WO2019148405A1 - ANTICORPS D'IL-4Rα ET SON UTILISATION - Google Patents

ANTICORPS D'IL-4Rα ET SON UTILISATION Download PDF

Info

Publication number
WO2019148405A1
WO2019148405A1 PCT/CN2018/074890 CN2018074890W WO2019148405A1 WO 2019148405 A1 WO2019148405 A1 WO 2019148405A1 CN 2018074890 W CN2018074890 W CN 2018074890W WO 2019148405 A1 WO2019148405 A1 WO 2019148405A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
tyr
antibody
leu
arg
Prior art date
Application number
PCT/CN2018/074890
Other languages
English (en)
Chinese (zh)
Inventor
许峥
李响
宋瑞
Original Assignee
北京凯因科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京凯因科技股份有限公司 filed Critical 北京凯因科技股份有限公司
Priority to KR1020207025183A priority Critical patent/KR102652133B1/ko
Priority to RU2020127107A priority patent/RU2758092C1/ru
Priority to PCT/CN2018/074890 priority patent/WO2019148405A1/fr
Publication of WO2019148405A1 publication Critical patent/WO2019148405A1/fr
Priority to ZA2020/04861A priority patent/ZA202004861B/en

Links

Images

Classifications

    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C07K2317/50—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]

Definitions

  • the present invention is in the field of antibody technology and relates to a humanized IL-4R antibody and the use of the antibody in the treatment of diseases associated with IL-4/IL-4R ⁇ signaling.
  • Allergic diseases can be well treated with antihistamines and specific immunological drugs, but for some more serious allergic diseases such as atopic dermatitis and asthma, there is no specific treatment. Non-specific immunosuppression remains the primary treatment modality.
  • asthma patients There are more than 235 million asthma patients worldwide, of which about 10-20% are not effectively controlled by existing drugs (asthma worsens) and the cost of treatment for this part of the population accounts for 80% of all asthma treatment expenditures.
  • Atopic dermatitis affects 10-20% of children and 3-10% of adults, of which about 20% are moderate to severe atopic dermatitis, and there is currently no effective treatment.
  • Oral/intravenous broad-spectrum immunosuppressive agents such as steroids, cyclosporin A, methotrexate, azathioprine and mycophenolate mofetil can effectively alleviate the symptoms of the disease.
  • the activity of these immunosuppressive agents is mainly derived from downstream regulators that target inflammation (such as transcription factors): such as steroid-binding glucocorticoid receptors and inhibition of key transcription factors that drive inflammation (such as NF-kB); cyclosporin A is a calcineurin inhibitor that blocks the expression of IL-2 required for T cell activation and proliferation by transcription factor activation of T cell nuclear factor (NFAT).
  • Systemic drug broad-spectrum immunosuppressive agents often cause a series of side effects such as fluid retention, glucose intolerance, hypertension, muscle weakness, osteoporosis, hypothalamic-pituitary-adrenal axis inhibition, and increased infection due to their pleiotropic effects. risk.
  • topical medications such as inhalants, topical drops, skin sprays, ointments, etc.
  • Euclisa a non-steroidal PDE4 inhibitor approved by Pfizer last December, is only effective in patients with mild to moderate atopic dermatitis.
  • the type 2 inflammatory pathway describes an inflammatory pathway in which Th2 cells play a key role.
  • Th2 cells play a key role in the type 2 inflammatory pathway by secreting the type 2 cytokines IL-4, IL-5 and IL-13.
  • IL-4 promotes the differentiation of Th cells into Th2 cells and proliferates, and Th2 cells further produce IL-4, IL-5 and IL-13.
  • IL-5 promotes eosinophil differentiation in bone marrow
  • IL-4, IL-5 and IL-13 promote the transfer of eosinophils to specific tissues
  • IL-4 and IL-13 also B cell serotypes Transformation leads to IgE, which also plays an important role in mucus secretion, goblet cell hyperplasia, smooth muscle contraction, and collagen production.
  • Typical markers of activation of type 2 inflammatory pathways are elevated IgE, eosinophils, and TARC. If type 2 immune responses are over-activated, severe allergic diseases can occur, with different disease manifestations in different tissues, depending on where they occur. Different can be divided into atopic dermatitis, chronic sinusitis, nasal polyps and asthma.
  • IL-4 and IL-13 are potent regulators of type 2 immune responses and exhibit corresponding functions depending on the binding receptor. Although IL-4 and IL-13 have only 25% amino acid homology, their receptor complexes (type I receptor and type II receptor) share a common component IL-4R ⁇ , depending on the expression distribution. Different cells play different roles. IL-4 can function through type I receptors and type II receptors, while IL-13 can only function through type II receptors. IL-4 binds with high affinity to IL-4R ⁇ and is independent of ⁇ -chain or IL-13R ⁇ 1, while IL-4R ⁇ increases the affinity of IL-13 for binding to 13R ⁇ 1.
  • IL-4 receptor alpha Human IL-4 receptor alpha (hIL-4R alpha) antibodies are described in U.S. Patents 5,707,072 and 7,186,809. Some methods of using hIL-4R antibodies are described in U.S. Patents 5,714,146, 5,985,280 and 6,716,587.
  • WO2005047331 relates to an IL-4R ⁇ recombinant protein Altrakincept developed by Immunex, which competitively binds to IL-4 by spraying, inhibits the binding of IL-4 to cell surface IL-4R ⁇ in vivo; US 6358509 discloses a one developed by GSK.
  • the IL-4 antibody Pascolizumab prevents IL-4 from binding to IL-4R ⁇ . Although these two IL-4 inhibitors showed good efficacy in early studies (preclinical, phase I clinical), they did not show efficacy in large-scale late-stage clinical studies, and development has stopped.
  • U.S. Patent No. 8,679,487 discloses that Amgen has also developed an IL-4 receptor alpha antibody, AMG-317, which is a fully human IL-4R ⁇ antibody that inhibits IL-4 and IL-13 activity but treats asthma. Phase II clinical failures have stopped development.
  • anti-IL-13 antibody molecules examples include: CAT-354 as disclosed in US07/0128192 or WO2005007699; TNX-650 disclosed in WO2005062967 and WO2005062972, and NTC00441818 published by Clinical Trails Gov. Identifier; QAX- disclosed by Clinical Trails Gov. Identifier 576 (NTC 532 233); US20060140948 or WO2006055638, filed in the name of Abgenix, U.S. Patent No. 6,465,528 to Amgen; a Centocor, Inc. as Applicant's WO2005091856; and as disclosed in WO2007080174, filed in the name of Glaxo, and in the name of Novartis, WO2007045477 Anti-IL-13 antibody.
  • the IL-13 antibody Tralokinumab developed by AstraZeneca, is expected to have Phase III clinical results for the treatment of severe asthma in the second half of 2017 and is expected to be the world's first marketed IL-13 antibody.
  • AstraZeneca and Abbott jointly developed a companion diagnostic method based on the biomarkers periostin and DPP4.
  • Tralokinmub has also completed Phase IIb clinical trials for the treatment of specific dermatitis.
  • blocking IL-4 and IL-13 can be achieved by at least two methods: one is IL-4R ⁇ antibody; the other is bispecific antibody of IL-4 and IL-13.
  • DVD-Ig dual variable domain Ig
  • WO2005023860 relates to an IL-4 mutant Pitrakinra developed by Aerovance, which binds IL-4R ⁇ with high affinity and is a dual antagonist of IL-4 and IL-13, which can significantly improve FEV1 (forced expiratory volume in one second) in asthma patients.
  • PEG-modified Pitrakinra was the first dual blocker to conduct a clinical trial of atopic dermatitis, although it showed a certain improvement in symptoms, but the improvement in disease endpoint was not statistically significant.
  • WO2010053751 relates to an IL-4R ⁇ antibody developed by Regeneron, which blocks both IL-4 and IL-13 to modulate type 2 immunity.
  • the antibody in this patent application involves six complementarity determining regions, the sequences of which are:
  • HCDR1 is: Gly-Phe-Thr-Phe-X5-X6-Tyr-X8, wherein X5 is Asp or Arg (preferably Arg), X6 is ASP or Ser (Asp), X8 is Ala or Gly (Ala)
  • variable region of an antibody comprises six complementarity determining regions (CDRs), namely the HCDR1, HCDR2, HCDR3 and light chain LCDR1, LCDR2, LCDR3 of the heavy chain.
  • CDRs complementarity determining regions
  • HCDR3 is the most highly variable in structure and sequence, and is also the key region that best reflects the diversity and specificity of antibody binding.
  • the complementarity determining region of an antibody is a key region for maintaining antibody activity.
  • the present invention provides a novel IL-4R ⁇ antibody having a binding activity significantly superior to that of piruzumab, which modulates type 2 by blocking IL-4 and IL-13 by binding to IL-4R ⁇ . Immunity.
  • One of the objects of the present invention is to provide an antibody or antigen-binding fragment thereof which specifically binds to human IL-4R ⁇ , which is associated with IL compared to the currently available dupilumab antibody.
  • the affinity of -4R has been further improved.
  • the Dubrizumab variant contains the following amino acid substitutions relative to the Dubrizumab: the 24th amino acid Arg mutation of LVR is Ala, or the 28th amino acid Ser is mutated to Arg, or the 32nd amino acid Ser is mutated to Arg or Lys; the 100th Arg mutation of HVR is Ala, or the 106th Arg is mutated to Ala or Glu, or the 108th Arg is mutated to Ala, Glu, Gln, Tyr or Leu.
  • one aspect of the invention relates to an antibody or antigen-binding fragment thereof which specifically binds to human interleukin-4 receptor alpha (hIL-4R alpha), comprising a heavy chain variable region (HVR) and a light chain A variable region (LVR), wherein the heavy chain variable region comprises: CDR1 (HCDR1) having the amino acid sequence Gly-Phe-Thr-Phe-Arg-Asp-Tyr-Ala; and the amino acid sequence is Ile-Ser-Gly-Ser- CDR2 (HCDR2) of Gly-Gly-Asn-Thr; and, the amino acid sequence is
  • CDR3 (HCDR3) of Ala-Lys-Asp-Xaa1-Leu-Ser-Ile-Thr-Ile-Xaa2-Pro-Xaa3-Tyr-Tyr-Gly-Leu-Asp-Val, wherein Xaa1 is Arg or Ala, Xaa2 is Arg, Ala or Glu, Xaa3 is Arg, Ala or Glu;
  • the light chain variable region comprises: the amino acid sequence is
  • CDR1 (LCDR1) of Gln-Xaa4-Leu-Leu-Tyr-Xaa5-Ile-Gly-Tyr-Asn-Tyr,
  • Xaa4 is Ser or Arg
  • Xaa5 is Ser, Arg or Lys; the amino acid sequence is
  • CDR3 (LCDR3) of Met-Gln-Ala-Leu-Gln-Thr-Pro-Tyr-Thr.
  • HCDR3 and LCDR1 are the following amino acid sequence (a) HCDR3:
  • the amino acid sequence of the HVR is SEQ ID NO: 1.
  • the amino acid sequence of the LVR is SEQ ID NO: 2.
  • the amino acid sequence of the LVR is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9.
  • the amino acid sequence of the HVR is selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21. And SEQ ID NO: 23 and SEQ ID NO: 25.
  • the HVR and LVR are selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3, SEQ ID NO: 1 and SEQ ID NO: 5, SEQ ID NO: 1 and SEQ ID NO: 7, SEQ ID NO: 1 and SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 2, SEQ ID NO: 13 and SEQ ID NO: 2, SEQ ID NO: 15 and SEQ ID NO: 2, SEQ ID NO: 17 and SEQ ID NO: 2, SEQ ID NO: 19 and SEQ ID NO: 2, SEQ ID NO: 21 and SEQ ID NO: 2, SEQ ID NO: 23 and SEQ ID NO: 2, or SEQ ID NO: 25 and SEQ ID NO: 2.
  • the HVR and LVR are selected from the group consisting of SEQ ID NO: 17 and SEQ ID NO: 2, or SEQ ID NO: 19 and SEQ ID NO: 2.
  • the present invention is also a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the present invention relates to an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention; an expression vector comprising the nucleic acid molecule of the present invention, and an expression vector comprising the same according to the present invention.
  • Host cells preferably eukaryotic cells.
  • the present invention relates to a method of producing an antibody or antigen-binding fragment thereof which specifically binds to human IL-4R, the method comprising expressing under conditions conducive for expression of the antibody or antigen-binding fragment thereof of the present invention
  • the nucleic acid molecule of the present invention, and the expressed antibody or antigen-binding fragment thereof is recovered.
  • the invention relates to the use of an antibody or antigen-binding fragment thereof for the preparation of a medicament for the prevention or treatment of a disease or condition associated with IL-4/IL-4R ⁇ signaling in humans.
  • the disease or condition is selected from the group consisting of: asthma, atopic dermatitis, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scar, Whipole disease, benign prostatic hyperplasia, COPD, Atopic dermatitis, idiopathic pulmonary fibrosis, allergic reaction, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymph Tissue hyperplasia syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis or kidney disease.
  • the invention relates to a method of preventing or treating a disease or condition associated with IL-4/IL-4R ⁇ signaling in a human comprising administering the antibody of any one of claims 1-8 to a subject. Or an antigen binding fragment thereof.
  • the disease or condition is selected from the group consisting of: asthma, atopic dermatitis, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scar, Whipole disease, benign prostatic hyperplasia, COPD, Atopic dermatitis, idiopathic pulmonary fibrosis, allergic reaction, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymph Tissue hyperplasia syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis or kidney disease.
  • the disease or condition is atopic dermatitis or asthma.
  • the antibody or antigen-binding fragment thereof is used in combination with other drugs for the treatment of autoimmune diseases.
  • the antibody or antigen-binding fragment thereof is used in combination with a hormonal drug, an immunosuppressant or an antihistamine.
  • the present invention relates to an article or kit comprising a container and a package insert, wherein the container contains the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition of the present invention,
  • the package insert contains the instruction manual for the drug.
  • the article or kit further comprises one or more containers containing one or more other agents or conditions for preventing or treating a disease or condition associated with IL-4/IL-4R ⁇ signaling in the human body. drug.
  • the other drug is a hormonal drug, an immunosuppressant or an antihistamine.
  • Figure 1 is an SDS-PAGE diagram of the dupilumab variant D1-D9: Lane 1 is the molecular weight marker, lane 2 is D1, lane 3 is D2, lane 4 is D3, lane 5 is D4, lane 6 is D5, lane 7 is D6, lane 8 is D7, lane 9 is D8, and lane 10 is D9.
  • Figure 2 is a SEC detection map of the Dubrizumab variant D8.
  • Figure 3 shows the biological effect of in vitro neutralization of IL-4 by Dubrizumab and Dubrizumab variants D5, D7, D8 and D9.
  • Dubrizumab only inhibits 92% of cell growth, whereas D8 and D9 have better inhibition, and the inhibition rate is 98%, which is obviously better than that of pirumab.
  • Figures 4A-4H show the results of the stability comparison of different storage conditions, such as high temperature, intense light irradiation, repeated freeze-thaw, and strong acid conditions of pirubizumab and pedizumab variants.
  • Fig. 4A is a graph showing the SEC results of three samples at 50 °C
  • Fig. 4B is a graph showing the results of ELISA of three samples at 50 °C.
  • Figure 4C is a SEC result plot of three samples under repeated freeze-thaw conditions
  • Figure 4D is a ELISA result plot of three samples under repeated freeze-thaw conditions.
  • Figure 4E is a graph of SEC results for three samples under strong acid conditions
  • Figure 4F is a graph of ELISA results for three samples under strong acid conditions
  • Figure 4G is a SEC result plot of three samples at 4500lx
  • Figure 4H is an ELISA result plot of three samples at 4500lx.
  • IL-4R CD124
  • IL-4R is a type I membrane-transparent glycoprotein composed of 825 amino acids (25 signal peptide domains, 207 extracellular domains, 24 membrane-penetrating portions, and 569 intracellular domains).
  • the extracellular domain has one CKR-SF domain and one type III fibrin domain, and there are six N-binding sugar chain adhesion sites.
  • T cells Distributed in T cells, B cells, NK cells, monocytes/macrophages, neutrophils, hematopoietic precursor cells, mast cells, endothelial cells, fibroblasts, keratinocytes, etc., IL-4R and IL-13R
  • the alpha chain is equivalent, and binding to IL-4 can cause several intracellular proteins to be phosphorylated by tyrosine.
  • Disease associated with IL-4/IL-4R ⁇ signaling means a disease in which biological function mediated by IL-4/IL-4R ⁇ signaling is involved.
  • diseases associated with IL-4/IL-4R ⁇ signaling include, for example, inflammatory diseases or conditions such as asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, multiple sclerosis, joints Inflammation, allergic rhinitis, eosinophilic esophagitis and psoriasis.
  • COPD chronic obstructive pulmonary disease
  • the disease associated with IL-4/IL-4R ⁇ signaling is airway inflammation. In some embodiments, the disease associated with IL-4/IL-4R ⁇ signaling is cutaneous inflammation or atopic dermatitis.
  • Allergic reactions are considered inflammatory diseases caused by the interaction of genetic and environmental factors.
  • Molecular and cellular immune mechanisms involved in the development of allergic diseases include changes in the expression of T cell immunoglobulin mucin regions caused by attenuated natural immune stimuli. Changes in the balance of helper T cells 1, 2 and regulatory T cell balance Change and many other factors.
  • atopic dermatitis, asthma, and allergic rhinitis are the most common inflammatory conditions in patients with allergies; these patients often suffer from multiple clinical symptoms.
  • the pathogenesis of allergy is associated with an abnormal immune response against exogenous antigens (Mueller et al.
  • IL-4/IL-13 receptor syste m a major factor in triggering allergic inflammation.
  • Abnormal type 2 T helper cell (Th2) polarization contributes to an increased IgE response.
  • Activated T cells produce interleukin-4 (IL-4) and interleukin-13 (IL-13), the major Th2 cytokines that initiate and maintain immune and inflammatory responses in allergic reactions.
  • IL-4 and IL-13 signaling are mediated through two different receptor complexes with a shared subunit IL-4 receptor alpha (IL-4Ralpha), which contributes to overlapping organisms between the two cytokines Answer.
  • IL-4R ⁇ forms two distinct heterodimeric receptor complexes that mediate the biological functions of IL-4 and IL-13 in a tissue and response-specific manner.
  • Dubrizumab is an antagonist monoclonal antibody directed against human IL-4R ⁇ that inhibits the biological activity induced by IL-4 and IL-13.
  • Dubrizumab blocks IL-4 signaling by preventing its binding to the receptor subunit, and inhibition of IL-13 signaling is likely to be mediated through interference with dimeric receptor interactions.
  • Dubrizumab a fully human monoclonal antibody against the shared IL-4R alpha subunit, was developed by Regeneron Pharmaceuticals, Inc. and is currently in the treatment of moderate to severe asthma and treatment of moderate to severe atopic dermatitis. In the test.
  • an "antibody” is an immunoglobulin molecule consisting of four peptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, each heavy chain comprising a heavy chain variable region ( HVR or VH) and heavy chain constant region, the heavy chain constant region comprises three domains of CH1, CH2 and CH3, each light chain comprising a light chain variable region (LVR or VL) and a light chain constant region, a light chain constant region Containing a domain (CL1), the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FR), each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the heavy chain constant region may be selected from IgG1, IgG2,
  • variable region is used herein to describe a region of a portion of an antibody that differs between antibody sequences and that relates to the binding and specificity of a particular antibody to its particular antigen.
  • variability is usually not evenly distributed throughout the variable region of the antibody. It typically focuses on three segments called the complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions.
  • CDRs complementarity determining regions
  • FR framework region
  • the variable regions of the native heavy and light chains each comprise four FRs, most of which adopt a beta-sheet conformation, joined by three CDRs that form a circular junction and in some cases form part of a beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR and together with the CDRs of the other chain contribute to the formation of the antigen binding site of the antibody.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits multiple effector functions, such as the involvement of antibodies in antibody-dependent cellular cytotoxicity.
  • CDR complementarity determining region
  • hypervariable region refers to one or more of the hypervariable regions or complementarity determining regions (CDRs) present in the variable regions of an antibody light or heavy chain (see, Kabat, EA et al, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These terms include the hypervariable regions defined by Kabat et al. ("Sequences of Proteins of Immunological Interest," Kabat E., et al., USD ept. of Health and Human Services, 1983) or hypervariable loops in the three-dimensional structure of antibodies (Chothia and Lesk, J Mol. Biol. 196901). -917 (1987)). The CDRs in each chain remain in close proximity through the framework regions and, together with the CDRs from other chains, promote the formation of antigen binding sites.
  • frame region and "FR” refer to one or more of the framework regions of the variable regions of the antibody light and heavy chains (see, Kabat, EA et al, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). . These terms include those amino acid sequences in the light and heavy chains of the antibody that are located between the amino terminus and the first CDR, those between the CDRs, and those between the third CDR and the start of the constant region.
  • CDR and FR residues can be defined according to standard sequences (Kabat et al, Sequences of Proteins of Immunological Interes, National Institutes of Health, Bethesda Md. (1987)) and structural definitions (Chothia and Lesk, J. Mot. Biol. 196:901-217 (1987)). determine.
  • Kabat utilizes methods for assigning residue numbers to each amino acid in the listed sequences, And this method of assigning residue numbers has become a standard in the art.
  • the specification follows the Kabat numbering scheme.
  • the Kabat numbering is not indicated, the sequential amino acid sequence numbering (ie, the amino acid use in the sequence is used) Sequential integers (1, 2, 3, etc.) are numbered from left to right in the direction from the amino terminus to the carboxy terminus.
  • affinity of an antibody to an antigen or epitope is a term well understood in the art and refers to the extent or strength of binding of an antibody to an epitope. Affinity can be measured and/or expressed in a variety of ways known in the art including, but not limited to, an equilibrium dissociation constant (KD or Kd, which can be defined as the ratio of the dissociation rate of the antibody to the rate of binding, ie, Koff / K on ), apparent equilibrium dissociation constant (KD' or Kd') and IC50 (the amount required to achieve 50% inhibition in competition assays); the relative affinity of the humanized antibody can also be correlated with the mouse or Combined with antibody to determine.
  • KD or Kd equilibrium dissociation constant
  • KD' or Kd' apparent equilibrium dissociation constant
  • IC50 the amount required to achieve 50% inhibition in competition assays
  • affinity is the average affinity of a particular population of antibodies that bind an antigen or epitope.
  • the affinity of the antibody can be measured using an enzyme-linked immunosorbent assay (ELISA) or a fluorescence activated cell sorting (FACS) assay.
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence activated cell sorting
  • Antibodies of the invention can be produced by any available method, such as recombinant expression techniques. Nucleic acids encoding light and heavy chain variable regions operably linked to a constant region can be inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. A DNA segment encoding an immunoglobulin chain can be operably linked to a control sequence of an expression vector that ensures expression of the immunoglobulin polypeptide.
  • Expression control sequences include, but are not limited to, promoters (eg, naturally-binding or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • the expression control sequence is a prokaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is introduced into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the antibody.
  • the expression vector can be replicable in any host organism, as an episome or as an integral part of the host chromosomal DNA.
  • the expression vector contains a selectable marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow detection of those cells that are transformed by the intended DNA sequence.
  • Expression vectors can be used to express antibodies of the invention from any host cell, including prokaryotic host cells (e.g., E. coli), yeast host cells, mammalian host cells, plant host cells, and insect host cells.
  • an antibody of the invention is produced using E. coli.
  • Other prokaryotic hosts suitable for this application include bacilli, such as Bacillus and other Enterobacteriaceae, such as Salmonella, S. genus, and various Pseudomonas species.
  • expression vectors can also be prepared, which expression vectors typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell.
  • expression control sequences e.g., origins of replication
  • any number of various known promoters will be present, such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from lambda phage. Promoters typically control expression, optionally together with an operator sequence, and have a ribosome binding site sequence, etc., for initiation and completion of transcription and translation.
  • yeast can also be used to express the antibodies of the invention.
  • the genus Saccharomyces can be used as a yeast host with a suitable promoter containing expression control sequences (e.g., promoters), origins of replication, termination sequences, and other sequences as desired.
  • Promoters for yeast expression techniques include 3-phosphoglycerate kinase and other glycolytic enzyme promoters.
  • Inducible yeast promoters include, but are not limited to, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • a mammalian tissue cell culture can be used to express and produce an antibody of the invention.
  • Any mammalian tissue cell can be used in such methods, and many suitable host cell lines capable of secreting heterologous proteins (eg, intact immunoglobulins) have been developed in the art, including mammalian BHK or CHO cell lines, various Cos. Cell lines, HeLa cell lines, preferably myeloma cell lines or transformed B cells or hybridomas. In one embodiment, the cells are non-human.
  • Expression vectors for mammalian cells can include expression control sequences, such as origins of replication, promoters and enhancers, as well as necessary processing signal sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription. Terminate the subsequence.
  • the expression control sequence is a promoter derived from an immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like.
  • a vector containing a polynucleotide sequence of interest can be transferred to a host cell by a known method, which varies depending on the type of cell host.
  • a polynucleotide sequence of interest e.g., heavy and light chain coding sequences and expression control sequences
  • a host cell can be transferred to a host cell by a known method, which varies depending on the type of cell host.
  • calcium chloride transfection is commonly used in prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biotrajective or viral transfection can be used in other cellular hosts.
  • Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection.
  • the transgene can be microinjected into the fertilized egg or can be introduced into the embryonic stem cell genome, and the nuclei of such cells are transferred into the enucleated oocyte.
  • the vector When a nucleic acid molecule encoding a heavy chain and a light chain is cloned into a separate expression vector, the vector can be co-transfected to obtain expression and assemble the intact immunoglobulin. Once expressed, intact antibodies, dimers thereof, individual light and heavy chains, or other immunoglobulin forms of antibodies can be purified according to standard methods in the art, including ammonium sulfate precipitation, affinity column, column chromatography, HPLC purification. , gel electrophoresis, etc. At least about 90 to 95% homogenous, substantially pure immunoglobulin can be prepared for pharmaceutical use. In another embodiment, substantially pure humanized antibodies of at least about 98 to 99% or greater homogeneity can be produced for use in pharmaceutical formulation and methods.
  • the invention provides methods of expressing an antibody of the invention comprising: (a) transforming a host cell with a nucleic acid molecule encoding an antibody described herein, and (b) culturing the transformed host cell under conditions permitting expression of the antibody.
  • Known techniques for including a selectable marker on a vector can be used such that the labeled host cell expressing the antibody can be readily selected.
  • an “antigen-binding fragment” of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen, such as hIL-4R. It has been demonstrated that the antigen binding function of antibodies can be achieved by certain fragments of full length antibodies.
  • antigen-binding fragments include, but are not limited to: (i) Fab fragments, ie, monovalent fragments consisting of VL, VH, CL1 and CH1 domains; (ii) F(ab')2 fragments, ie by hinges a divalent fragment consisting of two F(ab') fragments joined by a disulfide bond; (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) consisting of a one-armed VL and VH domain of the antibody Fv fragment; (v) a dAb fragment consisting of a VH domain; and (vi) a CDR.
  • Fab fragments ie, monovalent fragments consisting of VL, VH, CL1 and CH1 domains
  • F(ab')2 fragments ie by hinges a divalent fragment consisting of two F(ab') fragments joined by a disulfide bond
  • an Fd fragment consisting of a VH
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be joined together by a synthetic linker into a single linked strand, where the VL and VH regions are paired.
  • a monovalent molecule (called a single chain Fv, scFv) is formed.
  • single chain antibodies are also encompassed within the scope of "antigen-binding fragments" of antibodies.
  • the antibody of the present invention can be produced by a method comprising culturing a host cell containing a DNA sequence encoding the antibody and expressing the antibody under conditions permitting expression of the antibody, and then recovering the produced antibody from the culture. .
  • the medium used to culture the cells may be any conventional medium for culturing the host cells, such as a minimal medium or a compatible medium containing a suitable additive.
  • a suitable medium can be obtained by commercially available or a suitable medium can be prepared according to the published method.
  • the polypeptide produced by the cell can then be recovered from the culture medium by conventional methods, including isolating the host cell from the culture medium by centrifugation or filtration, and precipitating the supernatant or the protein component in the filtrate with a salt such as ammonium sulfate, according to
  • the type of the peptide of interest is selected by various chromatographic methods such as exchange chromatography, gel filtration chromatography, affinity chromatography, and the like.
  • the present invention provides a pharmaceutical composition comprising the anti-IL-4R ⁇ antibody of the present invention or an antigen-binding fragment thereof.
  • the pharmaceutical compositions of this invention will be administered with appropriate carriers, excipients, and other formulations. These formulations are included in the formulation to improve delivery and tolerability and the like. A large number of suitable formulations can be found in the pharmacopoeia well known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • the dose to be administered is adjusted depending on the age and size of the subject, the target disease, the symptoms, the route of administration, and the like.
  • the antibody of the present invention can be administered intravenously, usually in a single dose of from about 0.01 to about 20 mg/kg per kilogram of body weight. More preferably, it is from about 0.1 to about 15, from about 1 to about 10, or from about 3 to about 10 mg/kg of body weight.
  • the frequency and duration of treatment can be adjusted depending on the severity of the condition.
  • compositions of the invention such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing a variant virus, receptor-mediated endocytosis Role (see, eg, Wu et al. (1987), J. Biol. Chem. 262: 4429-4432).
  • Methods of administering a drug include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, dura, and oral.
  • compositions can be administered by any convenient route, such as by perfusion or intravenous bolus injection, epithelial and mucosal layers (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered with other bioactive agents.
  • the mode of administration can be systemic or topical.
  • the pharmaceutical composition can also be delivered by sacs, especially by liposome sac (see Langer (1990) Science 249: 1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein And Fidler (ed.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327).
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump can be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be employed (see Medical Applications of Controlled Release, Langer and Wise, ed., CRC Pres., Boca Raton, Florida (1974). For a discussion of other controlled release systems see Langer ( 1990) Science 249: 1527-1533.
  • Formulations for injection may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, drip, and the like. These injectable preparations can be prepared by the disclosed methods.
  • an injection preparation can be prepared by dissolving, suspending or emulsifying an antibody or a salt thereof in a sterile aqueous medium or an oil medium conventionally used for injection.
  • Aqueous media for injection are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like.
  • solubilizing agents such as alcohols (such as ethanol), polyols (such as propylene glycol, polyethylene glycol), nonionic surfactants [such as polysorbate 80, HCO-50 (polyoxygenated hydrogenated castor oil) Ethylene (50 mol) adduct) or the like is used in combination.
  • the oil medium used is, for example, sesame oil, soybean oil, and the like. They can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol and the like.
  • the injection prepared in this manner is preferably packaged in a suitable ampoule.
  • Monotherapy and Combination Therapy Antibodies and antibody fragments of the invention are useful for treating diseases and disorders that can be ameliorated, inhibited, or ameliorated by reducing IL-4 activity. These diseases include those characterized by aberrant expression or overexpression of IL-4 or a host's abnormal response to IL-4 production.
  • IL-4-related diseases treated with the antibodies or antibody fragments of the invention include, for example, arthritis (including septic arthritis), herpes, chronic primary urticaria, scleroderma, hypertrophic scars, Whipple's disease, benign Prostatic hyperplasia, lung disease such as asthma (mild, moderate and severe), inflammatory diseases such as inflammatory bowel disease, allergic reaction, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves's Disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis (sickness), atopic dermatitis, ulcerative colon Inflammation, fibrosis, and kidney disease (see U.S. Patent 7,186,809).
  • arthritis including septic arthritis
  • herpes chronic primary urticaria
  • scleroderma hypertrophic scars
  • the invention encompasses combination therapies wherein an anti-IL-4R ⁇ antibody or antibody fragment is administered in combination with one or more therapeutic agents (or second therapeutic agents).
  • Co-administration and combination therapy are not limited to simultaneous administration, but also include treatment regimens that administer at least one anti-IL-4R ⁇ antibody or antibody fragment in a course of treatment involving administration of at least one other therapeutic agent to the patient.
  • the second therapeutic agent may be another IL-4 antagonist, such as another antibody/antibody fragment, or a soluble cytokine receptor, an IgE antagonist, an anti-asthma that can be administered by inhalation or other suitable means.
  • an anti-IL-4R antibody or antibody fragment of the invention can be administered with an IL-1 antagonist, such as a rilonacept or an IL-13 antagonist.
  • the second agent may include one or more leukotriene receptor antagonists to treat conditions such as allergic inflammatory diseases such as asthma and allergies. Examples of leukotriene receptor antagonists include, but are not limited to, montelukast, pranlustat, and zafirlukast.
  • the second agent can include a cytokine inhibitor such as one or more TNF (etanercept, ENBRELTM), IL-9, IL-5 or IL-17 antagonist.
  • the invention also encompasses the use of any of the anti-IL-4R ⁇ antibodies or antigen-binding fragments described herein in the manufacture of a medicament for treating a disease or condition, wherein the disease or condition is by eliminating, inhibiting or reducing human interleukin-4 (hIL) -4) The activity is improved, improved or inhibited.
  • hIL human interleukin-4
  • diseases or symptoms include, for example, arthritis, herpes, chronic primary urticaria, scleroderma, hypertrophic scar, Whipple's disease, benign prostatic hyperplasia, lung disease, asthma, inflammatory disease, allergic reaction , Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett Esophagus, autoimmune uveitis, tuberculosis (sickness), kidney disease, atopic dermatitis, and asthma.
  • arthritis herpes, chronic primary urticaria, scleroderma, hypertrophic scar, Whipple's disease, benign prostatic hyperplasia, lung disease, asthma, inflammatory disease, allergic reaction , Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Graves' disease, pre-eclampsia, Sjogren's syndrome,
  • a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof of the invention that binds to human IL-4R, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable excipient refers to a non-toxic filler, stabilizer, diluent, carrier, solvent or other formulation excipient.
  • diluents such as microcrystalline cellulose, mannitol, etc.
  • fillers such as starch, sucrose, etc.
  • binders such as starch, cellulose derivatives, alginates, gelatin and/or polyethylene Pyrrolidone
  • a disintegrating agent such as calcium carbonate and/or sodium hydrogencarbonate
  • an absorption enhancer such as a quaternary ammonium compound
  • a surfactant such as cetyl alcohol
  • a carrier a solvent such as water, physiological saline, kaolin, soap clay, etc.
  • Lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, and the like.
  • the pharmaceutical composition of the present invention is preferably an injection.
  • the antibody or antigen-binding fragment thereof in the pharmaceutical composition of the invention is present at a concentration of from 1 mg/ml to 1000 mg/ml, preferably at a concentration of from 10 mg/ml to 1000 mg/ml, more preferably It is present at a concentration of from 50 mg/ml to 500 mg/ml, more preferably at a concentration of from 100 mg/ml to 300 mg/ml.
  • the pharmaceutical composition of the present invention preferably has a pH of from 3.0 to 9.0.
  • a buffer system, a preservative, a surface tension agent, a chelating agent, a stabilizer, and a surfactant may be further included.
  • the pharmaceutical composition of the invention is an aqueous formulation. This preparation is usually in solution or suspension.
  • the pharmaceutical composition is a stable aqueous solution.
  • the pharmaceutical composition is a lyophilized formulation which is dissolved by the physician or patient prior to use in a solvent and/or diluent.
  • HindIII cleavage site AAGCCT
  • KoZAK sequence GCCGCCACC
  • ATG signal peptide gene ATGGAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT
  • Dulbuzumab heavy chain coding gene SEQ ID NO: 35, including heavy chain variable region coding gene SEQ ID NO :27 and the constant region IgG4 encoding gene
  • the termination code TAATAATAA and the EcoRI encoding gene GAATCC were sequentially fused in tandem, and the gene fragment was obtained by chemical synthesis.
  • the above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.1 (+) by EcoRI and HindIII sites and verified by sequencing, and the expression plasmid PCDNA3.1(+)-DH for the antibody degree of the Pruzumab heavy chain was obtained.
  • HindIII cleavage site AAGCCT
  • KoZAK sequence GCCGCCACC
  • ATG signal peptide gene ATGGAGAGAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT
  • Dulbuzumab light chain coding gene SEQ ID NO: 38, including light chain variable region coding gene SEQ ID NO : 28 and constant region
  • the coding gene, the termination code TAATAATAA and the EcoRI coding gene GAATCC are sequentially fused in tandem, and the gene fragment is obtained by chemical synthesis.
  • the above fragment was inserted into the eukaryotic expression plasmid pCDNA 3.1 (+) by EcoRI and HindIII sites and verified by sequencing, and the expression plasmid PCDNA3.1(+)-DL for the antibody degree of the pruzumab light chain was obtained.
  • a series of expression plasmids of the variants of pirimumab were prepared, respectively: pCDNA 3.1 (+)-D1L, pCDNA 3.1 (+)-D2L, pCDNA 3.1 (+)-D3L, pCDNA 3.1 ( +)-D4L, pCDNA 3.1(+)-D5H, pCDNA3.1(+)-D6H, pCDNA 3.1(+)-D7H, pCDNA 3.1(+)-D8H, pCDNA3.1(+)-D9H, pCDNA 3.1( +)-D10H, pCDNA 3.1(+)-D11H, pCDNA 3.1(+)-D12H, pCDNA 3.1(+)-DH and pCDNA 3.1(+)-DL.
  • Variant D1 is the mutation of the 24th amino acid Arg of LVR of Dubrizumab to Ala, the gene sequence of LVR encoding D1 is SEQ ID NO: 4; and the mutant D2 is the 28th of LVR of Dubrizumab
  • the amino acid Ser is mutated to Arg, the gene sequence of the LVR encoding D2 is SEQ ID NO: 6; and the mutant D3 or D4 is the mutation of the 32nd amino acid Ser of the LVR of the Dubrizumab to Arg or Lys, encoding D3 and D4, respectively.
  • the gene sequences of the LVR are SEQ ID NOS: 8 and 10, respectively;
  • the mutant D5 is the mutation of the 100th Arg of the HVR of the Dubrizumab to Ala, and the gene sequence of the HVR encoding D5 is SEQ ID NO: 12;
  • the body D6 or D7 is mutated to Ala or Glu at position 106 of HVR of Dubrizumab, and the gene sequences of HVR encoding D6 or D7 are SEQ ID NOS: 14 and 16 respectively; mutants D8-D12 are respectively
  • the 108th Arg of HVR of the pirizumab is mutated to Ala, Glu, Gln, Tyr or Leu, and the gene sequence of the HVR encoding D8-D12 is SEQ ID NO: 18, 20, 22, 24 or 26, respectively.
  • the heavy chain constant region of each mutant is IgG4, and the light chain constant region is .
  • Example 1 DNA embodiment the antibody expressed by the eukaryotic cell expressing FreeStyle TM 293-F Cells (Invitrogen Corporation, R790-07). According FreeStyle TM 293 ExpressionSystem Operating Manual day cell density was adjusted to 1x10 6 cells / ml before transfection plasmid. On the day of plasmid transfection, the plasmids were combined according to the following table, mixed with the transfection reagent, and added to the cell culture medium. After continuous incubation for 5-7 days at 37 ° C, 8% CO 2 , the cell culture supernatant was collected for antibody purification.
  • FreeStyle TM 293-F Cells Invitrogen Corporation, R790-07
  • FreeStyle TM 293 ExpressionSystem Operating Manual day cell density was adjusted to 1x10 6 cells / ml before transfection plasmid.
  • the plasmids were combined according to the following table, mixed with the transfection reagent, and added to the cell culture medium. After continuous incubation for 5-7 days at 37
  • the expression supernatant was filtered through a 0.22 ⁇ M filter, and the expressed antibody was captured from the expression supernatant using a Mabpurix affinity chromatography column (purchased from Sepax, Inc., 65008), and equilibrated with an equilibration buffer (120 mM Tris + 100 mM NaCl, pH 7.5). After chromatography, the column was passed through an affinity column and eluted with an elution buffer (0.15 M glacial acetic acid, pH 2.8). The purified antibody was subjected to SDS PAGE and SEC detection, and the purity was 95% or more.
  • In vitro affinity assays were performed using the purified antibodies described in Example 2.
  • the plate was coated with 2 ⁇ g/ml of IL-4R (R&D, 7700-4R-050), 100 ⁇ l/well, and the plate was sealed, placed at 4 ° C overnight, and washed three times with the washing solution the next day.
  • IL-4R R&D, 7700-4R-050
  • a 10% bovine serum albumin solution was prepared by using a washing solution, and added to the microplate in 200 ⁇ l/well, and the plate was washed 3 times with a washing solution at 37 ° C for 2 hours; a gradient diluted antibody solution was added at 100 ⁇ l/well, 37 ° C 2 Hour, wash plate 3 times with washing solution; dilute solution (2% bovine serum albumin solution using washing solution) 1/5000 diluted HRP-labeled secondary antibody (No.
  • ab6858 product from Abcam
  • the plate was washed at 37 ° C for 1 hour; the plate was washed 5 times with the washing solution; the TMB coloring solution was added at 100 ⁇ l/well, and the reaction was allowed to stand at room temperature for 5 to 10 minutes, and the reaction was terminated with 50 ⁇ l/well of a stop solution.
  • the absorbance was measured at a wavelength of 450 nm.
  • Example 4 Biological effects of neutralizing IL-4 in vitro
  • TF-1 cells were cultured for 24 h at 37 ° C under 5% carbon dioxide using RPMI 1640 basal medium (C22400500BT from GIBCO). TF-1 cells were collected the next morning, then washed 3 times with RPMI1640 medium, and resuspended in complete medium (RPMI1640 basal medium containing 10% fetal bovine serum) at a cell concentration of 4.0 ⁇ 10 5 cells/ml. . Using a 96-well cell culture plate, a cell suspension, and a gradient-diluted antibody sample and IL-4 were added and mixed and mixed. Incubate for 2 days at 37 ° C under 5% carbon dioxide.
  • CCK-8 (CK04, product source Dojindo company) instruction manual
  • CCK-8 dilution solution incubated at 37 ° C, 5% carbon dioxide for 2.5 h, microplate reader detection, with 630 nm as reference wavelength, 450 nm for determination
  • the absorbance was measured at the wavelength, and the cell viability was measured.
  • the purity of the main peak after antibody storage was detected by SEC-HPLC method (SEC), and the relative activity of the antibody after storage was detected by ELISA.
  • SEC-HPLC method SEC-HPLC method
  • the purified antibody of the present invention and Dubrizumab were separately added to a buffer solution of 12.5 mM sodium acetate, pH: 6.15, to a final concentration of about 2.7 mg/ml.
  • 1.5ml sample was added with 35.1 ⁇ l 0.2M Tris-Base to reach pH 9.0, filtered and dispensed in 200 ⁇ l/branch, each sub-packaged 7; 2.5ml sample plus 288 ⁇ l 0.2M citric acid acidification To pH 3.0 or so, filter and dispense 200 ⁇ l/branch, and pack 12 pieces each; finally, the remaining sample was filtered and sterilized, 200 ⁇ l/piece, and each batch was 30 pieces. All samples were placed in clean, sterilized vials, covered with sterile rubber plugs, and sealed with aluminum lids.
  • SEC-HPLC detection Separation of different molecular weight substances by gel filtration chromatography to quantitatively analyze the purity of the sample.
  • the column is TSK G3000SWXL, mobile phase: 0.2 mol/L potassium phosphate buffer, 0.25 mol/L potassium chloride pH 6.2 ⁇ 0.1, flow rate 0.5 ml/min, wash time 30 min.
  • ELISA method microplate reader detection: 2 ⁇ g/ml IL-4R coated with the enzyme standard plate, the test sample 500ng/ml is the initial concentration 5 times diluted 8 concentration gradient, transferred to the enzyme plate and IL-4R combined, plus Goat anti-human Fab fragment secondary antibody (product of Jackson Immuno company), color development, reading plate, calculate the relative activity of the antibody of the present invention (relative activity means: the degree of the activity of the same plate in each test spot 1 time relative activity of the antibody of the invention).
  • Storage conditions include high temperature, strong light irradiation, repeated freezing and thawing, and strong acid.
  • the specific operations, test points, and test items are as follows:
  • High temperature test The sample was placed in a 50 ° C incubator for 20 days, and samples at 0, 5, 10, 15, and 20 were examined. The results are shown in Fig. 4A and Fig. 4B.
  • the results of SEC-HPLC experiments showed that the stability of the test samples was consistent with the change trend of the pirimizumab.
  • the purity of the sample at 0 o'clock was greater than that of the antibody of the present invention, and as the high temperature standing time was prolonged, less than 5% was reduced, and there was no significant difference in thermal stability.
  • the ELISA results neglect the abnormal results (the second point of HC-108A in Fig.
  • the activity of the antibody of the present invention is always higher than that of the puruzumab, and the gap becomes larger as the high temperature standing time is prolonged, indicating the binding activity of the antibody of the present invention.
  • the thermal stability is superior to that of piruzumab.
  • the ELISA showed that the antibody of the present invention is superior to the pirizumab in reducing the activity caused by repeated freezing and thawing, and the antibody of the present invention has a high degree of freezing and thawing under the same conditions. Relative activity.
  • the antibody of the present invention has better resistance than the Dubrizumab. Receptive, this is more advantageous in the later storage process.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Pulmonology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention se rapporte au domaine technique des anticorps. L'invention concerne un anticorps se liant de manière spécifique au récepteur de l'interleukine 4 humaine (hIL-4 R) ou un fragment de liaison à l'antigène de celui-ci, une composition pharmaceutique comprenant l'anticorps ou le fragment de liaison à l'antigène, et une utilisation associée. La présente invention concerne également une molécule d'acide nucléique pour coder l'anticorps, un vecteur et une cellule hôte comprenant la molécule d'acide nucléique, ainsi qu'un procédé de préparation de l'anticorps.
PCT/CN2018/074890 2018-02-01 2018-02-01 ANTICORPS D'IL-4Rα ET SON UTILISATION WO2019148405A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
KR1020207025183A KR102652133B1 (ko) 2018-02-01 2018-02-01 IL-4Rα 항체 및 그 용도
RU2020127107A RU2758092C1 (ru) 2018-02-01 2018-02-01 АНТИТЕЛО К IL-4Rα И ЕГО ПРИМЕНЕНИЕ
PCT/CN2018/074890 WO2019148405A1 (fr) 2018-02-01 2018-02-01 ANTICORPS D'IL-4Rα ET SON UTILISATION
ZA2020/04861A ZA202004861B (en) 2018-02-01 2020-08-05 Il-4rα antibody and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/074890 WO2019148405A1 (fr) 2018-02-01 2018-02-01 ANTICORPS D'IL-4Rα ET SON UTILISATION

Publications (1)

Publication Number Publication Date
WO2019148405A1 true WO2019148405A1 (fr) 2019-08-08

Family

ID=67479957

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/074890 WO2019148405A1 (fr) 2018-02-01 2018-02-01 ANTICORPS D'IL-4Rα ET SON UTILISATION

Country Status (4)

Country Link
KR (1) KR102652133B1 (fr)
RU (1) RU2758092C1 (fr)
WO (1) WO2019148405A1 (fr)
ZA (1) ZA202004861B (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113166259A (zh) * 2018-11-09 2021-07-23 亚洲大学校产学协力团 对人IL-4受体α具有高亲和力的人抗体及其用途
CN114605539A (zh) * 2020-12-09 2022-06-10 南京融捷康生物科技有限公司 人源化的抗IL-4Rα单域抗体及其应用
WO2023085978A1 (fr) * 2021-11-11 2023-05-19 Joint Stock Company «Biocad» Anticorps monoclonal ou fragment de liaison à l'antigène de celui-ci se liant de manière spécifique à il-4ra et son utilisation
RU2807060C1 (ru) * 2020-04-17 2023-11-09 Шанхаймабгикбиотех.Ко., Лтд. Антитело к альфа-рецептору интерлейкина-4 человека, способ его получения и его применение

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3147113A1 (fr) 2019-08-05 2021-02-11 Regeneron Pharmaceuticals, Inc. Methodes de traitement de la dermatite atopique par administration d'un antagoniste de l'il-4r

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102197052A (zh) * 2008-10-29 2011-09-21 瑞泽恩制药公司 抗人il-4受体的高亲和性人抗体
CN104788563A (zh) * 2007-12-21 2015-07-22 米迪缪尼有限公司 白介素-4受体α(IL-4Rα)-173的结合成员
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
CN105392497A (zh) * 2013-07-11 2016-03-09 瑞泽恩制药公司 以il-4r抑制剂治疗嗜酸性食管炎的方法
CN107474134A (zh) * 2016-06-08 2017-12-15 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

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU639754B2 (en) * 1989-12-20 1993-08-05 Schering Corporation Antibody antagonists of human interleukin-4
US5994511A (en) * 1997-07-02 1999-11-30 Genentech, Inc. Anti-IgE antibodies and methods of improving polypeptides
US7404957B2 (en) * 2003-08-29 2008-07-29 Aerovance, Inc. Modified IL-4 mutein receptor antagonists
ATE548388T1 (de) * 2003-11-07 2012-03-15 Immunex Corp An den interleukin-4-rezeptor bindende antikörper

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104788563A (zh) * 2007-12-21 2015-07-22 米迪缪尼有限公司 白介素-4受体α(IL-4Rα)-173的结合成员
CN102197052A (zh) * 2008-10-29 2011-09-21 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
CN105392497A (zh) * 2013-07-11 2016-03-09 瑞泽恩制药公司 以il-4r抑制剂治疗嗜酸性食管炎的方法
CN107474134A (zh) * 2016-06-08 2017-12-15 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

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113166259A (zh) * 2018-11-09 2021-07-23 亚洲大学校产学协力团 对人IL-4受体α具有高亲和力的人抗体及其用途
RU2807060C1 (ru) * 2020-04-17 2023-11-09 Шанхаймабгикбиотех.Ко., Лтд. Антитело к альфа-рецептору интерлейкина-4 человека, способ его получения и его применение
CN114605539A (zh) * 2020-12-09 2022-06-10 南京融捷康生物科技有限公司 人源化的抗IL-4Rα单域抗体及其应用
CN114605539B (zh) * 2020-12-09 2024-01-02 南京融捷康生物科技有限公司 人源化的抗IL-4Rα单域抗体及其应用
EP4151656A4 (fr) * 2020-12-09 2024-07-17 Regenecore Biotech Co Ltd Anticorps à domaine unique anti-il-4r alpha humanisé et son utilisation
WO2023085978A1 (fr) * 2021-11-11 2023-05-19 Joint Stock Company «Biocad» Anticorps monoclonal ou fragment de liaison à l'antigène de celui-ci se liant de manière spécifique à il-4ra et son utilisation

Also Published As

Publication number Publication date
ZA202004861B (en) 2021-08-25
KR20200116967A (ko) 2020-10-13
RU2758092C1 (ru) 2021-10-26
KR102652133B1 (ko) 2024-03-29

Similar Documents

Publication Publication Date Title
US10287348B2 (en) Antigen binding proteins capable of binding thymic stromal lymphopoietin
CN110105451B (zh) IL-4Rα抗体及其用途
ES2414460T3 (es) Anticuerpos para Dkk-1
CA2851322C (fr) Traitement de l'arthrite rhumatoide au moyen d'anticorps gm csfr alpha
TW201525004A (zh) 結合前蛋白轉化酶枯草桿菌蛋白酶加工酶９型之人類抗原結合蛋白
US20140271681A1 (en) High Affinity Human Antibodies to Human IL-4 Receptor
JP2005534622A (ja) 選択的ｏｐｇｌ経路インヒビターとしてのヒト抗ｏｐｇｌ中和抗体
KR20190082815A (ko) 중화 항-tl1a 단일 클론 항체
WO2019148405A1 (fr) ANTICORPS D'IL-4Rα ET SON UTILISATION
EP4063385A1 (fr) Anticorps gipr et protéine de fusion entre celui-ci et la glp-1, et composition pharmaceutique et application associée
EP3808771A1 (fr) Anticorps gipr et proteine de fusion glp-1 de ce dernier, et composition pharmaceutique et son application
US20240294655A1 (en) Antigen binding molecules that bind light
US20240026036A1 (en) Compounds and methods for treating pain
WO2024125445A1 (fr) PROTÉINE DE FUSION BISPÉCIFIQUE CIBLANT TNF-α ET IL-17A ET SON UTILISATION
JP5476310B2 (ja) ｈＧＭ−ＣＳＦに結合するモノクローナル抗体および前記抗体を含む医薬組成物
JP2012105657A (ja) ｈＧＭ−ＣＳＦに結合するモノクローナル抗体および前記抗体を含む医薬組成物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18903225

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20207025183

Country of ref document: KR

Kind code of ref document: A

122 Ep: pct application non-entry in european phase

Ref document number: 18903225

Country of ref document: EP

Kind code of ref document: A1