领域技术人员通常所理解的含义。
定义
如本文所用术语“抗体”，是指能够经由至少一个位于免疫球蛋白分子的可变区中的抗原识别位点特异性结合到标靶的免疫球蛋白分子。标靶包括但不限于碳水化合物、多聚核苷酸、脂质、多肽等。本文所使用的“抗体”不仅包括完整的(即全长的)抗体，而且还包括其抗原结合片段(例如Fab、Fab'、F(ab') 2、Fv)、其变异体、包含抗体部分的融合蛋白、人源化抗体、嵌合抗体、双抗体、线性抗体、单链抗体、多特异性抗体(例如双特异性抗体)及任何其他包含所需特异性的抗原识别位点的免疫球蛋白分子的修改配置，包括抗体的糖基化变体、抗体的氨基酸序列变体及共价修饰的抗体。
通常，完整或全长的抗体包含两个重链和两个轻链。每个重链含有重链变异区(VH)和第一、第二及第三恒定区(CH1、CH2及CH3)。每个轻链含有轻链变异区(VL)和恒定区(CL)。全长的抗体可以是任何种类的抗体，例如IgD、IgE、IgG、IgA或IgM(或上述的子类)，但抗体不需要属于任何特定的类别。根据重链的恒定域的抗体氨基酸序列，可以将免疫球蛋白指定为不同的类别。通常，免疫球蛋白有五种主要的类别：IgA、IgD、IgE、IgG及IgM，而且这些类别中有几个可以再被进一步区分成子类(同型)，例如IgG1、IgG2、IgG3、IgG4、IgA1及IgA2。对应于不同免疫球蛋白类别的重链恒定域分别称为α、δ、ε、γ、以及μ。不同类别的免疫球蛋白的子单元结构和三维结构是公知的。
如本文所用术语“抗原结合片段或抗原结合部分”，是指负责结合抗原的完整抗体分子的一部分或区域。抗原结合域可以包含重链变异区(VH)、轻链变异区(VL)或上述两者。VH和VL中的每个通常含有三个互补决定区CDR1、CDR2及CDR3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义。(参阅例如Kabat,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989))。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
抗原结合片段的实例包括但不限于：(1)Fab片段，其可以是具有VL-CL链和VH-CH1链的单价片段；(2)F(ab') 2片段，其可以是具有两个Fab'片段的二价片段，该两个Fab'片段由铰链区的二硫桥(即Fab'的二聚物)连接；(3)具有抗体的单臂的VL和VH域的Fv片段；(4)单链Fv(scFv)，其可以是由VH域和VL域经由胜肽连接符组成的单一多胜肽链；以及(5)(scFv) 2，其可以包含两个由胜肽连接符连接的VH域和两个VL域，该两个VL域是经由二硫桥与该两个VH域组合。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合。
本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(美国专利号4,816,567；和Morrison等，Proc.Natl.Acad.Sci.USA81:6851-6855(1984))。
在本文给出的核酸序列中涉及兼并碱基(除了A、T、C、G常规碱基之外)的使用，其含义与本领域技术人员通常理解的相同。例如，R代表A或G；Y代表C或T，M代表A或C；K代表G或T；S代表C或G；W代表A或T；H代表A或C或T；B代表C或G或T；V代表A或C或G；D代表A或G或T；N代表A或C或G或T。
第一方面，本申请提供了结合人IL-4R的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区和含LCDR1、LCDR2和LCDR3序列的轻链可变区，其中
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQSLLYSIGYNYLD，所述LCDR2序列为LGSNRAS，所述LCDR3序列为MQSFKAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSRNVIYGNGYNYLD，所述LCDR2序列为LGNNVAA，所述LCDR3序列为MQSLQAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQNVVYGNGYNYLD，所述LCDR2序列为LGTNVAA，所述LCDR3序列为MQSLQAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQNVVYGNGYNYLD，所述LCDR2序列为LGNNVAA，所述LCDR3序列为MQSLKAPYT；或者
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSHNLLYSNGYNYLD，所述LCDR2序列为LGSNRAY，所述LCDR3序列为MQALQSPYT；
所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQSLLYSNGYNYLD，所述LCDR2序列为LGSNRAS，所述LCDR3序列为MQALETPYA；
其中HCDR和LCDR序列根据Kabat定义。
在一些实施方案中，抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示。
在一些实施方案中，抗体的轻链可变区的氨基酸序列如SEQ ID NO:26、27、28、29、30或者31所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:26所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:27所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:28所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:29所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:30所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:31所示。
第二方面，本申请提供了结合人IL-4R的抗体，其中所述抗体的重链可变区的氨基酸序列与SEQ ID NO:18具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:26、27、28、29、30或者31中任何一项具有至少90％的一致性。
在第一方面至第二方面的一些实施方案中，所述抗体针对IL-4Rα。
在第一方面至第二方面的一些实施方案中，所述抗体能够结合重组人IL4R(SEQ ID NO:1)和重组猴IL4R(SEQ ID NO:3)，且结合重组人IL4R时的KD低于1nM。
在第一方面至第二方面的一些实施方案中，所述抗体能够以低于100pM的IC 50值抑制重组IL4(SEQ ID NO:4)对HEK-Blue IL-4/IL-13细胞的激活。
在第一方面至第二方面的一些实施方案中，所述抗体能够以低于50pM的IC 50值抑制重组IL13(SEQ ID NO:32)对HEK-Blue IL-4/IL-13细胞的激活。
在第一方面至第二方面的一些实施方案中，所述抗体能够以低于200pM的IC 50值抑制重组IL4(SEQ ID NO:4)诱导的TF-1细胞的增殖。
在第一方面至第二方面的一些实施方案中，所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)。
在第一方面至第二方面的一些实施方案中，所述抗体为全人源抗体。
在第一方面至第二方面的一些实施方案中，所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区和/或包含选自κ亚型或者λ亚型的轻链恒定区。
在第一方面至第二方面的一些实施方案中，所述抗体为单克隆抗体。
在第一方面至第二方面的一些实施方案中，所述抗体为中和抗体。
在第一方面至第二方面的一些实施方案中，所述抗体能结合并中和人IL4R，进而阻断IL4-IL4R和IL13-IL4R信号通路。
第三方面，本申请提供了核酸分子，其编码第一方面至第二方面所述的抗体或其抗原结合部分。
在一些实施方案中，所述核酸分子可操作地连接到调控序列，调控序列可以 被用所述载体转化过的宿主细胞识别。
第四方面，本申请提供了药物组合物，其包含第一方面至第二方面所述的抗体和药学可接受的赋形剂、稀释剂或载体。
在一些实施方案中，药物组合物还可包含下述中的一种或多种：润滑剂，如滑石粉、硬脂酸镁和矿物油；润湿剂；乳化剂；悬浮剂；防腐剂，如苯甲酸、山梨酸和丙酸钙；增甜剂和/或调味剂等。
在一些实施方案中，可将本申请中的药物组合物配制为片剂、丸剂、粉剂、锭剂、酏剂、悬液、乳剂、溶液、糖浆、栓剂或胶囊等形式。
在一些实施方案中，可以利用任何生理上可接受的给药方式递送本申请的药物组合物，这些给药方式包括但不限于：口服给药、肠胃外给药、经鼻给药、直肠给药、腹膜内给药、血管内注射、皮下给药、经皮给药、吸入给药等。
在一些实施方案中，可以通过混合具有所需纯度的试剂与视情况的药学上可接受的载体、赋形剂等，以冻干制剂或水溶液的形式配制用于治疗用途的药物组合物用于存储。
在一些实施方案中，所述药物组合物用于治疗IL-4R介导的疾病。
第五方面，本申请提供了第一方面至第二方面所述的抗体在制备用于预防或治疗IL-4R介导的疾病的药物中的用途。
第六方面，本申请提供了预防或治疗IL-4R介导的疾病的方法，包括向有需要的个体给予第一方面至第二方面所述的抗体或第四方面的药物组合物。
在第四方面、第五方面和第六方面的一些实施方案中，IL-4R介导的疾病为自身免疫性疾病。在一些具体实施方案中，自身免疫性疾病选自哮喘或过敏性皮炎。
在其他方面，本申请还提供包含编码本申请抗体或其抗原结合部分的分离的核酸分子的载体以及包含所述核酸分子或载体的宿主细胞。
在其他方面，本申请还提供产生本申请抗体的方法。在一些实施方案中，产生抗体的方法包括培养宿主细胞以便于表达核酸。在一些实施方案中，产生抗体的方法还包括从宿主细胞培养基中回收抗体。
应当理解，以上详细描述仅为了使本领域技术人员更清楚地了解本申请的内容，而并非意图在任何方面加以限制。本领域技术人员能够对所述实施方案进行各种改动和变化。
以下实施例仅用于说明而非限制本申请范围的目的。
实施例
实施例1.噬菌体展示抗体库的构建
本实施例的内容参照发明人之前的中国专利申请201610609651.X号(题目为：抗人PDL1抗体及其用途)以及201510097117.0号(题目为：抗人IL-17单克隆抗体)。通过引用的方式，将上述两篇专利申请的内容并入本文。
实施例2：重组蛋白的制备
制备和测试抗IL-4R单抗的过程中用到多种不同的重组蛋白，包括人IL-4R胞外区(hIL-4R，SEQ ID NO:1)、小鼠IL-4R胞外区(mIL-4R，SEQ ID NO:2)、猕猴IL-4R胞外区(mmIL-4R，SEQ ID NO:3)、人IL-4胞外区(hIL-4，SEQ ID NO:4)和人重组IL-13(SEQ ID NO:32)。这些蛋白都具有翻译后修饰(如：糖基化或者二硫键等)，因而利用哺乳动物细胞表达系统将更有利于保持重组蛋白的结构和功能。此外，在这些重组蛋白的C端添加了His标签(His，SEQ ID NO:5)或者人抗体IgG1的Fc段(Fc，SEQ ID NO:6)或者鼠抗体IgG2a的Fc段(mFc，SEQ ID NO:7)，更有利于重组蛋白的纯化和单克隆抗体功能的鉴定。抗体重链恒定区可以是人IgG1亚型(SEQ ID  NO:8)，人IgG2亚型(SEQ ID NO:9)、人IgG4亚型(SEQ ID NO:10)或者鼠IgG1亚型(SEQ ID NO:11)，鼠IgG2a亚型(SEQ ID NO:12)，轻链恒定区可以是人κ亚型(SEQ ID NO:13)、人λ亚型(SEQ ID NO:14)或者鼠κ亚型(SEQ ID NO:15)、鼠λ亚型(SEQ ID NO:16)。
根据Uniprot数据库的各种目的重组蛋白的氨基酸序列，设计并合成上述各种重组蛋白的基因(包含His标签或者Fc、mFc编码基因)。利用常规的分子生物学技术将合成的各种重组蛋白基因克隆至合适的真核表达载体(如invitrogen公司的pcDNA3.1等)，然后利用脂质体(如invitrogen公司的293fectin等)或者其他阳离子转染试剂(如PEI等)将制备的重组蛋白表达质粒转染入HEK293细胞(如invitrogen公司的HEK293F)，在无血清悬浮培养条件下培养3-4天。然后通过离心等方式收获培养上清。
His标签融合表达的重组蛋白利用金属螯合亲和层析柱(如GE公司的HisTrap FF等)对培养上清中的重组蛋白进行一步纯化。Fc和mFc融合表达的重组蛋白用ProteinA/G亲和层析柱(如GE公司的Mabselect SURE等)进行一步纯化。然后利用脱盐柱(如GE公司的Hitrap desaulting等)将重组蛋白保存缓冲液置换为PBS(pH7.0)或者其他合适的缓冲液。必要时，可以对抗体样品进行过滤除菌，然后分装保存于-20℃。
实施例3：利用噬菌体展示抗体库技术筛选抗人IL4R单抗
3.1 抗人IL4R单抗的筛选
以实施例2制备的重组hIL4R-his为抗原，利用固相筛选策略(实验方案参考噬菌体展示：通用实验指南/(美)克拉克森(Clackson,T.),(美)洛曼(Lowman,H.B.)编；马岚等译。化学工业出版社，2008.5)筛选实施例1制备的展示人单链抗体库的噬菌体库，获得3株序列不同，但均能特异性结合人IL4R的人抗体，包括克隆S1E6(氨基酸序列为SEQ ID NO:17，VH序列为SEQ ID NO:18，VL序列为SEQ ID NO:19)、S1H9(氨基酸序列为SEQ ID NO:20，VH序列为SEQ ID NO:21，VL序列为SEQ ID NO:22)、S2C2(氨基酸序列为SEQ ID NO:23，VH序列为SEQ ID NO:24，VL序列为SEQ ID NO:25)。
3.2 抗人IL4R单抗的初步功能分析(蛋白水平)
包被IL4R重组抗原，将S1E6、S1H9、S2C2这3株单克隆抗体制备纯化噬菌体(噬菌体-scFv)，用固定滴度的不同单克隆抗体噬菌体-scFv分别对重组蛋白IL4做一系列的浓度梯度，HRP-抗M13二抗检测IL4重组蛋白阻断3株单克隆抗体噬菌体-scFv结合IL4R的能力。结果(图1)显示，重组蛋白IL4可以和S1E6噬菌体-scFv竞争结合IL4R，说明S1E6单链抗体和IL4有相似的IL4R结合位点。
3.3 重组抗人IL4R单抗的初步功能分析(细胞水平)
HEK-Blue TM IL-4/IL-13细胞(InvivoGen,hkb-il413)为InvivoGen公司基于HEK293细胞开发的一个报告基因细胞株。该细胞株中稳转进人STAT6基因和SEAP(碱基磷酸酶)报告基因，当白介素-4(IL-4)或者白介素-13(IL-13)刺激细胞时，细胞内的STAT6信号通路被激活，并诱导SEAP报告基因表达、合成并分泌SEAP到细胞上清中，用酶标仪630nm定量分析SEAP浓度。本实施例用HEK-Blue TM IL-4/IL-13细胞评价不同重组抗IL4R单抗(人IgG4亚型重链恒定区)抑制IL-4和IL-13的能力。将96孔板的每孔接种5×10 4细胞，用一定浓度的IL-4(40pM)或IL-13(80pM)刺激HEK-Blue TM IL-4/IL-13细胞，加入一系列浓度梯度抗IL4R单克隆抗体阻断IL-4或IL-13。图2和图3的结果显示单抗S1E6阻断IL-4和IL-13的能力最强，S2C2弱 于S1E6，S1H9不能阻断IL-4和IL-13对HEK-Blue TM IL-4/IL-13细胞的刺激。
实施例4：抗人IL4R单抗的亲和力成熟
4.1 基于轻链CDR突变和轻链置换的策略对抗体S1E6进行体外亲和力成熟
基于双载体噬菌体展示系统，利用轻链CDR(LCDR)突变的策略(具体操作可参照申请人之前提交的中国专利第201510097117.0号中的实施例5)对抗体S1E6单抗进行体外亲和力成熟。利用经典重叠延伸PCR方法构建了库容量超过1.4×10E8的S1E6VK-CDR123突变库。其中在S1E6轻链(S1E6VK)的三个CDR中引入突变所需引物见表5。然后利用重组hIL4R-His为抗原，对此轻链突变库进行了三轮筛选。最后鉴定出4个高亲和力的轻链突变体L18D7(氨基酸序列为SEQ ID NO:26)、L28G5(氨基酸序列为SEQ ID NO:27)、L28F8(氨基酸序列为SEQ ID NO:28)、L28C9(氨基酸序列为SEQ ID NO:29)。
表5 构建S1E6轻链LCDR突变库所需引物
Figure PCTCN2018100263-appb-000001
同时，基于双载体噬菌体展示系统，以S1E6重链为基础，利用轻链置换策略(具体操作可参照本申请人之前提交的中国专利第201510097117.0号中的实施例4.3)对抗体S1E6进行了体外亲和力成熟研究，获得两个高亲和力轻链突变体L10B2(氨基酸序列为SEQ ID NO:30)、L10C2(氨基酸序列为SEQ ID NO:31)。
4.2 抗IL4R单抗重组蛋白水平功能分析
利用常规分子生物学方法，将4.1得到的高亲和力结合IL4R的轻链突变体制备重组人IgG4-κ形式全人源抗体。
用抗原IL4R-mFc包被96孔板(3μg/ml，100μl/孔)，4℃包被过夜。用固定浓度的IL4-his对各抗IL4R重组抗体进行梯度稀释，100μl/孔加入96孔板中，37℃孵育1h。HRP-小鼠-抗his IgG(康为世纪，CW0285M)检测IL4-his和IL4R-mFc的结合。ELISA分析结果(图4和图5)显示S1E6的6种轻链突变体能够有效阻断IL4R和IL4的结合，且能力优于S1E6，IC 50见表6和表7。
表6：S1E6轻链突变体抑制IL4结合IL4R的IC 50
L18D7 L28G5 L28F8 L28C9 S1E6
IC 50 9.682 8.304 10.78 3.755 16.6
表7：S1E6轻链突变体抑制IL4结合IL4R的IC 50
L10C2 L10B2 S1E6
IC 50 7.879 7.166 19.77
4.3 抗IL4R单抗的亲和力分析
利用Biacore X100测定各抗IL4R IgG4嵌合抗体的亲和力。氨基偶联试剂盒，人抗体捕获试剂盒，CM5芯片和pH7.4的10×HBS-EP等相关试剂和耗材均购自GE healthcare。根据试剂盒中的说明书，使用氨基偶联的方法将抗人Fc段的抗体偶联至CM5芯片表面，然后稀释抗体蛋白至合适浓度，保证100RU左右的抗体被抗人Fc的抗体捕获。将IL4R-his设置一系列的浓度梯度(100nm，33.3nm，11.1nm，3.7nm，1.23nm)流经固定相表面，用3M MgCl2对芯片表面进行再生，25℃测定各单克隆抗体的亲和力。使用Biacore X100 Evaluation软件(版本2.0.1)对biacore数据进行分析，拟合结果如表8和表9所示。
表8.抗IL4R单抗(轻链置换突变体)结合huIL4R亲和力常数
K on(1/Ms) K off(1/s) KD(M)
L10B2-IgG4 3.432E+5 1.520E-4 4.429E-10
L10C2-IgG4 3.531E+5 3.085E-4 8.736E-10
S1E6-IgG4 3.125E+5 9.227E-4 2.953E-9
表9.抗IL4R单抗(LCDR突变体)结合huIL4R亲和力常数
K on(1/Ms) K off(1/s) KD(M)
L18D7-IgG4 2.97E5 1.426E-4 4.8E-10
L28F8-IgG4 3.422E5 1.436E-4 4.196E-10
L28G5-IgG4 2.891E5 1.343E-4 4.645E-10
L28C9-IgG4 3.307E5 1.937E-4 5.857E-10
S1E6-IgG4 2.494E5 7.086E-4 2.841E-9
同样的，捕获抗IL4R单抗，将mmIL4R-mFc设置一系列的浓度梯度(50nm，16.7nm，5.56nm，31.85nm，0.62nm)，测得各抗IL4R单抗结合mmIL4R的亲和力数据如表10所示。
K on(1/Ms) K off(1/s) KD(M)
L18D7-IgG4 5.904E5 9.839E-4 1.667E-9
L28C9-IgG4 7.065E5 5.935E-3 8.401E-9
L28F8-IgG4 7.485E5 5.189E-3 6.932E-9
L28G5-IgG4 6.842E5 7.086E-3 1.036E-8
S1E6-IgG4 5.271E5 4.904E-3 9.305E-9
4.4 抗IL4R单抗的细胞学功能分析
4.4.1 基于HEK-Blue IL-4/IL-13细胞分析抗IL4R单抗的生物学活性
具体实施方法参见实施例3.3。结果(图6和图7)显示四种S1E6轻链突变体相对于S1E6生物活性显著提高，并且四种S1E6轻链突变体抑制IL-4或IL-13的IC 50值比较接近(表11和表12)。图8和图9的结果显示另外两种S1E6轻链突变体抑制IL-4和IL-13的活性增强，L10B2要优于L10C2，表13的IC 50值显示L10C2抑制IL-4的IC 50是L10B2的1.6倍，表14的IC 50值显示L10C2抑制IL-13的IC 50值是L10B2 的2.4倍。
表11：S1E6轻链突变体抑制IL-4的IC 50值
IL-4 S1E6-IgG4 L18D7-IgG4 L28C9-IgG4 L28G5-IgG4 L28F8-IgG4
IC 50(pM) ～ 48.39 49.40 48.41 55.02
表12：S1E6轻链突变体抑制IL-13的IC 50值
IL-13 S1E6-IgG4 L18D7-IgG4 L28C9-IgG4 L28G5-IgG4 L28F8-IgG4
IC 50(pM) ～ 10.20 12.12 11.51 12.07
表13：S1E6轻链突变体抑制IL-4的IC 50值
IL-4 S1E6-IgG4 L10B2-IgG4 L10C2-IgG4
IC 50(pM) ～ 206.7 332.8
表14：S1E6轻链突变体抑制IL-13的IC 50值
IL-13 S1E6-IgG4 L10B2-IgG4 L10C2-IgG4
IC 50(pM) ～ 13.05 31.42
4.4.2 基于TF-1细胞增殖分析抗IL4R单抗的生物学活性
人红细胞白血病细胞系(TF-1)由Kitamura等人在1989年建立，实验中所用TF-1来自ATCC细胞库(CRL-2003)。TF-1细胞生长完全依赖于GM-CSF或IL-3，红细胞生成素(EPO)也可以维持TF-1细胞的短期生长，但不能诱导TF-1细胞分化。此外多种细胞因子对TF-1都有作用，IL-4、IL-13等细胞因子可以刺激TF-1细胞增殖。将96孔板的每孔接种2×10 4细胞，用一定浓度的IL-4(80pM)刺激TF-1细胞，加入一系列浓度梯度抗IL-4R单克隆抗体(浓度范围65.536nM～0.25pM，四倍稀释)，用
Figure PCTCN2018100263-appb-000002
发光法细胞活力检测试剂盒(Promega,G7571)测定活细胞数量。图10结果显示四种S1E6轻链突变体抑制TF-1增殖的曲线几乎重合，表15的IC 50值无显著差异。
表15：S1E6轻链突变体抑制IL-4的IC 50值
L18D7-IgG4 L28C9-IgG4 L28G5-IgG4 L28F8-IgG4
IC 50(nM) 0.1159 0.1289 0.1181 0.1236
4.4.3 基于PBMC分析抗IL4R单抗的生物学活性
CD23(FcεRII)是一种对IgE亲和力较低的细胞表面受体，在多种炎症细胞表面都有表达，CD23表达上调会提高支气管粘膜对抗原的摄取和提呈导致过敏性反应。IL-4可以刺激单核细胞、巨噬细胞和B淋巴细胞表面CD23表达上调。利用Ficoll密度梯度离心法从健康人全血中分离PBMC，用一定浓度的IL-4(100pM)刺激PBMC细胞，加入一系列浓度梯度抗IL-4R单抗(最大浓度16384pM～0.25pM，四倍稀释)。将细胞在37℃5％CO 2环境下培养48h，收集细胞，用抗CD23-PE(BD Pharmingen,555711)染色，用流式细胞术(BD Accuri TMC6)测定PBMC上CD23的表达。图11的结果显示L18D7抑制PBMC CD23表达的能力强于L28C9，L28C9抑制IL-4的IC 50值是L18D7的2.3倍。
表16：S1E6轻链突变体抑制人PBMC CD23表达IC 50值
L18D7-IgG4 L28C9-IgG4
IC 50(pM) 26.82 63.48
参考文献
1.Howard,M.,et al.,Identification of a T cell-derived b cell growth factor distinct from interleukin 2.J Exp Med,1982.155(3):p.914-23.
2.LaPorte,S.L.,et al.,Molecular and structural basis of cytokine receptor pleiotropy in the interleukin-4/13 system.Cell,2008.132(2):p.259-72.
3.Nelms,K.,et al.,The IL-4 receptor:signaling mechanisms and biologic functions.Annu Rev Immunol,1999.17:p.701-38.4.Wynn,T.A.,IL-13 effector functions.Annu Rev Immunol,2003.21:p.425-56.
5.Punnonen,J.,et al.,Interleukin 13 induces interleukin 4-independent IgG4 and IgE synthesis and CD23 expression by human B cells.Proc Natl Acad Sci U S A,1993.90(8):p.3730-4.
6.Zurawski,G.and J.E.de Vries,Interleukin 13,an interleukin 4-like cytokine that acts on monocytes and B cells,but not on T cells.Immunol Today,1994.15(1):p.19-26.
7.Corren,J.,Role of interleukin-13 in asthma.Curr Allergy Asthma Rep,2013.13(5):p.415-20.
8.Obiri,N.I.,et al.,Receptor for interleukin 13.Interaction with interleukin 4 by a mechanism that does not involve the common gamma chain shared by receptors for interleukins 2,4,7,9,and 15.J Biol Chem,1995.270(15):p.8797-804.
9.Hilton,D.J.,et al.,Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.Proc Natl Acad Sci U S A,1996.93(1):p.497-501.
10.Kraich,M.,et al.,A modular interface of IL-4 allows for scalable affinity without affecting specificity for the IL-4 receptor.BMC Biol,2006.4:p.13.
11.Blakely,K.,M.Gooderham,and K.Papp,Dupilumab,A Monoclonal Antibody for Atopic Dermatitis:A Review of Current Literature.Skin Therapy Lett,2016.21(2):p.1-5.12.Esnault,S.,et al.,Differential spontaneous expression of mRNA for IL-4,IL-10,IL-13,IL-2 and interferon-gamma(IFN-gamma)in peripheral blood mononuclear cell(PBMC)from atopic patients.Clin Exp Immunol,1996.103(1):p.111-8.
13.Jujo,K.,et al.,Decreased interferon gamma and increased interleukin-4production in atopic dermatitis promotes IgE synthesis.J Allergy Clin Immunol,1992.90(3Pt 1):p.323-31.
14.Chan,L.S.,N.Robinson,and L.Xu,Expression of interleukin-4 in the epidermis of transgenic mice results in a pruritic inflammatory skin disease:an experimental animal model to study atopic dermatitis.J Invest Dermatol,2001.117(4):p.977-83.
15.Zheng,T.,et al.,Transgenic expression of interleukin-13 in the skin induces a pruritic dermatitis and skin remodeling.J Invest Dermatol,2009.129(3):p.742-51.
16.Howell,M.D.,et al.,Cytokine modulation of atopic dermatitis filaggrin skin expression.J Allergy Clin Immunol,2007.120(1):p.150-5.
17.Sehra,S.,et al.,IL-4regulates skin homeostasis and the predisposition toward allergic skin inflammation.J Immunol,2010.184(6):p.3186-90.
18.Larche,M.,D.S.Robinson,and A.B.Kay,The role of T lymphocytes in the pathogenesis of asthma.J Allergy Clin Immunol,2003.111(3):p.450-63；quiz 464.
19.Kotsimbos,T.C.,P.Ernst,and Q.A.Hamid,Interleukin-13 and interleukin-4 are coexpressed in atopic asthma.Proc Assoc Am Physicians,1996.108(5):p.368-73.
20.Wills-Karp,M.,Interleukin-13 in asthma pathogenesis.Curr Allergy Asthma Rep,2004.4(2):p.123-31.
21.Grunig,G.,et al.,Requirement for IL-13 independently of IL-4 in experimental asthma.Science,1998.282(5397):p.2261-3.
22.Wills-Karp,M.,et al.,Interleukin-13:central mediator of allergic asthma.Science,1998.282(5397):p.2258-61.

Claims (10)

  1. 一种结合人IL-4R的抗体，其包含含HCDR1、HCDR2和HCDR3序列的重链可变区和含LCDR1、LCDR2和LCDR3序列的轻链可变区，其中
    所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQSLLYSIGYNYLD，所述LCDR2序列为LGSNRAS，所述LCDR3序列为MQSFKAPYT；或者
    所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSRNVIYGNGYNYLD，所述LCDR2序列为LGNNVAA，所述LCDR3序列为MQSLQAPYT；或者
    所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQNVVYGNGYNYLD，所述LCDR2序列为LGTNVAA，所述LCDR3序列为MQSLQAPYT；或者
    所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQNVVYGNGYNYLD，所述LCDR2序列为LGNNVAA，所述LCDR3序列为MQSLKAPYT；或者
    所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSHNLLYSNGYNYLD，所述LCDR2序列为LGSNRAY，所述LCDR3序列为MQALQSPYT；
    所述HCDR1序列为GFTFSSYAMS，所述HCDR2序列为SITGGGGGIYYADSVKG，所述HCDR3序列为DRISITIRPRYFGLDF，所述LCDR1序列为RSSQSLLYSNGYNYLD，所述LCDR2序列为LGSNRAS，所述LCDR3序列为MQALETPYA；
    其中HCDR和LCDR序列根据Kabat定义。
  2. 根据权利要求1所述的抗体，其中所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示。
  3. 根据权利要求1所述的抗体，其中所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:26、27、28、29、30或者31所示。
  4. 根据权利要求1所述的抗体，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:26所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:27所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:28所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:29所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可 变区的氨基酸序列如SEQ ID NO:30所示；或者
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:18所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:31所示。
  5. 一种结合人IL-4R的抗体，所述抗体的重链可变区的氨基酸序列与SEQ ID NO:18具有至少90％的一致性，并且所述抗体的轻链可变区的氨基酸序列与SEQ ID NO:26、27、28、29、30或者31中任何一项具有至少90％的一致性。
  6. 如权利要求1-5中任一项所述的抗体，其中
    所述抗体能够结合重组人IL4R(SEQ ID NO:1)和重组猴IL4R(SEQ ID NO:3)，且结合重组人IL4R时的KD低于1nM；和/或
    所述抗体能够以低于100pM的IC 50值抑制重组IL4(SEQ ID NO:4)对HEK-Blue IL-4/IL-13细胞的激活；和/或
    所述抗体能够以低于50pM的IC 50值抑制重组IL13(SEQ ID NO:32)对HEK-Blue IL-4/IL-13细胞的激活；和/或
    所述抗体能够以低于200pM的IC 50值抑制重组IL4(SEQ ID NO:4)诱导的TF-1细胞的增殖；和/或
    所述抗体为全抗体、Fab片段、F(ab’) 2片段或单链Fv片段(scFv)，优选地，所述抗体为全人源抗体；和/或
    所述抗体还包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区和/或包含选自κ亚型或者λ亚型的轻链恒定区；和/或
    所述抗体为单克隆抗体；和/或
    所述抗体能结合并中和人IL4R，进而阻断IL4-IL4R和IL13-IL4R信号通路。
  7. 核酸分子，其编码权利要求1-6中任一项所述的抗体或其抗原结合部分。
  8. 药物组合物，其包含权利要求1-6中任一项所述的抗体和药学可接受的赋形剂、稀释剂或载体。
  9. 权利要求1-6中任一项所述的抗体在制备用于预防或治疗IL-4R介导的疾病的药物中的用途，例如所述IL-4R介导的疾病为自身免疫性疾病，例如哮喘或过敏性皮炎。
  10. 预防或治疗IL-4R介导的疾病的方法，包括向有需要的个体给予权利要求1-6中任一项所述的抗体或权利要求8所述的药物组合物，例如所述IL-4R介导的疾病为自身免疫性疾病，例如哮喘或过敏性皮炎。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111996172A (zh) * 2020-09-08 2020-11-27 中国食品药品检定研究院 一种用于测定il-4靶向治疗性抗体生物学活性的方法
RU2807060C1 (ru) * 2020-04-17 2023-11-09 Шанхаймабгикбиотех.Ко., Лтд. Антитело к альфа-рецептору интерлейкина-4 человека, способ его получения и его применение
US11939387B2 (en) 2020-04-17 2024-03-26 Shanghai Mabgeek Biotech. Co., Ltd Anti-human interleukin-4 receptor alpha antibody and preparation method and application thereof
WO2024061279A1 (en) * 2022-09-22 2024-03-28 Biosion Inc. Recombinant bispecific antibodies targeting tslp and il4r

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108373505B (zh) 2018-04-20 2019-08-20 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途
CN115991781A (zh) * 2018-08-24 2023-04-21 江苏恒瑞医药股份有限公司 结合人il-4r的抗体、其抗原结合片段及其医药用途
CN110872349A (zh) * 2018-09-04 2020-03-10 三生国健药业（上海）股份有限公司 结合人il-4r的抗体、其制备方法和用途
CN111494625B (zh) * 2018-12-25 2022-06-21 江苏荃信生物医药股份有限公司 用于治疗il-4和/或il-13介导的信号转导相关的疾病的药物组合物
US20230295312A1 (en) * 2018-12-27 2023-09-21 Akeso Biopharma, Inc Antibody against human il-4ra and use thereof
CN111592597B (zh) * 2019-05-29 2022-04-26 山东博安生物技术股份有限公司 白介素4受体(il-4r)结合蛋白及其用途
CN112279914A (zh) * 2019-07-25 2021-01-29 苏州丁孚靶点生物技术有限公司 Il4ra抗体、基因、载体、宿主细胞和il4ra拮抗剂
CN111825766B (zh) * 2019-10-31 2021-05-11 上海洛启生物医药技术有限公司 抗il-4r单域抗体及其应用
CN113549151A (zh) * 2020-04-24 2021-10-26 苏州康乃德生物医药有限公司 与人IL-4Rα中特定表位结合的抗体及其应用
CN117327181A (zh) * 2020-06-22 2024-01-02 南京融捷康生物科技有限公司 抗IL-4Rα的单域抗体以及应用和药物
KR20230048112A (ko) * 2020-08-21 2023-04-10 우시 바이올로직스 (상하이) 컴퍼니 리미티드 Cd40 효능성 항체 및 이의 사용 방법
TWI799959B (zh) * 2020-08-21 2023-04-21 大陸商上海藥明生物技術有限公司 Cd40激動劑抗體和使用方法
WO2023191665A1 (en) * 2022-03-31 2023-10-05 Milaboratory, Limited Liability Company ANTIBODIES TO HUMAN IL-4Rα HAVING REDUCED IMMUNOGENICITY AND APPLICATION THEREOF

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105377894A (zh) * 2013-06-04 2016-03-02 瑞泽恩制药公司 以il-4r抑制剂治疗过敏症及强化过敏原特异性免疫治疗的方法
CN106267190A (zh) * 2008-10-29 2017-01-04 瑞泽恩制药公司 抗人il‑4受体的高亲和性人抗体
CN108373505A (zh) * 2018-04-20 2018-08-07 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
PL2769992T3 (pl) * 2006-10-02 2021-08-02 Regeneron Pharmaceuticals, Inc. Przeciwciała ludzkie o wysokim powinowactwie względem ludzkiego receptora IL-4
US10059771B2 (en) * 2013-06-21 2018-08-28 Sanofi Biotechnology Methods for treating nasal polyposis by administering an IL-4R antagonist
CN113372446A (zh) 2016-06-08 2021-09-10 苏州康乃德生物医药有限公司 用于结合白细胞介素4受体的抗体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106267190A (zh) * 2008-10-29 2017-01-04 瑞泽恩制药公司 抗人il‑4受体的高亲和性人抗体
CN105377894A (zh) * 2013-06-04 2016-03-02 瑞泽恩制药公司 以il-4r抑制剂治疗过敏症及强化过敏原特异性免疫治疗的方法
CN108373505A (zh) * 2018-04-20 2018-08-07 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2807060C1 (ru) * 2020-04-17 2023-11-09 Шанхаймабгикбиотех.Ко., Лтд. Антитело к альфа-рецептору интерлейкина-4 человека, способ его получения и его применение
US11939387B2 (en) 2020-04-17 2024-03-26 Shanghai Mabgeek Biotech. Co., Ltd Anti-human interleukin-4 receptor alpha antibody and preparation method and application thereof
CN111996172A (zh) * 2020-09-08 2020-11-27 中国食品药品检定研究院 一种用于测定il-4靶向治疗性抗体生物学活性的方法
WO2024061279A1 (en) * 2022-09-22 2024-03-28 Biosion Inc. Recombinant bispecific antibodies targeting tslp and il4r

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