WO2017211319A1 - 用于结合白细胞介素4受体的抗体 - Google Patents

用于结合白细胞介素4受体的抗体 Download PDF

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Publication number
WO2017211319A1
WO2017211319A1 PCT/CN2017/087592 CN2017087592W WO2017211319A1 WO 2017211319 A1 WO2017211319 A1 WO 2017211319A1 CN 2017087592 W CN2017087592 W CN 2017087592W WO 2017211319 A1 WO2017211319 A1 WO 2017211319A1
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seq
variable region
chain variable
light chain
heavy chain
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PCT/CN2017/087592
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English (en)
French (fr)
Inventor
郑伟
潘武宾
杨欣
陈阳
张立民
蒋洁
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苏州康乃德生物医药有限公司
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Priority to AU2017276473A priority Critical patent/AU2017276473A1/en
Priority to CA3026568A priority patent/CA3026568A1/en
Application filed by 苏州康乃德生物医药有限公司 filed Critical 苏州康乃德生物医药有限公司
Priority to US16/307,930 priority patent/US20190177408A1/en
Priority to KR1020227022353A priority patent/KR102502988B1/ko
Priority to EP17809758.0A priority patent/EP3470430A4/en
Priority to JP2018564202A priority patent/JP7025356B2/ja
Priority to KR1020197000454A priority patent/KR102417217B1/ko
Priority to BR112018074325A priority patent/BR112018074325A2/pt
Priority to SG11201810855VA priority patent/SG11201810855VA/en
Priority to MX2018014941A priority patent/MX2018014941A/es
Priority to MYPI2018002201A priority patent/MY189035A/en
Priority to RU2019100002A priority patent/RU2774446C2/ru
Priority to IL263268A priority patent/IL263268B2/en
Publication of WO2017211319A1 publication Critical patent/WO2017211319A1/zh
Priority to PH12018502544A priority patent/PH12018502544A1/en
Priority to ZA2018/08209A priority patent/ZA201808209B/en
Priority to SA518400605A priority patent/SA518400605B1/ar
Priority to US16/809,411 priority patent/US10774141B2/en
Priority to US16/994,464 priority patent/US11866491B2/en
Priority to JP2022019435A priority patent/JP2022065063A/ja

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Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular to an antibody capable of binding to an interleukin 4 (IL-4) receptor (IL-4R) and uses thereof.
  • IL-4 interleukin 4
  • Interleukin-4 is a cytokine produced primarily by activated T cells, monocytes, basophils, mast cells, and eosinophils.
  • IL-4 is involved in a variety of biological processes, and its biological effects are known to stimulate the activation of activated B cells and T cells, and the differentiation of CD4 + T cells into type II helper T cells.
  • Studies have shown that IL-4 has multiple effects in the immune response of diseases such as allergic diseases, autoimmune diseases, infectious diseases, and tumors, and has therapeutic effects on tumors, autoimmune diseases, and infectious diseases, and IL-4 has a regulatory effect on the vaccine immune response. Therefore, IL-4 has always been one of the research hotspots.
  • IL-13 is also a cytokine produced by the secretion of activated T cells and has different functions in different cell types such as monocytes, B cells, mast cells and keratinocytes.
  • IL-13 can inhibit the release of inflammatory cytokines and chemical factors by monocytes, induce B cell proliferation and differentiation, and promote IgE synthesis.
  • IL-13 and IL-4 have many common functions in biological function, including inhibiting the release of inflammatory mediators by monocytes, inducing dendritic changes of macrophages, promoting the expression of CD23 on the surface of monocytes and stimulating the synthesis of immunoglobulin by B cells.
  • IL-13 also has its own biological functions, including: promoting human monocyte differentiation and cell surface antigen changes; inducing B cell proliferation, differentiation, promoting B cell secretion of antibodies; regulating IgE synthesis, and body allergy Related; inhibit tumor cell growth; inhibit HIV replication and the like.
  • IL-4R cell surface IL-4 receptor
  • Human IL-4R is a heterodimer formed by two polypeptide chains, one of which has a high affinity for IL-4 (hIL-4R ⁇ , UniProtKB: P24394).
  • IL-13R alpha chain also together with the IL-4R alpha chain constitutes another form of IL-4R complex.
  • the IL-4R ⁇ chain plays a leading role in IL-4 binding in the IL-4R complex, and it also involves other cytokines, the IL-4R ⁇ chain is currently being studied as a major subject, and for this target Human monoclonal antibodies have been clinically proven to be effective in relieving and treating asthma, eczema, and atopic dermatitis.
  • the human interleukin-4 receptor is known to produce a soluble form of the protein (shIL-4R ⁇ , SEQ ID NO. 94), a soluble form of the protein that inhibits IL-4 mediated cell proliferation and T cells. Mediated upregulation of IL-5. Two forms of this receptor are associated with allergic reactions, which manifest as allergic rhinitis, sinusitis, asthma or eczema. Blocking antibodies that target this protein are therefore useful in the treatment and alleviation of such diseases.
  • Asthma is a chronic airway inflammatory disease involving a variety of inflammatory cells such as eosinophils, mast cells and lymphocytes.
  • the specific pathogenesis is still unclear.
  • Cytokines such as IL-4 play an important role in the development of bronchial asthma.
  • the development of specific antibodies against IL-4 is one of the effective ways to solve asthma treatment.
  • Inhibition of IL-4/IL-4R ⁇ can have an effective immunomodulatory effect on asthma.
  • Allergic rhinitis has much in common with the pathogenesis of asthma, and all belong to type I allergy.
  • Drug therapy is currently the focus of AR treatment, in which nasal corticosteroids and antihistamines are at the core.
  • Atopic dermatitis also known as atopic dermatitis or atopic dermatitis, is a common dermatological disease, more common in children and adolescents, often with certain genetic allergic diseases such as allergic rhinitis, asthma, etc. Concurrent.
  • the immune factors involved in cytokines such as IL-4 and IL-13 are one of the main pathogenesis.
  • Eosinophilic esophagitis is a chronic immune inflammatory disease characterized by infiltration of eosinophils (EOS) in the esophageal wall. The onset of EoE is associated with Th2 cell dysfunction.
  • EOS eosinophils
  • specific specific protocols such as novel biological agents against IL-5 (such as mepolizumab) have become hotspots in research. Immunomodulatory therapy has yielded results in animal models, but human clinical trials still need to be explored.
  • Drugs such as PGD2 inhibitors, anti-TNF- ⁇ , and anti-IL-13 are under investigation.
  • hIL-4R targets have entered clinical trials, such as Dupilumab, and have shown good efficacy in the phase II clinical trial of atopic dermatitis.
  • Dupilumab monoclonal antibody drugs targeting hIL-4R targets have entered clinical trials, such as Dupilumab, and have shown good efficacy in the phase II clinical trial of atopic dermatitis.
  • other companies have applied for other monoclonal antibody patents for hIL-4R, such as US 7,186,809 and US 7,638,606.
  • the present invention obtains a specific antibody against IL-4R by antibody screening and optimization, and the antibody can act as a blocking agent for binding of IL-4 to IL-4R, and can be used for treating inflammation or allergy by binding to IL-4R. Sexual diseases, etc.
  • the antibody of the present invention has a serine (31Ser) at position 31 of the light chain variable region (VL), and refers to the sequence shown in SEQ ID NO: The amino acid residue numbering, the antibody having aspartic acid (103Asp) at position 103 of the heavy chain variable region (VH) and tyrosine (104Tyr) at position 104.
  • the 31Ser is correspondingly in the CDR1 of the light chain variable region of the antibody of the invention
  • the 103Asp and 104Tyr being correspondingly in the CDR3 of the heavy chain variable region of the antibody of the invention.
  • the light chain variable region of the antibody of the present invention comprises the CDR1 set forth in SEQ ID NO: 2
  • the heavy chain variable region of the antibody comprises the CDR3 set forth in SEQ ID NO: 19;
  • the light chain variable region of an antibody of the invention comprises a combination of CDR1, CDR2 and CDR3 selected from the group consisting of:
  • the heavy chain variable region of an antibody of the invention comprises a combination of CDR1, CDR2 and CDR3 selected from the group consisting of:
  • the antibody of the present invention comprises a light chain variable region selected from the amino acid sequence shown by the sequence: SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 57, and The antibody comprises a heavy chain variable region selected from the amino acid sequences set forth in the sequence: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ
  • the antibody of the invention comprises a combination of a light chain variable region and a heavy chain variable region selected from the group consisting of:
  • the invention provides an antibody capable of binding to an interleukin 4 (IL-4) receptor (IL-4R) comprising a light chain variable region (VL), said light chain variable region A combination comprising CDR1, CDR2 and CDR3 selected from the group consisting of:
  • IL-4R interleukin 4 receptor
  • VL light chain variable region
  • the antibody comprises a heavy chain variable region (VH) comprising a combination of CDR1, CDR2 and CDR3 selected from the group consisting of:
  • the light chain variable region of the antibody of the present invention comprises a combination of FR1, FR2, FR3 and FR4 selected from the group consisting of:
  • FR1 represented by SEQ ID NO: 9 FR2 represented by SEQ ID NO: 10, FR3 represented by SEQ ID NO: 12, and FR4 represented by SEQ ID NO: 13;
  • the heavy chain variable region of the antibody comprises a combination of FR1, FR2, FR3 and FR4 selected from the group consisting of:
  • FR1 represented by SEQ ID NO: 26 FR2 represented by SEQ ID NO: 32, FR3 represented by SEQ ID NO: 35, and FR4 represented by SEQ ID NO: 38;
  • FR1 shown in SEQ ID NO: 25 FR2 shown in SEQ ID NO: 32, FR3 shown in SEQ ID NO: 35, and FR4 shown in SEQ ID NO: 38;
  • FR1 represented by SEQ ID NO: 28 FR2 represented by SEQ ID NO: 32, FR3 represented by SEQ ID NO: 35, and FR4 represented by SEQ ID NO: 38;
  • FR1 represented by SEQ ID NO: 23 FR2 represented by SEQ ID NO: 32, FR3 represented by SEQ ID NO: 35, and FR4 represented by SEQ ID NO: 38;
  • FR1 represented by SEQ ID NO: 21 FR2 represented by SEQ ID NO: 32, FR3 represented by SEQ ID NO: 35, and FR4 represented by SEQ ID NO: 38.
  • the light chain variable region or heavy chain variable region of the antibody of the present invention is FR1-CDR1-FR2-CDR2-FR3-CDR3-, according to the domain composition of the antibody light chain variable region and the heavy chain variable region known in the art.
  • the sequence of FR4 comprises the above domain components, or (X)n-FR1-(X)n-CDR1-(X)n-FR2-(X)n-CDR2-(X)n-FR3-(X)
  • the sequence of n-CDR3-(X)n-FR4-(X)n comprises the above domain components, wherein X is any amino acid residue and n is an integer equal to or greater than zero.
  • the antibody provided by the present invention comprises a light chain variable region selected from the amino acid sequences shown by the following sequences:
  • the antibody provided by the present invention comprises a heavy chain variable region selected from the amino acid sequences shown by the following sequences:
  • the antibodies provided herein comprise a combination of a light chain variable region and a heavy chain variable region selected from the group consisting of:
  • (31) a light chain variable region of SEQ ID NO. 57 and a heavy chain variable region of SEQ ID NO.
  • the antibody provided by the present invention is capable of binding to IL-4R and functions as an antagonist of IL-4R.
  • the antibody is capable of binding to IL-4R ⁇ , preferably to mammalian IL-4R ⁇ , more preferably to human IL-4R ⁇ , even more preferably to human soluble IL-4R ⁇ .
  • the binding affinity of the antibodies provided by the present invention to IL-4R ⁇ can be determined by Biacore or ELISA methods.
  • the antibody was determined to bind IL-4R ⁇ with an affinity of less than 100 nM, less than 10 nM, less than 1 nM, less than 0.5 nM, and even less than 0.1 nM.
  • the ratio of expression of the antibody and the reference antibody provided by the present invention is 0.1-3:1, excellent It is selected from 0.3 to 3:1, more preferably from 0.4 to 3:1, still more preferably from 0.5 to 3:1, still more preferably from 0.6 to 3:1, still more preferably from 0.7 to 3:1, still more preferably from 1 to 3:1.
  • the antibody provided by the present invention may be a monoclonal antibody, a fully or partially humanized antibody or a chimeric antibody;
  • the antibody is an immunoglobulin, preferably IgA, IgD, IgE, IgG or IgM, more preferably an IgGl, IgG2, IgG3 or IgG4 subtype, more preferably an IgG2 or IgG4 subtype.
  • the invention provides a fusion protein or conjugate comprising an antibody of the invention.
  • the fusion protein or conjugate further comprises a cell surface receptor, an active protein and a polypeptide, a small molecule compound such as an amino acid and a saccharide, a small molecule polymer, or a method of the present invention, which is chemically or physically bound to the antibody of the present invention.
  • a cell surface receptor an active protein and a polypeptide
  • a small molecule compound such as an amino acid and a saccharide
  • a small molecule polymer or a method of the present invention, which is chemically or physically bound to the antibody of the present invention.
  • Other parts of the antibody that are chemically modified, and the like.
  • the invention provides a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of an antibody provided herein;
  • the nucleic acid sequence is capable of encoding the heavy and/or light chains of the antibodies provided herein.
  • the invention also provides a vector comprising a nucleic acid sequence provided herein.
  • the vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, or the like.
  • the above vectors or nucleic acid sequences can be used for transformation or transfection of host cells for purposes such as preservation or antibody expression. Accordingly, the invention also provides a host cell transformed or transfected with the nucleic acid sequence or vector.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
  • the antibodies provided herein can be obtained using any method known in the art.
  • the heavy chain variable region and/or the light chain variable region of the antibody can be obtained first from the nucleic acid sequence provided by the present invention, or the heavy and/or light chain of the antibody can be obtained, and then Other domains are selected for assembly into antibodies; alternatively, the host cell provided by the present invention is allowed to express the heavy chain variable region and/or the light chain variable region of the antibody or the heavy and/or light chain of the antibody to assemble In the case of the antibody, the host cell is cultured.
  • the method further comprises the step of recovering the produced antibody.
  • the antibody, fusion protein or conjugate, nucleic acid sequence, vector, host cell or antibody produced by the above method provided by the present invention may be contained in a pharmaceutical composition, more specifically, in a pharmaceutical preparation, thereby according to actual needs. Used for a variety of purposes. Accordingly, in a further aspect, the invention provides a pharmaceutical composition comprising an antibody, fusion protein or conjugate of the invention, a nucleic acid sequence, a vector, a host cell and/or by the method described above Produced antibodies.
  • the pharmaceutical composition may be a pharmaceutical preparation.
  • the pharmaceutical preparation is, for example, an injection dosage form.
  • the pharmaceutical or pharmaceutical formulation further comprises a pharmaceutically acceptable carrier or excipient, depending on the particular dosage form.
  • At least one drug selected from the group consisting of an antiasthmatic drug such as salbutamol, an antihistamine such as loratadine, an immunosuppressive agent such as tacrolimus, and pyridin may be further included.
  • MeMos et al M receptor blockers such as ipratropium bromide, leukotriene receptor blockers such as montelukast, phosphodiesterase inhibitors such as theophylline, non-steroidal anti-inflammatory drugs Such as 5-aminosalicylic acid, etc., hormones such as beclomethasone, budesonide, etc., that is, the antibody, fusion protein or conjugate provided by the present invention, nucleic acid sequence, vector, host cell or antibody produced by the above method can be Use in combination with other drugs as needed.
  • the invention provides the antibody, fusion protein or conjugate, nucleic acid sequence, vector, host cell and/or antibody produced by the method for the preparation of a prophylactic, therapeutic or ameliorating inflammatory or allergic disease Use in
  • the inflammatory or allergic disease includes autoimmune diseases such as atopic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis and the like.
  • autoimmune diseases such as atopic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis and the like.
  • the invention provides a method of preventing, treating or ameliorating an inflammatory or allergic disease in a subject, the method comprising administering to a subject in need thereof an antibody, fusion protein or fusion provided by the invention An antibody, a nucleic acid sequence, a vector, a host cell, and/or an antibody produced by the method.
  • the subject is a mammal; more preferably, the subject is a human.
  • the inflammatory or allergic disease includes autoimmune diseases such as atopic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis and the like.
  • autoimmune diseases such as atopic dermatitis, asthma, eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyps, rheumatoid arthritis and the like.
  • the method further comprises administering to the subject at least one drug selected from the group consisting of antiasthmatic drugs such as salbutamol, anti-group Amine drugs such as loratadine, immunosuppressive agents such as tacrolimus, pimecrolimus, etc., M receptor blockers such as ipratropium bromide, etc., leukotriene receptor blockers such as montelukast
  • antiasthmatic drugs such as salbutamol
  • anti-group Amine drugs such as loratadine
  • immunosuppressive agents such as tacrolimus, pimecrolimus, etc.
  • M receptor blockers such as ipratropium bromide, etc.
  • leukotriene receptor blockers such as montelukast
  • phosphodiesterase inhibitors such as theophylline
  • non-steroidal anti-inflammatory drugs such as 5-aminosalicylic acid, and the like, such as beclomethasone, budesonide, and the like.
  • the medicament is administered simultaneously or sequentially with the antibodies, fusion proteins or conjugates, nucleic acid sequences, vectors, host cells and/or antibodies produced by the methods provided herein.
  • the present invention also provides a kit comprising the anti-invention provided by the present invention Body, fusion protein or conjugate, nucleic acid sequence, vector, host cell and/or antibody produced by the method.
  • Figure 1 shows the pharmacokinetic profile of the antibody of the present invention in mice
  • Figure 2 is a graph showing the pharmacokinetic profile of the antibody of the present invention in cynomolgus monkeys
  • 3A-3C show, by FACS, the specific binding of the antibody of the present invention to TF-1 cells expressing IL-4R ⁇ , wherein 3A shows the fluorescent signal when the antibody L1012H1031 of the present invention is not added, and 3B shows the addition of the antibody L1012H1031 of the present invention. After the fluorescent signal, 3C shows the signal superposition comparison of 3A-3B.
  • 4A-4D show by FACS that the specific binding of the antibody of the present invention to IL-4R ⁇ -expressing TF-1 cells is blocked by sIL-4R ⁇ present in the system, wherein 4A shows that the antibody L1012H1031 of the present invention is not added.
  • the fluorescent signal 4B shows the fluorescent signal after addition of the antibody L1012H1031 of the present invention
  • 4C shows the fluorescent signal when the antibodies L1012H1031 and sIL-4R ⁇ of the present invention are added
  • 4D shows the signal superposition comparison of 4A-4C.
  • 5A-5B show the effect of the antibody of the present invention on inhibiting TARC and MDC release by ELISA, wherein 5A shows that the antibody L1020H1031 of the present invention inhibits the release of TARC factor, and 5B shows that the antibody L1020H1031 of the present invention inhibits the release of MDC factor.
  • test materials used in the following examples were purchased from conventional biochemical reagent stores.
  • Example 1 Preparation of antibody of the present invention and determination of expression amount by non-reducing SDS-PAGE gel electrophoresis
  • the coding sequence of the variable region of the antibody light chain was inserted into the pFUSE2ss-CLIg-hK vector (Invivogen, accession number: pfuse2ss-hclk) using the EcoRI and BsiWI digestion sites to construct a light chain expression vector; the coding sequence of the heavy chain variable region was utilized.
  • the EcoRI and NheI restriction sites were inserted into the pFUSEss-CHIg-hG2 vector (Invivogen, Cat. No.: pfusess-hchg2) or the pFUSEss-CHIg-hG4 vector (Invivogen, Cat. No.: pfusess-hchg4) to constitute a heavy chain expression vector.
  • Cell culture and transfection Expi293 performed (NO: A14635) according to Invitrogen Corporation Expi293 TM Expression System Kit manual. The cell density was adjusted to 2 ⁇ 10 6 /ml at the time of transfection, and 0.6 ⁇ g of the light chain expression vector and 0.4 ⁇ g of the heavy chain expression vector were added per ml of cells, and the culture supernatant was collected four days later.
  • the cell culture supernatant was subjected to non-reducing SDS-PAGE gel electrophoresis according to the method described in Appendix 8 of the Third Guide to Molecular Cloning.
  • Example 2 Detection of the antibody of the present invention inhibits the proliferation of TF-1 cells by hIL-4 or hIL-13
  • hIL-4 (Invivogen, article number: rhil-4) solution: Dissolve hIL-4 with 100 ⁇ l of PBS solution containing 0.1% BSA (Beyotime, Cat. No. ST023) to obtain a concentration of 100 ⁇ g/ml, and dissolve hIL-4 according to 5 ⁇ l of each tube was dispensed into a 1.5 ml (Nunc) centrifuge tube and stored in a -20 ° C refrigerator.
  • BSA Beyotime, Cat. No. ST023
  • WST-1 (Beyotime, Cat. No. C0036) solution Add 5ml of electron coupling agent (C0036-2) to WST-1 powder (C0036-1), completely dissolve into WST-1 solution, according to the volume of 620 ⁇ l per tube Pack into a 1.5ml centrifuge tube and store in a -20 ° C refrigerator.
  • C0036-2 electron coupling agent
  • the frozen TF-1 (ATCC: CRL- 2003TM ) cells in liquid nitrogen were taken out and shaken in a 37 ° C water bath to dissolve rapidly. Transfer the dissolved cell suspension to a 15 ml centrifuge tube, add 1640 medium to 10 ml, centrifuge at 800 rpm for 5 min, aspirate the supernatant, retain the cell pellet, repeat the wash once, add 10 ml of 10% FBS and 2 ng/ml GM-CSF. (Sino Biological, Cat. No.
  • the plasmids carrying the different sets of antibody genes were transfected into Expi293 cells, and 200 ⁇ l of the cell culture supernatant was taken 4 days after the transfection, and centrifuged at 800 rpm for 5 min, and the supernatant was filtered through a 0.22 ⁇ m pore size filter to be used for the proliferation blocking experiment.
  • a cell culture supernatant expressing the antibody of the present invention is passed through a 0.22 ⁇ m filter and purified by GE MabSelect Sure (Cat Number: 11003494) Protein A affinity chromatography column, and the purification system is GE AKTA. Purifier 10, the antibody collected after purification was concentrated and quantified using an Amicon ultrafiltration concentrator (Cat Number: UFC903096). At the time of detection, the antibody was diluted with PBS to 0 to 1 ⁇ g/ml for the proliferation blocking experiment.
  • the cells in the T75 cell flask were well-growth, transferred into a 15 ml centrifuge tube, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cell pellet was taken.
  • the cells were resuspended in 10 ml of PBS, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and a cell pellet was taken.
  • the cells were resuspended in 10 ml of 1640 medium (without GM-CSF) containing 10% FBS, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and a cell pellet was taken.
  • the cells were resuspended in 5 ml of 1640 medium containing 10% FBS (without GM-CSF), and after the cells were counted, the medium was supplemented to adjust the cell density to 5 ⁇ 10 5 /ml.
  • the cell suspension was added to a 96-well plate at a volume of 80 ⁇ l per well (reserved outer ring anti-volatile cells without cells).
  • 10 ⁇ l of the prepared purified antibodies or 10 ⁇ l of the cell culture supernatant were added to 96-well cells (3 replicate wells). Then hIL-4 was diluted to 50 ng/ml with 1640 medium containing 10% FBS.
  • hIL-4 50 ng/ml of hIL-4 was added to the corresponding cell wells of a 96-well plate in a volume of 10 ⁇ l per well, so that the final cell density was 4 ⁇ 10 5 /ml, and the concentration of hIL-4 was 5 ng/ml.
  • 96-well plate volume per well was 100 ⁇ l, no hIL-4 was added, no antibody was added, only the same number of cells and the same volume of the culture medium negative control group (3 replicate wells) were added, and no antibody was added, supplemented with the same In the volume culture medium, only the positive control group (3 replicate wells) of the same concentration of hIL-4 was added to the cells.
  • 200 ⁇ l of PBS was added to each well of a 96-well cell plate to prevent evaporation of the inner ring liquid.
  • the 96-well cell plates were placed in a 37 ° C, 5% CO 2 incubator for static growth.
  • 96-well cell plates were statically cultured in a 5% CO2 incubator for 72 h and then added to each well of the cells. 10 ⁇ l of WST-1 solution. The 96-well cell plates were further placed in a 37 ° C, static culture in a 5% CO 2 incubator. After 24 h, the 96-well plate was placed in a flexstation 3 (Molecular Devices) and the value of OD450-OD650 was read.
  • the blocking effect of the antibody of the present invention on the proliferation of TF-1 cells of hIL-4 or hIL-13 is shown in Tables 3a-3b below.
  • the measured OD450-OD650 data was input into the prism5 software, the negative control value was set to the lowest value, the positive control value was set to the highest value, and the antibody concentration was taken as the logarithm value by prism5
  • the software fits the logarithm of the antibody concentration to the OD450-OD650 curve, and the calculated IC50 is shown in Table 4 below.
  • Example 3 ELISA assay for the binding ability of the antibody of the present invention to sIL-4R ⁇
  • sIL-4R ⁇ PEPRO TECH, Cat. No.: 200-04R
  • solution Take a sIL-4R ⁇ , add 1ml ddH2O, mix upside down, ie 100 ⁇ g/ml solution, then store in -20 °C refrigerator.
  • Sample to be tested Take the Expi293 cell culture supernatant expressing the antibody of the present invention after transient staining (the medium is Expi293 Expression Medium, Invitrogen, Cat. No. A1435102; in a 8% CO2 incubator, suspension culture at 100 rpm for 4 days) 10 ⁇ l and 2 ⁇ l, respectively, were added to 990 ⁇ l and 998 ⁇ l of PBS solution to prepare a sample of the antibody to be tested diluted 1:100 and 1:500.
  • the medium is Expi293 Expression Medium, Invitrogen, Cat. No. A1435102; in a 8% CO2 incubator, suspension culture at 100 rpm for 4 days
  • Control sample normal cell culture supernatant (Expi293 cells were not transfected, the medium was Expi293 Expression Medium (Invitrogen, Cat. No. A1435102; in 8% CO2 incubator, suspension culture at 100 rpm for 4 days) was also performed 1:100 and 1 :500 dilution, as a negative control sample.
  • the diluted antibody to be tested was sequentially added to the corresponding well, and the normal cell culture supernatant was added as a negative control sample, and each sample was made into three duplicate wells of 100 ⁇ l per well.
  • the enzyme plate was wrapped (or capped) with plastic wrap and incubated in a 10 ° C incubator for 1 h.
  • the 96-well microtiter plate was taken out, and the solution was discarded.
  • TMB solution (Solarbio, Cat Number: PR1200) was added to the 96-well microtiter plate row by row, 100 ⁇ l per well. After standing at room temperature for 5 minutes, the reaction was immediately terminated by adding a 2 M H 2 SO 4 solution to a 96-well microtiter plate.
  • the 96-well microtiter plate was placed in a flexstation 3 (Molecular Devices), the value of OD450 was read, and data collection was calculated and analyzed. The results are shown relative to the affinity of Control Antibody 1, see Tables 5a-5c below.
  • Antibody number OD 450/OD450 control antibody 1 Control antibody 1 1 L1021H1000 2.42 L1020H1000 2.27 L1019H1000 1.79 L1001H1000 1.56 L1012H1000 1.22 L1000H1031 1.14 L1020H1031 1.12 L1000H1014 1.06 L1020H1029 1.01
  • Antibody number OD 450/OD450 control antibody 1 L1024H1000 0.69 L1015H1000 0.67 L1000H1015 0.65 L1009H1000 0.64 L1021H1016 0.63 L1000H1023 0.63 L1000H1016 0.61 L1000H1009 0.56 L1000H1032 0.53 L1017H1000 0.49 L1021H1007 0.44 L1000H1027 0.44 L1020H1007 0.40 L1020H1027 0.40 L1012H1007 0.40 L1000H1013 0.38 L1000H1007 0.37 L1000H1021 0.35 L1000H1028 0.33 L1021H1027 0.29 L1016H1000 0.27 L1020H1020 0.26
  • mice To further screen for antibodies, a series of pharmacokinetic experiments were performed in mice.
  • the blood sample was not anticoagulated, and the serum was separated by centrifugation at room temperature 1500 g for 10 min within 2 h after the blood was taken. The collected supernatant was immediately transferred to a new labeled centrifuge tube and the serum was stored at -70 °C for temporary storage. Determination of antibody concentration in mice using ELISA:
  • sIL-4R ⁇ PEPRO TECH, Cat. No.: 200-04R
  • solution Take a sIL-4R ⁇ , add 1ml ddH2O, mix upside down, ie 100 ⁇ g/ml solution, then store in -20 °C refrigerator.
  • Samples to be tested 1 ⁇ l of each serum collected in different time periods was added to 999 ⁇ l of PBS solution containing 1% BSA to form a 1:1000 dilution of the serum sample to be tested.
  • the antibody to be detected was diluted to 0.1 ⁇ g/ml with a PBS solution containing 1% BSA and 0.1% normal animal serum (Beyotime, Cat. No. ST023).
  • 0.1 ⁇ g/ml of the antibodies to be detected 800, 600, 400, 200, 100, 50, 10, and 0 ⁇ l, respectively, were added to a PBS solution containing 1% BSA and 0.1% normal animal serum, 200, 400, 600, 800, 900, respectively.
  • 950, 990, 1000 ⁇ l were formulated into antibody standards of the present invention having final concentrations of 80, 60, 40, 20, 10, 5, 1, 0 ng/ml, respectively.
  • sIL-4R ⁇ solution 250 ⁇ l of 100 ⁇ g/ml sIL-4R ⁇ solution was added to 9.75 ml of PBS coating solution, and mixed upside down to obtain 2.5 ⁇ g/ml of antigen coating solution.
  • the prepared antigen coating solution was added to a 96-well enzymatic plate for centrifugation at 100 ⁇ l per well.
  • the 96-well microtiter plate was wrapped (or capped) with plastic wrap and incubated overnight in a 4 °C refrigerator. On the next day, the 96-well microtiter plate was taken out, the solution was discarded, and a PBS solution containing 2% BSA was added to 300 ⁇ l per well.
  • the plate was wrapped (or covered) with plastic wrap and incubated for 2 h in a refrigerator at 4 °C.
  • the 96-well microtiter plate was taken out, the solution was discarded, and the PBST was washed three times.
  • the diluted standard antibody and the serum sample to be tested are sequentially added to the corresponding wells, and each sample is made into three duplicate wells, 100 ⁇ l per well.
  • the plate was wrapped (or capped) with plastic wrap and incubated for 1 h at room temperature. The solution was discarded and washed 3 times with PBST.
  • TMB solution (Solarbio, Cat Number: PR1200) was added to the 96-well microtiter plate row by row, 100 ⁇ l per well.
  • Example 5 Pharmacokinetics of the antibodies of the invention in cynomolgus monkeys
  • the 3-5 year old cynomolgus monkey was selected and weighed 2-5 Kg, and the antibody was subcutaneously injected in an amount of 5 mg (antibody of the present invention or control antibody 2) / kg (crab monkey weight).
  • the antibody or control antibody 2 to be administered is accurately extracted with a disposable sterile syringe, and multiple injections are performed subcutaneously on the inner side of the animal's thigh, and the injection volume per point is not more than 2 ml.
  • the whole blood was collected from the subcutaneous vein of the hind limb of the animal at the time points of 0.5, 2, 4, 8, 24, 48, 72, 120, 168, 240, 336h, 432h, 504h, 600h, and 672h. Each blood volume was 1.5 ml.
  • the blood sample was not anticoagulated, and the serum was separated by centrifugation at room temperature 1500 g for 10 min within 2 h after the blood was taken.
  • the collected supernatant was immediately transferred to a new labeled centrifuge tube and the serum was stored at -70 °C for temporary storage.
  • the antibody concentration in the cynomolgus monkey was determined by the method described in Example 4. The pharmacokinetic results are shown in Figure 2 and Table 7 below.
  • Example 6 FACS detection of binding of an antibody of the invention to TF-1 cells
  • the frozen TF-1 (ATCC: CRL- 2003TM ) cells were taken out in liquid nitrogen and gently shaken in a 37 ° C water bath to dissolve rapidly. Add the dissolved cell suspension to a 15 ml centrifuge tube, add 1640 medium (Hyclone, Cat. No. SH30809.01B) to 10 ml, centrifuge at 800 rpm for 5 min, aspirate the supernatant, retain the cell pellet, repeat the wash once, add 10%.
  • FBS Hyclone, Cat. No.: SV30184.02
  • 2ng/ml GM-CSF (Sino Biological, Cat. No.
  • the cells were cultured in a T75 cell culture flask (Nunc) at 37 ° C in a 5% CO 2 incubator (Thermo). Every 2-3 days, the cell suspension was taken, centrifuged at 800 rpm for 5 min, the cells were resuspended in 10 ml of medium, and 1 ⁇ 10 6 cells of the cells were counted and transferred into a new T75 cell bottle, and the medium was added to 10 ml for continuous passage. After 2-3 times, the experiment can be carried out after the cells are in good condition (the cells are translucent and the cells are slightly suspended in a slightly irregular shape).
  • TF-1 cells were taken and counted by microscopy.
  • the cells were divided into three groups and placed in three 1.5 ml centrifuge tubes, each of which had a number of cells of 1 ⁇ 10 6 .
  • the cells were centrifuged at 800 rpm for 5 min, and the cells were resuspended in 1 ml of cold 1% BSA in PBS and washed once. Centrifuge the cells at 800 rpm for 5 min, add 45 ⁇ l of cold PBS containing 1% BSA to each centrifuge tube, and add 5 ⁇ l (500 ⁇ g/ml) of the L1021H1011, L1020H1031 or L1012H1031 antibody of the present invention to the first centrifuge tube.
  • Example 7 FACS detection of soluble hIL-4R ⁇ (sIL-4R ⁇ ) blocking the binding of antibodies to TF-1 cells
  • 10 ⁇ l (100 ⁇ g/ml) of sIL-4R ⁇ and 5 ⁇ l (500 ⁇ g/ml) of the L1021H1011, L1020H1031 or L1012H1031 antibody of the present invention were uniformly mixed in a 1.5 ml centrifuge tube at a molar ratio of 2:1.
  • 10 ⁇ l of PBS was mixed with 5 ⁇ l of the L1021H1011, L1020H1031 or L1012H1031 antibody of the present invention as a positive control, and 15 ⁇ l of PBS was used as a negative control.
  • the tube was placed in a 37 ° C incubator for 1 h.
  • TF-1 cells were taken and counted by microscopy.
  • the cells were divided into 4 groups and placed in three 1.5 ml centrifuge tubes, each of which had a number of cells of 1 ⁇ 10 6 .
  • the cells were centrifuged at 800 rpm for 5 min, and the cells were resuspended in 1 ml of cold 1% BSA in PBS and washed once.
  • the cells were centrifuged at 800 rpm for 5 min, and after resuspending the cells with 35 ⁇ l of cold 1% BSA in each centrifuge tube, 15 ⁇ l of the different antibody mixture and the control in step 3 were added.
  • the cells were centrifuged at 800 rpm for 5 min, and the cells were resuspended in 1 ml of cold 1% BSA in PBS and washed once.
  • the cells were centrifuged at 800 rpm for 5 min, and 499 ⁇ l of cold PBS containing 1% BSA was added to each centrifuge tube, and then 1 ⁇ l of FITC-labeled goat anti-human IgG (H+L) (Beyotime, Cat. No. A0556) was added.
  • H+L FITC-labeled goat anti-human IgG
  • an anti-human Fc (AHC) antibody (GE Healthcare) was coupled to a CM5 chip, and then the antibody of the present invention was captured by AHC, and different concentrations of human sIL-4R ⁇ were flowed through the surface of the chip on which the antibody of the present invention was captured, wherein The antibody of the invention was diluted to 2 ⁇ g/ml, and the antigen sIL-4R ⁇ was diluted to 0.39. 0.78, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100.0 nM. The experimental method is then established in the Biacore T200 control software, after which the experimental program is run. The results are shown in Table 8 below.
  • Example 9 ELISA method to examine the effect of the antibodies of the invention on the inhibition of TARC and MDC release
  • Fresh heparin anticoagulation 10 ml (donation) was taken, mixed with PBS solution at room temperature 1:1, and carefully added to a prepared 20 ml human lymphocyte separation solution (Solarbio, Cat. No.: P8610). Centrifuge at 1500 rpm for 30 min at room temperature. After centrifugation, the PBMC layer was carefully aspirated and washed twice with PBS. The cells were finally diluted with 1640 medium containing 10% FBS, 200 IU/ml IL-2, and added to a 24-well plate for 1 x 10 6 cells per well.
  • L1020H1031 antibody was added to each well at a final concentration of 1000, 300, 100, 30, 10 ng/ml, and then a final concentration of 10 ng/ml of IL-4 (or 100 ng/ml of IL-13) was added. After 72 hours of culture, the assay was carried out according to the method provided by Human TARC ELISA kit (Abeam, Cat. No. ab183366) or Human MDC ELISA kit (Abeam, Cat. No. ab179885).
  • Example 10 Effect of specific amino acids on the metabolic kinetics and expression levels of the antibodies of the invention
  • the 103rd position (in CDR3) of the antibody heavy chain H1031 (SEQ ID NO. 91) sequence is Asp (103Asp), and the 104th position is Tyr (104Tyr).
  • the antibody of the present invention having 103Asp and 104Tyr has a 2- to 4-fold higher area under the drug-time curve and a clearance rate of about 70% compared to an antibody having no 103Asp and 104Tyr in the heavy chain.
  • the expression levels of the antibodies of the invention were further tested and compared to examine the effect that a particular amino acid at a particular location may have on the expression of the antibody.
  • the culture and transfection of Expi293 cells were the same as in Example 1.
  • the collected culture supernatant was then passed through a 0.22 ⁇ m filter and purified by GE MabSelect Sure (Cat. No. 11003494) Protein A affinity chromatography column.
  • the purification system was GE AKTA purifier 10
  • the antibody collected after purification was concentrated and quantified using an Amicon ultrafiltration concentrating tube (Cat. No. UFC903096). The quantitative results are shown in Table 10 below.
  • the 31st position (in CDR1) of the sequences of the antibody light chain L1012 (SEQ ID NO. 44), L1020 (SEQ ID NO. 55) and L1023 (SEQ ID NO. 51) is Ser. (31Ser).
  • the expression level of the antibody of the present invention having 31Ser is increased by about 2 to 5 times compared to an antibody having no 31Ser in the light chain.

Abstract

一种能够结合白细胞介素4(IL-4)受体(IL-4R)的抗体。编码所述抗体的核酸序列，包含所述核酸序列的载体以及用所述载体转化或转染的宿主细胞。提供用于生产所述抗体的方法，所述抗体的医学用途以及包含所述抗体的试剂盒。

Description

用于结合白细胞介素4受体的抗体 技术领域
本发明涉及生物制药领域，具体而言，本发明涉及能够结合白细胞介素4(IL-4)受体(IL-4R)的抗体及其用途。
背景技术
白细胞介素-4(IL-4)是主要由活化的T细胞、单核细胞、嗜碱粒细胞、肥大细胞和嗜酸粒细胞产生的细胞因子。IL-4涉及多种生物学过程，已知其生物作用包括刺激活化B细胞和T细胞增殖、CD4+T细胞分化成II型辅助T细胞。研究表明，IL-4在介导过敏性疾病、自身免疫性疾病、感染性疾病、肿瘤等疾病的免疫反应中有多重作用，对肿瘤、自身免疫性疾病和感染性疾病等有治疗作用，并且IL-4对疫苗免疫应答具有调节作用，因此，IL-4一直是人们关注的研究热点之一。
IL-13也是由活化的T细胞分泌产生的一种细胞因子，在不同的细胞类型如单核细胞、B细胞、肥大细胞和角质形成细胞中具有不同功能。IL-13能抑制单核细胞释放炎性细胞因子和化学因子，诱导B细胞增殖和分化，促进IgE合成。IL-13与IL-4在生物功能方面有许多共性，包括抑制单核细胞释放炎症介质，诱导巨噬细胞树突样变，促进单核细胞表面表达CD23及刺激B细胞合成免疫球蛋白。同时IL-13也具有自身的生物学功能特点，主要包括：促进人单核细胞分化及细胞表面抗原发生改变；诱导B细胞增殖，分化，促进B细胞分泌抗体；调节IgE合成，与机体变态反应相关；抑制肿瘤细胞生长；抑制HIV的复制等。
IL-4的生物活性是由特异的细胞表面IL-4受体(IL-4R，在人类中为“hIL-4R”)介导的。人IL-4R是由两条多肽链形成的异二聚体，其中一条α链(hIL-4Rα，UniProtKB:P24394)对IL-4有很高的亲和力。并且研究表明，IL-13的细胞表面受体α链(IL-13Rα链)也和IL-4Rα链共同组成另一种形式的IL-4R复合体。由于在IL-4R复合物中IL-4Rα链对IL-4的结合起主导作用，并且其还涉及其他细胞因子，因此目前人们把IL-4Rα链作为主要对象开展研究，并且针对该靶点的人单克隆抗体已经在临床上证明可以有效缓解和治疗哮喘、湿疹以及特应性皮炎等症状。
已知人白细胞介素-4受体可产生一种可溶形式的蛋白(shIL-4Rα，SEQ ID NO.94)，这种可溶形式的蛋白可以抑制IL-4介导的细胞增殖和T细胞介导的IL-5上调。该受体的两种形式与过敏反应相关，其表现为过敏性鼻炎、鼻窦炎、哮喘或湿疹等病症。因此将该蛋白作为靶点的阻断抗体有助于治疗和缓解此类疾病。
哮喘(Asthma)是一种由多种炎症细胞如嗜酸性粒细胞、肥大细胞和淋巴细胞共同参与的慢性气道炎症性疾病，其具体发病机制目前尚不清楚。IL-4等细胞因子在支气管哮喘发生发展中起着十分重要的作用，研制IL-4的特异性抗体是解决哮喘治疗的有效途径之一。抑制IL-4/IL-4Rα可以对哮喘起到有效的免疫调节作用。
过敏性鼻炎(allergic rhinitis，AR)与哮喘的发病机制有许多共同之处，均属于I型变态反应。研究发现，IL-4、IL-17和IgE在过敏性鼻炎的发病机制中扮演了重要的角色。药物治疗是目前AR治疗的重点，其中，鼻用皮质类固醇和抗组胺药物处于核心地位。
特应性皮炎(atopic dermatitis，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。IL-4和IL-13等细胞因子参与的免疫因素是其主要发病机制之一。
嗜酸粒细胞性食管炎(eosinophilic esophagitis，EoE)是以食管壁全层嗜酸粒细胞(eosinophils，EOS)浸润为特征的慢性免疫性炎性疾病。EoE发病与Th2细胞功能紊乱相关。目前特异性强的方案，如新型生物制剂抗IL-5(如美泊利单抗)成为研究的热点。免疫调节治疗已在动物模型中取得成果，但人类临床试验仍需探索。PGD2抑制剂、抗TNF-α、抗IL-13等药物正在探索中。
目前针对hIL-4R靶点的单克隆抗体药物进入临床试验，如Dupilumab，在治疗特应性皮炎的II期临床中表现出了较好疗效。除Dupilumab以外，亦有公司申请了针对hIL-4R的其他单克隆抗体专利，如US 7,186,809和US 7,638,606。
发明内容
本发明通过抗体筛选和优化获得了针对IL-4R的特异性抗体，所述抗体能够作为IL-4与IL-4R结合的阻断剂，通过与IL-4R结合，可以用于治疗炎症或过敏性疾病等。
参照SEQ ID NO:58所示序列的氨基酸残基编号，本发明的抗体在包含的轻链可变区(VL)的第31位具有丝氨酸(31Ser)，并且参照SEQ ID NO:93所示序列的氨基酸残基编号，所述抗体在包含的重链可变区(VH)的第103位具有天冬氨酸(103Asp)，在第104位具有酪氨酸(104Tyr)。其中，该31Ser相应地在本发明抗体的轻链可变区的CDR1中，该103Asp和104Tyr相应地在本发明抗体的重链可变区的CDR3中。
优选地，本发明的抗体的轻链可变区包含SEQ ID NO:2所示的CDR1，并且所述抗体的重链可变区包含SEQ ID NO:19所示的CDR3；
优选地，本发明的抗体的轻链可变区包含选自下述的CDR1、CDR2和CDR3组合：
(1)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:5所示的CDR3；
(2)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:5所示的CDR3；
(3)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:6所示的CDR3；
(4)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:7所示的CDR3；
(5)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:8所示的CDR3；
(6)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:6所示的CDR3；
(7)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:7所示的CDR3；和
(8)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:8所示的CDR3；并且
本发明的抗体的重链可变区包含选自下述的CDR1、CDR2和CDR3组合：
(1)SEQ ID NO:14所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
(2)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3；
(3)SEQ ID NO:15所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ  ID NO:19所示的CDR3；
(4)SEQ ID NO:15所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3；
(5)SEQ ID NO:16所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3；和
(6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
更优选地，本发明的抗体包含选自下述序列所示氨基酸序列的轻链可变区：SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:54、SEQ ID NO:55和SEQ ID NO:57，并且所述抗体包含选自下述序列所示氨基酸序列的重链可变区：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:91和SEQ ID NO:92；
进一步优选地，本发明的抗体包含选自下述的轻链可变区和重链可变区组合：
(1)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(2)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.63所示的重链可变区；
(3)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.59所示的重链可变区；
(4)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.60所示的重链可变区；
(5)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.61所示的重链可变区；
(6)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.67所示的重链可变区；
(7)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.65所示的重链可变区；
(8)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.66所示的重链可变 区；
(9)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.64所示的重链可变区；
(10)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(11)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.74所示的重链可变区；
(12)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.75所示的重链可变区；
(13)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.76所示的重链可变区；
(14)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.77所示的重链可变区；
(15)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.78所示的重链可变区；
(16)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.80所示的重链可变区；
(17)SEQ ID NO.40所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(18)SEQ ID NO.41所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(19)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(20)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(21)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(22)SEQ ID NO.49所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(23)SEQ ID NO.50所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(24)SEQ ID NO.45所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(25)SEQ ID NO.46所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(26)SEQ ID NO.47所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(27)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(28)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(29)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(30)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(31)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(32)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.91所示的重链可变区；和
(33)SEQ ID NO.51所示的轻链可变区和SEQ ID NO.92所示的重链可变区。
另一方面，本发明提供一种能够结合白细胞介素4(IL-4)受体(IL-4R)的抗体，所述抗体包含轻链可变区(VL)，所述轻链可变区包含选自下述的CDR1、CDR2和CDR3组合：
(1)SEQ ID NO:1所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:5所示的CDR3；
(2)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:5所示的CDR3；
(3)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:5所示的CDR3；
(4)SEQ ID NO:1所示的CDR1，SEQ ID NO.4所示的CDR2和SEQ ID NO:5所示的CDR3；
(5)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:6所示的CDR3；
(6)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:7所示的CDR3；
(7)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:8所示的CDR3；
(8)SEQ ID NO:1所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:6所示的CDR3；
(9)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:6所示的CDR3；
(10)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:8所示的CDR3；
(11)SEQ ID NO:1所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:8所示的CDR3；和
(12)SEQ ID NO:1所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:8所示的CDR3；
和/或
所述抗体包含重链可变区(VH)，所述重链可变区包含选自下述的CDR1、CDR2和CDR3组合：
(1)SEQ ID NO:14所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
(2)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3；
(3)SEQ ID NO:14所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:20所示的CDR3；
(4)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:20所示的CDR3；
(5)SEQ ID NO:15所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
(6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；和
(7)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3。
进一步地，关于本发明的上述抗体的框架区，优选地，本发明的抗体的轻链可变区包含选自下述的FR1、FR2、FR3和FR4组合：
(1)SEQ ID NO:9所示的FR1，SEQ ID NO:10所示的FR2，SEQ ID NO:12所示的FR3和SEQ ID NO:13所示的FR4；和
(2)SEQ ID NO:9所示的FR1，SEQ ID NO:11所示的FR2，SEQ ID NO:12所示的FR3和SEQ ID NO:13所示的FR4。
优选地，所述抗体的重链可变区包含选自下述的FR1、FR2、FR3和FR4组合：
(1)SEQ ID NO:21所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(2)SEQ ID NO:22所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(3)SEQ ID NO:23所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(4)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(5)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
(6)SEQ ID NO:25所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(7)SEQ ID NO:26所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(8)SEQ ID NO:27所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(9)SEQ ID NO:29所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(10)SEQ ID NO:30所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(11)SEQ ID NO:24所示的FR1，SEQ ID NO:33所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(12)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:36所示的FR3和SEQ ID NO:38所示的FR4；
(13)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:37所示的FR3和SEQ ID NO:38所示的FR4；
(14)SEQ ID NO:31所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(15)SEQ ID NO:27所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO: 35所示的FR3和SEQ ID NO:38所示的FR4；
(16)SEQ ID NO:26所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
(17)SEQ ID NO:25所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
(18)SEQ ID NO:28所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
(19)SEQ ID NO:28所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
(20)SEQ ID NO:23所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
(21)SEQ ID NO:22所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；和
(22)SEQ ID NO:21所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4。
按照本领域公知的抗体轻链可变区、重链可变区的结构域组成，本发明抗体的轻链可变区或重链可变区以FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的顺序包含上述结构域组分，或者以(X)n-FR1-(X)n-CDR1-(X)n-FR2-(X)n-CDR2-(X)n-FR3-(X)n-CDR3-(X)n-FR4-(X)n的顺序包含上述结构域组分，其中X为任意氨基酸残基，n为零或大于零的整数。
优选地，本发明提供的抗体包含选自下述序列所示氨基酸序列的轻链可变区：
SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；
和/或
本发明提供的抗体包含选自下述序列所示氨基酸序列的重链可变区：
SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、 SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92和SEQ ID NO:93。
根据本发明的具体实施方式，本发明提供的抗体包含选自下述的轻链可变区和重链可变区组合：
(1)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(2)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.63所示的重链可变区；
(3)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.59所示的重链可变区；
(4)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.60所示的重链可变区；
(5)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.61所示的重链可变区；
(6)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.67所示的重链可变区；
(7)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.65所示的重链可变区；
(8)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.66所示的重链可变区；
(9)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.64所示的重链可变区；
(10)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
(11)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.69所示的重链可变区；
(12)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.70所示的重链可变区；
(13)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.71所示的重链可变区；
(14)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.72所示的重链可 变区；
(15)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.73所示的重链可变区；
(16)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.89所示的重链可变区；
(17)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.88所示的重链可变区；
(18)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.87所示的重链可变区；
(19)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.86所示的重链可变区；
(20)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.83所示的重链可变区；
(21)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
(22)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.81所示的重链可变区；
(23)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
(24)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.84所示的重链可变区；
(25)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(26)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.90所示的重链可变区；
(27)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.74所示的重链可变区；
(28)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.75所示的重链可变区；
(29)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.76所示的重链可变区；
(30)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.77所示的重链可变区；
(31)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.78所示的重链可变区；
(32)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.80所示的重链可变区；
(33)SEQ ID NO.39所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(34)SEQ ID NO.40所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(35)SEQ ID NO.41所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(36)SEQ ID NO.42所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(37)SEQ ID NO.43所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(38)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(39)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(40)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
(41)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.72所示的重链可变区；
(42)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
(43)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
(44)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(45)SEQ ID NO.48所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(46)SEQ ID NO.49所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(47)SEQ ID NO.50所示的轻链可变区和SEQ ID NO.92所示的重链可 变区；
(48)SEQ ID NO.45所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(49)SEQ ID NO.46所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(50)SEQ ID NO.47所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(51)SEQ ID NO.56所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(52)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(53)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(54)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
(55)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.72所示的重链可变区；
(56)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
(57)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
(58)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(59)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(60)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
(61)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
(62)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.72所示的重链可变区；
(63)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
(64)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
(65)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(66)SEQ ID NO.53所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(67)SEQ ID NO.51所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(68)SEQ ID NO.52所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(69)SEQ ID NO.52所示的轻链可变区和SEQ ID NO.62所示的重链可变区；和
(70)SEQ ID NO.52所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(71)SEQ ID NO.58所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
(72)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.93所示的重链可变区；
(73)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.93所示的重链可变区；
(74)SEQ ID NO.58所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
(75)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.93所示的重链可变区；和
(76)SEQ ID NO.51所示的轻链可变区和SEQ ID NO.93所示的重链可变区。
本发明提供的抗体能够结合IL-4R，作为IL-4R的拮抗剂发挥作用。优选地，所述抗体能够结合IL-4Rα，优选结合哺乳动物类IL-4Rα，更优选结合人IL-4Rα，甚至更优选结合人可溶性IL-4Rα。
可以通过Biacore或ELISA方法测定本发明提供的抗体与IL-4Rα的结合亲和力。测得所述抗体能够以小于100nM、小于10nM、小于1nM、小于0.5nM和甚至小于0.1nM亲和力结合IL-4Rα。
在相同条件下，本发明提供的抗体与参照抗体的表达量比为0.1-3:1，优 选0.3-3:1，更优选0.4-3:1，更优选0.5-3:1，更优选0.6-3:1，更优选0.7-3:1，更优选1-3:1。
就抗体类别而言，本发明提供的抗体可以是单克隆抗体、完全或部分人源化抗体或嵌合抗体；
或者优选地，所述抗体为免疫球蛋白，优选为IgA、IgD、IgE、IgG或IgM，更优选为IgG1、IgG2、IgG3或IgG4亚型，更优选为IgG2或IgG4亚型。
另一方面，本发明提供一种融合蛋白或偶联物，所述融合蛋白或偶联物包含本发明所述的抗体。该融合蛋白或偶联物还包含通过化学或物理方法结合于本发明所述抗体的细胞表面受体、活性蛋白及多肽、小分子化合物如氨基酸和糖类、小分子聚合物或对本发明所述抗体进行化学修饰的其它部分等。
又一方面，本发明提供一种核酸序列，所述核酸序列能够编码本发明所提供抗体的重链可变区和/或轻链可变区；
优选地，所述核酸序列能够编码本发明提供抗体的重链和/或轻链。
再一方面，本发明还提供一种包含本发明提供的核酸序列的载体。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。
上述载体或核酸序列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，本发明还提供一种采用所述核酸序列或载体转化或转染的宿主细胞。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序列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、融合蛋白或偶联物、核酸序列、载体、宿主细胞或者通过上述方法生产的抗体可以被包含在药物组合物中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。因此，在又一方面，本发明还提供一种药物组合物，所述药物组合物包含本发明所述的抗体、融合蛋白或偶联物、核酸序列、载体、宿主细胞和/或通过上述方法生产的抗体。
可选地，所述药物组合物可以是药物制剂。所述药物制剂例如为注射剂剂型。
取决于特定剂型，所述药物组合物或药物制剂还包含药学上可接受的载体或赋形剂。
在药物组合物或药物制剂中，还可以包含至少一种选自下述的药物：平喘药如沙丁胺醇等，抗组胺药如氯雷他定等，免疫抑制剂如他克莫司、吡美莫司等，M受体阻断剂如异丙托溴铵等，白三烯受体阻断药如孟鲁司特等，磷酸二酯酶抑制剂如茶碱等，非甾体抗炎药如5-氨基水杨酸等，激素类如倍氯米松，布地奈德等，即本发明提供的抗体、融合蛋白或偶联物、核酸序列、载体、宿主细胞或者通过上述方法生产的抗体可以根据需要与其他药物联合使用。
再一方面，本发明还提供所述抗体、融合蛋白或偶联物、核酸序列、载体、宿主细胞和/或通过所述方法生产的抗体在制备用于预防、治疗或改善炎症或过敏性疾病中的用途；
优选地，所述炎症或过敏性疾病包括自身免疫学疾病，例如过敏性皮炎、哮喘、嗜酸性粒细胞食管炎、湿疹、过敏性鼻炎、鼻息肉、类风湿性关节炎等。
还一方面，本发明提供一种预防、治疗或改善受试者的炎症或过敏性疾病的方法，所述方法包括给有此需要的受试者施用本发明提供的抗体、融合蛋白或偶联物、核酸序列、载体、宿主细胞和/或通过所述方法生产的抗体。
优选地，所述受试者为哺乳类动物；更优选地，所述受试者为人。
优选地，所述炎症或过敏性疾病包括自身免疫学疾病，例如过敏性皮炎、哮喘、嗜酸性粒细胞食管炎、湿疹、过敏性鼻炎、鼻息肉、类风湿性关节炎等。
在预防、治疗或改善炎症或过敏性疾病时，还可以联合应用其他药物，例如所述方法还包括给受试者施用至少一种选自下述的药物：平喘药如沙丁胺醇等，抗组胺药如氯雷他定等，免疫抑制剂如他克莫司、吡美莫司等，M受体阻断剂如异丙托溴铵等，白三烯受体阻断药如孟鲁司特等，磷酸二酯酶抑制剂如茶碱等，非甾体抗炎药如5-氨基水杨酸等，激素类如倍氯米松、布地奈德等。
优选地，所述药物与本发明提供的抗体、融合蛋白或偶联物、核酸序列、载体、宿主细胞和/或通过所述方法生产的抗体同时或顺序施用。
再一方面，本发明还提供一种试剂盒，所述试剂盒包括本发明提供的抗 体、融合蛋白或偶联物、核酸序列、载体、宿主细胞和/或通过所述方法生产的抗体。
附图说明
结合本发明非限制性实施例和附图可以更好地理解本发明，在附图中：
图1示出了本发明的抗体在小鼠体内的药代动力学曲线；
图2示出了本发明的抗体在食蟹猴体内的药代动力学曲线；
图3A-3C通过FACS示出了本发明的抗体与表达IL-4Rα的TF-1细胞特异性结合，其中3A示出没有加入本发明抗体L1012H1031时的荧光信号，3B示出加入本发明抗体L1012H1031后的荧光信号，3C示出3A-3B的信号叠加比较。
图4A-4D通过FACS示出了本发明的抗体与表达IL-4Rα的TF-1细胞的特异性结合被体系中存在的sIL-4Rα所阻断，其中4A示出没有加入本发明抗体L1012H1031时的荧光信号，4B示出加入本发明抗体L1012H1031后的荧光信号，4C示出加入本发明抗体L1012H1031及sIL-4Rα时的荧光信号，4D示出4A-4C的信号叠加比较。
图5A-5B通过ELISA示出了本发明的抗体抑制TARC和MDC释放的作用，其中5A示出本发明抗体L1020H1031抑制TARC因子释放结果，5B示出本发明抗体L1020H1031抑制MDC因子释放结果。
实施发明的最佳方式
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试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
在下述实施例中，示例性地提供并验证了表1中所示抗体的作用。
表1本发明提供的示例性抗体
编号 轻链可变区和重量可变区的序列编号
L1000H1007 SEQ ID NO.57+SEQ ID NO.62
L1000H1008 SEQ ID NO.57+SEQ ID NO.63
L1000H1009 SEQ ID NO.57+SEQ ID NO.59
L1000H1010 SEQ ID NO.57+SEQ ID NO.60
L1000H1011 SEQ ID NO.57+SEQ ID NO.61
L1000H1012 SEQ ID NO.57+SEQ ID NO.67
L1000H1013 SEQ ID NO.57+SEQ ID NO.65
L1000H1014 SEQ ID NO.57+SEQ ID NO.66
L1000H1015 SEQ ID NO.57+SEQ ID NO.64
L1000H1016 SEQ ID NO.57+SEQ ID NO.68
L1000H1017 SEQ ID NO.57+SEQ ID NO.69
L1000H1018 SEQ ID NO.57+SEQ ID NO.70
L1000H1019 SEQ ID NO.57+SEQ ID NO.71
L1000H1020 SEQ ID NO.57+SEQ ID NO.72
L1000H1021 SEQ ID NO.57+SEQ ID NO.73
L1000H1022 SEQ ID NO.57+SEQ ID NO.89
L1000H1023 SEQ ID NO.57+SEQ ID NO.88
L1000H1024 SEQ ID NO.57+SEQ ID NO.87
L1000H1025 SEQ ID NO.57+SEQ ID NO.86
L1000H1026 SEQ ID NO.57+SEQ ID NO.83
L1000H1027 SEQ ID NO.57+SEQ ID NO.82
L1000H1028 SEQ ID NO.57+SEQ ID NO.81
L1000H1029 SEQ ID NO.57+SEQ ID NO.85
L1000H1030 SEQ ID NO.57+SEQ ID NO.84
L1000H1031 SEQ ID NO.57+SEQ ID NO.91
L1000H1032 SEQ ID NO.57+SEQ ID NO.90
L1000H1033 SEQ ID NO.57+SEQ ID NO.74
L1000H1034 SEQ ID NO.57+SEQ ID NO.75
L1000H1035 SEQ ID NO.57+SEQ ID NO.76
L1000H1036 SEQ ID NO.57+SEQ ID NO.77
L1000H1037 SEQ ID NO.57+SEQ ID NO.78
L1000H1038 SEQ ID NO.57+SEQ ID NO.80
L1007H1000 SEQ ID NO.39+SEQ ID NO.92
L1008H1000 SEQ ID NO.40+SEQ ID NO.92
L1009H1000 SEQ ID NO.41+SEQ ID NO.92
L1010H1000 SEQ ID NO.42+SEQ ID NO.92
L1011H1000 SEQ ID NO.43+SEQ ID NO.92
L1012H1000 SEQ ID NO.44+SEQ ID NO.92
L1012H1007 SEQ ID NO.44+SEQ ID NO.62
L1012H1016 SEQ ID NO.44+SEQ ID NO.68
L1012H1020 SEQ ID NO.44+SEQ ID NO.72
L1012H1027 SEQ ID NO.44+SEQ ID NO.82
L1012H1029 SEQ ID NO.44+SEQ ID NO.85
L1012H1031 SEQ ID NO.44+SEQ ID NO.91
L1013H1000 SEQ ID NO.48+SEQ ID NO.92
L1014H1000 SEQ ID NO.49+SEQ ID NO.92
L1015H1000 SEQ ID NO.50+SEQ ID NO.92
L1016H1000 SEQ ID NO.45+SEQ ID NO.92
L1017H1000 SEQ ID NO.46+SEQ ID NO.92
L1018H1000 SEQ ID NO.47+SEQ ID NO.92
L1019H1000 SEQ ID NO.56+SEQ ID NO.92
L1020H1000 SEQ ID NO.55+SEQ ID NO.92
L1020H1007 SEQ ID NO.55+SEQ ID NO.62
L1020H1016 SEQ ID NO.55+SEQ ID NO.68
L1020H1020 SEQ ID NO.55+SEQ ID NO.72
L1020H1027 SEQ ID NO.55+SEQ ID NO.82
L1020H1029 SEQ ID NO.55+SEQ ID NO.85
L1020H1031 SEQ ID NO.55+SEQ ID NO.91
L1021H1000 SEQ ID NO.54+SEQ ID NO.92
L1021H1007 SEQ ID NO.54+SEQ ID NO.62
L1021H1016 SEQ ID NO.54+SEQ ID NO.68
L1021H1020 SEQ ID NO.54+SEQ ID NO.72
L1021H1027 SEQ ID NO.54+SEQ ID NO.82
L1021H1029 SEQ ID NO.54+SEQ ID NO.85
L1021H1031 SEQ ID NO.54+SEQ ID NO.91
L1022H1000 SEQ ID NO.53+SEQ ID NO.92
L1023H1000 SEQ ID NO.51+SEQ ID NO.92
L1024H1000 SEQ ID NO.52+SEQ ID NO.92
L1024H1007 SEQ ID NO.52+SEQ ID NO.62
L1024H1031 SEQ ID NO.52+SEQ ID NO.91
L1001H1000 SEQ ID NO.58+SEQ ID NO.92
L1000H1001 SEQ ID NO.57+SEQ ID NO.93
L1012H1001 SEQ ID NO.44+SEQ ID NO.93
L1001H1031 SEQ ID NO.58+SEQ ID NO.91
L1020H1001 SEQ ID NO.55+SEQ ID NO.93
L1023H1001 SEQ ID NO.51+SEQ ID NO.93
L1000H1000 SEQ ID NO.57+SEQ ID NO.92
L1001H1001 SEQ ID NO.58+SEQ ID NO.93
实施例1：本发明抗体的制备与非还原SDS-PAGE凝胶电泳法测定表达量
将抗体轻链可变区的编码序列利用EcoRI和BsiWI酶切位点插入到pFUSE2ss-CLIg-hK载体(Invivogen，货号：pfuse2ss-hclk)构成轻链表达载体；重链可变区的编码序列利用EcoRI和NheI酶切位点插入到pFUSEss-CHIg-hG2载体(Invivogen，货号：pfusess-hchg2)或pFUSEss-CHIg-hG4载体(Invivogen，货号：pfusess-hchg4)构成重链表达载体。
Expi293细胞的培养和转染按照Invitrogen公司Expi293TM Expression System Kit手册进行(货号：A14635)。转染时细胞密度调整为2×106个/ml，每毫升细胞加入上述0.6μg轻链表达载体和0.4μg重链表达载体，四天后收集培养上清。
细胞培养上清按照《分子克隆实验指南》第三版附录8所述方法进行非还原SDS-PAGE凝胶电泳。
使用东方君意凝胶扫描成像系统拍照并使用Gel-PRO ANALYZER软件进行凝胶定量，以测定抗体瞬转的表达量。结果以相对于对照抗体1(根据专利US 7,186,809构建，其轻链可变区为US 7,186,809的SEQ ID No.10，重链可变区为US 7,186,809的SEQ ID No.12，下同)的表达量表示(对照抗体2根据专利US 7,638,606构建，其轻链可变区为US 7,638,606的SEQ ID No.6，重链可变区为US 7,638,606的SEQ ID No.42，下同)，见下表2a-2c。
表2a本发明抗体的瞬转表达量(表达量较对照抗体1显著提高的抗体)：
Figure PCTCN2017087592-appb-000001
表2b本发明抗体的瞬转表达量(表达量较对照抗体1略有降低的抗体)：
Figure PCTCN2017087592-appb-000002
表2c本发明抗体的瞬转表达量(表达量较对照抗体1有大幅降低的抗体)：
Figure PCTCN2017087592-appb-000003
Figure PCTCN2017087592-appb-000004
实施例2：检测本发明的抗体抑制hIL-4或hIL-13对TF-1细胞的增殖作用
1.试剂的配制
hIL-4(Invivogen，货号：rhil-4)溶液：用100μl含0.1％BSA(Beyotime，货号：ST023)的PBS溶液溶解hIL-4，得到100μg/ml的浓度，将溶解后的hIL-4按照5μl每管的体积分装至1.5ml(Nunc)离心管内，并存放于-20℃冰箱内。
WST-1(Beyotime，货号：C0036)溶液：把5ml电子耦合剂(C0036-2)加入到WST-1粉末(C0036-1)中，完全溶解即成WST-1溶液，按照每管620μl体积分装至1.5ml离心管内，并存放于-20℃冰箱内。
2.TF-1细胞培养
取出液氮中冻存的TF-1(ATCC：CRL-2003TM)细胞，在37℃水浴中震荡，使其迅速溶解。将溶解后的细胞悬液移至15ml离心管内，加1640培养基至10ml，800rpm离心5min，吸去上清，保留细胞沉淀，重复洗涤一次，加入10ml含10％FBS和2ng/ml GM-CSF(Sino Biological，货号：10015-H01H)的1640培养基，调整细胞密度为1×105～1×106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1×106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，单一悬浮的略微不规则形态细胞)后可进行增殖实验。
3.抗体的制备和纯化
a)本发明抗体细胞培养上清样品：
将带有不同组抗体基因的质粒转染Expi293细胞，转染后4天取细胞培养上清200μl，800rpm离心5min，将上清经0.22μm孔径滤膜过滤后，用于增殖阻断实验。
b)纯化的本发明抗体样品：取表达本发明抗体的细胞培养上清，过0.22μm滤膜，经GE MabSelect Sure(Cat Number：11003494)Protein A亲和层析柱纯化，纯化系统为GE AKTA purifier 10，纯化后收集的抗体使用Amicon超滤浓缩管(Cat Number：UFC903096)浓缩并定量。检测时，用PBS稀释抗体至0至1μg/ml用于增殖阻断实验。
4.抑制增殖实验
取T75细胞瓶中生长状态良好细胞，移入15ml离心管内，800rpm离心5min，弃上清，取细胞沉淀。用10ml PBS重悬细胞，800rpm离心5min，弃上清，取细胞沉淀。用10ml含10％FBS的1640培养基(不含GM-CSF)重悬细胞，800rpm离心5min，弃上清，取细胞沉淀。用5ml含10％FBS的1640培养基(不含GM-CSF)重悬细胞，细胞计数后，补加培养基，调整细胞密度至5×105个/ml。将细胞悬液按每孔80μl体积加入96孔板内(预留外圈防挥发孔不加细胞)。将准备好的不同浓度的纯化抗体10μl或细胞培养上清10μl加入96孔的细胞内(3个重复孔)。然后用含10％FBS的1640培养基稀释hIL-4至50ng/ml。将50ng/ml的hIL-4按照10μl每孔的体积分别加入96孔板上对应的细胞孔内，使得最终的细胞密度为4×105个/ml，hIL-4浓度为5ng/ml，最终96孔板每孔体积为100μl，设置不添加hIL-4，不添加抗体，仅加入相同数量细胞与同体积培养液的阴性对照组(3个重复孔)，另设置不添加抗体，补加相同体积培养液，细胞中仅加入相同浓度hIL-4的阳性对照组(3个重复孔)。在96孔细胞板外圈每孔加入200μl PBS防止内圈液体挥发。将96孔细胞板置于37℃，5％CO2培养箱中静置培养。
使用500ng/ml的hIL-13(加入细胞后的终浓度为50ng/ml)，按照相同过程重复上述实验。
5.数据统计
96孔细胞板在5％CO2培养箱中静置培养72h后，在每孔细胞内加入 10μl WST-1溶液。将96孔细胞板继续置于37℃，5％CO2培养箱中静置培养。24h后，将96孔板置于flexstation 3(Molecular Devices)中，读取OD450-OD650的值。
对于本发明抗体培养上清，测得的OD450-OD650数值(OD值)与阳性对照组和阴性对照组的数值进行计算，抑制百分率＝(转染后细胞上清OD值-阳性对照组OD值)/(阴性对照组OD值-阳性对照组OD值)×100％。本发明的抗体对hIL-4或hIL-13的TF-1细胞增殖作用的阻断作用结果见下表3a-3b。
对于经过纯化的不同浓度的本发明抗体，测得的OD450-OD650数据输入prism5软件中，将阴性对照组数值设为最低值，阳性对照组数值设为最高值，抗体浓度取对数值，由prism5软件拟合抗体浓度对数值与OD450-OD650的曲线，计算得到的IC50见下表4。
表3a本发明的抗体抑制TF-1细胞增殖活性的筛选结果(抑制率较对照抗体1增加的抗体)
Figure PCTCN2017087592-appb-000005
Figure PCTCN2017087592-appb-000006
表3b本发明的抗体抑制TF-1细胞增殖活性的筛选结果(抑制率较对照抗体1等同或降低的抗体)
Figure PCTCN2017087592-appb-000007
Figure PCTCN2017087592-appb-000008
Figure PCTCN2017087592-appb-000009
表4抗体600-900ml瞬转纯化产物活性数据
Figure PCTCN2017087592-appb-000010
实施例3：ELISA检测本发明的抗体与sIL-4Rα的结合能力
1.试剂的配制
sIL-4Rα(PEPRO TECH，货号：200-04R)溶液：取一支sIL-4Rα，加入1ml ddH2O，上下颠倒混匀，即为100μg/ml溶液，然后分装后于-20℃冰箱中保存。
待测样品：取瞬染后表达本发明抗体的Expi293细胞培养物上清液(培养基为Expi293Expression Medium，Invitrogen，货号：A1435102；在8％CO2培养箱中，连续100rpm悬浮培养4天)10μl和2μl，分别加至990μl和998μl的PBS溶液中，配制成为1:100和1:500稀释的待测抗体样品。
对照样品：正常细胞培养上清(Expi293细胞不经过转染，培养基为Expi293Expression Medium(Invitrogen，货号：A1435102；在8％CO2培养箱中，连续100rpm悬浮培养4天)同样进行1:100和1:500稀释，作为阴性对照样品。
2.ELISA检测
取100μl 100μg/ml sIL-4Rα溶液，加入到9.90ml PBS包被液中，上下颠倒混匀，即为1.0μg/ml抗原包被液。将配好的抗原包被液加入到96孔酶标 板(corning)中，每孔100μl。将96孔酶标板置于4℃冰箱中孵育过夜。第二天，弃去其中溶液，向96孔酶标板中逐排加入含2％BSA的PBS溶液，每孔300μl。4℃冰箱中孵育2h。弃去2％BSA，使用PBST洗涤3次。将稀释好的待测抗体依次加入到相应的孔中，同时加入正常细胞培养上清作为阴性对照样品，每个样品做三个复孔，每孔100μl。将酶标板用保鲜膜包裹(或加盖)后，置于10℃恒温培养箱中孵育1h。将96孔酶标板取出，弃去其中溶液，PBST洗涤3次后向96孔酶标板中逐排加入TMB溶液(Solarbio，Cat Number：PR1200)，每孔100μl。室温放置5分钟后，立即向96孔酶标板中加入2M H2SO4溶液终止反应。将96孔酶标板置于flexstation 3(Molecular Devices)中，读取OD450的值，数据收集计算分析。结果以相对于对照抗体1的亲和力示出，见下表5a-5c。
表5a本发明的抗体对sIL-4Rα的亲和力(亲和力显著大于对照抗体1的抗体)
抗体编号 OD 450/OD450对照抗体1
对照抗体1 1
L1021H1000 2.42
L1020H1000 2.27
L1019H1000 1.79
L1001H1000 1.56
L1012H1000 1.22
L1000H1031 1.14
L1020H1031 1.12
L1000H1014 1.06
L1020H1029 1.01
表5b本发明的抗体对sIL-4Rα的亲和力(亲和力较对照抗体1等同或略有降低的抗体)
Figure PCTCN2017087592-appb-000011
Figure PCTCN2017087592-appb-000012
表5c本发明的抗体对sIL-4Rα的亲和力(亲和力较对照抗体1显著降低的抗体)
抗体编号 OD 450/OD450对照抗体1
L1024H1000 0.69
L1015H1000 0.67
L1000H1015 0.65
L1009H1000 0.64
L1021H1016 0.63
L1000H1023 0.63
L1000H1016 0.61
L1000H1009 0.56
L1000H1032 0.53
L1017H1000 0.49
L1021H1007 0.44
L1000H1027 0.44
L1020H1007 0.40
L1020H1027 0.40
L1012H1007 0.40
L1000H1013 0.38
L1000H1007 0.37
L1000H1021 0.35
L1000H1028 0.33
L1021H1027 0.29
L1016H1000 0.27
L1020H1020 0.26
L1000H1024 0.26
L1021H1020 0.24
L1013H1000 0.24
L1024H1007 0.23
L1000H1020 0.23
L1000H1035 0.22
L1000H1008 0.21
L1000H1025 0.21
L1000H1030 0.20
L1000H1012 0.18
L1000H1022 0.18
L1022H1000 0.16
L1012H1031 0.16
L1000H1017 0.13
L1000H1010 0.11
L1000H1026 0.11
L1012H1020 0.11
L1012H1029 0.11
L1000H1036 0.10
L1000H1018 0.09
L1000H1034 0.09
L1000H1011 0.09
L1000H1033 0.08
L1012H1016 0.08
L1018H1000 0.06
L1023H1000 0.05
L1000H1038 0.04
L1000H1019 0.02
L1012H1027 0.02
L1014H1000 0.01
L1000H1037 -0.01
实施例4：本发明的抗体在小鼠体内的药代动力学
为了进一步筛选抗体，在小鼠体内进行了一系列的药代动力学实验。
选择6-8周龄SPF级Balb/c小鼠，以5mg(本发明的抗体或对照抗体2)/kg(小鼠体重)的量进行抗体皮下注射。于药前(0h)，药后2、8、24、48、72、120、168、216、264、336h的时间点采集血样。取样为动物用异氟烷吸入麻醉后，眼眶静脉丛取血，每只取血量约0.1mL；药后336h动物首先用异氟烷吸入麻醉，下腔静脉取血安乐死。
血样不抗凝，取血后2h之内室温1500g离心10min以分离血清。收集的上清液立即转移至新的标记好的离心管，血清保存于-70℃以下暂存。使用ELISA方法测定小鼠体内的抗体浓度：
1.试剂的配制
sIL-4Rα(PEPRO TECH，货号：200-04R)溶液：取一支sIL-4Rα，加入1ml ddH2O，上下颠倒混匀，即为100μg/ml溶液，然后分装后于-20℃冰箱中保存。
待测样品：取不同时间段采集的血清各1μl，加入至999μl含1％BSA的PBS溶液中，即成1:1000稀释的待测血清样品。
标准品：将待检测抗体用含1％BSA和0.1％正常动物血清(Beyotime，货号：ST023)的PBS溶液稀释成0.1μg/ml。分别取0.1μg/ml的待检测抗体800、600、400、200、100、50、10、0μl，分别加入含1％BSA和0.1％正常动物血清的PBS溶液200、400、600、800、900、950、990、1000μl，配成最终浓度分别为80、60、40、20、10、5、1、0ng/ml的本发明抗体标准品。
2.ELISA检测
取250μl 100μg/ml sIL-4Rα溶液,加入到9.75ml PBS包被液中，上下颠倒混匀，即为2.5μg/ml抗原包被液。将配好的抗原包被液加入入96孔酶标板(corning)中，每孔100μl。将96孔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孔酶标板取出，弃去其中溶液，加入含2％BSA的PBS溶液，每孔300μl。将酶标板用保鲜膜包裹(或加盖)后，于4℃冰箱中孵育2h。将96孔酶标板取出，弃去其中溶液，PBST重复洗涤3次。将稀释好的标准抗体及待测血清样品依次加入到相应的孔中，每个样品做三个复孔，每孔100μl。将酶标板用保鲜膜包裹(或加盖)后，室温孵育1h。弃去其中溶液，PBST洗涤3次。向96孔酶标板中逐排加入TMB溶液(Solarbio，Cat Number：PR1200)，每孔100μl。室温放置5分钟，立 即向96孔酶标板中加入2M H2SO4溶液终止反应。将96孔酶标板置于flexstation 3(Molecular Devices)中，读取OD450的值，收集数据使用Winnonlin软件计算结果。药代动力学结果见图1与下表6。
表6本发明的抗体在小鼠体内的药代动力学结果
Figure PCTCN2017087592-appb-000013
实施例5：本发明的抗体在食蟹猴体内的药代动力学
为了进一步筛选抗体，在食蟹猴体内进行了一系列的药代动力学实验。
选择3-5岁食蟹猴，体重2-5Kg，以5mg(本发明的抗体或对照抗体2)/kg(食蟹猴体重)的量进行抗体皮下注射。用一次性无菌注射器准确抽取要施用的抗体或对照抗体2，于动物大腿内侧皮下进行多点注射，每点注射容量不超过2ml。于药前(0h)，药后0.5、2、4、8、24、48、72、120、168、240、336h、432h、504h、600h、672h的时间点从动物后肢皮下静脉采集全血，每只取血量1.5ml。
血样不抗凝，取血后2h之内室温1500g离心10min以分离血清。收集的上清液立即转移至新的标记好的离心管，血清保存于-70℃以下暂存。采用实施例4所述方法测定食蟹猴体内的抗体浓度。药代动力学结果见图2与下表7。
表7本发明的抗体在食蟹猴体内的药代动力学结果
Figure PCTCN2017087592-appb-000014
Figure PCTCN2017087592-appb-000015
实施例6：FACS检测本发明的抗体与TF-1细胞的结合
1.细胞培养
在液氮中取出冻存的TF-1(ATCC：CRL-2003TM)细胞，在37℃水浴中不停轻柔震荡，使其迅速溶解。加溶解后的细胞悬液移至15ml离心管内，加1640培养基(Hyclone，货号：SH30809.01B)至10ml，800rpm离心5min，吸去上清，保留细胞沉淀，重复洗涤一次，加入含10％FBS(Hyclone，货号：SV30184.02)、2ng/ml GM-CSF(Sino Biological，货号：10015-H01H)的1640培养基，调整细胞密度为1×105-1×106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1×106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，单一悬浮的略微不规则形态细胞)后可进行实验。
2.细胞处理
取TF-1细胞，通过显微镜计数，将细胞分为3组，分别置于3个1.5ml离心管内，每组细胞个数为1×106个。800rpm 5min离心细胞，用冷的含1％BSA的PBS 1ml重悬细胞，重复洗涤一次。800rpm 5min离心细胞，每个离心管内加入冷的含1％BSA的PBS 45μl重悬细胞后，其中第一个离心管内加入本发明L1021H1031、L1020H1031或L1012H1031抗体5μl(500μg/ml)，第二个离心管内加入PBS 5μl作为阴性对照。冰上静置45min后，800rpm 5min离心细胞，用冷的含1％BSA的PBS 1ml重悬细胞，重复洗涤一次。800rpm 5min离心细胞，每个离心管内加入冷的含1％BSA的PBS 499μl重悬细胞后，加入1μl FITC标记山羊抗人IgG(H+L)(Beyotime，货号：A0556)。冰上静置45min后，800rpm 5min离心细胞，用冷的含1％BSA的PBS 1ml重悬细胞，重复洗涤一次，细胞待测。
3.FACS上机检测
按正确流程打开仪器和操作系统，设置正确的参数后，将细胞加入检测管内，检测FL1通道的荧光信号，使用FlowJo 7.6软件进行分析，结果见图3A-3C，显示本发明的抗体能够与表达IL-4Rα的TF-1细胞特异性结合。
实施例7：FACS检测可溶性hIL-4Rα(sIL-4Rα)对抗体与TF-1细胞结合的阻断作用
1.试剂的配制
按照实施例1中所述进行hIL-4(Invivogen，货号：rhil-4)溶液的配制。
2.细胞培养
按照实施例6中所述进行TF-1(ATCC：CRL-2003TM)的细胞培养。
3.sIL-4Rα与本发明的抗体的混合
取sIL-4Rα10μl(100μg/ml)与本发明的L1021H1031、L1020H1031或L1012H1031抗体5μl(500μg/ml)以2:1的摩尔比在1.5ml离心管内混合均匀。同时取PBS 10μl与本发明L1021H1031、L1020H1031或L1012H1031抗体5μl混合作为阳性对照，取PBS 15μl作为阴性对照。将离心管放37℃培养箱内静置1h。
4.细胞处理
取TF-1细胞，通过显微镜计数，将细胞分为4组，分别置于3个1.5ml离心管内，每组细胞个数为1×106个。800rpm 5min离心细胞，用冷的含1％BSA的PBS 1ml重悬细胞，重复洗涤一次。800rpm 5min离心细胞，每个离心管内加入冷的含1％BSA的PBS 35μl重悬细胞后，加入步骤3中不同抗体混合物与对照15μl。冰上静置45min后，800rpm 5min离心细胞，用冷的含1％BSA的PBS 1ml重悬细胞，重复洗涤一次。800rpm 5min离心细胞，每个离心管内加入冷的含1％BSA的PBS 499μl重悬细胞后，加入1μl FITC标记山羊抗人IgG(H+L)(Beyotime，货号：A0556)。冰上静置45min后，800rpm 5min离心细胞，用冷的含1％BSA的PBS 1ml重悬细胞，重复洗涤一次，细胞待测。
5.FACS上机检测
按正确流程打开仪器和操作系统，设置正确的参数后，将细胞加入检测管内，检测FL1通道的荧光信号，使用FlowJo 7.6软件进行分析，结果见图4A-4D，显示sIL-4Rα能够特异性地有效阻断本发明抗体与TF-1细胞的特异性结合。
实施例8：Bicore抗体亲和力测定
首先将抗人Fc(AHC)抗体(GE Healthcare)偶联到CM5芯片上，然后用AHC捕获本发明抗体，再将不同浓度的人sIL-4Rα流过捕获了本发明抗体的芯片表面，其中本发明抗体稀释到2μg/ml，抗原sIL-4Rα稀释至0.39、 0.78、1.56、3.13、6.25、12.5、25.0、50.0和100.0nM。然后在Biacore T200 control software软件中建立实验方法，之后运行实验程序检测。结果见下表8。
表8抗体亲和力检测结果
Figure PCTCN2017087592-appb-000016
实施例9：ELISA方法考察本发明的抗体抑制TARC和MDC释放的作用
取新鲜肝素抗凝血10ml(捐献)，与室温下的PBS溶液1:1混匀，小心加入预先准备好的20ml人淋巴分离液(Solarbio，货号：P8610)中。室温下1500rpm离心30min，离心完成后小心吸取PBMC层，并用PBS清洗2次。最终用含10％FBS、200IU/ml IL-2的1640培养基稀释细胞后，加入24孔板中备用，每孔1×106个细胞。各孔中分别加入终浓度为1000、300、100、30、10ng/ml的L1020H1031抗体，然后加入终浓度为10ng/ml的IL-4(或是100ng/ml的IL-13)。培养72小时后按照Human TARC ELISA试剂盒(Abcam，货号：ab183366)或Human MDC ELISA试剂盒(Abcam，货号：ab179885)提供的方法进行检测。
结果见图5A-5B，显示本发明抗体能够有效抑制TARC和MDC释放，并且随着浓度增加，对释放的抑制作用也增大。
实施例10：特定氨基酸对本发明抗体代谢动力学与表达量的影响
进一步检测和比较了本发明抗体的体内代谢动力学，以考察在特定位置的特定氨基酸可能给抗体在动物体内的代谢动力学带来的影响。具体实验方法同实施例4，结果见下表9。
表9抗体体内代谢动力学检测结果
Figure PCTCN2017087592-appb-000017
Figure PCTCN2017087592-appb-000018
由具体序列可知，抗体重链H1031(SEQ ID NO.91)序列的第103位(CDR3中)是Asp(103Asp)，第104位是Tyr(104Tyr)。相比于在重链中不具有103Asp和104Tyr的抗体，具有103Asp和104Tyr的本发明抗体的药时曲线下面积高2-4倍，而清除率降低约70％。
还进一步检测和比较了本发明抗体的表达水平，以考察在特定位置的特定氨基酸可能给抗体的表达带来的影响。Expi293细胞的培养和转染同实施例1，然后将收集的培养上清过0.22μm滤膜，经GE MabSelect Sure(货号：11003494)Protein A亲和层析柱纯化，纯化系统为GE AKTA purifier 10，纯化后收集的抗体使用Amicon超滤浓缩管(货号：UFC903096)浓缩并定量。定量结果见下表10。
表10抗体表达水平检测结果
Figure PCTCN2017087592-appb-000019
由具体序列可知，抗体轻链L1012(SEQ ID NO.44)、L1020(SEQ ID NO.55)和L1023(SEQ ID NO.51)序列的第31位(CDR1中)是Ser (31Ser)。相比于在轻链中不具有31Ser的抗体，具有31Ser的本发明抗体的表达量提高约2-5倍。
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 (16)

  1. 一种能够结合白细胞介素4(IL-4)受体(IL-4R)的抗体，所述抗体的特征在于，参照SEQ ID NO:58所示序列的氨基酸残基编号，所述抗体在包含的轻链可变区(VL)的第31位具有丝氨酸(31Ser)，并且参照SEQ ID NO:93所示序列的氨基酸残基编号，所述抗体在包含的重链可变区(VH)的第103位具有天冬氨酸(103Asp)，在第104位具有酪氨酸(104Tyr)。
  2. 根据权利要求1所述的抗体，其特征在于，所述抗体的轻链可变区包含SEQ ID NO:2所示的CDR1，并且所述抗体的重链可变区包含SEQ ID NO:19所示的CDR3；
    优选地，所述抗体的轻链可变区包含选自下述的CDR1、CDR2和CDR3组合：
    (1)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:5所示的CDR3；
    (2)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:5所示的CDR3；
    (3)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:6所示的CDR3；
    (4)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:7所示的CDR3；
    (5)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:8所示的CDR3；
    (6)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:6所示的CDR3；和
    (7)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:7所示的CDR3；和
    (8)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:8所示的CDR3；并且
    所述抗体的重链可变区包含选自下述的CDR1、CDR2和CDR3组合：
    (1)SEQ ID NO:14所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
    (2)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ  ID NO:19所示的CDR3；
    (3)SEQ ID NO:15所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
    (4)SEQ ID NO:15所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3；
    (5)SEQ ID NO:16所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3；和
    (6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
    更优选地，所述抗体包含选自下述序列所示氨基酸序列的轻链可变区：SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:54、SEQ ID NO:55和SEQ ID NO:57，并且所述抗体包含选自下述序列所示氨基酸序列的重链可变区：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:91和SEQ ID NO:92；
    进一步优选地，所述抗体包含选自下述的轻链可变区和重链可变区组合：
    (1)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (2)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.63所示的重链可变区；
    (3)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.59所示的重链可变区；
    (4)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.60所示的重链可变区；
    (5)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.61所示的重链可变区；
    (6)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.67所示的重链可变区；
    (7)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.65所示的重链可变 区；
    (8)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.66所示的重链可变区；
    (9)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.64所示的重链可变区；
    (10)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (11)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.74所示的重链可变区；
    (12)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.75所示的重链可变区；
    (13)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.76所示的重链可变区；
    (14)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.77所示的重链可变区；
    (15)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.78所示的重链可变区；
    (16)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.80所示的重链可变区；
    (17)SEQ ID NO.40所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (18)SEQ ID NO.41所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (19)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (20)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (21)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (22)SEQ ID NO.49所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (23)SEQ ID NO.50所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (24)SEQ ID NO.45所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (25)SEQ ID NO.46所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (26)SEQ ID NO.47所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (27)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (28)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (29)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (30)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (31)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (32)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.91所示的重链可变区；和
    (33)SEQ ID NO.51所示的轻链可变区和SEQ ID NO.92所示的重链可变区。
  3. 一种能够结合白细胞介素4(IL-4)受体(IL-4R)的抗体，所述抗体的特征在于，所述抗体包含轻链可变区(VL)，所述轻链可变区包含选自下述的CDR1、CDR2和CDR3组合：
    (1)SEQ ID NO:1所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:5所示的CDR3；
    (2)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:5所示的CDR3；
    (3)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:5所示的CDR3；
    (4)SEQ ID NO:1所示的CDR1，SEQ ID NO.4所示的CDR2和SEQ ID NO:5所示的CDR3；
    (5)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:6所示的CDR3；
    (6)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:7所示的CDR3；
    (7)SEQ ID NO:2所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:8所示的CDR3；
    (8)SEQ ID NO:1所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:6所示的CDR3；
    (9)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:6所示的CDR3；
    (10)SEQ ID NO:2所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:8所示的CDR3；
    (11)SEQ ID NO:1所示的CDR1，SEQ ID NO:4所示的CDR2和SEQ ID NO:8所示的CDR3；和
    (12)SEQ ID NO:1所示的CDR1，SEQ ID NO:3所示的CDR2和SEQ ID NO:8所示的CDR3；
    和/或
    所述抗体包含重链可变区(VH)，所述重链可变区包含选自下述的CDR1、CDR2和CDR3组合：
    (1)SEQ ID NO:14所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
    (2)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3；
    (3)SEQ ID NO:14所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:20所示的CDR3；
    (4)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:20所示的CDR3；
    (5)SEQ ID NO:15所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；
    (6)SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:19所示的CDR3；和
    (7)SEQ ID NO:14所示的CDR1，SEQ ID NO:18所示的CDR2和SEQ ID NO:19所示的CDR3。
  4. 根据权利要求1至3中任一项所述的抗体，其特征在于，所述抗体的轻链可变区包含选自下述的FR1、FR2、FR3和FR4组合：
    (1)SEQ ID NO:9所示的FR1，SEQ ID NO:10所示的FR2，SEQ ID NO:12所示的FR3和SEQ ID NO:13所示的FR4；和
    (2)SEQ ID NO:9所示的FR1，SEQ ID NO:11所示的FR2，SEQ ID NO:12所示的FR3和SEQ ID NO:13所示的FR4；
    和/或
    所述抗体的重链可变区包含选自下述的FR1、FR2、FR3和FR4组合：
    (1)SEQ ID NO:21所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (2)SEQ ID NO:22所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (3)SEQ ID NO:23所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (4)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (5)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
    (6)SEQ ID NO:25所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (7)SEQ ID NO:26所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (8)SEQ ID NO:27所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (9)SEQ ID NO:29所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (10)SEQ ID NO:30所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (11)SEQ ID NO:24所示的FR1，SEQ ID NO:33所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (12)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:36所示的FR3和SEQ ID NO:38所示的FR4；
    (13)SEQ ID NO:24所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:37所示的FR3和SEQ ID NO:38所示的FR4；
    (14)SEQ ID NO:31所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO: 34所示的FR3和SEQ ID NO:38所示的FR4；
    (15)SEQ ID NO:27所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
    (16)SEQ ID NO:26所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
    (17)SEQ ID NO:25所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
    (18)SEQ ID NO:28所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
    (19)SEQ ID NO:28所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:34所示的FR3和SEQ ID NO:38所示的FR4；
    (20)SEQ ID NO:23所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
    (21)SEQ ID NO:22所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；和
    (22)SEQ ID NO:21所示的FR1，SEQ ID NO:32所示的FR2，SEQ ID NO:35所示的FR3和SEQ ID NO:38所示的FR4；
    优选地，所述抗体包含选自下述序列所示氨基酸序列的轻链可变区：
    SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57和SEQ ID NO:58；
    和/或
    所述抗体包含选自下述序列所示氨基酸序列的重链可变区：
    SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92和SEQ ID NO:93；
    更优选地，所述抗体包含选自下述的轻链可变区和重链可变区组合：
    (1)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (2)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.63所示的重链可变区；
    (3)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.59所示的重链可变区；
    (4)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.60所示的重链可变区；
    (5)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.61所示的重链可变区；
    (6)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.67所示的重链可变区；
    (7)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.65所示的重链可变区；
    (8)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.66所示的重链可变区；
    (9)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.64所示的重链可变区；
    (10)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
    (11)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.69所示的重链可变区；
    (12)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.70所示的重链可变区；
    (13)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.71所示的重链可变区；
    (14)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.72所示的重链可变区；
    (15)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.73所示的重链可变区；
    (16)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.89所示的重链可变区；
    (17)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.88所示的重链可变区；
    (18)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.87所示的重链可变区；
    (19)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.86所示的重链可变区；
    (20)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.83所示的重链可变区；
    (21)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
    (22)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.81所示的重链可变区；
    (23)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
    (24)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.84所示的重链可变区；
    (25)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (26)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.90所示的重链可变区；
    (27)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.74所示的重链可变区；
    (28)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.75所示的重链可变区；
    (29)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.76所示的重链可变区；
    (30)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.77所示的重链可变区；
    (31)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.78所示的重链可变区；
    (32)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.80所示的重链可变区；
    (33)SEQ ID NO.39所示的轻链可变区和SEQ ID NO.92所示的重链可 变区；
    (34)SEQ ID NO.40所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (35)SEQ ID NO.41所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (36)SEQ ID NO.42所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (37)SEQ ID NO.43所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (38)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (39)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (40)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
    (41)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.72所示的重链可变区；
    (42)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
    (43)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
    (44)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (45)SEQ ID NO.48所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (46)SEQ ID NO.49所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (47)SEQ ID NO.50所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (48)SEQ ID NO.45所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (49)SEQ ID NO.46所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (50)SEQ ID NO.47所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (51)SEQ ID NO.56所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (52)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (53)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (54)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
    (55)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.72所示的重链可变区；
    (56)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
    (57)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
    (58)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (59)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (60)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (61)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.68所示的重链可变区；
    (62)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.72所示的重链可变区；
    (63)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.82所示的重链可变区；
    (64)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.85所示的重链可变区；
    (65)SEQ ID NO.54所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (66)SEQ ID NO.53所示的轻链可变区和SEQ ID NO.92所示的重链可 变区；
    (67)SEQ ID NO.51所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (68)SEQ ID NO.52所示的轻链可变区和SEQ ID NO.92所示的重链可变区；
    (69)SEQ ID NO.52所示的轻链可变区和SEQ ID NO.62所示的重链可变区；
    (70)SEQ ID NO.52所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (71)SEQ ID NO.58所示的轻链可变区和SEQ ID NO.92所示的重链可变区；和
    (72)SEQ ID NO.57所示的轻链可变区和SEQ ID NO.93所示的重链可变区；
    (73)SEQ ID NO.44所示的轻链可变区和SEQ ID NO.93所示的重链可变区；
    (74)SEQ ID NO.58所示的轻链可变区和SEQ ID NO.91所示的重链可变区；
    (75)SEQ ID NO.55所示的轻链可变区和SEQ ID NO.93所示的重链可变区；和
    (76)SEQ ID NO.51所示的轻链可变区和SEQ ID NO.93所示的重链可变区。
  5. 根据权利要求1至4中任一项所述的抗体，其特征在于，所述抗体能够结合白细胞介素4(IL-4)受体(IL-4R)；
    优选地，所述抗体能够结合IL-4Rα，优选结合哺乳动物类IL-4Rα，优选结合人IL-4Rα，更优选结合人可溶性IL-4Rα；
    更优选地，所述抗体能够以小于100nM、小于10nM、小于1nM、小于0.5nM或甚至小于0.1nM的亲和力结合IL-4Rα。
  6. 根据权利要求1至5中任一项所述的抗体，其特征在于，所述抗体为单克隆抗体、完全或部分人源化抗体或嵌合抗体；
    优选地，所述抗体为免疫球蛋白，优选为IgA、IgD、IgE、IgG或IgM，更优选为IgG1、IgG2、IgG3或IgG4亚型。
  7. 一种融合蛋白或偶联物，所述融合蛋白或偶联物包含权利要求1至6中任一项所述的抗体。
  8. 一种核酸序列，所述核酸序列编码权利要求1至6中任一项所述的抗体的重链可变区和/或轻链可变区；
    优选地，所述核酸序列编码权利要求1至6中任一项所述的抗体的重链和/或轻链。
  9. 一种包含权利要求8所述的核酸序列的载体。
  10. 一种采用权利要求8所述的核酸序列或权利要求9所述的载体转化或转染的宿主细胞。
  11. 一种用于生产权利要求1至6中任一项所述的抗体的方法，所述方法包括：由权利要求10所述的核酸序列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，并与所述抗体的任选其他结构域组装成抗体；或者
    所述方法包括：在允许权利要求12所述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。
  12. 一种药物组合物，所述药物组合物包含权利要求1至6中任一项所述的抗体、权利要求7所述的融合蛋白或偶联物、权利要求8所述的核酸序列、权利要求9所述的载体、权利要求10所述的宿主细胞和/或通过权利要求10所述的方法生产的抗体；
    优选地，所述药物组合物为药物制剂，所述药物制剂优选为注射剂剂型；
    优选地，所述药物组合物或药物制剂还包含药学上可接受的载体或赋形剂。
    优选地，所述药物组合物或药物制剂还包含至少一种选自下述的药物：平喘药如沙丁胺醇等，抗组胺药如氯雷他定等，免疫抑制剂如他克莫司、吡美莫司等，M受体阻断剂如异丙托溴铵等，白三烯受体阻断药如孟鲁司特等，磷酸二酯酶抑制剂如茶碱等，非甾体抗炎药如5-氨基水杨酸等，激素类如倍氯米松，布地奈德等。
  13. 权利要求1至6中任一项所述的抗体、权利要求7所述的融合蛋白或偶联物、权利要求8所述的核酸序列、权利要求9所述的载体、权利要求10所述的宿主细胞和/或通过权利要求11所述的方法生产的抗体在制备用于预防、治疗或改善炎症或过敏性疾病中的用途；
    优选地，所述炎症或过敏性疾病包括自身免疫学疾病，例如过敏性皮炎、哮喘、嗜酸性粒细胞食管炎、湿疹、过敏性鼻炎、鼻息肉、类风湿性关节炎 等。
  14. 一种预防、治疗或改善受试者的炎症或过敏性疾病的方法，所述方法包括给有此需要的受试者施用权利要求1至6中任一项所述的抗体、权利要求7所述的融合蛋白或偶联物、权利要求8所述的核酸序列、权利要求9所述的载体、权利要求10所述的宿主细胞和/或通过权利要求11所述的方法生产的抗体；
    优选地，所述受试者为哺乳类动物；
    更优选地，所述受试者为人；
    优选地，所述炎症或过敏性疾病包括自身免疫学疾病，例如过敏性皮炎、哮喘、嗜酸性粒细胞食管炎、湿疹、过敏性鼻炎、鼻息肉、类风湿性关节炎等。
  15. 根据权利要求14所述的方法，其特征在于，所述方法还包括给受试者施用至少一种选自下述的药物：平喘药如沙丁胺醇等，抗组胺药如氯雷他定等，免疫抑制剂如他克莫司、吡美莫司等，M受体阻断剂如异丙托溴铵等，白三烯受体阻断药如孟鲁司特等，磷酸二酯酶抑制剂如茶碱等，非甾体抗炎药如5-氨基水杨酸等，激素类如倍氯米松、布地奈德等；
    优选地，所述药物与权利要求1至6中任一项所述的抗体、权利要求7所述的融合蛋白或偶联物、权利要求8所述的核酸序列、权利要求9所述的载体、权利要求10所述的宿主细胞和/或通过权利要求11所述的方法生产的抗体同时或顺序施用。
  16. 一种试剂盒，所述试剂盒包括权利要求1至6中任一项所述的抗体、权利要求7所述的融合蛋白或偶联物、权利要求8所述的核酸序列、权利要求9所述的载体、权利要求10所述的宿主细胞和/或通过权利要求11所述的方法生产的抗体。
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