WO2024099310A1 - 抗il-13长效纳米抗体序列及其应用 - Google Patents

抗il-13长效纳米抗体序列及其应用 Download PDF

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WO2024099310A1
WO2024099310A1 PCT/CN2023/130165 CN2023130165W WO2024099310A1 WO 2024099310 A1 WO2024099310 A1 WO 2024099310A1 CN 2023130165 W CN2023130165 W CN 2023130165W WO 2024099310 A1 WO2024099310 A1 WO 2024099310A1
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antibody
nanobody
seq
present
antibodies
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PCT/CN2023/130165
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French (fr)
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万亚坤
朱敏
盖军伟
李光辉
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上海洛启生物医药技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • A61K47/6845—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 cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—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
    • C07K2317/30—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
    • C07K2317/50—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
    • C07K2317/76—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
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5437—IL-13
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    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-IL-13 long-acting nano antibody sequence and application thereof.
  • Interleukin-13 is an important member of the interleukin family. It is a protein encoded by the IL-13 gene with a molecular weight of about 10KD. IL-13 can be secreted by a variety of cells, such as CD4+T cells, CD8+T cells, mast cells, basophils, eosinophils and natural killer cells. Especially type 2 helper T cells (Th2). IL-13 has a variety of biological effects on mononuclear macrophages, B lymphocytes, NK cells and vascular endothelial cells, and is a mediator of the body's immune response.
  • Th2 cells produce a series of cytokines such as IL-4, IL-5, IL-9 and IL-13, among which IL-13 is considered to be the most core factor and is involved in almost all concurrent processes of Th2 inflammation.
  • IL-13 plays a central role in the pathogenesis of Th2 inflammation.
  • IL-13 is associated with autoimmune diseases related to Th2 immune response, such as asthma, atopic dermatitis (AD), chronic obstructive pulmonary disease (COPD), allergic rhinitis, inflammatory bowel disease, etc.
  • IL-13 has been involved in clinical research related biological agents, such as Adtralza (Tralokinumab, already on the market) from LEO Pharma, which is under investigation for atopic dermatitis, alopecia areata, asthma, ulcerative colitis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease; lebrikizumab (clinical phase III under study) from Roche, which is under investigation for atopic dermatitis, eczema, asthma, Hodgkin's lymphoma, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease; and Cendakimab (clinical phase II under study) from BMS, which is
  • IL-13 and its signaling pathway have demonstrated their important clinical value as promising therapeutic targets.
  • a nanobody (Nanobody) derived from a naturally missing light chain in camelids is the smallest known antigen-binding fragment.
  • Antibody drug research based on its characteristic advantages has gradually attracted attention. Therefore, the development of nanobodies targeting IL-13 targets will have great clinical value and provide new ideas for the treatment of autoimmune diseases such as asthma and atopic dermatitis.
  • the purpose of the present invention is to provide a high-affinity and high-specificity anti-IL-13 long-acting nano antibody.
  • Another object of the present invention is to provide the application of anti-IL-13 long-acting nanobody.
  • an anti-IL-13 nanobody wherein the complementary determining region (CDR) region of the VHH chain in the nanobody is one or more selected from the following groups:
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the VHH chain further comprises a framework region FR, and the framework region FR is one or more selected from the following group:
  • the CDR region of the nanoantibody VHH chain comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% to any one of SEQ ID NO: 2, 4 and 6, SEQ ID NO: 11, 13 and 15.
  • the amino acid sequence of the CDR region of the nanoantibody VHH chain contains one or more amino acid substitutions compared to any one of SEQ ID NO: 2, 4 and 6, SEQ ID NO: 11, 13 and 15, preferably conservative amino acid substitutions.
  • any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and/or substituted with at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to specifically bind to IL-13.
  • the nanobody can specifically bind to IL-13.
  • the IL-13 is IL-13 from human or non-human mammals.
  • the IL-13 is IL-13 from humans, mice, rats, or non-human primates (such as monkeys).
  • the nanobodies include humanized antibodies, camel-derived antibodies, and chimeric antibodies.
  • amino acid sequence of the VHH chain of the nanoantibody is selected from the following group: SEQ ID NO: 8, SEQ ID NO: 17, or a combination thereof.
  • the anti-IL-13 nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the anti-IL-13 nanobody comprises one or more sequences having SEQ ID NO: 8.
  • the anti-IL-13 nanoantibody comprises two VHH chains having amino acid sequences shown in SEQ ID NO:8 and SEQ ID NO:17.
  • the VHH chains are connected via a connecting peptide.
  • sequence of the connecting peptide is shown as SEQ ID NO:28.
  • an anti-IL-13 antibody which is an antibody against an IL-13 epitope and has the anti-IL-13 nanobody described in the first aspect of the present invention.
  • the anti-IL-13 antibody comprises one or more anti-IL-13 nanobodies.
  • the anti-IL-13 antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the anti-IL-13 antibody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:17.
  • the anti-IL-13 antibody comprises two VHH chains having an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO:17.
  • the antibody can specifically bind to IL-13.
  • the antibody is capable of effectively blocking the release of SEAP in IL-13-induced HEK-BlueTM IL-4/IL-13 cells, and the blocking activity is significantly better than that of the control antibody Lebrikizumab.
  • the antibody can effectively inhibit the proliferation of HEK-BlueTM IL-4/IL-13 cells, and its inhibitory activity is better than that of the control antibody 4B06.
  • the antibody is a nanobody.
  • a polynucleotide which encodes a protein selected from the following group: the anti-IL-13 nanobody described in the first aspect of the present invention or the anti-IL-13 antibody described in the second aspect of the present invention.
  • polynucleotides are in a combined form.
  • polynucleotide sequence contains one or more of the sequences shown in SEQ ID NO:9 or 18.
  • the polynucleotide includes RNA, DNA or cDNA.
  • an expression vector is provided, wherein the expression vector contains the polynucleotide described in the third aspect of the present invention.
  • the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, Transposons, other gene transfer systems, or combinations thereof.
  • the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, or retrovirus.
  • a host cell contains the expression vector described in the fourth aspect of the present invention, or the polynucleotide described in the third aspect of the present invention is 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, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.
  • the host cell is Pichia pastoris.
  • a method for producing an anti-IL-13 nanobody comprising the steps of:
  • step (c) Optionally, purifying and/or modifying the anti-IL-13 nanobody obtained in step (b).
  • an immunoconjugate comprising:
  • a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.
  • the radioactive nuclides include:
  • a diagnostic isotope selected from the group consisting of 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 isotopes wherein the therapeutic isotopes are selected from the group consisting of 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 following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.
  • examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors
  • typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or a combination thereof.
  • PBDs pyrrolo[1,4]benzodiazepines
  • indolinobenzodiazepines and oxazolidinobenzodiazepines vinca alkaloids, or a combination thereof.
  • the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansyl, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, leuprorelin, dactinomycin ...
  • auristatins e.g., auristatin E, auristatin F, MMAE and MMAF
  • chlortetracycline maytansyl
  • ricin e.g., auristatin E, auri
  • the coupling moiety is a detectable label.
  • the coupling portion is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, 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
  • a multispecific antibody comprises: the anti-IL-13 nanobody described in the first aspect of the present invention, or the anti-IL-13 antibody described in the second aspect of the present invention.
  • the multispecific antibody further comprises an antibody Fc segment.
  • a recombinant protein is provided, wherein the recombinant protein has:
  • the tag sequence includes an Fc tag, an HA tag and a 6His tag.
  • the recombinant protein specifically binds to the IL-13 protein.
  • a pharmaceutical composition comprising:
  • the conjugated portion of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
  • the pharmaceutical composition also contains other drugs for treating immune system diseases or tumor diseases.
  • the other drugs for treating immune system diseases or tumor diseases are selected from the following group: budesonide, fluticasone, beclomethasone, mometasone furoate, salbutamol, theophylline, formoterol, tiotropium bromide, sulfasalazine, methotrexate, cyclophosphamide, fluorouracil, bleomycin, anastrozole, or a combination thereof.
  • the pharmaceutical composition is used to prepare a drug for preventing and/or treating a disease or condition associated with IL-13.
  • the disease or disorder associated with IL-13 includes an immune system disease.
  • the immune system disease is selected from the following group: asthma, atopic dermatitis (AD), chronic obstructive pulmonary disease (COPD), allergic rhinitis, inflammatory bowel disease, alopecia areata, ulcerative colitis, idiopathic pulmonary fibrosis, eczema, Hodgkin's lymphoma, idiopathic pulmonary fibrosis, eosinophilic esophagitis, or a combination thereof.
  • AD atopic dermatitis
  • COPD chronic obstructive pulmonary disease
  • allergic rhinitis rhinitis
  • inflammatory bowel disease alopecia areata
  • ulcerative colitis idiopathic pulmonary fibrosis
  • eczema Hodgkin's lymphoma
  • idiopathic pulmonary fibrosis eosinophilic esophagitis
  • the disease or disorder associated with IL-13 includes an immune system disease.
  • the immune system disease is selected from the group consisting of asthma, atopic dermatitis (AD), Chronic obstructive pulmonary disease (COPD), allergic rhinitis, inflammatory bowel disease, alopecia areata, ulcerative colitis, idiopathic pulmonary fibrosis, eczema, Hodgkin's lymphoma, idiopathic pulmonary fibrosis, eosinophilic esophagitis, or a combination thereof.
  • AD atopic dermatitis
  • COPD Chronic obstructive pulmonary disease
  • allergic rhinitis rhinitis
  • inflammatory bowel disease alopecia areata
  • ulcerative colitis idiopathic pulmonary fibrosis
  • eczema Hodgkin's lymphoma
  • idiopathic pulmonary fibrosis eosinophilic esophagitis
  • the IL-13 is human IL-13.
  • the reagent is a diagnostic reagent.
  • the diagnostic agent is a contrast agent
  • the reagent is used to detect IL-13 protein or a fragment thereof in a sample.
  • the detection includes flow cytometry detection and cell immunofluorescence detection.
  • the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
  • a method for detecting IL-13 protein in a sample comprising the steps of:
  • the method is a non-diagnostic and non-therapeutic method.
  • an IL-13 protein detection reagent comprising:
  • the conjugated portion of the immunoconjugate is a diagnostic isotope.
  • test-acceptable carrier is a non-toxic, inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.
  • the detection reagent is used for in vivo detection.
  • the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal tablet, inhaler).
  • kits for detecting IL-13 protein comprising the immunoconjugate according to the seventh aspect of the present invention or the detection reagent according to the thirteenth aspect of the present invention, and instructions.
  • the instructions state that the kit is used for non-invasively detecting the expression of IL-13 in a subject to be tested.
  • the fifteenth aspect of the present invention there is provided a use of the immunoconjugate described in the seventh aspect of the present invention for preparing a contrast agent for detecting IL-13 protein in vivo.
  • the detection is used for the diagnosis or prognosis of a disease or disorder associated with IL-13.
  • a method for treating a disease associated with IL-13 comprising administering to a subject in need thereof the anti-IL-13 nanobody described in the first aspect of the present invention, the anti-IL-13 antibody described in the second aspect of the present invention, the immunoconjugate described in the seventh aspect of the present invention, the multispecific antibody described in the eighth aspect of the present invention, the recombinant protein described in the ninth aspect of the present invention, or the pharmaceutical composition described in the tenth aspect of the present invention.
  • the subject includes a human or a non-human mammal.
  • the non-human mammals include rodents (such as mice and rabbits) and non-human primates (such as monkeys).
  • FIG1 shows the ELISA method for detecting the binding activity of candidate antibodies to human IL-13 and cynomolgus monkey IL-13.
  • the results show that MY9217 and MY9219 can bind to human IL-13 and cynomolgus monkey IL-13 at the same time.
  • the inventors have conducted extensive and in-depth research and, after a large number of screenings, unexpectedly obtained anti-IL-13 nanoantibodies with excellent affinity and specificity.
  • the experimental results show that the nanobody of the present invention has excellent functional activity, can effectively block the release of SEAP in HEK-BlueTM IL-4/IL-13 cells induced by IL-13, and has better activity than the control antibodies Lebrikizumab and 4B06.
  • the nanobody of the present invention has a good half-life in vivo, can effectively bind to human serum albumin, and has better binding activity than the control antibody 3 (HuNb3-11).
  • the nanobody of the present invention can simultaneously bind to human IL-13 and cynomolgus monkey IL-13. On this basis, the inventors have completed the present invention.
  • binding refers to a soluble receptor or a fragment or an analog thereof, or an antibody or a fragment or an analog thereof that can bind to a target.
  • the "IL-13 binder” of the present invention refers to an antibody or a fragment or an analog thereof that can specifically recognize IL-13 and bind to the IL-13 antigen.
  • the term “optional” or “optionally” means that the event or situation described subsequently may occur but need not occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may have but need not have, and may be 1, 2 or 3.
  • Interleukin-13 (IL-13)
  • Interleukin-13 is an important member of the interleukin family. It is a protein encoded by the IL-13 gene with a molecular weight of about 10KD. IL-13 has multiple biological effects on monocytes, macrophages, B lymphocytes, NK cells and vascular endothelial cells, and is a mediator of the body's immune response.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. 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.
  • VL variable region
  • Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
  • the terms "single domain”, “VHH”, “nanobody”, “recombinant “Single domain antibody, sdAb, or nanobody” have the same meaning and can be used interchangeably, referring to cloning the variable region of the antibody heavy chain to construct a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions.
  • a nanobody VHH
  • an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.
  • the term "antigen-binding fragment” refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment that has antigen-binding activity.
  • the Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but does not have a constant region, and is the smallest antibody fragment with all antigen-binding sites.
  • the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.
  • antigenic determinant refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombinant techniques well known in the art.
  • the term "monoclonal antibody” refers to an antibody secreted from a clone of a single cell source. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope.
  • the cell may be a eukaryotic, prokaryotic, or phage clone.
  • chimeric antibody refers to an antibody molecule that is spliced from the V region gene of a mouse antibody and the C region gene of a human antibody, and then inserted into a vector and transfected into a host cell for expression. It retains the high specificity and affinity of the parent mouse antibody, and enables its human Fc segment to effectively mediate biological effect functions.
  • humanized antibody is a variable region modified form of the mouse antibody of the present invention, having a CDR region derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody), and a FR region and a constant region substantially derived from a human antibody sequence; that is, the CDR region sequence of the mouse antibody is grafted onto different types of human germline antibody framework sequences. Because the CDR sequence is responsible for most of the antibody-antigen interaction, recombinant antibodies that mimic the properties of specific naturally occurring antibodies can be expressed by constructing an expression vector. In the present invention, antibodies can be monospecific, bispecific, trispecific, or more multispecific.
  • variable means that certain parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conservative parts of the variable region are called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • the variable regions of natural heavy and light chains each contain four FR regions, which are generally in a b-fold configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial b-fold structure.
  • the CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)).
  • the constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments thereof of the present invention to form conjugates.
  • the present invention also includes cell surface markers or antigens combined with the anti-IL-13 antibodies 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 above-mentioned heavy chain variable region and heavy chain constant region.
  • antibodies of the present invention are used interchangeably, and all refer to polypeptides that specifically bind to IL-13 proteins, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.
  • the present invention also provides other proteins or fusion expression products having the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.
  • variable region This segment is divided into four framework regions (FR).
  • FR framework regions
  • the amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the ⁇ -folds formed by the FRs in between are close to each other in spatial structure.
  • the CDRs on the heavy chain and the CDRs on the corresponding 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 region.
  • variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.
  • the present invention includes not only complete antibodies, but also fragments of antibodies or antibodies and 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 antibodies of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a
  • the antibody of the present invention refers to a polypeptide having IL-13 binding activity and comprising the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides comprising the above-mentioned CDR region and having the same function as the antibody of the present invention. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • substitution is made with amino acids with similar or similar properties, the function of the protein is usually not changed.
  • adding one or several amino acids at 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 present invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof.
  • the invention also includes fragments of nanobodies of the invention.
  • the fragment has at least about 50 consecutive amino acids of an antibody of the 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 antibodies of the present invention” refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table A.
  • the present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein.
  • the polynucleotide of the present invention can be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or artificially synthesized DNA.
  • the DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may include a polynucleotide further including additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned 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 relatively low ionic strength and relatively high temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short.
  • a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • biomolecules nucleic acids, proteins, etc.
  • the biomolecules involved in the present invention include biomolecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • 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 such as CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be carried out using 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 CaCl2 method, the steps used are well known in the art.
  • Another method is to use MgCl2.
  • transformation can also be carried out using electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow 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, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. 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 protein precipitants (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.
  • Therapeutic agents that can be combined or coupled with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • Nanoantibodies of the present invention As used herein, the terms “Nanoantibodies of the present invention”, “Nanoantibodies of the present invention”, “anti-IL-13 Nanoantibodies of the present invention”, “IL-13 Nanoantibodies of the present invention”, “anti-IL-13 Nanoantibodies”, and “IL-13 Nanoantibodies” have the same meaning and are used interchangeably, all referring to Nanoantibodies that specifically recognize and bind to IL-13 (including human IL-13).
  • IL-13 refers to the IL-13 protein, whose amino acid sequence is shown in SEQ ID No:1.
  • the anti-IL-13 nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the anti-IL-13 nanobody comprises two VHH chains having amino acid sequences as shown in SEQ ID NO:8 and SEQ ID NO:17.
  • the VHH chains are connected via a connecting peptide.
  • sequence of the connecting peptide is GGGGSGGGGSGGGGSGGGGS.
  • the CDR region of the nanoantibody VHH chain comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% to any one of SEQ ID NO: 2, 4 and 6, SEQ ID NO: 11, 13 and 15.
  • the amino acid sequence of the CDR region of the nanoantibody VHH chain contains one or more amino acid substitutions compared to any one of SEQ ID NO: 2, 4 and 6, SEQ ID NO: 11, 13 and 15, preferably conservative amino acid substitutions.
  • the nanobody carries a detectable marker. More preferably, the marker is selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker.
  • colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the nanobody against IL-13 is labeled with colloidal gold to obtain a colloidal gold labeled nanobody.
  • any method suitable for producing antibodies can be used to produce the nanobodies of IL-13 of the present invention.
  • animals can be immunized with linked or naturally occurring IL-13 or fragments thereof.
  • Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • IL-13 can be used as an immunogen (antigen) to produce non-human antibodies specific for IL-13 and screen the biological activity of the antibodies.
  • the stimulating immunogen can be recombinant IL-13 or a fragment thereof.
  • the immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.
  • the immunogen can be purified from a natural source or produced in a genetically modified cell.
  • the DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin.
  • the DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to: an adenoviral vector, an adeno-associated viral vector, a baculovirus vector, a plasmid and a non-viral vector.
  • a suitable genetic vector including but not limited to: an adenoviral vector, an adeno-associated viral vector, a baculovirus vector, a plasmid and a non-viral vector.
  • Example 1 An exemplary method for producing an anti-IL-13 antibody of the invention is described in Example 1.
  • the antibodies of the present invention can be selected from any type of immunoglobulin of any species, including IgG and IgE.
  • Preferred antibodies are IgG antibodies, such as IgG1 subtype. Optimization of the necessary constant domain sequences is easily achieved by screening antibodies with the biological assays described in the examples below to produce the desired biological activity.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof can be used in the compounds and methods of the invention.
  • sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
  • the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.
  • the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize the relevant sequence, especially when the fragment length is short.
  • a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.
  • the step of transforming host cells with recombinant DNA described in the present invention can be carried out by techniques well known in the art.
  • the obtained transformants can be cultured by conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention.
  • conventional culture medium is used to cultivate under suitable conditions.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention.
  • the antibodies of the present invention can be purified by conventional separation and purification methods well known to those skilled in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
  • the resulting monoclonal antibodies can be characterized by conventional means.
  • the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the present invention also provides a pharmaceutical composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the properties of the formulated substance and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, or intracavitary injection.
  • the term "pharmaceutical composition” means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to more stably exert the therapeutic effect. These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while also protecting the multifunctional groups of the protein to prevent its degradation (including but not limited to aggregation, deamination or oxidation).
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned bispecific antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically 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 an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably 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.
  • the bispecific antibodies of the present invention can also
  • a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases does not exceed about 50 mg/kg body weight, preferably the dose is about 10 ⁇ g/kg body weight to about 10 mg/kg body weight.
  • the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.
  • the present invention also relates to a method for detecting IL-13 protein.
  • the method generally comprises the following steps: obtaining cells and/or tissues dissolving the sample in a medium; and detecting the level of IL-13 protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.
  • the antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.
  • the sample (specimen) used includes cells, tissue samples and biopsy specimens.
  • the term "biopsy” used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or fragment thereof) of the present invention.
  • the kit further comprises a container, instructions for use, a buffer, etc.
  • the antibody of the present invention can be fixed to a detection plate.
  • the nanobodies of the present invention have a wide range of biological and clinical application values, and their applications involve the diagnosis and treatment of diseases or conditions related to IL-13, basic medical research, biological research, and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy of IL-13.
  • Nanobodies of the present invention have good functional activity, and their activity is better than that of the control antibody Lebrikizumab.
  • the nanoantibody of the present invention can effectively block the release of SEAP in HEK-BlueTM IL-4/IL-13 cells induced by IL-13, and its activity is superior to that of the control antibody Lebrikizumab.
  • Nanobodies of the present invention can be produced using a microbial expression system with low production costs.
  • the high-purity protein IL-13-Fc was mixed with an immune adjuvant to immunize Xinjiang Bactrian camels. After 7 immunizations, Camel peripheral blood was collected to detect the antibody titer in the serum. When the antibody titer was greater than 1:1000, the immunization was considered successful.
  • PBMC was isolated from camel peripheral blood and total RNA was extracted. After reverse transcription into cDNA, VHH fragments were amplified using nested PCR, cloned and connected into phage display vectors, and transfected into TG1 cells to construct a phage display nanoantibody library. After three rounds of "adsorption-washing-binding" process, IL-13 specific nanoantibody phage was enriched. 600 monoclonal clones were randomly selected for supernatant ELISA identification.
  • the above 39 nanobodies were purified from Escherichia coli strains to obtain high-purity antibodies, and the functional activity of the antibodies was detected using HEK-BlueTM IL4/IL-13 cells (purchased from Invivogen).
  • HEK-Blue TM IL-4/IL-13 cells produce SEAP under IL-13 stimulation, and the level of STAT6-induced SEAP in the supernatant is measured using QUANTI-Blue TM Solution.
  • the stimulation of HEK-Blue TM IL-4/IL-13 cells by recombinant human IL-13 can be blocked by neutralizing IL-13 antibodies.
  • the functional activity of the above 39 candidate antibodies was detected using this method.
  • HEK-Blue TM IL-4/IL-13 cells were resuspended in DMEM medium containing 1% FBS, counted, and dispensed into 96-well plates. Pre-mixed antibodies and IL-13 proteins were added and cultured in an incubator for 24 hours. The cultured cell supernatant was removed and nine times the amount of QUANTI-Blue reagent was added and incubated at 37°C for 3 hours. The absorbance at 620 nm was detected by an enzyme reader.
  • control VHH1 is the antibody sequence derived from 4B06 in patent WO2021116182A1.
  • the above antibodies with good cell activity were humanized and designed.
  • the humanization method was based on the method of Example 4 in patent CN2018101517526.
  • the humanized sequence of the framework region of each nanobody was humanized, and the variable region was kept unchanged.
  • the humanized antibody sequence was optimized according to the Pichia pastoris codon.
  • the base sequence of was synthesized into the yeast expression vector pPICZaA.
  • the recombinant plasmid with the antibody gene inserted was linearized and electroporated into Pichia pastoris X33 competent cells. Then, a single clone was picked from the resistance plate, induced by methanol, and the expression level of the antibody in the culture medium was detected.
  • the test results are shown in Table 2.
  • the nanoantibodies were purified from the Pichia pastoris fermentation supernatant and used to identify the functional activity of HEK-BlueTM IL-4/IL-13 cells.
  • the above 8 humanized antibodies were combined into bivalents in pairs, and a total of 64 bivalent antibodies were constructed.
  • the two monomers were connected by a GS20 linker (amino acid sequence such as SEQ ID NO: 28).
  • the bivalent antibody base sequence optimized by Pichia pastoris codon was cloned into the pPICZaA vector.
  • the linearized recombinant plasmid was electroporated into Pichia pastoris X33 competent cells, and then a single clone was picked from the resistance plate, induced by methanol, and the expression of the antibody in the culture medium was detected.
  • the above 11 humanized bivalent nanoantibodies were purified from the Pichia pastoris fermentation supernatant and used for the functional activity identification of HEK-BlueTM IL-4/IL-13 cells.
  • nanobodies In order to make the above nanobodies have a good half-life in vivo, anti-human serum albumin nanobody HuNb3-11 was added to them. Long-acting nanobodies with different structures were constructed, and their yield and activity were evaluated after expression. The preparation and evaluation methods were the same as in Example 4.
  • MY9217 and MY9219 are preferred for IL-13 functional activity identification and serum albumin binding activity assessment.
  • the trivalent nanobody purified from the fermentation broth is first subjected to IL-13 functional activity identification, and the detection method is the same as in Example 2.
  • results are shown in Table 2.
  • the results show that the two nanoantibodies can effectively block the release of SEAP in HEK-BlueTM IL-4/IL-13 cells induced by IL-13, and their activity is better than that of the control antibody 2 (Lebrikizumab, Roche).
  • MY9217 and MY9219 can effectively bind to human serum albumin, and their binding activity is better than that of the control antibody 3 (HuNb3-11).
  • the binding activity of candidate antibodies to human IL-13 and cynomolgus monkey IL-13 was detected by ELISA.
  • Human IL-13 and cynomolgus monkey IL-13 proteins were coated on ELISA plates (4°C overnight) and the blocking reaction was followed by Add the gradient diluted antibodies MY9127 and MY9219 to react at 37°C for 1 hour, then add the diluted HRP-sheep anti-nanoantibody polyclonal antibody (1:2000 dilution) to react at 37°C for 1 hour, add TMB colorimetric solution and stop with H 2 SO 4 before reading the absorbance.

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Abstract

一种抗IL-13长效纳米抗体，编码抗IL-13纳米抗体的编码序列、相应的表达载体和能够表达该纳米抗体的宿主细胞，以及纳米抗体的生产方法。

Description

抗IL-13长效纳米抗体序列及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及抗IL-13长效纳米抗体序列及其应用。
背景技术
白细胞介素-13(Interleukin-13)是白介素家族的重要成员，是由IL-13基因编码生成的蛋白质，分子量约10KD。IL-13可由多种细胞分泌，如CD4+T细胞、CD8+T细胞、肥大细胞、嗜碱性粒细胞、嗜酸性粒细胞和自然杀伤细胞。特别是2型辅助性T细胞(Th2)。IL-13对单核巨噬细胞、B淋巴细胞、NK细胞和血管内皮细胞等具有多种生物学作用，是机体免疫反应的调节的介质。在Th2炎症中，Th2细胞产生一系列细胞因子如IL-4、IL-5、IL-9和IL-13等，其中IL-13被认为是最为核心的因子，几乎参与到Th2炎症所有的并发过程。目前已经有大量试验证明，IL-13在Th2炎症发病机制中起到中轴的作用。
多项研究表明IL-13与Th2免疫应答相关的自身免疫性疾病有关，例如哮喘、特异性皮炎(AD)、慢性阻塞性肺疾病(COPD)、变应性鼻炎、炎性肠病等。IL-13作为自身免疫性疾病的靶点之一，已有相关的生物制剂进入临床研究，例如：LEO Pharma公司的Adtralza(Tralokinumab，已上市)，在研适应症为特应性皮炎、斑秃、哮喘、溃疡性结肠炎、特发性肺纤维化、慢性阻塞性肺疾病；罗氏的lebrikizumab(临床III期在研)，在研适应症为特应性皮炎、湿疹、哮喘、霍奇金淋巴瘤、特发性肺纤维化、慢性阻塞性肺疾病；BMS的Cendakimab(临床II期在研)，在研适应症为嗜酸粒细胞性食管炎、哮喘。
众多因子中，IL-13及其信号途径作为有前景的治疗靶位点展现了其在临床上的重要价值。基于此，应用IL-13的对抗物阻断Th2相关疾病中大量表达的IL-13的生物活性，对与改善Th2炎症应该是一个好的方法。来源于骆驼科动物体内的一种天然缺失轻链的纳米抗体(Nanobody)，是目前已知最小抗原结合片段，基于其特性优势开展的抗体药物研究逐渐受到关注。因此，研发针对IL-13靶点的纳米抗体，将具有较大的临床价值，为哮喘、特异性皮炎等自身免疫性疾病的治疗提供新思路。
综上所述，本领域仍需开发出一种高亲和力和高特异性的抗IL-13长效纳米抗体。
发明内容
本发明的目的是提供一种高亲和力和高特异性的抗IL-13长效纳米抗体。
本发明的另一目的是提供抗IL-13长效纳米抗体的应用。
在本发明的第一方面，提供了一种抗IL-13纳米抗体，所述纳米抗体中的VHH链的互补决定区CDR区为选自下组的一种或多种：
(1)SEQ ID NO:2所示的CDR1、SEQ ID NO:4所示的CDR2、和SEQ ID NO:6所示的CDR3；和
(2)SEQ ID NO:11所示的CDR1、SEQ ID NO:13所示的CDR2、和SEQ ID NO:15所示的CDR3。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述VHH链还包括框架区FR，所述的框架区FR为选自下组的一种或多种：
(1)SEQ ID NO:1所示的FR1、SEQ ID NO:3所示的FR2、SEQ ID NO:5所示的FR3、和SEQ ID NO:7所示的FR4；和
(2)SEQ ID NO:10所示的FR1、SEQ ID NO:12所示的FR2、SEQ ID NO:14所示的FR3、和SEQ ID NO:16所示的FR4。
在另一优选例中，所述的纳米抗体VHH链的CDR区包含与SEQ ID NO:2、4和6、SEQ ID NO:11、13和15任一具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述的纳米抗体VHH链CDR区的氨基酸序列与SEQ ID NO:2、4和6、SEQ ID NO:11、13和15中任一相比包含一个或多个氨基酸取代，优选保守氨基酸取代。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与IL-13特异性结合能力的衍生序列。
在另一优选例中，所述纳米抗体能够特异性结合IL-13。
在另一优选例中，所述IL-13为人或非人哺乳动物的IL-13。
在另一优选例中，所述IL-13为人、小鼠、大鼠、或非人灵长类动物(如猴)的IL-13。
在另一优选例中，所述的纳米抗体包括人源化抗体、骆驼源抗体、嵌合抗体。
在另一优选例中，所述纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:8、SEQ ID NO:17、或其组合。
在另一优选例中，所述抗IL-13纳米抗体包括单体、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-13纳米抗体包括一条或多条具有如SEQ ID NO: 8、SEQ ID NO:17所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL-13纳米抗体包括两个具有如SEQ ID NO:8、SEQ ID NO:17所示的氨基酸序列的VHH链。
在另一优选例中，所述VHH链之间通过连接肽进行连接。
在另一优选例中，所述连接肽选自以下序列：(GaSb)x，其中a，b，x＝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，x＝4)。
在另一优选例中，所述连接肽的序列如SEQ ID NO:28所示。
在本发明的第二方面，提供了一种抗IL-13抗体，它是针对IL-13表位的抗体，并且具有本发明第一方面所述的抗IL-13纳米抗体。
在另一优选例中，所述抗IL-13抗体包括一个或多个抗IL-13纳米抗体。
在另一优选例中，所述抗IL-13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-13抗体包括一条或多条具有如SEQ ID NO:8或SEQ ID NO:17所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL-13抗体包括两条具有如SEQ ID NO:8或SEQ ID NO:17所示的氨基酸序列的VHH链。
在另一优选例中，所述的抗体能够特异性结合IL-13。
在另一优选例中，所述的抗体具有能够有效阻断IL-13诱导的HEK-BlueTM IL-4/IL-13细胞中SEAP的释放，且阻断活性显著优于对照抗体Lebrikizumab。
在另一优选例中，所述的抗体能够有效抑制HEK-BlueTM IL-4/IL-13细胞的增殖，其抑制活性优于对照抗体4B06。
在另一优选例中，所述抗体为纳米抗体。
在本发明的第三方面，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：本发明第一方面所述的抗IL-13纳米抗体或本发明第二方面所述的抗IL-13抗体。
在另一优选例中，所述多核苷酸为组合形式。
在另一优选例中，所述多核苷酸序列包含SEQ ID NO:9或18所示序列中的一种或多种。
在另一优选例中，所述多核苷酸包括RNA、DNA或cDNA。
在本发明的第四方面，提供了一种表达载体，所述表达载体含有本发明第三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。
在另一优选例中，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。
在本发明的第五方面，提供了一种宿主细胞，所述宿主细胞含有本发明第四方面所述的表达载体，或其基因组中整合有本发明第三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，所述的宿主细胞为毕赤酵母。
在本发明的第六方面，提供了一种产生抗IL-13纳米抗体的方法，包括步骤：
(a)在适合产生纳米抗体的条件下，培养本发明第五方面所述的宿主细胞，从而获得含所述抗IL-13纳米抗体的培养物；
(b)从所述培养物中分离或回收所述的抗IL-13纳米抗体；以及
(c)任选地，纯化和/或修饰得步骤(b)中获得的抗IL-13纳米抗体。
在本发明的第七方面，提供了一种免疫偶联物，所述免疫偶联物含有：
(a)本发明第一方面所述的抗IL-13纳米抗体、或本发明第二方面所述的抗IL-13抗体；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。
在本发明的第八方面，提供了一种多特异性抗体，所述的多特异性抗体包含：本发明第一方面所述的抗IL-13纳米抗体，或本发明第二方面所述的抗IL-13抗体。
在另一优选例中，所述多特异性抗体还包含抗体的Fc段。
在本发明的第九方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)本发明第一方面所述的抗IL-13纳米抗体、或本发明第二方面所述的抗IL-13抗体；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-13蛋白。
在本发明的第十方面，提供了一种药物组合物，所述药物组合物含有：
(i)本发明第一方面所述的抗IL-13纳米抗体、或本发明第二方面所述的抗IL-13抗体、或本发明第七方面所述的免疫偶联物或本发明第八方面所述的多特异性抗体或本发明第九方面所述的重组蛋白；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其他的治疗免疫系统疾病或肿瘤疾病的药物。
在另一优选例中，所述其他的治疗免疫系统疾病或肿瘤疾病的药物选自下组：布地奈德、氟替卡松、倍氯米松、糠酸莫米松、沙丁胺醇、茶碱、福莫特罗、噻托溴铵、柳氮磺胺吡啶、甲氨蝶呤、环磷酰胺、氟尿嘧啶、博来霉素、阿那曲唑、或其组合。
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-13相关的疾病或病症的药物。
在另一优选例中，所述与IL-13相关的疾病或病症包括免疫系统疾病。
在另一优选例中，所述免疫系统疾病选自下组：哮喘、特异性皮炎(AD)、慢性阻塞性肺疾病(COPD)、变应性鼻炎、炎性肠病、斑秃、溃疡性结肠炎、特发性肺纤维化、湿疹、霍奇金淋巴瘤、特发性肺纤维化、嗜酸粒细胞性食管炎、或其组合。
在本发明的第十一方面，提供了一种如本发明第一方面所述的抗IL-13纳米抗体、本发明第二方面所述的抗IL-13抗体、本发明第七方面所述的免疫偶联物或本发明第八方面所述的多特异性抗体或本发明第九方面所述的重组蛋白或本发明第十方面所述的药物组合物的用途，用于：
(a)制备预防和/或治疗与IL-13相关的疾病的药物；和/或
(b)制备检测IL-13的试剂、检测板或试剂盒。
在另一优选例中，所述与IL-13相关的疾病或病症包括免疫系统疾病。
在另一优选例中，所述免疫系统疾病选自下组：哮喘、特异性皮炎(AD)、 慢性阻塞性肺疾病(COPD)、变应性鼻炎、炎性肠病、斑秃、溃疡性结肠炎、特发性肺纤维化、湿疹、霍奇金淋巴瘤、特发性肺纤维化、嗜酸粒细胞性食管炎、或其组合。
在另一优选例中，所述IL-13为人IL-13。
在另一优选例中，所示试剂为诊断试剂。
在另一优选例中，所示诊断试剂为造影剂
在另一优选例中，所述试剂用于检测样品中的IL-13蛋白或其片段。
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。
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。
在本发明的第十二方面，提供了一种检测样品中IL-13蛋白的方法，所述方法包括步骤：
(1)将样品与本发明第一方面所述的抗IL-13纳米抗体、或如本发明第二方面所述的抗IL-13抗体、或本发明第七方面所述的免疫偶联物或本发明第八方面所述的多特异性抗体或本发明第九方面所述的重组蛋白接触；
(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-13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。
在本发明的第十三方面，提供了一种IL-13蛋白检测试剂，所述的检测试剂包含：
(i)本发明第一方面所述的抗IL-13纳米抗体、或本发明第二方面所述的抗IL-13抗体、或本发明第七方面所述的免疫偶联物或本发明第八方面所述的多特异性抗体或本发明第九方面所述的重组蛋白；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)。
在本发明的第十四方面，提供了一种检测IL-13蛋白的试剂盒，所述试剂盒含有本发明第七方面所述的免疫偶联物或本发明第十三所述的检测试剂，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-13的表达。
在本发明的第十五方面，提供了一种本发明第七方面所述的免疫偶联物的用途，用于制备体内检测IL-13蛋白的造影剂。
在另一优选例中，所述检测用于与IL-13相关的疾病或病症的诊断或预后。
在本发明的第十六方面，提供了一种治疗与IL-13相关的疾病的方法，所述方法包括，给需要的对象施用本发明第一方面所述的抗IL-13纳米抗体、本发明第二方面所述的抗IL-13抗体、本发明第七方面所述的免疫偶联物或本发明第八方面所述的多特异性抗体或本发明第九方面所述的重组蛋白或本发明第十方面所述的药物组合物。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如鼠、兔)、非人灵长类动物(如猴)。
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为利用ELISA方法检测候选抗体对人IL-13和食蟹猴IL-13的结合活性，结果表明MY9217和MY9219能同时结合人IL-13和食蟹猴IL-13。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，意外地获得具有优异的亲和力和特异性的抗IL-13纳米抗体。
实验结果表明，本发明的纳米抗体具有优异的功能活性，能够有效阻断IL-13诱导的HEK-BlueTM IL-4/IL-13细胞中SEAP的释放，且活性优于对照抗体Lebrikizumab和4B06。本发明纳米抗体具有良好的体内半衰期，能够有效结合人血清白蛋白，且结合活性优于对照抗体3(HuNb3-11)。本发明的纳米抗体能同时结合人IL-13和食蟹猴IL-13。在此基础上，本发明人完成了本发明。
术语
如本文所用，术语“结合物”是指能够与靶点结合的可溶性受体或其片段或其类似物，或抗体或其片段或其类似物。本发明所述的“IL-13结合物”，是指能特异性识别IL-13并与IL-13抗原结合的抗体或其片段或其类似物。
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且意图不是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
白细胞介素-13(IL-13)
白细胞介素-13(Interleukin-13，IL-13)是白介素家族的重要成员，是由IL-13基因编码生成的蛋白质，分子量约10KD。IL-13对单核巨噬细胞、B淋巴细胞、NK细胞和血管内皮细胞等具有多种生物学作用，是机体免疫反应的调节的介质。
抗体
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”、“VHH”、“纳米抗体(nanobody)”、“重 链抗体”(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纳米抗体(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纳米抗体(VHH)。
如本文所用，术语“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab'片段，F(ab')2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
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。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
如本文所用，术语“嵌合抗体”是由鼠源性抗体的V区基因与人抗体的C区基因拼接为嵌合基因，然后插入载体，转染宿主细胞表达的抗体分子。既保留了亲本鼠抗体的高特异性和亲和力，又使其人源Fc段能有效介导生物学效应功能。
如本文所用，术语“人源化抗体”，是本发明鼠抗的一种可变区改造形式，具有源自(或基本上源自)非人类抗体(优选小鼠单克隆抗体)的CDR区，和基本源自人源抗体序列的FR区和恒定区；即将鼠抗的CDR区序列嫁接到不同类型的人种系抗体构架序列上。因为CDR序列负责大部分的抗体-抗原相互作用，所以可以通过构建表达载体来表达模拟特定天然存在的抗体性质的重组抗体。在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
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-13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-13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-13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，如包含纳米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纳米抗体的片段。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A进行氨基酸替换而产生。
表A
编码核酸和表达载体
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CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。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。
抗IL-13纳米抗体
如本文所用，术语“本发明纳米抗体”、“本发明的纳米抗体”、“本发明的抗IL-13纳米抗体”、“本发明IL-13纳米抗体”、“抗IL-13纳米抗体”、“IL-13纳米抗体”具有相同的含义，可互换使用，均指特异性识别和结合于IL-13(包括人IL-13)的纳米抗体。
如本文所用，术语“IL-13”是指IL-13蛋白，其氨基酸序列如SEQ ID No:1所示。
在本发明的一个优选例中，所述抗IL-13纳米抗体包括单体、二价体(二价抗体)、四价体(四价抗体)、和/或多价体(多价抗体)。
典型的，所述抗IL-13纳米抗体包括两条具有如SEQ ID NO:8、SEQ ID NO:17中所示的氨基酸序列的VHH链。
在另一优选例中，所述VHH链之间通过连接肽进行连接。
在另一优选例中，所述连接肽选自以下序列：(GaSb)x，其中a，b，x＝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，x＝4)。
在另一优选例中，所述连接肽的序列为GGGGSGGGGSGGGGSGGGGS。
在另一优选例中，所述的纳米抗体VHH链的CDR区包含与SEQ ID NO:2、4和6、SEQ ID NO:11、13和15任一具有至少80％、优选地至少90％、更优选地至少95％、甚至更优选地至少99％的序列相似性的氨基酸序列。
在另一优选例中，所述的纳米抗体VHH链CDR区的氨基酸序列与SEQ ID NO:2、4和6、SEQ ID NO:11、13和15中任一相比包含一个或多个氨基酸取代，优选保守氨基酸取代。
标记的纳米抗体
在本发明的一个优选例中，所述纳米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-13的纳米抗体用胶体金标记，得到胶体金标记的纳米抗体。
抗体的制备
任何适于产生抗体的方法都可用于产生本发明的IL-13的纳米抗体。例如，可以用连接或天然存在的IL-13或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的IL-13都可以作为免疫原(抗原)，用于产生对IL-13特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是重组IL-13或其片段。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于：腺病毒载体、腺相关病毒载体、杆状病毒载体、质料和非病毒载体。
一种生产本发明的抗IL-13抗体的示例性方法，描述于实施例1。
本发明的抗体可以选自任何物种的任何种类的免疫球蛋白，包括IgG和IgE。优选的抗体是IgG抗体，如IgG1亚型。通过用下文实施例中描述的生物学测定筛选抗体易于实现必需恒定结构域序列的最优化，以产生所需生物学活性。
同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本发明的化合物和方法中是可以用的。
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
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。
此外，还可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。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。
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。优选的动物细胞包括(但并不限于)：CHO-S、CHO-K1、HEK-293细胞。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本发明的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常 规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
药物组合物和应用
本发明还提供了一种药物组合物。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)：静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射、或腔内注射。
本发明中，术语“药物组合物”是指本发明的双特异性抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
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％)的本发明上述的双特异性抗体(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，本发明的双特异性抗体还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的双特异性抗体或其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。
检测方法
本发明还涉及检测IL-13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-13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。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)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。在优选例中，本发明的抗体可以固定于检测板。
应用
如上所述，本发明的纳米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-13相关的疾病或病症的诊断和治疗、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-13的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明的纳米抗体具有良好的功能活性，且活性优于对照抗体Lebrikizumab。
(b)本发明的纳米抗体能够有效阻断IL-13诱导的HEK-BlueTM IL-4/IL-13细胞中SEAP的释放，且活性优于对照抗体Lebrikizumab。
(c)本发明的纳米抗体可用微生物表达系统生产，生产成本低。
下面结合具体实施例，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等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)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，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
实施例1免疫文库构建及筛选
将高纯度的蛋白IL-13-Fc与免疫佐剂混合后免疫新疆双峰驼，7次免疫后 取骆驼外周血检测血清中抗体滴度，当抗体滴度大于1：1000时认为免疫成功。从骆驼外周血中分离PBMC并提取总RNA，经反转录成cDNA后利用巢式PCR扩增VHH片段，克隆连接入噬菌体展示载体，转染TG1细胞构建成噬菌体展示纳米抗体文库。经三轮“吸附-洗涤-结合”的过程，使IL-13特异性纳米抗体噬菌体富集。随机挑选600个单克隆进行上清液ELISA鉴定。
最终获得了39株CDR3区序列不同的纳米抗体。
实施例2 EK-BlueTM IL4/IL-13细胞活性法筛选阻断型抗体
将以上39株纳米抗体从大肠杆菌菌株中纯化获得高纯度抗体，利用HEK-BlueTM IL4/IL-13细胞(购自Invivogen)检测抗体的功能活性。HEK-BlueTMIL-4/IL-13细胞在IL-13刺激下产生SEAP，使用QUANTI-BlueTMSolution测定上清液中STAT6诱导的SEAP水平。重组人IL-13对HEK-BlueTMIL-4/IL-13细胞的刺激可以被中和性IL-13抗体阻断。利用该方法检测以上39株候选抗体功能活性。
将HEK-BlueTMIL-4/IL-13细胞用含1％FBS的DMEM培养基重悬，计数后分装至96孔板，加入预先混合好的抗体及IL-13蛋白后于培养箱培养24加入小时，将培养的细胞上清取出，并加入九倍量的QUANTI-Blue试剂于37℃孵育3小时，酶标仪检测620nm处吸光值。
结果如表1所示，其中对照VHH1为源自专利WO2021116182A1中4B06的抗体序列。
表1单价抗体的阻断活性
实施例3人源化抗体活性鉴定
对以上细胞活性良好的抗体进行人源化设计，人源化方法参考专利CN2018101517526中实施例4的方法。针对每个纳米抗体的骨架区序列进行人源化改造，保持可变区不变。将人源化后的抗体序列按照毕赤酵母密码子优化 的碱基序列合成至酵母表达载体pPICZaA。将插入抗体基因的重组质粒线性化后电转至毕赤酵母X33感受态细胞，随后从抗性平板上挑取单克隆，经甲醇诱导表达，检测培养液中抗体的表达量。检测结果见表2。
同时将纳米抗体从毕赤酵母发酵上清液中纯化出来，用于HEK-BlueTM IL-4/IL-13细胞功能活性鉴定。
结果见表2，8株人源化抗体具有良好的功能活性。
表2人源化抗体产量及活性评估
实施例4二价抗体构建及表达
为了进一步提升抗体的活性，将以上8个人源化抗体进行两两组合成二价体，共构建64个二价抗体。两个单体之间均由GS20连接子(氨基酸序列如SEQ ID NO:28)进行连接。将毕赤酵母密码子优化的二价抗体碱基序列克隆至pPICZaA载体上。将线性化的重组质粒线电转化至毕赤酵母X33感受态细胞，然后从抗性平板上挑取单克隆，经甲醇诱导表达，检测培养液中抗体的表达量。
经产量评估，11个候选二价抗体的产量高于2g/L，见表3。
表3二价纳米抗体的产量评估

实施例5二价抗体的活性鉴定
将以上11株人源化二价纳米抗体从毕赤酵母发酵上清液中纯化出来，用于HEK-BlueTM IL-4/IL-13细胞功能活性鉴定。
结果见表4，二价纳米抗体的活性显著提升，且优于对照抗体1(源自专利WO2021116182A1)和对照抗体2(Lebrikizumab)。
表4二价人源化抗体的功能活性鉴定
实施例6长效型抗体的构建
为了使以上纳米抗体具有良好的体内半衰期，对其添加抗人血清白蛋白纳米抗体HuNb3-11。构建成不同结构的长效型纳米抗体，表达后评估其产量及活性。制备和评估方法同实施例4。
其结构及表达产量评估结果见表5。
表5长效纳米抗体的产量评估
表6氨基酸序列及核苷酸序列
实施例7长效型抗体的活性评估
优选MY9217和MY9219进行IL-13功能活性鉴定及血清白蛋白结合活性评估。将从发酵液纯化出的三价纳米抗体先进行IL-13功能活性鉴定，检测方法与实施例2相同。
结果如表2所示，结果表明，2株纳米抗体能有效阻断IL-13诱导的HEK-BlueTM IL-4/IL-13细胞中SEAP的释放，且活性优于对照抗体2(Lebrikizumab，罗氏)。此外，MY9217和MY9219能够有效结合人血清白蛋白，且结合活性优于对照抗体3(HuNb3-11)。
表7抗IL-13长效纳米抗体的活性评估
表8抗体MY9217和MY9219的氨基酸序列和核苷酸序列
实施例8种属结合活性
利用ELISA方法检测候选抗体对人IL-13和食蟹猴IL-13的结合活性。将人IL-13及食蟹猴IL-13蛋白分别包被在酶标板上(4℃过夜)，封闭反应后加 入梯度稀释的待测抗体MY9127和MY9219于37℃反应1小时，再加入稀释的HRP-羊抗纳米抗体多抗(1:2000稀释)于37℃反应1小时，加入TMB显色液并用H2SO4终止后读取吸光值。
结果如图1所示，表明MY9217和MY9219能同时结合人IL-13和食蟹猴IL-13。
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。

Claims (15)

  1. 一种抗IL-13纳米抗体，其特征在于，所述纳米抗体中的VHH链的互补决定区CDR区为选自下组的一种或多种：
    (1)SEQ ID NO:2所示的CDR1、SEQ ID NO:4所示的CDR2、和SEQ ID NO:6所示的CDR3；和
    (2)SEQ ID NO:11所示的CDR1、SEQ ID NO:13所示的CDR2、和SEQ ID NO:15所示的CDR3。
  2. 如权利要求1所述的抗IL-13纳米抗体，其特征在于，所述纳米抗体的VHH链还包括框架区FR，所述的框架区FR为选自下组的一种或多种：
    (1)SEQ ID NO:1所示的FR1、SEQ ID NO:3所示的FR2、SEQ ID NO:5所示的FR3、和SEQ ID NO:7所示的FR4；和
    (2)SEQ ID NO:10所示的FR1、SEQ ID NO:12所示的FR2、SEQ ID NO:14所示的FR3、和SEQ ID NO:16所示的FR4。
  3. 如权利要求1所述的抗IL-13纳米抗体，其特征在于，所述纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:8、SEQ ID NO:17、或其组合。
  4. 一种抗IL-13抗体，其特征在于，它是针对IL-13表位的抗体，并且具有权利要求1所述的抗IL-13纳米抗体。
  5. 如权利要求1所述的抗IL-13抗体，其特征在于，所述抗IL-13抗体包括一个或多个抗IL-13纳米抗体。
  6. 如权利要求4或5所述的抗IL-13抗体，其特征在于，所述抗IL-13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)。
  7. 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-13纳米抗体或权利要求4所述的抗IL-13抗体。
  8. 一种表达载体，其特征在于，所述表达载体含有权利要求7所述的多核苷酸。
  9. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求8所述的表达载体，或其基因组中整合有权利要求7所述的多核苷酸。
  10. 一种产生抗IL-13纳米抗体的方法，其特征在于，包括步骤：
    (a)在适合产生纳米抗体的条件下，培养权利要求9所述的宿主细胞，从而获得含所述抗IL-13纳米抗体的培养物；
    (b)从所述培养物中分离或回收所述的抗IL-13纳米抗体；以及
    (c)任选地，纯化和/或修饰得步骤(b)中获得的抗IL-13纳米抗体。
  11. 一种免疫偶联物，所述免疫偶联物含有：
    (a)如权利要求1所述的抗IL-13纳米抗体、或如权利要求4所述的抗IL-13 抗体；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。
  12. 一种多特异性抗体，所述的多特异性抗体包含：如权利要求1所述的抗IL-13纳米抗体、或如权利要求4所述的抗IL-13抗体。
  13. 一种重组蛋白，所述的重组蛋白具有：
    (i)如权利要求1所述的抗IL-13纳米抗体、或如权利要求4所述的抗IL-13抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  14. 一种药物组合物，所述药物组合物含有：
    (i)如权利要求1所述的抗IL-13纳米抗体、或如权利要求4所述的抗IL-13抗体、或如权利要求11所述的免疫偶联物、或如权利要求12所述的多特异性抗体、或如权利要求13所述的重组蛋白；以及
    (ii)药学上可接受的载体。
  15. 如权利要求1所述的抗IL-13纳米抗体、或如权利要求4所述的抗IL-13抗体、或如权利要求11所述的免疫偶联物、或如权利要求12所述的多特异性抗体、或如权利要求13所述的重组蛋白、或如权利要求14所述的药物组合物的用途，用于：
    (a)制备预防和/或治疗与IL-13相关的疾病的药物；和/或
    (b)制备检测IL-13的试剂、检测板或试剂盒。
PCT/CN2023/130165 2022-11-08 2023-11-07 抗il-13长效纳米抗体序列及其应用 WO2024099310A1 (zh)

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