WO2021082573A1 - 抗il-4r单域抗体及其应用 - Google Patents

抗il-4r单域抗体及其应用 Download PDF

Info

Publication number
WO2021082573A1
WO2021082573A1 PCT/CN2020/106311 CN2020106311W WO2021082573A1 WO 2021082573 A1 WO2021082573 A1 WO 2021082573A1 CN 2020106311 W CN2020106311 W CN 2020106311W WO 2021082573 A1 WO2021082573 A1 WO 2021082573A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain antibody
single domain
seq
antibody
present
Prior art date
Application number
PCT/CN2020/106311
Other languages
English (en)
French (fr)
Inventor
万亚坤
朱敏
盖军伟
沈晓宁
Original Assignee
上海洛启生物医药技术有限公司
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 上海洛启生物医药技术有限公司 filed Critical 上海洛启生物医药技术有限公司
Priority to US17/772,350 priority Critical patent/US12012456B2/en
Priority to JP2022525051A priority patent/JP7387206B2/ja
Priority to AU2020377355A priority patent/AU2020377355A1/en
Priority to BR112022007851A priority patent/BR112022007851A2/pt
Priority to EP20880575.4A priority patent/EP4053161A1/en
Priority to CA3156084A priority patent/CA3156084A1/en
Priority to KR1020227018317A priority patent/KR20220091566A/ko
Priority to IL292622A priority patent/IL292622A/en
Publication of WO2021082573A1 publication Critical patent/WO2021082573A1/zh

Links

Images

Classifications

    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-IL-4R single domain antibody and its application.
  • Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1) is a cytokine mainly produced by activated T cells, monocytes, basophils, mast cells and eosinophils. All the biological functions of IL-4 are mediated by IL-4R on the surface of effector cells. IL-4R is composed of ⁇ and ⁇ subunits. Interleukin-4 receptor ⁇ subunit (IL-4R ⁇ ) is located on chromosome 16p12.1 -PI1.2 area, which is also an asthma susceptible area, and genetic polymorphisms in this area are associated with allergies and elevated serum IgE levels. In addition, IL-4R ⁇ is also a common component of IL-4 and IL-13 gene receptor complexes. Studies have shown that IL-4R ⁇ gene polymorphisms are related to asthma, elevated serum IgE levels, and allergic dermatitis.
  • BSF-1 B cell stimulating factor
  • Asthma is a common respiratory disease and a frequently-occurring disease. Asthma is a chronic inflammatory disease of the airway involving a variety of cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, airway epithelial cells, etc.) and cellular components. It has many different molecular diseases Phenotypes of science and physiology. Asthma is a heterogeneous disease, which can be divided into two types: allergic and non-allergic asthma. In allergic asthma, the abnormally high expression of type II helper T cytokine (Th2) in the bronchi was found.
  • Th2 type II helper T cytokine
  • Th2 cytokine comprehensively mediates the occurrence and development of inflammatory reactions and promotes pathological changes in the respiratory tract. It is an ideal target for anti-asthma. Th2 subgroup mainly produces IL-4, IL-5, IL-6, IL-10, IL-13 and so on. IL-4R plays an important biological role in asthma diseases mainly through its combination with IL-4 and IL-13.
  • Atopic dermatitis is the most common inflammatory skin disease, with an incidence of 15%-30% in children and 2%-10% in adults. It has the characteristics of early onset, long duration, and severe pruritus. It will have a serious impact on the patient's quality of life. Th2 type cytokines are significantly increased in patients' skin lesions, and promote the secretion of IL-4 and IL-13, and mediate downstream signal transduction through the co-receptor IL-4R ⁇ .
  • Dupilumab is an anti-IL-4R ⁇ monoclonal antibody that can block the binding of IL-4 and IL-13 to IL-4R ⁇ , thereby blocking downstream signal transduction pathways.
  • CBP201 a drug under investigation by Suzhou Conrad Biomedical Co., Ltd.
  • It is also an anti-IL-4R ⁇ monoclonal antibody. It has filed an application in Australia and started the first phase of clinical trials. .
  • single domain antibody that is, VHH (variable domain of heavy chain of heavy-chain antibody)
  • VHH variable domain of heavy chain of heavy-chain antibody
  • HCAb heavy-chain antibodies
  • the single-domain antibody that consists of only one heavy chain variable region is obtained by cloning its variable region. It is currently available with complete The smallest unit that is functionally stable and can bind to an antigen.
  • Single-domain antibodies have the characteristics of high stability, good water solubility, simple humanization, high targeting, and strong penetration. They play a huge function beyond imagination in immunological experiments, diagnosis and treatment. Single-domain antibodies are gradually becoming an emerging force in the diagnosis and treatment of a new generation of antibodies.
  • an anti-IL-4R single domain antibody that has better specificity, blocking activity, better clinical efficacy, and is simple to produce, can reduce production costs, and reduce the burden of medication for patients, which has become an urgent need solved problem.
  • the purpose of the present invention is to provide an anti-IL-4R single domain antibody and its application.
  • the purpose of the present invention is to provide a single domain antibody that can specifically bind to IL-4R protein.
  • the first aspect of the present invention provides a complementarity determining region CDR region of an anti-IL-4R single domain antibody VHH chain.
  • the complementarity determining region CDR of the VHH chain includes CDR1 shown in SEQ ID NO:1 and SEQ ID NO: CDR2 shown in: 2 and CDR3 shown in SEQ ID NO: 3.
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4.
  • the second aspect of the present invention provides a VHH chain of an anti-IL-4R single domain antibody.
  • the VHH chain includes the framework region FR and the complementarity determining region CDR described in the first aspect of the present invention.
  • the framework region FR includes:
  • VHH chain of the anti-IL-4R single domain antibody is shown in SEQ ID NO: 8 or 14.
  • the heavy chain variable region includes CDR1 shown in SEQ ID NO:1 and CDR2 shown in SEQ ID NO:2. And CDR3 shown in SEQ ID NO: 3.
  • the third aspect of the present invention provides an anti-IL-4R single domain antibody, which is a single domain antibody directed against the interleukin 4 receptor (IL-4R) epitope and has the second aspect of the present invention.
  • the VHH chain is a single domain antibody directed against the interleukin 4 receptor (IL-4R) epitope and has the second aspect of the present invention.
  • the anti-IL-4R single domain antibody includes monomer, bivalent (bivalent antibody), tetravalent (tetravalent antibody), and/or multivalent (multivalent antibody).
  • the anti-IL-4R single domain antibody includes one or more VHH chains having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 14.
  • VHH chain sequence of the anti-IL-4R single domain antibody is shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody includes two VHH chains having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody includes four VHH chains having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody has a VHH chain with an amino acid sequence as shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
  • the two VHH chains with the amino acid sequence shown in SEQ ID NO: 14 are connected by a linker.
  • the four VHH chains having the amino acid sequence shown in SEQ ID NO: 14 are connected by a linker.
  • the linker is selected from the following group: GGGGSGGGS (SEQ ID NO: 18).
  • amino acid sequence of the anti-IL-4R single domain antibody is shown in SEQ ID NO: 19.
  • the fourth aspect of the present invention provides an anti-IL-4R single domain antibody Fc fusion protein, the structure of the fusion protein from the N-terminus to the C-terminus is as shown in Formula Ia or Ib:
  • A is the anti-IL-4R single domain antibody according to the third aspect of the present invention.
  • B is the Fc fragment of IgG.
  • L is no or flexible joint.
  • the flexible linker is a peptide linker.
  • the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.
  • the Fc fragment of IgG includes the Fc fragment of human IgG.
  • the peptide linker has the structure of (GGGGS)n, where n is a positive integer of 1-5.
  • the Fc fragment of IgG includes the Fc fragment of human IgG.
  • the Fc fragment of IgG is selected from the following group: IgG1, IgG2, IgG3, IgG4 Fc fragment, or a combination thereof.
  • the Fc fragment of IgG is IgG4.
  • amino acid sequence of the Fc fragment is shown in positions 120 to 346 in SEQ ID NO: 16.
  • amino acid sequence of the fusion protein is shown in SEQ ID NO: 16 or SEQ ID NO: 19.
  • the fusion protein is a single domain antibody Fc fusion protein directed against the IL-4R epitope.
  • the fifth aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of: the CDR region of the anti-IL-4R single domain antibody VHH chain of the first aspect of the present invention, The VHH chain of the anti-IL-4R single domain antibody of the second aspect of the present invention, or the anti-IL-4R single domain antibody of the third aspect of the present invention, or the anti-IL-4R single domain antibody of the fourth aspect of the present invention 4R single domain antibody Fc fusion protein.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 9 or 15.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO:20.
  • the polynucleotide includes DNA or RNA.
  • the sixth aspect of the present invention provides an expression vector containing the polynucleotide of the fifth aspect of the present invention.
  • the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.
  • the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a viral vector such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • the seventh aspect of the present invention provides a host cell containing the expression vector of the sixth aspect of the present invention, or the polynucleotide of the fifth aspect of the present invention integrated into its genome.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.
  • the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, fission yeast, Trichoderma, or a combination thereof.
  • the eukaryotic cells are selected from the group consisting of insect cells such as armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.
  • the host cell is preferably a mammalian cell, more preferably HEK293 cell, CHO cell, BHK cell, NSO cell or COS cell.
  • the host cell is Pichia pastoris.
  • the eighth aspect of the present invention provides a method for producing an anti-IL-4R single domain antibody or an Fc fusion protein thereof, including the steps:
  • step (c) Optionally, purify and/or modify the anti-IL-4R single domain antibody or its Fc fusion protein obtained in step (b).
  • the anti-IL-4R single domain antibody has an amino acid sequence as shown in SEQ ID NO: 8 or 14.
  • the anti-IL-4R single domain antibody has an amino acid sequence as shown in SEQ ID NO: 19.
  • the ninth aspect of the present invention provides an immunoconjugate, which contains:
  • a coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof .
  • the radionuclide includes:
  • Diagnostic isotopes said diagnostic isotopes are selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • Therapeutic isotope said therapeutic isotope is selected from the following group: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or a combination thereof.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatin ( auristatins), camptothecins, docarmycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 DM1 and DM4
  • benzodiazepines or benzodiazepine containing drugs such as pyrrolo[1,4] benzodiazepines (PBDs), indole Indolinobenzodiazepines (indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PBDs pyrrolo[1,4] benzodiazepines
  • indole Indolinobenzodiazepines indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • the toxin is selected from the following group: auristatin (for example, auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, mettancilol, a toxin, and Ephedrine A-chain, combstatin, docarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, Tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthracisin dione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia Toxin, acacia toxin A chain, capsulatoxin A chain, ⁇ -sarcin, white tree toxin, mitogellin, retstrictocin
  • the coupling portion is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or capable of producing Detectable enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, magnetic nanoparticles , Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: a multivalent (such as bivalent or tetravalent) VHH chain of the anti-IL-4R single domain antibody according to the second aspect of the present invention, as described in the present invention
  • a multivalent (such as bivalent or tetravalent) VHH chain of the anti-IL-4R single domain antibody according to the second aspect of the present invention as described in the present invention
  • the anti-IL-4R single domain antibody of the third aspect of the present invention, or the anti-IL-4R single domain antibody Fc fusion protein of the fourth aspect of the present invention is a multivalent (such as bivalent or tetravalent) VHH chain of the anti-IL-4R single domain antibody according to the second aspect of the present invention, as described in the present invention
  • the anti-IL-4R single domain antibody of the third aspect of the present invention, or the anti-IL-4R single domain antibody Fc fusion protein of the fourth aspect of the present invention as described in the present invention.
  • the multivalent means that the amino acid sequence of the immunoconjugate contains a plurality of repeated VHH chains of the anti-IL-4R single domain antibody according to the second aspect of the present invention, such as The anti-IL-4R single domain antibody according to the third aspect of the present invention, or the anti-IL-4R single domain antibody Fc fusion protein according to the fourth aspect of the present invention.
  • the tenth aspect of the present invention provides the VHH chain of the anti-IL-4R single domain antibody of the second aspect of the present invention, the anti-IL-4R single domain antibody of the third aspect of the present invention, or the first aspect of the present invention.
  • the use of the anti-IL-4R single domain antibody Fc fusion protein in the four aspects is used to prepare a medicine for preventing or treating diseases or disorders related to IL-4/IL-4R signal transduction.
  • the eleventh aspect of the present invention provides a pharmaceutical composition, which contains:
  • the coupling part of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
  • the pharmaceutical composition also contains other drugs for the treatment of asthma, atopic dermatitis, arthritis, allergic rhinitis and/or eczema, such as corticosteroids (TCS), nedocromil sodium, Cromolyn, theophylline, leukotriene receptor antagonist, or a combination thereof.
  • TCS corticosteroids
  • nedocromil sodium Cromolyn
  • theophylline theophylline
  • leukotriene receptor antagonist or a combination thereof.
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare drugs for preventing and/or treating diseases or disorders related to IL-4/IL-4R signal transduction.
  • the disease or condition includes, but is not limited to: asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema and the like.
  • the twelfth aspect of the present invention provides one of the anti-IL-4R single domain antibody of the third aspect of the present invention and/or the anti-IL-4R single domain antibody Fc fusion protein of the fourth aspect of the present invention One or more uses; for preparation
  • the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
  • the thirteenth aspect of the present invention provides an antibody comprising one or more VHH chains of the anti-IL-4R single domain antibody according to the second aspect of the present invention.
  • the antibody includes two VHH chains of the anti-IL-4R single domain antibody described in the second aspect of the present invention.
  • the antibody has the VHH of the anti-IL-4R single domain antibody as described in the second aspect of the present invention.
  • the antibody has the heavy chain variable region VHH as described in the second aspect of the present invention.
  • the antibody can specifically target IL-4R protein with the correct spatial structure.
  • the antibody can recognize IL-4R of human and marmoset, but not IL-4R of mouse.
  • the antibody can effectively inhibit the proliferation inhibition of TF-1 cells, and the inhibitory activity is significantly higher than that of the marketed monoclonal antibody Dupilumab.
  • the antibody can effectively inhibit the activation of the pSTAT6 signaling pathway in cells, and the inhibitory activity is significantly higher than that of the marketed monoclonal antibody Dupilumab.
  • the antibody is a single domain antibody.
  • the fourteenth aspect of the present invention provides a recombinant protein, the recombinant protein having:
  • the tag sequence includes Fc tag, HA tag and 6His tag.
  • the recombinant protein specifically binds to IL-4R protein.
  • the reagent, detection plate or kit is used to: detect IL-4R protein in the sample;
  • the medicament is used to treat and/or prevent diseases or disorders related to IL-4/IL-4R signaling.
  • the disease or condition includes, but is not limited to: asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema and the like.
  • the detection includes flow cytometry and cellular immunofluorescence detection.
  • the sixteenth aspect of the present invention provides a method for treating diseases, the method comprising administering the single domain antibody according to the third aspect of the present invention or the single domain antibody according to the fourth aspect of the present invention to a subject in need Anti-IL-4R single domain antibody Fc fusion protein, or the immunoconjugate according to the ninth aspect of the present invention.
  • the subject includes mammals, such as humans and marmosets.
  • the seventeenth aspect of the present invention provides a method for detecting IL-4R protein in a sample, the method comprising the steps:
  • the method is a non-diagnostic and non-therapeutic method.
  • an IL-4R protein detection reagent comprising:
  • the coupling part of the immunoconjugate is an isotope for diagnosis.
  • the detection-acceptable carrier is a non-toxic and inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the following group: isotope tracer, contrast agent, flow detection reagent, cellular immunofluorescence detection reagent, nano magnetic particle and imaging Agent.
  • the detection reagent is used for in vivo detection.
  • the dosage form of the detection reagent is liquid or powder (such as liquid, injection, lyophilized powder, tablet, buccal, or mist).
  • the nineteenth aspect of the present invention provides a kit for detecting IL-4R protein, the kit containing the immunoconjugate according to the ninth aspect of the present invention or the detection according to the eighteenth aspect of the present invention Reagents, and instructions.
  • the instructions stated that the kit is used to non-invasively detect the expression of IL-4R in the test subject.
  • the twentieth aspect of the present invention provides a use of the immunoconjugate according to the ninth aspect of the present invention to prepare a contrast agent for detecting IL-4R protein in vivo.
  • the test is used for the diagnosis or prognosis of asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, etc.
  • the twenty-first aspect of the present invention provides a framework region FR of an anti-IL-4R single domain antibody VHH chain.
  • the framework region FR of the VHH chain is represented by FR1 shown in SEQ ID NO: 4, SEQ ID NO : FR2 shown in SEQ ID NO: 6, FR3 shown in SEQ ID NO: 7, FR4 shown in SEQ ID NO: 7; or FR2 shown in SEQ ID NO: 10, FR1, SEQ ID NO: 11 Composition of FR3 shown in SEQ ID NO: 12 and FR4 shown in SEQ ID NO: 13.
  • the twenty-second aspect of the present invention provides a method for treating diseases or disorders related to IL-4/IL-4R signaling, and administers the pharmaceutical composition according to the eleventh aspect of the present invention to a subject in need.
  • the subject includes mammals, such as humans.
  • Figure 1 shows the storage capacity of the phage-displayed IL-4R single domain antibody library.
  • the constructed library was diluted to spread the number of clones on the plate, and the library volume was calculated to be 1.1 ⁇ 10 9 CFU.
  • Figure 2 shows the fragment insertion rate of the phage-displayed IL-4R single domain antibody library.
  • the single clones in the library were randomly selected for PCR detection, and the library insertion rate was calculated to be 95.8%.
  • Figure 3 shows the blocking activity of blocking IL-4R single domain antibodies identified by flow cytometry.
  • Figure 4 shows the inhibitory effect of blocking IL-4R single domain antibodies identified by flow cytometry on the proliferation of TF-1 cells under IL-4 induction conditions.
  • Figure 5 shows the inhibitory effect of blocking IL-4R single domain antibodies on TF-1 cell proliferation under IL-13 induction conditions identified by flow cytometry.
  • Figure 6 shows the binding properties of IL-4R single domain antibody Nb103 to IL-4R of different species detected by ELISA.
  • Candidate antibody Nb103 can recognize human IL-4R, but not murine and cynomolgus IL-4R.
  • Figure 7 shows the binding properties of IL-4R single domain antibody Nb103 to different types of monkey IL-4R detected by ELISA.
  • Candidate antibody Nb103 can recognize marmoset IL-4R but not rhesus IL-4R.
  • Figure 8 shows the detection of the binding activity of humanized IL-4R single domain antibody to human IL-4R by flow cytometry.
  • Figure 9 shows the detection of IL-4/IL-4R blocking activity of humanized IL-4R single domain antibodies by flow cytometry.
  • Figure 10 shows a schematic diagram of the structure of a humanized tetravalent antibody.
  • Figure 11 shows the purity of the humanized tetravalent antibody identified by SEC-HPLC.
  • the expressed humanized tetravalent antibody tet-huNb103 has a purity of 90.69%.
  • Figure 12 shows the killing effect of humanized tetravalent antibody on TF-1 cells under IL-4 induction conditions.
  • Figure 13 shows the killing effect of humanized tetravalent antibody on TF-1 cells under IL-13 induction conditions.
  • Figure 14 shows the inhibitory effect of the humanized tetravalent antibody on the pSTAT6 signaling pathway in HEK-BlueTM IL-4/IL-13 cells under IL-4 induction conditions.
  • Figure 15 shows the inhibitory effect of the humanized tetravalent antibody on the pSTAT6 signaling pathway in HEK-BlueTM IL-4/IL-13 cells under IL-13 induction conditions.
  • Figure 16 shows the inhibition of the humanized tetravalent antibody at two doses of 5mpk and 25mpk on the content of OVA-specific IgE in the serum of the transgenic mouse OVA model.
  • Figure 17 shows that the humanized tetravalent antibody at two doses of 5mpk and 25mpk can effectively reduce lung cancer.
  • the inventors unexpectedly discovered a type of IL-4R single domain antibody for the first time.
  • Experimental results show that the single domain antibody of the present invention can specifically recognize IL-4R and has good specificity. ; It can recognize IL-4R of humans and marmosets, but not IL-4R of mice; and can effectively inhibit the proliferation of TF-1 cells and effectively inhibit the activation of the pSTAT6 signaling pathway in the cells.
  • the single domain antibody of the present invention is easy to produce. This completes the present invention.
  • the present invention uses human IL-4R protein to immunize camels to obtain a high-quality immune single domain antibody gene library. Then the IL-4R protein molecule is coupled to the ELISA plate to display the correct spatial structure of the IL-4R protein.
  • the antigen in this form is used to screen the immune single domain antibody gene library (camel heavy chain antibody phage display gene library using phage display technology). ), thus obtaining IL-4R specific single domain antibody gene.
  • the gene was transferred to mammalian cells, and a single domain antibody strain with high specificity that can be expressed in mammalian cells was obtained. Then the IL-4R single domain antibody with blocking activity was identified by ELISA, flow cytometry, luciferase reporter gene detection system and other methods.
  • the terms "single domain antibody of the present invention”, “single domain antibody of the present invention”, “anti-IL-4R single domain antibody of the present invention”, “IL-4R single domain antibody of the present invention”, “anti-IL- “4R single domain antibody” and “IL-4R single domain antibody” have the same meaning and can be used interchangeably, and both refer to single domain antibodies that specifically recognize and bind to IL-4R (including human IL-4R).
  • the variable region of the single domain antibody of the present invention has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3.
  • the framework region of the single domain antibody of the present invention has (a) FR1 shown in SEQ ID NO: 4, FR1 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and SEQ ID FR4 shown in NO: 7, or (b) FR1 shown in SEQ ID NO: 10, FR1 shown in SEQ ID NO: 11, FR2 shown in SEQ ID NO: 12, and shown in SEQ ID NO: 13 FR4.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.
  • single domain antibody As used herein, the terms “single domain antibody”, “VHH”, “nanobody”, “single domain antibody” (single domain antibody, sdAb, or nanobody) have the same meaning and can be used interchangeably, Refers to the cloning of the variable region of the heavy chain of an antibody to construct a single domain antibody (VHH) consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete functions. Usually, after obtaining an antibody with naturally missing light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • variable means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a b-sheet configuration and are connected by three CDRs forming a connecting loop, which can form a partial b-sheet structure in some cases.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments of the present invention to form ⁇ conjugate.
  • the present invention also includes cell surface markers or antigens that bind to the anti-IL-4R antibody or fragments thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the heavy chain variable region and the heavy chain constant region described above.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to a polypeptide that specifically binds to IL-4R protein, such as a protein with a heavy chain variable region. Or polypeptide. They may or may not contain the starting methionine.
  • the present invention also provides other proteins or fusion expression products with the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention.
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homology.
  • variable regions which are divided into 4 framework regions (FR), 4 FR amino acids
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ sheets formed by the FRs between them are close to each other in space structure, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified here. Sex.
  • the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogues of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or proprotein sequence, or with Fusion protein formed by 6His tag
  • the antibody of the present invention refers to a polypeptide having IL-4R binding activity and including the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides containing the above-mentioned CDR regions that have the same functions as the antibodies of the present invention. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the present invention also provides other polypeptides, such as fusion proteins containing single domain antibodies or fragments thereof.
  • the present invention also includes fragments of single domain antibodies of the present invention.
  • the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 compared to the amino acid sequence of the antibody of the present invention. Two amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table 1 through amino acid substitutions.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence or fragments of the antibody of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be carried out by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the present invention can be used alone, or can be combined or coupled with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or those capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • Interleukin-4 (IL-4)
  • Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1) is a cytokine mainly produced by activated T cells, monocytes, basophils, mast cells and eosinophils. IL-4 can respond to low concentrations of antibodies against surface immunoglobulins and can stimulate B cell proliferation. It has been confirmed that IL-4 has a wide range of biological activities, including stimulating the growth of T cells, mast cells, granulocytes, megakaryocytes and red blood cells. IL-4 induces the expression of class II major histocompatibility complex molecules in resting B cells and enhances the secretion of IgE and IgG1 isotypes by stimulated B cells.
  • BSF-1 B cell stimulating factor
  • Interleukin-4 receptor alpha IL-4R ⁇
  • Human IL-4R ⁇ subunit is a 140kDa type I membrane protein that binds human IL-4 with high affinity. IL-4R ⁇ is not expressed in high numbers on many cell types, such as peripheral blood T cells, monocytes, airway epithelial cells, B cells, and lung fibroblasts.
  • Interleukin-4 receptor alpha subunit (IL-4R ⁇ ) is located in the region of chromosome 16p12.1-PI1.2, which is also a susceptible area for asthma. Genetic polymorphisms in this area are associated with allergies and elevated serum IgE levels .
  • IL-4R ⁇ is also a common component of IL-4 and IL-13 gene receptor complexes. Studies have shown that IL-4R ⁇ gene polymorphisms are related to asthma, elevated serum IgE levels, and allergic dermatitis.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind IL-4R protein molecules, and thus can be used to treat asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema and the like.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned single domain antibody (or conjugate) of the present invention and a pharmaceutical Acceptable carrier or excipient.
  • a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injections, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases, does not exceed about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the anti-IL-4R single domain antibody includes monomer, bivalent (bivalent antibody), tetravalent (tetravalent antibody), and/or multivalent (multivalent antibody).
  • the anti-IL-4R single domain antibody includes one, two or more VHH chains having the amino acid sequence shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody includes two VHH chains having the amino acid sequence shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody includes four VHH chains having the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody has a VHH chain with an amino acid sequence as shown in SEQ ID NO: 8 and/or SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody includes two VHH chains having the amino acid sequence shown in SEQ ID NO: 14.
  • the anti-IL-4R single domain antibody includes four VHH chains having the amino acid sequence shown in SEQ ID NO: 14.
  • the two VHH chains having the amino acid sequence shown in SEQ ID NO: 8 are connected by a linker.
  • the two VHH chains having the amino acid sequence shown in SEQ ID NO: 14 are connected by a linker.
  • the linker is selected from the following group: GGGGSGGGS (SEQ ID NO: 18).
  • amino acid sequence of the bivalent anti-IL-4R single domain antibody is shown in SEQ ID NO: 16.
  • amino acid sequence of the tetravalent anti-IL-4R single domain antibody is shown in SEQ ID NO: 19.
  • the single domain antibody carries a detectable label. More preferably, the label is selected from the following group: isotope, colloidal gold label, colored label or fluorescent label.
  • Colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the IL-4R single domain antibody is labeled with colloidal gold to obtain the colloidal gold labeled single domain antibody.
  • the novel IL-4R single domain antibody of the present invention has good specificity and high titer.
  • the invention also relates to a method for detecting IL-4R protein.
  • the method steps are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; and detect the level of IL-4R protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a sample containing cells in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention.
  • the kit further includes a container, instructions for use, buffers, and the like.
  • the present invention also provides a detection kit for detecting the level of IL-4R.
  • the kit includes an antibody that recognizes IL-4R protein, a lysis medium used to dissolve the sample, and general reagents and buffers required for detection, such as various Buffer, detection label, detection substrate, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the single domain antibody of the present invention has a wide range of biological application value and clinical application value, and its application relates to the diagnosis and treatment of diseases related to IL-4R, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy for IL-4R.
  • the single domain antibody of the present invention specifically binds to IL-4R protein with the correct spatial structure.
  • the single domain antibody of the present invention can recognize IL-4R of human and marmoset, but not IL-4R of mouse.
  • the single domain antibody of the present invention has stronger binding activity and blocking activity.
  • the single domain antibody of the present invention can effectively inhibit the proliferation inhibition of TF-1 cells, and its inhibitory activity is significantly higher than that of the marketed monoclonal antibody Dupilumab.
  • the single domain antibody of the present invention can effectively inhibit the activation of the pSTAT6 signaling pathway in cells, and its inhibitory activity is significantly higher than that of the marketed monoclonal antibody Dupilumab.
  • Example 1 Screening and expression of anti-human IL-4R single domain antibody
  • mammalian cells HEK293F were first used to transiently express human IL-4R protein, which was then used for camel immunization after affinity purification.
  • the specific method please refer to the description of the method in embodiment 1 and embodiment 2 of patent CN2018101517526. Briefly, 1 Xinjiang Bactrian camel was immunized with the purified IL-4R protein.
  • the volume of the constructed library as shown in Figure 1 is 1.1 ⁇ 10 9 CFU, and the insertion rate of the library as shown in Figure 2 is 95.8%.
  • library screening was performed, and phage enrichment containing antibody genes was obtained after 3 rounds of screening. 300 clones were selected from the library for PE-ELISA identification, the positive clones obtained were sequenced, and single domain antibodies with different sequences were transiently expressed using the E. coli system.
  • the expression method please refer to the method description in Example 4 of patent CN2018101521076.
  • Example 2 Screening of IL-4R single domain antibodies with blocking activity by FACS
  • Example 3 FACS detection of the inhibitory effect of candidate single domain antibodies on the proliferation of TF-1 cells
  • the method for detecting the inhibitory effect of the candidate single domain antibody on the proliferation of TF-1 cells induced by IL-4 is as follows: (1) Take a certain number of TF-1 cells, centrifuge at 1000 rpm for 5 min, discard the supernatant; add PBS and resuspend at 1000 rpm Centrifuge for 5 min and wash once; then add an appropriate amount of PBS to resuspend, count, dilute the cell solution concentration to 6 ⁇ 10 5 /mL, and distribute the cells to 96-well plates.
  • IC 50 Nb103 0.149 ug/mL
  • IC 50 Dupilumab 0.566 ug/mL. Therefore, the candidate single domain antibody Nb103 has a significantly stronger inhibitory effect on the proliferation of TF-1 cells than the control antibody Dupilumab.
  • the method for detecting the inhibitory effect of the candidate single domain antibody on the proliferation of TF-1 cells induced by IL-13 is the same as above.
  • the diluted antibody to be tested is mixed with IL-13 at a working concentration of 10 ng/mL and added to the cells.
  • Example 4 ELISA to detect the species specificity of candidate single domain antibodies
  • the candidate antibody Nb103 can recognize the marmoset IL-4R but not the rhesus IL- 4R.
  • the candidate antibody is humanized and modified, keeping the variable regions unchanged, and humanized design is performed for the four framework region sequences.
  • modification method please refer to the method of Example 4 in the patent CN2018101517526.
  • the humanized antibody sequence is constructed on the pFUSE vector, and the humanized single domain antibody is fused with the Fc sequence to form huNb103 (see SEQ ID NO: 16 for the fusion protein sequence, and SEQ ID for the coding nucleotide sequence. NO: 17), expressed using the HEK293F system, and the expressed protein can be used for subsequent verification.
  • Method of expression refer to Example 3 of the patent CN2018101517526.
  • the modified sequence is shown in Table 2 below:
  • Example 8 Construction and preparation of humanized tetravalent antibody
  • Example 9 Inhibitory effect of humanized tetravalent antibody on the proliferation of TF-1 cells
  • the humanized tetravalent antibody has a significant inhibitory effect on the proliferation of TF-1 cells induced by IL-13.
  • concentration of IL-13 added during detection is 10ng/mL, and the rest of the steps are the same as above.
  • the results are shown in Figure 13:
  • Example 10 Detection of the inhibitory effect of humanized tetravalent antibody on the pSTAT6 signaling pathway in HEK-Blue TM IL-4/IL-13 cells
  • the humanized tetravalent antibody has a significant inhibitory effect on the pSTAT6 signaling pathway of HEK-Blue TM IL-4/IL-13 cells induced by IL-13.
  • concentration of IL-13 added during detection is 10ng/mL, and the rest of the steps are the same as above.
  • Example 11 Using hIL4/hIL4Ra transgenic mouse OVA model to verify the efficacy of humanized tetravalent antibody
  • the 15 animals were randomly divided into 3 groups according to their body weights, each with 5 animals.
  • Group 1 was the negative control group
  • group 2 was the test product low-dose group (5mpk)
  • group 3 was the test product high-dose group (25mpk).
  • Animals in groups 1-3 were all sensitized and administered with OVA: 200 ⁇ g/mL OVA was prepared, sensitized by intraperitoneal injection, 200 ⁇ L/head. The sensitization time was 0, 7, and 14 days. On days 21-25, aerosol inhalation of 2% OVA was used for 30 minutes each time for 5 consecutive days.
  • the test substance was administered on the 20th and 23rd day, and samples were collected for detection and analysis on the day after the excitation operation on the 25th day.
  • FIG. 17A and 17B 5mpk 25mpk and two doses of tetravalent humanized antibodies were effective in reducing the number and proportion of eosinophils lungs.
  • SEQ ID NO: 16 SEQ ID NO: 14 + Fc fragment
  • SEQ ID NO: 17 (nucleotide sequence encoding huNb103)
  • SEQ ID NO: 19 SEQ ID NO: 14 + SEQ ID NO: 18 + SEQ ID NO: 14 + Fc fragment

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Pulmonology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种IL-4R单域抗体及其应用。具体为一种IL-4R单域抗体及其VHH链，编码上述单域抗体或其VHH链的编码序列、相应的表达载体和能够表达该单域抗体的宿主细胞，以及该单域抗体的生产方法。所述单域抗体能够特异性识别人和狨猴的IL-4R，不识别小鼠的IL-4R，具有良好的特异性；能够有效抑制TF-1细胞的增殖抑制以及细胞中pSTAT6信号通路的激活。

Description

抗IL-4R单域抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及一种抗IL-4R单域抗体及其应用。
背景技术
白细胞介素-4(IL-4，也成为B细胞刺激因子或BSF-1)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的所有生物功能都是通过效应细胞表面IL-4R介导的，IL-4R由α和γ亚单位组成，白介素-4受体α亚单位(IL-4Rα)定位于染色体16p12.1-PI1.2区域，该区域也是哮喘易感区，该区基因多态性与过敏症和升高的血清IgE水平相关。此外，IL-4Rα还是IL-4和IL-13基因受体复合物的共同组成部分，研究表明IL-4Rα基因多态性与哮喘、升高的血清中IgE水平及过敏性皮炎有关。
哮喘是常见的呼吸道疾病，也是一种多发病。哮喘是由多种细胞(如嗜酸性粒细胞、肥大细胞、T淋巴细胞、中性粒细胞气道上皮细胞等)和细胞组分参与的气道慢性炎症性疾病，它有很多不同的分子病理学和生理学的表型。哮喘是一种异质性的疾病，可分为过敏性和非过敏性哮喘两种。在过敏性哮喘中，发现II型辅助性T细胞因子(Th2)在支气管中的异常高表达，同时已证实Th2细胞因子全面介导了炎症反应的发生和发展并促进了呼吸道的病理改变等，是抗哮喘的理想靶点。Th2亚群主要产生IL-4、IL-5、IL-6、IL-10、IL-13等。IL-4R主要通过与IL-4、IL-13等的结合在哮喘疾病中发挥重要生物学作用。
特应性皮炎(AD)是最常见的炎症性皮肤病，在儿童中的发病率15％-30％，成人2％-10％，具有发病年龄早，持续时间久、瘙痒症剧烈的特点，对患者的生活质量会产生严重的影响。Th2型细胞因子在患者皮损中显著提高，并促进IL-4和IL-13分泌，通过共受体IL-4Rα介导下游信号转导。
目前已有针对hIL-4R靶点的抗体药物进入临床试验，如Dupilumab为抗IL-4Rα单抗，能阻断IL-4和IL-13与IL-4Rα结合，从而阻断下游的信号传导通路来抑制皮炎慢性炎症的发展，其在治疗特应性皮炎的II期临床中表现了较好的疗效。除了Dupilumab以外，苏州康乃德生物医药有限公司的在研药物CBP201，公开于专利CN201610399254.4中，也是一种抗IL-4Rα的单克隆抗体，已在澳大利亚递交申请，开始了第一阶段的临床试验。
截至目前，市场上尚未有针对hIL-4R靶点的单域抗体药物公布，而单域抗体(nanobody，Nb)，即重链单域抗体VHH(variable domain of heavy chain of heavy-chain antibody)—骆驼体内存在着天然缺失轻链的重链抗体(heavy-chain antibody，HCAb)，克隆其可变区而得到的只由一个重链可变区组成的单域抗体，是目前可以得到的具有完整功能的稳定的可结合抗原的最小单位。单域抗体具有稳定性高、水溶性好、人源化简单、靶向性高、穿透性强等特点，在免疫实验、诊断与治疗中，发挥着超乎想象的巨大功能。单域抗体正逐渐成为新一代抗体诊断及治疗中的新兴力量。
因此，开发一种抗IL-4R单域抗体，使其具有较好的特异性、阻断活性，更佳的临床药效，并且生产简便，能够降低生产成本，减轻患者用药负担，已经成为亟待解决的问题。
发明内容
本发明的目的在于提供一种抗IL-4R单域抗体及其应用。
具体地，本发明的目的在于提供一种能够特异性结合IL-4R蛋白的单域抗体。
本发明的第一方面，提供了一种抗IL-4R单域抗体VHH链的互补决定区CDR区，所述VHH链的互补决定区CDR包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3。
在另一优选例中，所述的CDR1、CDR2和CDR3被框架区FR1、FR2、FR3和FR4所隔开。
本发明的第二方面，提供了一种抗IL-4R单域抗体的VHH链，所述的VHH链包括框架区FR和本发明的第一方面所述的互补决定区CDR。
在另一优选例中，所述的框架区FR包括：
(a)SEQ ID NO:4所示的FR1、SEQ ID NO:5所示的FR2、SEQ ID NO:6所示的FR3、和SEQ ID NO:7所示的FR4；或
(b)SEQ ID NO:10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。
在另一优选例中，所述的抗IL-4R单域抗体的VHH链如SEQ ID NO:8或14所示。
此外，还提供一种新型抗IL-4R单域抗体的重链可变区，所述的重链可变 区包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3。
本发明的第三方面，提供了一种抗IL-4R单域抗体，它是针对白细胞介素4受体(IL-4R)表位的单域抗体，并且具有本发明的第二方面所述的VHH链。
在另一优选例中，所述抗IL-4R单域抗体包括单体、二价体(二价抗体)、四价体(四价抗体)、和/或多价体(多价抗体)。
在另一优选例中，所述抗IL-4R单域抗体包括一条或多条具有如SEQ ID NO:8或SEQ ID NO:14所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL-4R单域抗体的VHH链序列如SEQ ID NO:8和/或SEQ ID NO:14所示。
在另一优选例中，所述抗IL-4R单域抗体包括两条具有如SEQ ID NO:8或SEQ ID NO:14中所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL-4R单域抗体包括四条具有如SEQ ID NO:8或SEQ ID NO:14中所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL-4R单域抗体具有如SEQ ID NO:8和/或SEQ ID NO:14中所示的氨基酸序列的VHH链。
在另一优选例中，所述两条具有如SEQ ID NO:14中所示的氨基酸序列的VHH链之间通过连接子进行连接。
在另一优选例中，所述四条具有如SEQ ID NO:14中所示的氨基酸序列的VHH链之间通过连接子进行连接。
在另一优选例中，所述连接子选自以下序列：(G aS b) x—(G mS n) y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
在另一优选例中，所述连接子选自下组：GGGGSGGGS(SEQ ID NO:18)。
在另一优选例中，所述抗IL-4R单域抗体的氨基酸序列如SEQ ID NO:19所示。
本发明第四方面，提供了一种抗IL-4R单域抗体Fc融合蛋白，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
A-L-B(Ia)；
B-L-A(Ib)；
其中，
A为本发明第三方面所述的抗IL-4R单域抗体；
B为IgG的Fc片段；和
L为无或柔性接头。
在另一优选例中，所述的柔性接头为肽接头。
在另一优选例中，所述的肽接头具有1-50个氨基酸，较佳地1-20个氨基酸。
在另一优选例中，所述IgG的Fc片段包括人的IgG的Fc片段。
在另一优选例中，所述的肽接头具有(GGGGS)n的结构，其中n为1-5的正整数。
在另一优选例中，所述IgG的Fc片段包括人的IgG的Fc片段。
在另一优选例中，所述IgG的Fc片段选自下组：IgG1、IgG2、IgG3、IgG4的Fc片段、或其组合。
在另一优选例中，所述IgG的Fc片段为IgG4。
在另一优选例中，所述Fc片段的氨基酸序列如SEQ ID NO:16中第120-346位所示。
在另一优选例中，所述融合蛋白的氨基酸序列如SEQ ID NO:16或SEQ ID NO:19所示。
在另一优选例中，所述融合蛋白为针对IL-4R表位的单域抗体Fc融合蛋白。
本发明的第五方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：本发明的第一方面所述的抗IL-4R单域抗体VHH链的CDR区、本发明的第二方面所述的抗IL-4R单域抗体的VHH链、或本发明的第三方面所述的抗IL-4R单域抗体、或本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白。
在另一优选例中，所述多核苷酸具有如SEQ ID NO:9或15所示的核苷酸序列。
在另一优选例中，所述多核苷酸具有如SEQ ID NO:20所示的核苷酸序列。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
本发明的第六方面，提供了一种表达载体，所述表达载体含有本发明第五方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质 粒、转座子、其他基因转移系统、或其组合。
优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
本发明的第七方面，提供了一种宿主细胞，所述宿主细胞含有本发明的第六方面所述的表达载体，或其基因组中整合有本发明的第五方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞、噬菌体、或其组合。
在另一优选例中，所述原核细胞选自下组：大肠杆菌、枯草杆菌、乳酸菌、链霉菌、奇异变形菌、或其组合。
在另一优选例中，所述真核细胞选自下组：毕赤酵母、酿酒酵母、裂殖酵母、木霉、或其组合。
在另一优选例中，所示真核细胞选自下组：草地粘虫等昆虫细胞、烟草等植物细胞、BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞、或其组合。
在另一优选例中，所述宿主细胞优选为哺乳动物细胞，更优选HEK293细胞、CHO细胞、BHK细胞、NSO细胞或COS细胞。
在另一优选例中，所述的宿主细胞为毕赤酵母。
本发明的第八方面，提供了一种产生抗IL-4R单域抗体或其Fc融合蛋白的方法，包括步骤：
(a)在适合产生单域抗体或其Fc融合蛋白的条件下，培养本发明的第七方面所述的宿主细胞，从而获得含所述抗IL-4R单域抗体或其Fc融合蛋白的培养物；
(b)从所述培养物中分离或回收所述的抗IL-4R单域抗体或其Fc融合蛋白；以及
(c)任选地，纯化和/或修饰得步骤(b)中获得的抗IL-4R单域抗体或其Fc融合蛋白。
在另一优选例中，所述的抗IL-4R单域抗体具有如SEQ ID NO:8或14所示的氨基酸序列。
在另一优选例中，所述的抗IL-4R单域抗体具有如SEQ ID NO:19所示的氨基酸序列。
本发明的第九方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第二方面所述的抗IL-4R单域抗体的VHH链、或如本发明第三方面所述的抗IL-4R单域抗体、或如本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177、或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。
在另一优选例中，特别有用的细胞毒性药物的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))、长春花生物碱(vinca alkaloids)、或其组合。
在另一优选例中，所述的毒素选自下组：耳他汀类(例如，耳他汀E、耳他 汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素、或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联部分选自下组：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))或任何形式的纳米颗粒。
在另一优选例中，所述免疫偶联物含有：多价(如二价或四价)的如本发明的第二方面所述的抗IL-4R单域抗体的VHH链、如本发明的第三方面所述的抗IL-4R单域抗体、或本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明的第二方面所述抗IL-4R单域抗体的VHH链、如本发明的第三方面所述的抗IL-4R单域抗体、或本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白。
本发明的第十方面，提供了本发明的第二方面所述的抗IL-4R单域抗体的VHH链、本发明的第三方面所述的抗IL-4R单域抗体、或本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白的用途，用于制备：用于预防或治疗与IL-4/IL-4R信号传导相关的疾病或病症的药物。
本发明的第十一方面，提供了一种药物组合物，所述药物组合物含有：
(i)如本发明的第一方面所述的抗IL-4R单域抗体VHH链的互补决定区CDR区、如本发明的第二方面所述的抗IL-4R单域抗体的VHH链、如本发明的第三方面所述的抗IL-4R单域抗体、或本发明第四方面所述的抗IL-4R单域抗体Fc融 合蛋白、或如本发明的第九方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为药物、毒素、和/或治疗用同位素。
在另一优选例中，所述的药物组合物中还含有治疗哮喘、特应性皮炎、关节炎、过敏性鼻炎和/或湿疹的其他药物，如皮质类固醇(TCS)、奈多罗米钠、色甘酸钠、茶碱、白三烯受体拮抗剂、或其组合。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备预防和/或治疗与IL-4/IL-4R信号传导相关的疾病或病症的药物。
在另一优选例中，所述疾病或病症包括但不限于：哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹等。
本发明的第十二方面，提供了本发明的第三方面所述的抗IL-4R单域抗体、和/或本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白的一种或多种的用途；用于制备
(a)用于预防和/或治疗与IL-4/IL-4R信号传导相关的疾病或病症的药物；
(b)用于检测人IL-4R分子；
(c)用于流式检测；
(d)用于细胞免疫荧光检测；
在另一优选例中，所述用途为诊断性和/或非诊断性的，和/或治疗性和/或非治疗性的。
本发明的第十三方面，提供了一种抗体，所述抗体包括一条或多条如本发明的第二方面所述的抗IL-4R单域抗体的VHH链。
在另一优选例中，所述抗体包括两条本发明的第二方面所述的抗IL-4R单域抗体的VHH链。
在另一优选例中，所述抗体具有如本发明的第二方面所述的抗IL-4R单域抗体的VHH。
在另一优选例中，所述抗体具有如本发明的第二方面所述的重链可变区VHH。
在另一优选例中，所述的抗体能够特异性针对具有正确空间结构的IL-4R 蛋白。
在另一优选例中，所述的抗体能够识别人、狨猴的IL-4R，不识别小鼠的IL-4R。
在另一优选例中，所述的抗体能够有效抑制TF-1细胞的增殖抑制，且抑制活性显著高于上市单抗Dupilumab。
在另一优选例中，所述的抗体能够有效抑制细胞中pSTAT6信号通路的激活，且抑制活性显著高于上市单抗Dupilumab。
在另一优选例中，所述抗体为单域抗体。
本发明的第十四方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明的第二方面所述的VHH链、或如本发明的第三方面所述的抗IL-4R单域抗体、或如本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括Fc标签、HA标签和6His标签。
在另一优选例中，所述的重组蛋白特异性结合于IL-4R蛋白。
在本发明的第十五方面，提供了如本发明的第二方面所述的抗IL-4R单域抗体的VHH链、如本发明的第三方面所述的抗IL-4R单域抗体、或如本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白、或如本发明的第九方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；
其中，所述试剂、检测板或试剂盒用于：检测样品中IL-4R蛋白；
其中，所述药剂用于治疗和/或预防与IL-4/IL-4R信号传导相关的疾病或病症。
在另一优选例中，所述疾病或病症包括但不限于：哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹等。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
本发明的第十六方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用本发明的第三方面所述的单域抗体、或如本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白、或本发明的第九方面所述的免疫偶联物。
在另一优选例中，所述的对象包括哺乳动物，如人、狨猴。
本发明的第十七方面，提供了一种检测样品中IL-4R蛋白的方法，所述方法包括步骤：
(1)将样品与本发明的第二方面所述的VHH链、本发明的第三方面所述的单域抗体、或本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白、或本发明的第九方面所述的免疫偶联物接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在IL-4R蛋白。
在另一优选例中，所述方法为非诊断和非治疗性的方法。
在本发明的第十八方面，提供了一种IL-4R蛋白检测试剂，所述的检测试剂包含：
(i)本发明的第二方面所述的VHH链、本发明的第三方面所述的单域抗体、或本发明第四方面所述的抗IL-4R单域抗体Fc融合蛋白、或本发明的第九方面所述的免疫偶联物；以及
(ii)检测学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。
在另一优选例中，所述的检测学上可接受的载体为无毒的、惰性的水性载体介质。
在另一优选例中，所述的检测试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述的检测试剂用于体内检测。
在另一优选例中，所述的检测试剂的剂型为液态或粉状(如水剂，针剂，冻干粉，片剂，含服剂，吸雾剂)。
本发明的第十九方面，提供一种检测IL-4R蛋白的试剂盒，所述试剂盒含有本发明的第九方面所述的免疫偶联物或本发明的第十八方面所述的检测试剂，以及说明书。
在另一优选例中，所述的说明书记载，所述的试剂盒用于非侵入性地检测待测对象的IL-4R表达。
本发明的第二十方面，提供了一种本发明的第九方面所述的免疫偶联物的 用途，用于制备体内检测IL-4R蛋白的造影剂。
在另一优选例中，所述检测用于哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹等的诊断或预后。
本发明的第二十一方面，提供了一种抗IL-4R单域抗体VHH链的框架区FR，所述的VHH链的框架区FR由SEQ ID NO:4所示的FR1，SEQ ID NO:5所示的FR2，SEQ ID NO:6所示的FR3，SEQ ID NO:7所示的FR4组成；或由SEQ ID NO:10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组成。
本发明第二十二方面，提供了一种治疗与IL-4/IL-4R信号传导相关的疾病或病症的方法，给所需要的对象施用本发明第十一方面所述的药物组合物。
在另一优选例中，所述的对象包括哺乳动物，如人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了噬菌体展示IL-4R单域抗体文库的库容。构建的文库经过梯度稀释涂布平板生长克隆数目，计算文库库容为1.1×10 9CFU。
图2显示了噬菌体展示IL-4R单域抗体文库的片段插入率。经随机挑选文库中单克隆进行PCR检测，计算文库插入率为95.8％。
图3显示了流式细胞术鉴定的阻断型IL-4R单域抗体的阻断活性。IL-4R单域抗体Nb103的阻断活性优于对照抗体Dupilumab(度匹鲁单抗)(IC 50  Nb103＝0.274ug/mL，IC 50 Dupilumab＝0.511ug/mL)。
图4显示了流式细胞术鉴定的阻断型IL-4R单域抗体在IL-4诱导条件下对TF-1细胞的增殖抑制作用。候选单域抗体Nb103对TF-1细胞的增殖抑制作用显著强于对照抗体Dupilumab(IC 50 Nb103＝0.149ug/mL，IC 50 Dupilumab＝0.566ug/mL)。
图5显示了流式细胞术鉴定的阻断型IL-4R单域抗体在IL-13诱导条件下对TF-1细胞的增殖抑制作用。候选单域抗体Nb103对TF-1细胞的增殖抑制作用显著强于对照抗体Dupilumab(IC 50 Nb103＝0.066ug/mL，IC 50 Dupilumab＝0.740ug/mL)。
图6显示了ELISA检测IL-4R单域抗体Nb103对不同种属IL-4R的结合特性。候选抗体Nb103能够识别人IL-4R，而不能识别鼠和食蟹猴IL-4R。
图7显示了ELISA检测IL-4R单域抗体Nb103对不同类型猴子IL-4R的结合特性。候选抗体Nb103能够识别狨猴IL-4R，不能识别恒河猴IL-4R。
图8显示了流式细胞术检测人源化IL-4R单域抗体与人IL-4R的结合活性。人源化抗体huNb103的EC 50＝0.286ug/mL，对照抗体Dupilumab的EC 50＝0.360ug/mL。
图9显示了流式细胞术检测人源化IL-4R单域抗体对IL-4/IL-4R的阻断活性。人源化抗体huNb103的IC 50＝0.474ug/mL，对照抗体Dupilumab的IC 50＝1.126ug/mL。
图10显示了人源化四价抗体的结构示意图。
图11显示了SEC-HPLC鉴定的人源化四价抗体的纯度。表达的人源化四价抗体tet-huNb103纯度为90.69％。
图12显示了人源化四价抗体在IL-4诱导条件下对TF-1细胞的杀伤作用。人源化四价抗体能够有效抑制IL-4诱导的TF-1细胞的增殖抑制，其抑制活性IC 50 tet-huNb103＝0.020ug/mL，其活性是人源化二价抗体的1-2倍(IC 50 huNb103＝0.032ug/mL)，也显著优于对照抗体对TF-1细胞的增殖抑制作用(IC 50 Dupilumab＝0.238ug/mL)。
图13显示了人源化四价抗体在IL-13诱导条件下对TF-1细胞的杀伤作用。人源化四价抗体能够有效抑制IL-13诱导的TF-1细胞的增殖抑制，其抑制活性IC 50 tet-huNb103＝0.026ug/mL，其活性是人源化二价抗体的2-3倍(IC 50 huNb103＝0.059ug/mL)，也显著优于对照抗体对TF-1细胞的增殖抑制作用(IC 50 Dupilumab＝0.180ug/mL)。
图14显示了在IL-4诱导条件下人源化四价抗体对HEK-Blue TM IL-4/IL-13细胞中pSTAT6信号通路的抑制作用。人源化四价抗体能够有效抑制IL-4诱导的HEK-Blue TM IL-4/IL-13细胞的pSTAT6信号通路，其抑制活性IC50 tet-huNb103＝0.044ug/mL，其活性是人源化二价抗体的2-3倍(IC 50  huNb103＝0.109ug/mL)，也显著优于对照抗体对该细胞的pSTAT6信号通路抑制作用(IC 50 Dupilumab＝0.346ug/mL)。
图15显示了在IL-13诱导条件下人源化四价抗体对HEK-Blue TM IL-4/IL-13细胞中pSTAT6信号通路的抑制作用。人源化四价抗体能够有效抑制IL-13诱导的HEK-Blue TM IL-4/IL-13细胞的pSTAT6信号通路，其抑制活性IC 50  tet-huNb103＝0.017ug/mL，其活性是人源化二价抗体的2倍(IC 50  huNb103＝0.034ug/mL)，也显著优于对照抗体对HEK-Blue TM IL-4/IL-13细胞的pSTAT6信号通路抑制作用(IC 50 Dupilumab＝0.120ug/mL)。
图16显示了 5mpk和25mpk两个剂量的人源化四价抗体对转基因小鼠OVA 模型中血清中的OVA特异性的IgE的含量的抑制。
图17显示了5mpk和25mpk两个剂量的人源化四价抗体均能够有效减少肺 部嗜酸性粒细胞数目(图17A)和比例(图17B)。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的筛选，首次意外地发现一类IL-4R单域抗体，实验结果表明，本发明单域抗体能够特异性识别IL-4R，具有良好的特异性；能够识别人、狨猴的IL-4R，不识别小鼠的IL-4R；且能够有效抑制TF-1细胞的增殖以及有效抑制细胞中pSTAT6信号通路的激活。本发明单域抗体生成简便。由此完成了本发明。
具体地，本发明利用人源的IL-4R蛋白免疫骆驼，获得高质量的免疫单域抗体基因文库。然后将IL-4R蛋白分子偶联在酶标板上，展示IL-4R蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫单域抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了IL-4R特异性的单域抗体基因。再将此基因转至哺乳动物细胞中，从而获得了能在哺乳动物细胞中高效表达的、且特异性高的的单域抗体株。然后通过ELISA、流式细胞术、荧光素酶报告基因检测系统等方法鉴定出具有阻断活性的IL-4R单域抗体。
术语
如本文所用，术语“本发明单域抗体”、“本发明的单域抗体”、“本发明的抗IL-4R单域抗体”、“本发明IL-4R单域抗体”、“抗IL-4R单域抗体”、“IL-4R单域抗体”具有相同的含义，可互换使用，均指特异性识别和结合于IL-4R(包括人IL-4R)的单域抗体。优选地，本发明的单域抗体的可变区具有SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3。更优选地，本发明的单域抗体的框架区具有(a)SEQ ID NO:4所示的FR1、SEQ ID NO:5所示的FR2、SEQ ID NO:6所示的FR3、和SEQ ID NO:7所示的FR4， 或(b)SEQ ID NO:10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。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体”、“VHH”、“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb，或纳米抗体nanobody)具有相同的含义并可互换使用，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈b-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分b折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的抗IL-4R抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合IL-4R蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似 物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有IL-4R结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含单域抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1) 在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体 包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))等。
白细胞介素-4(IL-4)
白细胞介素-4(IL-4，也成为B细胞刺激因子或BSF-1)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能够响应低浓度的针对表面免疫球蛋白的抗体，能够刺激B细胞增殖。已证实IL-4具有广泛的生物活性，包括刺激T细胞、肥大细胞、粒细胞、巨核细胞和红细胞的生长。IL-4诱导静止B细胞中II类主要组织相容性复合体分子的表达，增强受激B细胞对IgE和IgG1同种型的分泌。
白细胞介素-4受体α(IL-4Rα)
人的IL-4Rα亚单位是一个以高亲和力结合人IL-4的140kDa I型膜蛋白质。IL-4Rα在许多种细胞类型上的表达数量不高，例如外周血T细胞、单核细胞、气道上皮细胞、B细胞和肺成纤维细胞。白介素-4受体α亚单位(IL-4Rα)定位于染色体16p12.1-PI1.2区域，该区域也是哮喘易感区，该区基因多态性与过敏症和升高的血清IgE水平相关。此外，IL-4Rα还是IL-4和IL-13基因受体复合物的共同组成部分，研究表明IL-4Rα基因多态性与哮喘、升高的血清中IgE水平及过敏性皮炎有关。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合IL-4R蛋白分子，因而可用于治疗哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹等。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50 毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
抗IL-4R单域抗体
在本发明中，所述抗IL-4R单域抗体包括单体、二价体(二价抗体)、四价(四价抗体)、和/或多价体(多价抗体)。
在本发明的一个优选例中，所述抗IL-4R单域抗体包括一条、两条或多条具有如SEQ ID NO:8和/或SEQ ID NO:14所示的氨基酸序列的VHH链。
典型的，所述抗IL-4R单域抗体包括两条具有如SEQ ID NO:8和/或SEQ ID NO:14中所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL-4R单域抗体包括四条具有如SEQ ID NO:8或SEQ ID NO:14中所示的氨基酸序列的VHH链。
典型的，所述抗IL-4R单域抗体具有如SEQ ID NO:8和/或SEQ ID NO:14中所示的氨基酸序列的VHH链。
典型的，所述抗IL-4R单域抗体包括两条具有如SEQ ID NO:14中所示的氨基酸序列的VHH链。
典型的，所述抗IL-4R单域抗体包括四条具有如SEQ ID NO:14中所示的氨基酸序列的VHH链。
在本发明的一个优选例中，所述两条具有如SEQ ID NO:8中所示的氨基酸序列的VHH链之间通过连接子进行连接。
在本发明的一个优选例中，所述两条具有如SEQ ID NO:14中所示的氨基酸序列的VHH链之间通过连接子进行连接。
在本发明的一个优选例中，所述连接子选自以下序列：(G aS b) x—(G mS n) y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
在本发明的一个优选例中，所述连接子选自下组：GGGGSGGGS(SEQ ID NO:18)。
在本发明的一个优选例中，所述二价抗IL-4R单域抗体的氨基酸序列如SEQ ID NO:16所示。
在本发明的一个优选例中，所述四价抗IL-4R单域抗体的氨基酸序列如SEQ ID NO:19所示。
标记的单域抗体
在本发明的一个优选例中，所述单域抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，IL-4R的单域抗体用胶体金标记，得到胶体金标记的单域抗体。
本发明的新型IL-4R单域抗体具有很好的特异性，很高的效价。
检测方法
本发明还涉及检测IL-4R蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中IL-4R蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测IL-4R水平的检测试剂盒，该试剂盒包括识别IL-4R蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的单域抗体有广泛生物应用价值和临床应用价值，其应用涉及到与IL-4R相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对IL-4R的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明单域抗体特异性结合具有正确空间结构的IL-4R蛋白。
(b)本发明单域抗体能够识别人、狨猴的IL-4R，不识别小鼠的IL-4R。
(c)本发明单域抗体相较于上市单抗Dupilumab具有更强的结合活性和阻断活性。
(d)本发明单域抗体能够有效抑制TF-1细胞的增殖抑制，且抑制活性显著高于上市单抗Dupilumab。
(e)本发明单域抗体能够有效抑制细胞中pSTAT6信号通路的激活，且抑制活性显著高于上市单抗Dupilumab。
(f)本发明单域抗体生产简便。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：抗人IL-4R单域抗体的筛选及表达
为了获得特异性针对人IL-4R的单域抗体，首先利用哺乳动物细胞HEK293F瞬转表达人IL-4R蛋白，经亲和纯化后用于骆驼免疫。具体方法可参见专利CN2018101517526实施例1和实施例2中的方法描述。简要地，将纯化的IL-4R蛋白免疫1只新疆双峰驼，7次免疫结束后从骆驼外周血中分离total RNA，随后反转录和PCR扩增出VHH基因，再将VHH基因克隆至噬菌体载体pMECS上，转化至TG1中构建成噬菌体展示文库。构建的文库库容如图1所示为1.1×10 9CFU，文库插入率如图2所示为95.8％。随后进行文库筛选，经3轮筛选获得含抗体基因的噬菌体富集。从文库挑选300颗克隆进行PE-ELISA鉴定，将获得的阳性克隆进行测序，再将序列不同的单域抗体利用E.coli系统瞬时表达，表达方法参见专利CN2018101521076实施例4中的方法描述。
实施例2：FACS筛选具有阻断活性的IL-4R单域抗体
(1)取一定数量的瞬转48h的293F/IL-4R细胞，1000rpm离心5min，弃 上清，用PBS重悬洗涤细胞一次，计数，分装100uL/孔至96孔板中，然后再3000rpm，4℃离心4min。(2)吸去上清，加入梯度稀释好的实施例1中序列不同的单域抗体(从40ug/mL开始2倍梯度稀释)，每孔加入50uL，再加入50uL 5ug/mL的IL-4-biotin混匀，4℃孵育20min；(3)3000rpm，4℃离心4min，弃上清，每孔加入200uL PBS洗涤细胞，再次离心弃上清；加入稀释好的SA-PE染色液于4℃孵育20min；(4)3000rpm，4℃离心4min，弃上清，每孔加入200uL PBS洗涤细胞，再次离心弃上清；再加入200uL PBS重悬细胞，上流式细胞仪检测样本PE信号。结果如图3所示，候选出一株阻断活性良好的IL-4R单域抗体Nb103(IC 50 Nb103＝0.274ug/mL)，其阻断活性优于对照抗体Dupilumab(IC 50 Dupilumab＝0.511ug/mL)。
实施例3：FACS检测候选单域抗体对TF-1细胞的增殖抑制作用
候选单域抗体对IL-4诱导的TF-1细胞的增殖抑制作用的检测方法如下：(1)取一定数量的TF-1细胞，1000rpm，5min离心，弃上清；加入PBS重悬，1000rpm离心5min洗涤一次；再加入适量PBS重悬，计数，稀释细胞溶液浓度至6×10 5/mL，分装细胞至96孔板。(2)分别将50uL梯度稀释(从20ug/mL开始三倍梯度稀释)的IgG1(阴性对照)、Dupilumab(阳性对照)、IL-4Ra Nb103抗体与50uL浓度为200ng/mL的IL-4蛋白混合，重悬收集的细胞，同时将所有细胞周边孔加100uL PBS，以防止有细胞孔溶液蒸发。(4)培养箱培养3天，3天后取出细胞加入CCK8溶液10uL/孔，置于37℃，显色4h。(5)显色结束后，用酶标仪读取各孔的OD450。结果如图4所示：IC 50 Nb103＝0.149ug/mL，IC 50  Dupilumab＝0.566ug/mL，因此候选单域抗体Nb103对TF-1细胞的增殖抑制作用显著强于对照抗体Dupilumab。
候选单域抗体对IL-13诱导的TF-1细胞的增殖抑制作用的检测方法同上，将待测稀释的抗体与工作浓度为10ng/mL的IL-13混合加入细胞。经检测其结果如图5所示：IC 50 Nb103＝0.066ug/mL，IC 50 Dupilumab＝0.740ug/mL，因此候选单域抗体Nb103对TF-1细胞的增殖抑制作用显著强于对照抗体Dupilumab。
实施例4：ELISA检测候选单域抗体的种属特异性
利用ELISA验证候选抗体能否与不同种属IL-4R蛋白发生交叉反应，方法如下：(1)将1ug/mL人IL-4R、鼠IL-4R、食蟹猴IL-4R加入酶标板中包被过夜，4℃，100uL/孔；(2)用PBST洗涤5次后，每孔加入300uL 1％BSA 室温封闭2小时；(3)用PBST洗涤5次后，加入100uL 2ug/mL待测抗体于37℃下孵育1小时；(4)用PBST洗涤5次后，加入100uL稀释的anti-HA antibody(1:2000稀释)于37下孵育1小时；(4)再用PBST洗涤5次后，加入100uL稀释的anti-mouse IgG antibody(1:2000稀释)于37℃下孵育30分钟；(5)用PBST洗涤5次后，加入加入PA显色液100uL，37℃显色10min，酶标仪405nm波长下测定吸收值。结果如图6所示：候选抗体Nb103能够识别人IL-4R，而不能识别鼠和食蟹猴IL-4R。
再次利用ELISA验证候选抗体能否与不同类型猴子的IL-4R蛋白发生交叉反应，检测方法同上，结果如图7所示：候选抗体Nb103能够识别狨猴IL-4R，不能识别恒河猴IL-4R。
实施例5：候选单域抗体的人源化改造
将候选抗体进行人源化改造，保持可变区不变，针对四个骨架区序列进行人源化设计，改造方法参见专利CN2018101517526中实施例4的方法。再将人源化后的抗体序列构建在pFUSE载体上，使人源化的单域抗体与Fc序列融合，形成huNb103(融合蛋白序列见SEQ ID NO:16，其编码核苷酸序列见SEQ ID NO:17)，利用HEK293F系统表达，表达后的蛋白可用于后续验证。表达的方法参见专利CN2018101517526实施例3。改造后的序列如下表2所示：
表2
Figure PCTCN2020106311-appb-000001
实施例6：FACS检测人源化抗体的结合活性
(1)收集高表达IL-4Rα的HEK293F稳转细胞，1000rpm离心5min，弃上清，用5mL PBS重悬，1000rpm离心5min，弃上清，再加2mL PBS重悬细胞，计数，将细胞分装至96孔板中，每孔3×10 5个细胞；(2)将人源化抗体huNb103(氨基酸序列见SEQ ID NO:16)和Dupilumab进行2倍梯度稀释(40ug/mL、20ug/mL、10ug/mL、5ug/mL、2.5ug/mL、1.25ug/mL、0.625ug/mL、0.313ug/mL、0.156ug/mL、0.078ug/mL)，用稀释好的抗体重悬分装在96孔板中的细胞，于4℃孵育20min；(3)3000rpm，4℃离心4min，加入200ul PBS/孔，重悬后再于3000rpm，4℃离心4min；(4)加入稀释好的anti-human Fc-FITC抗体(1:200稀释使用)，4℃孵育20min；(5)3000rpm，4℃离心4min，弃上清，加入200uL PBS/孔洗涤细胞，洗涤2次，再加入200uL PBS重悬细胞，转移至流式管中，流式细胞仪检测每个样本FITC信号。
结果如图8所示，人源化抗体huNb103的EC 50＝0.286ug/mL，对照抗体Dupilumab的EC 50＝0.360ug/mL。
实施例7：FACS检测人源化抗体的阻断活性
(1)收集高表达IL-4Rα的HEK293F稳转细胞，1000rpm离心5min，弃上清，用5mL PBS重悬，1000rpm离心5min，弃上清，再加2mL PBS重悬细胞，计数，将细胞分装至96孔板中，每孔3×10 5个细胞；(2)将人源化抗体huNb103和Dupilumab进行2倍梯度稀释(40ug/mL、20ug/mL、10ug/mL、5ug/mL、2.5ug/mL、1.25ug/mL、0.625ug/mL、0.313ug/mL、0.156ug/mL、0.078ug/mL)，将稀释的抗体分别与0.5ug IL-4-Biotin混合，然后重悬分装在96孔板中的细胞，于4℃孵育20min；(3)3000rpm，4℃离心4min，加入200ul PBS/孔，重悬后再于3000rpm，4℃离心4min；(4)加入稀释好的SA-PE染色液(0.3:100稀释使用)，4℃孵育20min；(5)3000rpm，4℃离心4min，弃上清，加入200uL PBS/孔洗涤细胞，洗涤2次，再加入200uL PBS重悬细胞，转移至流式管中，流式细胞仪检测每个样本PE信号。
结果如图9所示，人源化抗体huNb103的IC 50＝0.474ug/mL，对照抗体Dupilumab的IC 50＝1.126ug/mL。
实施例8：人源化四价抗体的构建及制备
为了进一步提高抗体的活性，设计进行多价体制备，构建的四价单域抗体结构如图10所示，对应的氨基酸序列如SEQ ID NO:19所示，其编码核苷酸序 列为SEQ ID NO:20。将含有SEQ ID NO:20所示的核苷酸序列合成至pCDNA3.1+载体上，然后用合成的质粒转染HEK293F细胞，转染方法参见专利CN2018101517526中实施例3。表达后的抗体用SEC-HPLC进行纯度检测，结果如图11所示，抗体纯度为90.69％，可用于后续研究。
实施例9：人源化四价抗体对TF-1细胞的增殖抑制作用
(1)将复苏培养的TF-1细胞于1000rpm离心5min，弃上清；用5mL PBS重悬，1000rpm再离心5min；5mL PBS重悬细胞计数，稀释细胞溶液浓度至6×10 5/mL，分装60uL/孔到96孔板中；(2)分别将50uL梯度稀释的IL-4Ra抗体(50ug/mL、12.5ug/mL、3.125ug/mL、0.781ug/mL、0.195ug/mL、0.049ug/mL、0.012ug/mL、0.003ug/mL)均与50uL 5ng/mL的IL-4蛋白混合后，取40uL混合液加到细胞溶液中；(3)同时向所有细胞周边孔补加200uL PBS，以防止有细胞孔溶液蒸发，置于37℃，5％CO 2培养箱培养72h；(4)取出细胞培养板，加入CCK8溶液10uL/孔，置于37℃，显色4h；(5)显色结束后，用酶标仪读取各孔的OD450值。
结果如图12所示：人源化四价抗体能够有效抑制IL-4诱导的TF-1细胞的增殖抑制，其抑制活性IC 50 tet-huNb103＝0.020ug/mL，其活性是人源化二价抗体的1-2倍(IC 50 huNb103＝0.032ug/mL)，也显著优于对照抗体对TF-1细胞的增殖抑制作用(IC 50 Dupilumab＝0.238ug/mL)。
同样地，人源化四价抗体对由IL-13诱导的TF-1细胞的增殖抑制作用也十分显著。检测时加入的IL-13浓度为10ng/mL，其余步骤同上。结果如图13所示：人源化四价抗体能够有效抑制IL-13诱导的TF-1细胞的增殖抑制，其抑制活性IC 50 tet-huNb103＝0.026ug/mL，其活性是人源化二价抗体的2-3倍(IC 50  huNb103＝0.059ug/mL)，也显著优于对照抗体对TF-1细胞的增殖抑制作用(IC 50  Dupilumab＝0.180ug/mL)。
实施例10：人源化四价抗体对HEK-Blue TM IL-4/IL-13细胞中pSTAT6信号通路的抑制作用检测
(1)将HEK-IL-4/IL-13 TM细胞于37℃5％CO 2条件下培养，待长至一定数目后收集细胞，用20mL PBS洗涤细胞两遍，再用10mL DMEM完全培养基重悬，取50uL台盼蓝染色液和50uL细胞溶液混合计数，稀释HEK-IL-4/IL-13 TM细胞溶液浓度至3×10 5/mL，分装160uL/孔至96孔细胞培养板；(2)将梯度稀 释的待测抗体(40ug/mL、10ug/mL、0.625ug/mL、0.156ug/mL、0.039ug/mL、0.010ug/mL、0.002ug/mL)分别与等体积5ng/mL IL-4蛋白混合；(3)取40uL混合液加入到分装好的细胞板中，同时向所有细胞周边孔补加200uL PBS，以防止有细胞孔溶液蒸发，置于37℃，5％CO 2培养箱孵育22h；(4)将QUANTI-Blue试剂加入96孔板中，180uL/孔，再加入20uL培养的细胞上清液于37℃孵育2h，酶标仪检测655nm处读值。
结果如图14所示：人源化四价抗体能够有效抑制IL-4诱导的HEK-Blue TM IL-4/IL-13细胞的pSTAT6信号通路，其抑制活性IC 50 tet-huNb103＝0.044ug/mL，其活性是人源化二价抗体的2-3倍(IC 50 huNb103＝0.109ug/mL)，也显著优于对照抗体对该细胞的pSTAT6信号通路作用(IC 50 Dupilumab＝0.346ug/mL)。
同样地，人源化四价抗体对由IL-13诱导的HEK-Blue TM IL-4/IL-13细胞的pSTAT6信号通路抑制作用也十分显著。检测时加入的IL-13浓度为10ng/mL，其余步骤同上。结果如图15所示：人源化四价抗体能够有效抑制IL-13诱导的HEK-Blue TM IL-4/IL-13细胞的pSTAT6信号通路，其抑制活性IC 50 tet-huNb103＝0.017ug/mL，其活性是人源化二价抗体的2倍(IC 50 huNb103＝0.034ug/mL)，也显著优于对照抗体对HEK-Blue TM IL-4/IL-13细胞的pSTAT6信号通路作用(IC 50  Dupilumab＝0.120ug/mL)。
实施例11：用hIL4/hIL4Ra转基因小鼠OVA模型验证人源化四价抗体药效
将15只动物按照体重随机分为3组，每组5只，组1为阴性对照组，组2为受试品低剂量组(5mpk)，组3为受试品高剂量组(25mpk)。1-3组的动物都进行OVA致敏及给药：配制200μg/mL OVA，腹腔注射致敏，200μL/只。致敏时间为第0、7、14天。在第21-25天，雾化吸入2％OVA激发，每次30min，连续5天。受试品在第20、23天给药，第25天激发操作结束次日采集样本检测分析。
结果如图16所示： 5mpk和25mpk两个剂量的人源化四价抗体均能够有效 抑制转基因小鼠OVA模型中血清中的OVA特异性的IgE的含量。
如图17A和17B所示： 5mpk和25mpk两个剂量的人源化四价抗体均能够 有效减少肺部嗜酸性粒细胞数目和比例。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献 被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明序列信息：
SEQ ID NO:1
Figure PCTCN2020106311-appb-000002
SEQ ID NO:2
Figure PCTCN2020106311-appb-000003
SEQ ID NO:3
Figure PCTCN2020106311-appb-000004
SEQ ID NO:4
Figure PCTCN2020106311-appb-000005
SEQ ID NO:5
Figure PCTCN2020106311-appb-000006
SEQ ID NO:6
Figure PCTCN2020106311-appb-000007
SEQ ID NO:7
Figure PCTCN2020106311-appb-000008
SEQ ID NO:8
Figure PCTCN2020106311-appb-000009
SEQ ID NO:9
Figure PCTCN2020106311-appb-000010
SEQ ID NO:10
Figure PCTCN2020106311-appb-000011
SEQ ID NO:11
Figure PCTCN2020106311-appb-000012
SEQ ID NO:12
Figure PCTCN2020106311-appb-000013
SEQ ID NO:13
Figure PCTCN2020106311-appb-000014
SEQ ID NO:14
Figure PCTCN2020106311-appb-000015
SEQ ID NO:15
Figure PCTCN2020106311-appb-000016
SEQ ID NO:16(SEQ ID NO:16＝SEQ ID NO:14+Fc片段)
Figure PCTCN2020106311-appb-000017
SEQ ID NO:17(编码huNb103的核苷酸序列)
Figure PCTCN2020106311-appb-000018
SEQ ID NO:18
Figure PCTCN2020106311-appb-000019
SEQ ID NO:19(SEQ ID NO:19＝SEQ ID NO:14+SEQ ID NO:18+SEQ ID NO:14+Fc片段)
Figure PCTCN2020106311-appb-000020
Figure PCTCN2020106311-appb-000021
SEQ ID NO: 20
Figure PCTCN2020106311-appb-000022

Claims (10)

  1. 一种抗IL-4R单域抗体VHH链的互补决定区CDR区，其特征在于，所述VHH链的互补决定区CDR包括SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3。
  2. 一种抗IL-4R单域抗体的VHH链，其特征在于，所述的VHH链包括框架区FR和权利要求1所述的互补决定区CDR，优选地，所述的框架区FR包括：
    (a)SEQ ID NO:4所示的FR1、SEQ ID NO:5所示的FR2、SEQ ID NO:6所示的FR3、和SEQ ID NO:7所示的FR4；或
    (b)SEQ ID NO:10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。
  3. 一种抗IL-4R单域抗体，其特征在于，它是针对白细胞介素4受体(IL-4R)表位的单域抗体，并且具有权利要求2所述的VHH链。
  4. 如权利要求3所述的抗IL-4R单域抗体，其特征在于，所述单域抗体包含一条或多条具有如SEQ ID NO:8或SEQ ID NO:14所示的氨基酸序列的VHH链。
  5. 一种抗IL-4R单域抗体Fc融合蛋白，其特征在于，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
    A-L-B(Ia)；
    B-L-A(Ib)；
    其中，
    A为权利要求3所述的抗IL-4R单域抗体；
    B为IgG的Fc片段；和
    L为无或柔性接头。
  6. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白质：权利要求1所述的抗IL-4R单域抗体VHH链的CDR区、权利要求2所述的抗IL-4R单域抗体的VHH链、权利要求3所述的抗IL-4R单域抗体、或权利要求5所述的抗IL-4R单域抗体Fc融合蛋白。
  7. 一种表达载体，其特征在于，所述表达载体含有权利要求6所述的多核苷酸。
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的表达载体， 或其基因组中整合有权利要求6所述的多核苷酸。
  9. 一种产生抗IL-4R单域抗体或其Fc融合蛋白的方法，其特征在于，包括步骤：
    (a)在适合产生单域抗体或其Fc融合蛋白的条件下，培养权利要求8所述的宿主细胞，从而获得含所述抗IL-4R单域抗体或其Fc融合蛋白的培养物；
    (b)从所述培养物中分离或回收所述的抗IL-4R单域抗体或其Fc融合蛋白；以及
    (c)任选地，纯化和/或修饰得步骤(b)中获得的抗IL-4R单域抗体或其Fc融合蛋白。
  10. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求2所述的抗IL-4R单域抗体的VHH链、或如权利要求3所述的抗IL-4R单域抗体、或如权利要求5所述的抗IL-4R单域抗体Fc融合蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
PCT/CN2020/106311 2019-10-31 2020-07-31 抗il-4r单域抗体及其应用 WO2021082573A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
US17/772,350 US12012456B2 (en) 2019-10-31 2020-07-31 Anti-interleukin-4 receptor (IL-4R) single-domain antibody, encoding polynucleotide and methods of making and using the antibody for IL-4R detection and disease treatment
JP2022525051A JP7387206B2 (ja) 2019-10-31 2020-07-31 抗ｉｌ－４ｒ単一ドメイン抗体およびその適用
AU2020377355A AU2020377355A1 (en) 2019-10-31 2020-07-31 Anti-IL-4R single-domain antibody and use thereof
BR112022007851A BR112022007851A2 (pt) 2019-10-31 2020-07-31 Anticorpo de domínio único anti-il-4r e uso do mesmo
EP20880575.4A EP4053161A1 (en) 2019-10-31 2020-07-31 Anti-il-4r single-domain antibody and use thereof
CA3156084A CA3156084A1 (en) 2019-10-31 2020-07-31 Anti-il-4r single-domain antibody and use thereof
KR1020227018317A KR20220091566A (ko) 2019-10-31 2020-07-31 항il-4r 단일 도메인 항체 및 이의 응용
IL292622A IL292622A (en) 2019-10-31 2020-07-31 Anti-il-4r antibodies targeting a single compound and their use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911054787.9 2019-10-31
CN201911054787.9A CN111825766B (zh) 2019-10-31 2019-10-31 抗il-4r单域抗体及其应用

Publications (1)

Publication Number Publication Date
WO2021082573A1 true WO2021082573A1 (zh) 2021-05-06

Family

ID=72912582

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/106311 WO2021082573A1 (zh) 2019-10-31 2020-07-31 抗il-4r单域抗体及其应用

Country Status (9)

Country Link
EP (1) EP4053161A1 (zh)
JP (1) JP7387206B2 (zh)
KR (1) KR20220091566A (zh)
CN (1) CN111825766B (zh)
AU (1) AU2020377355A1 (zh)
BR (1) BR112022007851A2 (zh)
CA (1) CA3156084A1 (zh)
IL (1) IL292622A (zh)
WO (1) WO2021082573A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113307869B (zh) * 2020-10-30 2023-01-06 上海洛启生物医药技术有限公司 抗il5纳米抗体及其应用
KR20240038841A (ko) * 2022-09-16 2024-03-26 연세대학교 산학협력단 신규한 인간 인터류킨-4 수용체 결합 나노바디 및 이의 용도

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092340A2 (en) * 2000-05-26 2001-12-06 Immunex Corporation Use of interleukin-4 antagonists and compositions thereof
CN105753987A (zh) * 2016-05-11 2016-07-13 贵阳中医学院 抗il-4r单链抗体的制备方法及其在抗肿瘤上的应用
CN105924531A (zh) * 2016-05-30 2016-09-07 贵阳中医学院 一种抗il-4r单链抗体与蜂毒肽融合蛋白及其应用
CN106267190A (zh) * 2008-10-29 2017-01-04 瑞泽恩制药公司 抗人il‑4受体的高亲和性人抗体
CN108373505A (zh) * 2018-04-20 2018-08-07 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途
CN108409860A (zh) * 2017-02-10 2018-08-17 上海麦济生物技术有限公司 抗人白细胞介素-4受体α单克隆抗体、其制备方法和应用
CN110105451A (zh) * 2018-02-01 2019-08-09 北京凯因科技股份有限公司 IL-4Rα抗体及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8808015D0 (en) * 1988-04-06 1988-05-05 Ritter M A Chemical compounds
EP4374919A2 (en) * 2012-09-07 2024-05-29 Regeneron Pharmaceuticals, Inc. Methods for treating atopic dermatitis by administering an il-4r antagonist

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001092340A2 (en) * 2000-05-26 2001-12-06 Immunex Corporation Use of interleukin-4 antagonists and compositions thereof
CN106267190A (zh) * 2008-10-29 2017-01-04 瑞泽恩制药公司 抗人il‑4受体的高亲和性人抗体
CN105753987A (zh) * 2016-05-11 2016-07-13 贵阳中医学院 抗il-4r单链抗体的制备方法及其在抗肿瘤上的应用
CN105924531A (zh) * 2016-05-30 2016-09-07 贵阳中医学院 一种抗il-4r单链抗体与蜂毒肽融合蛋白及其应用
CN108409860A (zh) * 2017-02-10 2018-08-17 上海麦济生物技术有限公司 抗人白细胞介素-4受体α单克隆抗体、其制备方法和应用
CN110105451A (zh) * 2018-02-01 2019-08-09 北京凯因科技股份有限公司 IL-4Rα抗体及其用途
CN108373505A (zh) * 2018-04-20 2018-08-07 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, CSHL PRESS

Also Published As

Publication number Publication date
US20220411519A1 (en) 2022-12-29
CN111825766B (zh) 2021-05-11
JP7387206B2 (ja) 2023-11-28
EP4053161A1 (en) 2022-09-07
IL292622A (en) 2022-07-01
CA3156084A1 (en) 2021-05-06
AU2020377355A1 (en) 2022-06-09
KR20220091566A (ko) 2022-06-30
JP2023501182A (ja) 2023-01-18
BR112022007851A2 (pt) 2022-07-05
CN111825766A (zh) 2020-10-27

Similar Documents

Publication Publication Date Title
WO2021042694A1 (zh) 抗vegf单域抗体及其应用
WO2022089108A1 (zh) 抗il5纳米抗体及其应用
WO2020119728A1 (zh) 抗人白细胞介素5(il-5)单克隆抗体及其应用
WO2023142309A1 (zh) 抗tslp纳米抗体及其应用
WO2021082573A1 (zh) 抗il-4r单域抗体及其应用
WO2023169583A1 (zh) 基于Pep42构建的双特异性细胞接合器分子的制备及其应用
WO2023274365A1 (zh) 抗trop2单域抗体及其应用
WO2021047386A1 (zh) 靶向caix抗原的纳米抗体及其应用
US12012456B2 (en) Anti-interleukin-4 receptor (IL-4R) single-domain antibody, encoding polynucleotide and methods of making and using the antibody for IL-4R detection and disease treatment
WO2024099310A1 (zh) 抗il-13长效纳米抗体序列及其应用
WO2023116751A1 (zh) 抗人血管生成素3纳米抗体及其应用
WO2023020551A1 (zh) 抗ptk7单域抗体及其应用
WO2023133842A1 (zh) 一种靶向IL-18Rβ的抗体及其应用
CN115873113B (zh) 靶向CaSR的纳米抗体及其制备方法和应用
WO2023125975A1 (zh) 一种新型靶向人flt3的嵌合抗原受体修饰的t细胞的构建及应用
WO2023143535A1 (zh) 一种靶向il-18bp的抗体及其应用
WO2024032750A1 (zh) 抗cgrp抗体及用途
WO2023208104A1 (zh) 抗人il-4ra的抗体及其用途
WO2023134767A1 (zh) 一种靶向IL-18Rβ的抗体及其应用
CN116554322A (zh) 一种靶向IL-18Rβ的抗体及其应用
CN117186226A (zh) 抗cd38纳米抗体及其应用

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: 20880575

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3156084

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022525051

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 292622

Country of ref document: IL

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112022007851

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20227018317

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020377355

Country of ref document: AU

Date of ref document: 20200731

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2020880575

Country of ref document: EP

Effective date: 20220531

ENP Entry into the national phase

Ref document number: 112022007851

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20220425