WO2023142309A1 - 抗tslp纳米抗体及其应用 - Google Patents

抗tslp纳米抗体及其应用 Download PDF

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WO2023142309A1
WO2023142309A1 PCT/CN2022/093166 CN2022093166W WO2023142309A1 WO 2023142309 A1 WO2023142309 A1 WO 2023142309A1 CN 2022093166 W CN2022093166 W CN 2022093166W WO 2023142309 A1 WO2023142309 A1 WO 2023142309A1
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tslp
seq
antibody
nanobody
present
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PCT/CN2022/093166
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English (en)
French (fr)
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万亚坤
李光辉
朱敏
盖军伟
沈晓宁
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上海洛启生物医药技术有限公司
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Priority to JP2023540115A priority Critical patent/JP2024512858A/ja
Priority to KR1020237025523A priority patent/KR20230125042A/ko
Priority to AU2022434181A priority patent/AU2022434181A1/en
Priority to CA3209675A priority patent/CA3209675A1/en
Priority to EP22912783.2A priority patent/EP4257605A1/en
Priority to IL303474A priority patent/IL303474A/en
Publication of WO2023142309A1 publication Critical patent/WO2023142309A1/zh

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    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • 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
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    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • 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
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • 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
    • G—PHYSICS
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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]
    • G01N2333/5418—IL-7

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically relates to anti-TSLP nanobody and its application.
  • Antibody drugs used for the treatment of severe asthma mostly target IgE (omalizumab), IL5 (mepolizumab, relizumab), IL5R (benralizumab), IL4R in the Th2 pathway (Dupilumab), etc. All the above drugs have a good control effect on hypereosinophilic asthma, and they are all treated by subcutaneous or intravenous injection.
  • Thymic stromal lymphopoietin is a short-chain cytokine with a four-helix bundle structure and belongs to the IL-2 cytokine family.
  • TSLP is an epithelial cytokine produced in response to pro-inflammatory stimuli, such as pulmonary allergens, viruses, and other pathogens, and plays a key role in the initiation and persistence of airway inflammation.
  • TSLP drives the release of downstream Th2 cytokines, including IL-4, IL-5, and IL-13, leading to inflammation and asthma symptoms.
  • Th2 cytokines including IL-4, IL-5, and IL-13, leading to inflammation and asthma symptoms.
  • TSLP also activates various cell types involved in non-Th2-driven inflammation.
  • TSLP's early upstream activity in the inflammatory cascade has been identified as a potential target in a broad population of asthmatic patients.
  • Blocking TSLP prevents immune cells from releasing pro-inflammatory cytokines, thereby preventing asthma exacerbations, improving asthma control, and related diseases such as COPD.
  • Nanobody that is, heavy chain nanobody VHH (variable domain of heavy chain of heavy-chain antibody) - there is a heavy-chain antibody (heavy-chain antibody, HCAb) that naturally lacks light chains in camels, and its clone Nanobodies, which are composed of only one heavy chain variable region, are currently available as the smallest unit of stable antigen-binding with complete functions. Nanobodies have the characteristics of high stability, good water solubility, simple humanization, high targeting, and strong penetrability. They play a huge role beyond imagination in immune experiments, diagnosis, and treatment. Nanobodies are gradually becoming an emerging force in the new generation of antibody therapy.
  • the purpose of the present invention is to provide a kind of anti-TSLP nanobody with better blocking activity, better clinical efficacy and easy production.
  • an anti-TSLP Nanobody is provided, and the CDR region of the VHH chain in the Nanobody is one or more selected from the group consisting of:
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the VHH chain further includes a framework region FR, and the framework region FR is one or more selected from the following group:
  • the CDR region of the VHH chain of the Nanobody comprises at least 80% of any of SEQ ID NO:1-3, SEQ ID NO:14-16, SEQ ID NO:27-29, Amino acid sequences with at least 90%, more preferably at least 95%, even more preferably at least 99% sequence similarity are preferred.
  • the amino acid sequence of the CDR region of the VHH chain of the Nanobody comprises a or multiple amino acid substitutions, preferably conservative amino acid substitutions.
  • any amino acid sequence in the above amino acid sequence also includes at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid derivative sequence that can retain the specific binding ability to TSLP.
  • the Nanobody can specifically bind TSLP.
  • the nanobody can effectively block the interaction between TSLP and TSLPR.
  • the TSLP is human or non-human mammalian TSLP.
  • the TSLP is the TSLP of human, mouse, rat, or non-human primate (such as monkey).
  • the nanobodies include humanized antibodies, camelid antibodies, and chimeric antibodies.
  • amino acid sequence of the VHH chain of the Nanobody is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO: 34. SEQ ID NO: 38, or a combination thereof.
  • the anti-TSLP nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the anti-TSLP nanobody comprises one or more nanobodies such as SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or The VHH chain of the amino acid sequence shown in SEQ ID NO:38.
  • the anti-TSLP nanobody comprises two compounds having such as SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID The VHH chain of the amino acid sequence shown in NO:38.
  • VHH chains are linked by linking peptides.
  • sequence of the connecting peptide is: GGGGSGGGGSGGGGSGGGGS.
  • the second aspect of the present invention provides an anti-TSLP antibody, which is an antibody against TSLP epitopes, and has the anti-TSLP nanobody described in the first aspect of the present invention.
  • the anti-TSLP antibody includes one or more anti-TSLP nanobodies.
  • the anti-TSLP antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the anti-TSLP antibody includes one or more antibodies having such as SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 34 or SEQ ID NO: The VHH chain of the amino acid sequence shown in ID NO:38.
  • the anti-TSLP antibody comprises two antibodies having such as SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO : VHH chain of the amino acid sequence shown in 38.
  • the antibody can specifically bind TSLP.
  • the antibody can specifically target the TSLP protein with the correct spatial structure.
  • the antibody can effectively block the interaction between TSLP and TSLPR.
  • the affinity (KD value) of the antibody to TSLP is less than 3.77nM.
  • the antibody has good TSLP/TSLPR blocking activity, and the blocking activity is significantly better than that of the control antibody Tezepelumab, wherein the control antibody Tezepelumab is obtained from AstraZeneca or Amgen )company.
  • the antibody can effectively inhibit the proliferation of BaF3/TSLPR-IL7R cells, and its inhibitory activity is better than that of the control antibody Tezepelumab.
  • the antibody is a Nanobody.
  • the third aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of the anti-TSLP nanobody described in the first aspect of the present invention or the anti-TSLP antibody described in the second aspect of the present invention .
  • polynucleotides are in combination.
  • polynucleotide sequence comprises one or more of the sequences shown in SEQ ID NO: 9, 13, 22, 26, 35 or 39.
  • the polynucleotide includes RNA, DNA or cDNA.
  • the fourth aspect of the present invention provides an expression vector, which 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, transposon, other gene transfer systems, or combinations thereof.
  • the expression vector includes a viral vector, such as lentivirus, adenovirus, AAV virus, and retrovirus.
  • the fifth aspect of the present invention provides a host cell containing the expression vector of the fourth aspect of the present invention, or the polynucleotide of the third aspect of the present invention integrated in its genome.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, phage, or combinations thereof.
  • the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.
  • the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, fission yeast, Trichoderma, or combinations thereof.
  • the host cell is Pichia pastoris.
  • the sixth aspect of the present invention provides a method of producing anti-TSLP nanobody, comprising the steps of:
  • step (c) Optionally, purifying and/or modifying the anti-TSLP Nanobody obtained in step (b).
  • the seventh aspect of the present invention provides an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof .
  • the radionuclides include:
  • isotopes for diagnosis are 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 combinations thereof; and/or
  • the isotope for treatment is 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 drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, 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, auristatin ( auristatins, camptothecins, duocarmycins/duocarmycins, etoposides, maytansines, and maytansinoids (such as DM1 and DM4 ), taxanes, benzodiazepines, or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indole Indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • auristatin auristatins, camptothecins, duocarmycins/duocarmycins, etoposides
  • maytansines such as DM1 and DM4
  • taxanes such as benzodiazepines, or benz
  • the toxin is selected from the group consisting of auristatins (for example, auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinol, ricin, grate Anesthetic toxin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, Tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthraxin diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, acacia Toxin, abrin toxin A chain, lotus root toxin A chain, ⁇ -sarcinia, gelonin, mitogellin
  • the coupling moiety 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 (computer X-ray tomography) contrast agents, or capable of producing Detectable products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles , prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), or nanoparticles in any form.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • the eighth aspect of the present invention provides a multispecific antibody, said multispecific antibody comprising: the anti-TSLP nanobody of the first aspect of the present invention, or the anti-TSLP antibody of the second aspect of the present invention.
  • the multispecific antibody further comprises an Fc segment of the antibody.
  • the ninth aspect of the present invention provides a recombinant protein, which has:
  • the tag sequence includes Fc tag, HA tag and 6His tag.
  • the recombinant protein specifically binds to TSLP protein.
  • the tenth aspect of the present invention provides a pharmaceutical composition, which contains:
  • the coupling moiety 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 group consisting of 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 medicines for preventing and/or treating diseases or diseases related to TSLP.
  • the diseases or conditions related to TSLP include immune system diseases or tumor diseases.
  • the immune system disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergy, ulcerative colitis, Crohn's disease , rhinitis, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis, hypersensitivity pneumonitis, allergic granulomatous vasculitis, nasal polyposis, or combinations thereof.
  • COPD chronic obstructive pulmonary disease
  • the tumor disease is selected from the group consisting of breast cancer, pancreatic cancer, cervical cancer, multiple myeloma, colorectal cancer, lung cancer, thyroid cancer, ovarian cancer, liver cancer, or a combination thereof.
  • the eleventh aspect of the present invention provides the anti-TSLP nanobody described in the first aspect of the present invention, the anti-TSLP antibody described in the second aspect of the present invention, the immunoconjugate as described in the seventh aspect of the present invention, or the anti-TSLP nanobody described in the seventh aspect of the present invention.
  • the use of the multispecific antibody described in the eighth aspect or 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 (a) for the preparation of prevention and/or treatment of TSLP-related Drugs for diseases; and/or (b) preparation of reagents, detection plates or kits for detecting TSLP.
  • the diseases or conditions related to TSLP include immune system diseases or tumor diseases.
  • the immune system disease is selected from the group consisting of asthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD), allergic conjunctivitis, food allergy, ulcerative colitis, Crohn's disease , rhinitis, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis, hypersensitivity pneumonitis, allergic granulomatous vasculitis, nasal polyposis, or combinations thereof.
  • COPD chronic obstructive pulmonary disease
  • the tumor disease is selected from the group consisting of breast cancer, pancreatic cancer, cervical cancer, multiple myeloma, colorectal cancer, lung cancer, thyroid cancer, ovarian cancer, liver cancer, or a combination thereof.
  • the TSLP is human TSLP.
  • the indicated reagent is a diagnostic reagent.
  • the diagnostic reagent shown is a contrast agent
  • the reagent is used to detect TSLP protein or fragments thereof in a sample.
  • the detection includes flow detection and cell immunofluorescence detection.
  • the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
  • the twelfth aspect of the present invention provides a method for detecting TSLP protein in a sample, the method comprising the steps of:
  • the method is a non-diagnostic and non-therapeutic method.
  • the thirteenth aspect of the present invention provides a TSLP protein detection reagent, said detection reagent comprising:
  • the coupling moiety of the immunoconjugate is an isotope for diagnosis.
  • the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the group consisting of isotopic tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles 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 aqueous solution, injection, freeze-dried powder, tablet, buccal preparation, aerosol).
  • the fourteenth aspect of the present invention provides a kit for detecting TSLP protein, which contains 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 describe that the kit is used to non-invasively detect the expression of TSLP in the subject.
  • the fifteenth aspect of the present invention provides the use of the immunoconjugate described in the seventh aspect of the present invention for preparing a contrast agent for detecting TSLP protein in vivo.
  • the detection is used for the diagnosis or prognosis of diseases or diseases related to TSLP.
  • the sixteenth aspect of the present invention provides a method for treating diseases, the method comprising: administering the anti-TSLP nanobody described in the first aspect of the present invention, the anti-TSLP antibody described in the second aspect of the present invention, The immunoconjugate according to the seventh aspect of the present invention or the multispecific antibody according to the eighth aspect of the present invention or the recombinant protein according to the ninth aspect of the present invention or the pharmaceutical composition according to the tenth aspect of the present invention.
  • the subject includes a human or a non-human mammal.
  • non-human mammals include rodents (such as mice, rabbits), and non-human primates (such as monkeys).
  • Figure 1A and Figure 1B are the results of flow cytometry blocking activity detection of 31 candidate antibodies. The results showed that the blocking activity of 14 antibodies among the 31 candidate antibodies was significantly better than that of the control antibody Tezepelumab.
  • Fig. 2 is the detection result of binding kinetics of 14 strains of blocking TSLP nanobodies.
  • Figure 3 is the SDS-PAGE results of the shake flask expression supernatants of bivalent single domain antibodies Bi-HuNb5-31, Bi-HuNb7-54, and Bi-HuNb10-63 in Pichia pastoris, and the yields are 475ug/mL and 310ug respectively /mL, 510ug/mL.
  • Fig. 4 is the result of detecting the blocking activity of the humanized bivalent antibody by ELISA. The results showed that the blocking activity of the three humanized bivalent antibodies was significantly better than that of the control antibody Tezepelumab.
  • Fig. 5 is the results of the inhibitory effect of the humanized bivalent antibody on the proliferation of BaF3/TSLPR-IL7R cells. The results showed that the inhibitory effect of two humanized antibodies on the pSTAT5 signaling pathway in BaF3/TSLPR-IL7R cells was better than that of the control antibody Tezepelumab.
  • a class of anti-TSLP nanobody was unexpectedly discovered for the first time, and the experimental results showed that the nanobody of the present invention can effectively block the interaction between TSLP and TSLPR, and the blocking activity is significantly better than that of the control antibody Tezepelumab; the nanobody of the present invention can effectively inhibit The inhibitory activity of the Baf3/TSLPR-IL7R cell proliferation is better than that of the control antibody Tezepelumab; the expression yield of the nanobody of the present invention in Pichia pastoris can reach 17-23g/L, which is significantly higher than the industry level. On this basis, the present inventors have completed the present invention.
  • Nanobody of the invention As used herein, the terms “Nanobody of the invention”, “Nanobody of the invention”, “anti-TSLP Nanobody of the invention”, “TSLP Nanobody of the invention”, “anti-TSLP Nanobody”, “TSLP Nanobody” Having the same meaning and being used interchangeably, both refer to Nanobodies that specifically recognize and bind to TSLP (including human TSLP).
  • antibody or "immunoglobulin” is a heterotetrameric protein of about 150,000 Daltons with identical structural features, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by 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 the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain .
  • VL variable region
  • Specific amino acid residues form the interface between the variable domains of the light and heavy chains.
  • the terms “single domain”, “VHH”, “nanobody”, “heavy chain antibody” (single domain antibody, sdAb, or nanobody nanobody) have the same meaning and are 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 full functionality.
  • VHH Single domain antibody
  • sdAb single domain antibody, sdAb, or nanobody nanobody
  • variable means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable domains are called the framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each contain four FR regions in a roughly b-sheet configuration connected by three CDRs that form connecting loops, which in some cases may form partial b-sheet structures.
  • the CDRs in each chain are in close proximity through the FR regions 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, Vol. I, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example involved in the antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form of conjugates.
  • the present invention also includes cell surface markers or antigens that bind to the anti-TSLP antibody or fragment 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.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to polypeptides that specifically bind to TSLP proteins, such as proteins or polypeptides with heavy chain variable regions . They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) 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% homologous.
  • the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR), which is separated into four framework regions (FR), four FR amino acids
  • CDR variable region
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a ring structure, and the ⁇ sheets formed by the FRs in between are close to each other in the spatial structure.
  • the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody.
  • Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • 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 the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein sex.
  • 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. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having 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 having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • the antibody of the present invention refers to a polypeptide that has TSLP binding activity and includes the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations 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 , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • 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 invention.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with the DNA encoding 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 invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the invention also includes fragments of the Nanobodies of the invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “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 amino acid sequences compared with the amino acid sequence of the antibody of the present invention.
  • An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • a polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 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 fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
  • biomolecules nucleic acid, protein, etc.
  • the biomolecules involved in the present invention include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may 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 are: 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, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl2 . Transformation can also be performed by electroporation, if desired.
  • DNA transfection methods can be used: calcium phosphate co-precipitation 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 medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, 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 invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any 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 substances capable of producing a detectable product. enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 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 biphenylhydrolase-like protein (BPHL)), etc.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • Thymic stromal lymphopoietin TSLP
  • Thymic stromal lymphopoietin is a short-chain cytokine with a four-helix bundle structure and belongs to the IL-2 cytokine family.
  • TSLP is commonly expressed in epithelial cells lining the lung, skin, and intestinal barrier surfaces.
  • Animal experiments showed that TSLP was highly expressed in the lungs of allergen-induced asthma model mice, while TSLP receptor-deficient mice showed significantly reduced asthma, and lung-specific TSLP transgenic mice showed Th2-type inflammation and increased IgE airway inflammation and highly reactive. Further studies suggest that TSLP activates bone marrow-derived dendritic cells and upregulates co-stimulatory molecules to produce Th2-type cell chemokine CCL17.
  • TSLP is an important factor and necessary condition for initiating airway allergic inflammation. It is an upstream regulator of multiple inflammatory pathways in various diseases, including asthma, and is critical for the initiation and persistence of airway inflammation.
  • TSLP is also a cytokine related to the pathogenesis of atopic dermatitis (AD). What's more, researchers have found that in different types of tumors, TSLP levels are increased, and TSLP can induce tumor cells to express another protein called BCL-2, which can protect tumors from death. . TSLP is therefore also crucial for tumor survival.
  • TSLPR Thymic stromal lymphopoietin receptor
  • TSLPR receptors are type I transmembrane proteins and belong to the hematopoietic cytokine receptor family.
  • a functional TSLPR complex is composed of TSLPR and IL-7Ra.
  • TSLPR is also known as cytokine receptor-like molecule 2 or type I cytokine receptor ⁇ 1.
  • TSLP initiates intracellular type 2 signaling by binding to its high-affinity heterodimeric receptor complex consisting of its specific receptor TSLPR, which interacts with IL-2, IL-4, IL
  • TSLPR specific receptor
  • IL-2 IL-2
  • IL-4 IL-4
  • IL-7R ⁇ subunit in cells that co-express TSLPR and IL-7R ⁇ (CD127).
  • TSLP initially binds to TSLPR and subsequently recruits the IL-7R ⁇ chain.
  • the present invention also provides a 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 materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • 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 TSLP protein molecules, and thus can be used to treat diseases or disorders related to TSLP (including immune system diseases or tumor diseases).
  • diseases or disorders related to TSLP including immune system diseases or tumor diseases.
  • other therapeutic agents may also be used concomitantly.
  • 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 Nanobody (or its conjugate) of the present invention and pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the invention can also be used with other therapeutic agents.
  • 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 no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • amino acid sequence of the VHH chain of the anti-TSLP nanobody is selected from SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:34 or SEQ ID One or more of NO:38.
  • the anti-TSLP nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).
  • the anti-TSLP nanobody comprises two compounds having the following characteristics such as SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 38
  • SEQ ID NO: 8 SEQ ID NO: 12
  • SEQ ID NO: 21 SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 34 or SEQ ID NO: 38
  • VHH chains are linked by linking peptides.
  • sequence of the connecting peptide is GGGGSGGGGSGGGGSGGGGS.
  • the Nanobody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
  • colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the nanobody of TSLP is labeled with colloidal gold to obtain a nanobody labeled with colloidal gold.
  • the present invention also relates to methods for detecting TSLP proteins.
  • the steps of the method are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; detect the level of TSLP protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or its fragment) or detection plate of the present invention.
  • the kit further includes a container, instructions for use, buffer and the like.
  • the present invention also provides a detection kit for detecting the level of TSLP, which includes an antibody for recognizing TSLP protein, a lysis medium for dissolving samples, general reagents and buffers required for detection, such as various buffers, detection labeling, detection substrates, etc.
  • the test kit may be an in vitro diagnostic device.
  • the nanobody of the present invention has a wide range of biological and clinical application values, and its application involves the diagnosis and treatment of diseases or diseases related to TSLP, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy against TSLP.
  • Nanobody of the present invention can effectively block the interaction between TSLP and TSLPR.
  • the nanobody of the present invention has stronger blocking activity.
  • the nanobody of the present invention can be expressed in Pichia pastoris, the expression yield of the shake flask is relatively high, and the yield of the fermenter can reach 17-23g/L.
  • human TSLP protein was purified after transfection into HEK293F cells.
  • human TSLP protein was purified after transfection into HEK293F cells.
  • four Xinjiang Bactrian camels were immunized, once a week, and peripheral blood was collected after seven times of immunization, RNA was isolated and VHH gene fragment was amplified, and then cloned into pMECS vector, Electroporation into TG1 competent cells to establish a high-quality phage display nanobody library. After testing, the storage capacity of the four libraries all reached above 1x10 9 CFU, and the fragment insertion rate was above 80%.
  • Nanobody clones with different sequences above were inoculated in TB medium and induced overnight with IPTG, and the supernatant was collected by lysing the cells for blocking activity detection.
  • the cultured CHOZEN/TSLPR stably transfected cells were divided into 96-well plates, with 3E5 cells per well, centrifuged at 3000rpm for 3 minutes to remove the supernatant, and the lysate of each antibody and TSLP-Biotin protein were added to incubate for 20 minutes. Discard the supernatant by centrifugation, add diluted SA-PE antibody, and incubate at 4°C for 20min.
  • the above 31 purified antibodies were tested for blocking activity by flow cytometry again. Aliquot the cultured HEK293F/TSLPR transient cells into a 96-well plate, 3E5 cells per well, centrifuge at 3000rpm for 3min to remove the supernatant, and add the diluted antibodies (2-fold serial dilution starting from 20ug/mL) And TSLP-Biotin protein incubation for 20min. In this experiment, Tezepelumab was used as the control antibody. Then centrifuge the supernatant, add diluted SA-PE antibody, and incubate at 4°C for 20min.
  • the binding kinetics of 14 blocking TSLP nanobodies were detected by bio-layer interferometry (BLI).
  • the candidate antibody was diluted to 5ug/mL with PBST buffer, and the TSLP-Fc antigen was diluted 2 times with PBST buffer for six concentration gradients (2 times serial dilution starting from 20nM), and the operating conditions of the instrument were set: temperature 30°C, Shake speed 1000rpm.
  • the protein A-coated probe was used to capture the antibody, the capture time was 60s; the binding time was 240s to the antigen diluted in gradient; the dissociation time was 300s; 10mM glycine (pH1.7) was regenerated twice, each 5s.
  • Fortebio Analysis version 9.0 was used for analysis, the Global mode was used for fitting, and the association rate (Kon), dissociation rate (Kdis) and dissociation constant KD were calculated. The result is shown in Figure 2.
  • the above humanized antibody was constructed into a bivalent form, connected by a linker (G 4 S)4, and the sequence after connection is shown in SEQ ID NO.:40, SEQ ID NO.:41 and SEQ ID NO.:42 , followed by expression in Pichia pastoris.
  • the expression method is as follows: (1) construct the above TSLP nanobody bivalent sequence into pPICZaA vector; (2) electrotransform into X-33 competent cells after linearization with Sac I restriction endonuclease; ( 3) Spread the electroporated samples on the YPD plate medium containing different concentrations of bleomycin resistance, and culture them in a 30°C incubator for 3-4 days; After cloning, pick single clones on plates with different concentrations and place them in BMGY medium.
  • the above purified bivalent humanized antibody was subjected to ELISA to detect blocking activity.
  • For good antibody samples add 50uL 0.02ug/mL biotinylated TSLPR protein to each well, and incubate at 37°C for 1 hour; after washing 5 times with PBST, add 100uL of SA-HRP (1:100000 dilution), and incubate at 37°C for 1 hour hours; after washing with PBST for 5 times, add 100uL of TMB color developing solution, develop color at 37°C for 10min, add 2M H 2 SO 4 50uL/well to terminate the reaction, and measure the absorbance at a wavelength of 450nm with a microplate
  • Well-grown BaF3/TSLPR-IL7R cells were centrifuged at 1000rpm for 5min, resuspended in PBS, and distributed into 96-well plates at 1x105 cell wells per well.
  • Mix 25uL diluted human TSLP factor with serially diluted bivalent humanized antibody or Tezepelumab incubate at 37°C for 30 minutes, then add the mixture to the cells at 37°C, 5% CO 2 for 20 minutes. After washing the cells with PBS, add formaldehyde to fix them, and then add pre-cooled methanol to incubate for 10 minutes. Finally, an anti-phosphorylated STAT5-PE-labeled antibody was added and incubated for 30 minutes.
  • the expression strains of the above three antibodies, Glycerol bacteria were respectively amplified and cultivated as primary seeds at a ratio of 1:100, and then transferred to fresh medium for secondary seed cultivation. After the secondary seed culture was qualified, they were placed in a 7L fermenter for fermentation For cultivation, ammonia water was added automatically to adjust the pH of the fermentation culture to 6.0. During the cultivation process, regular samples were taken to detect the pH of the fermentation broth, the wet weight of the bacteria, the OD value of the bacteria solution, etc., and the stirring speed, tank pressure and oxygen ventilation were adjusted according to the change of dissolved oxygen. According to the wet weight of fermentation cells and the change of dissolved oxygen, glycerol feed medium and methanol feed medium were added at different stages of fermentation culture.

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Abstract

本发明提供了抗TSLP纳米抗体及其应用体。本发明提供了一种抗TSLP的纳米抗体，本发明还提供了编码上述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，以及本发明纳米抗体的生产方法。本发明纳米抗体具有良好的TSLP/TSLPR阻断活性；本发明纳米抗体利用毕赤酵母表达，其发酵罐表达产量可达17-23g/L。

Description

抗TSLP纳米抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及抗TSLP纳米抗体及其应用。
背景技术
近年来，中重度哮喘治疗途径都聚焦于试图控制Th2细胞的响应。用于重度哮喘治疗的抗体药物多为靶向Th2通路中的IgE(奥马珠单抗)、IL5(美泊利单抗、瑞利珠单抗)、IL5R(贝那利珠单抗)、IL4R(杜匹鲁单抗)等。以上药物均对高嗜酸性粒细胞性哮喘具有良好控制效果，均以皮下或是静脉注射方式进行治疗。大约三分之一的严重哮喘患者没有Th2炎症通路激活的特征，目前对于这些非Th2驱动的疾病在既定标准的护理治疗中不能控制的患者没有生物治疗选择，因此亟待开发新的治疗途径。
胸腺基质淋巴生成素(thymic stromal lymphopoietin，TSLP)是具有四螺旋束折叠结构的短链型细胞因子，属于IL-2细胞因子家族成员。TSLP是一种针对促炎性刺激(例如肺内过敏原、病毒及其他病原体)产生的上皮细胞因子，在气道炎症的发生和持续中起着关键作用。TSLP驱动下游Th2细胞因子的释放，包括IL-4、IL-5和IL-13，导致炎症和哮喘症状。TSLP也能激活参与非Th2驱动炎症的多种类型细胞。因此，TSLP在炎症级联反应的早期上游活动已被确定为在广泛哮喘患者群体中的一个潜在靶点。阻断TSLP可阻止免疫细胞释放促炎细胞因子，从而预防哮喘恶化、改善哮喘控制，以及慢阻肺等相关疾病。
纳米抗体(nanobody，Nb)，即重链纳米抗体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纳米抗体，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。纳米抗体具有稳定性高、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。纳米抗体正逐渐成为新一代抗体治疗的新兴力量。
因此，本领域迫切需要开发一种具有较好阻断活性，较好的临床药效，并且生产简便的抗TSLP纳米抗体。
发明内容
本发明的目的就是提供一种具有较好阻断活性，较好的临床药效，并且生产简便的抗TSLP纳米抗体。
在本发明的第一方面，提供了一种抗TSLP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:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3；
(2)SEQ ID NO:14所示的CDR1、SEQ ID NO:15所示的CDR2、和SEQ ID NO:16所示的CDR3；和
(3)SEQ ID NO:27所示的CDR1、SEQ ID NO:28所示的CDR2、和SEQ ID NO:29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: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；
(2)SEQ ID NO:10所示的FR1、SEQ ID NO:5所示的FR2、SEQ ID NO:6所示的FR3、和SEQ ID NO:11所示的FR4；
(3)SEQ ID NO:17所示的FR1、SEQ ID NO:18所示的FR2、SEQ ID NO:19所示的FR3、和SEQ ID NO:20所示的FR4；
(4)SEQ ID NO:23所示的FR1、SEQ ID NO:18所示的FR2、SEQ ID NO:19所示的FR3、和SEQ ID NO:24所示的FR4；
(5)SEQ ID NO:30所示的FR1、SEQ ID NO:31所示的FR2、SEQ ID NO:32所示的FR3、和SEQ ID NO:33所示的FR4；和
(6)SEQ ID NO:36所示的FR1、SEQ ID NO:31所示的FR2、SEQ ID NO:32所示的FR3、和SEQ ID NO:37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:1-3、SEQ ID NO:14-16、SEQ ID NO:27-29中任一具有至少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:1-3、SEQ ID NO:14-16、SEQ ID NO:27-29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TSLP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TSLP。
在另一优选例中，所述纳米抗体能够有效阻断TSLP与TSLPR的相互作用。
在另一优选例中，所述TSLP为人或非人哺乳动物的TSLP。
在另一优选例中，所述TSLP为人、小鼠、大鼠、或非人灵长类动物(如猴)的TSLP。
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:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34、SEQ ID NO:38、或其组合。
在另一优选例中，所述抗TSLP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TSLP纳米抗体包括一条或多条具有如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34或SEQ ID NO:38所示的氨基酸序列的VHH链。
在另一优选例中，所述抗TSLP纳米抗体包括两个具有如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34、SEQ ID NO:38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：(G aS b) 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。
本发明第二方面提供了一种抗TSLP抗体，它是针对TSLP表位的抗体，并且具有本发明第一方面所述的抗TSLP纳米抗体。
在另一优选例中，所述抗TSLP抗体包括一个或多个抗TSLP纳米抗体。
在另一优选例中，所述抗TSLP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TSLP抗体包括一条或多条具有如SEQ ID NO:8、 SEQ ID NO:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34或SEQ ID NO:38所示的氨基酸序列的VHH链。
在另一优选例中，所述抗TSLP抗体包括两条具有如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34或SEQ ID NO:38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TSLP。
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TSLP蛋白。
在另一优选例中，所述抗体能够有效阻断TSLP与TSLPR的相互作用。
在另一优选例中，所述的抗体对TSLP的亲和力(KD值)小于3.77nM。
在另一优选例中，所述的抗体具有良好的TSLP/TSLPR阻断活性，且阻断活性显著优于对照抗体Tezepelumab，其中，对照抗体Tezepelumab获自阿斯利康(AstraZeneca)或安进(Amgen)公司。
在另一优选例中，所述的抗体能够有效抑制BaF3/TSLPR-IL7R细胞的增殖，其抑制活性优于对照抗体Tezepelumab。
在另一优选例中，所述抗体为纳米抗体。
本发明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TSLP纳米抗体或本发明第二方面所述的抗TSLP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、13、22、26、35或39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。
本发明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。
本发明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。
本发明第六方面提供了一种产生抗TSLP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TSLP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TSLP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TSLP纳米抗体。
本发明第七方面提供了一种免疫偶联物，该免疫偶联物含有：
(a)本发明第一方面所述的抗TSLP纳米抗体、或本发明第二方面所述的抗TSLP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。
本发明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TSLP纳米抗体，或本发明第二方面所述的抗TSLP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。
本发明第九方面提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的抗TSLP纳米抗体、或如本发明第二方面所述的抗TSLP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TSLP蛋白。
本发明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TSLP纳米抗体、或本发明第二方面所述的抗TSLP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TSLP相关的疾病或病症的药物。
在另一优选例中，所述与TSLP相关的疾病或病症包括免疫系统疾病或肿瘤疾病。
在另一优选例中，所述免疫系统疾病选自下组：哮喘、特应性皮炎、慢性阻塞性肺疾病(COPD)、过敏性结膜炎、食物过敏、溃疡性结肠炎、克罗恩病、鼻炎、强直性脊柱炎、系统性红斑狼疮、类风湿性关节炎、过敏性肺炎、变应性肉芽肿性血管炎、鼻息肉病、或其组合。
在另一优选例中，所述肿瘤疾病选自下组：乳腺癌、胰腺癌、宫颈癌、多发性骨髓瘤、结直肠癌、肺癌、甲状腺癌、卵巢癌、肝癌、或其组合。
本发明第十一方面提供了本发明第一方面所述的抗TSLP纳米抗体、本发明第二方面所述的抗TSLP抗体、如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，(a)用于制备预防和/或治疗与TSLP相关的疾病的药物；和/或(b)用于制备检测TSLP的试剂、检测板或试剂盒。
在另一优选例中，所述与TSLP相关的疾病或病症包括免疫系统疾病或肿瘤疾病。
在另一优选例中，所述免疫系统疾病选自下组：哮喘、特应性皮炎、慢性阻塞性肺疾病(COPD)、过敏性结膜炎、食物过敏、溃疡性结肠炎、克罗恩病、鼻炎、强直性脊柱炎、系统性红斑狼疮、类风湿性关节炎、过敏性肺炎、变应性肉芽肿性血管炎、鼻息肉病、或其组合。
在另一优选例中，所述肿瘤疾病选自下组：乳腺癌、胰腺癌、宫颈癌、多发性骨髓瘤、结直肠癌、肺癌、甲状腺癌、卵巢癌、肝癌、或其组合。
在另一优选例中，所述TSLP为人TSLP。
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TSLP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。
本发明第十二方面提供了一种检测样品中TSLP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TSLP纳米抗体、或如本发明第二方面所述的抗TSLP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TSLP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。
本发明第十三方面提供了一种TSLP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TSLP纳米抗体、或如本发明第二方面所述的抗TSLP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)。
本发明第十四方面提供了一种检测TSLP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TSLP表达。
本发明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TSLP蛋白的造影剂。
在另一优选例中，所述检测用于与TSLP相关的疾病或病症的诊断或预后。
本发明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第一方面所述的抗TSLP纳米抗体、本发明第二方面所述的抗TSLP抗体、如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1A和图1B是31株候选抗体进行流式细胞术阻断活性检测的结果。结果表明：31株候选抗体中的14株抗体的阻断活性显著优于对照抗体Tezepelumab。
图2是14株阻断型TSLP纳米抗体的结合动力学检测结果。
图3是二价单域抗体Bi-HuNb5-31、Bi-HuNb7-54、Bi-HuNb10-63在毕赤酵母中的摇瓶表达上清SDS-PAGE结果，其产量分别为475ug/mL、310ug/mL、510ug/mL。
图4是ELISA检测人源化二价抗体阻断活性的结果。结果表明，三株人源化二价抗体的阻断活性显著优于对照抗体Tezepelumab。
图5是人源化二价抗体对BaF3/TSLPR-IL7R细胞的增殖抑制作用的结果。结果表明，其中两株人源化抗体对BaF3/TSLPR-IL7R细胞中pSTAT5信号通路的抑制作用优于对照抗体Tezepelumab。
具体实施方式
首次意外地发现一类抗TSLP纳米抗体，实验结果表明，本发明的纳米抗体能够有效阻断TSLP与TSLPR的相互作用，且阻断活性显著优于对照抗体Tezepelumab；本发明的纳米抗体能够有效抑制Baf3/TSLPR-IL7R细胞的增殖，其抑制活性优于对照抗体Tezepelumab；本发明纳米抗体在毕赤酵母中的发酵罐表达产量可达17-23g/L，显著高于行业水平。在此基础上，本发明人完成了本发明。
术语
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TSLP纳米抗体”、“本发明TSLP纳米抗体”、“抗TSLP纳米抗体”、“TSLP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TSLP(包括人TSLP)的纳米抗体。
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“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TSLP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TSLP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TSLP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
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。
胸腺基质淋巴生成素(thymic stromal lymphopoietin，TSLP)
胸腺基质淋巴生成素是具有四螺旋束折叠结构的短链型细胞因子，属于IL-2细胞因子家族成员。作为一种新型细胞因子，TSLP通常在肺、皮肤和肠道屏障表面的上皮细胞中表达。动物实验表明TSLP在过敏原诱导的哮喘模型小鼠肺中高表达，而TSLP受体缺陷小鼠哮喘表现明显减轻，肺脏特异性TSLP转基因小鼠表现出以Th2型炎症以及IgE增高的气道炎症和高反应性。进一步研究提示，TSLP激活骨髓来源的树突状细胞并上调共刺激分子，产生Th2型细胞趋化因子CCL17。因此，TSLP是启动气道过敏性炎症的重要因子和必要条件。它是包括哮喘在内的各种疾病中多种炎症途径的上游调节子，对气道炎症的发生和持续进行至关重要。另外，研究表明，TSLP也是一种与特应性皮炎(AD)发病机理有关的细胞因子。更重要的是，已有研究人员发现在许可 不同类型肿瘤中，TSLP水平会升高，TSLP又能够诱导肿瘤细胞表达另一种称为BCL-2的蛋白质，这种蛋白质可以保护肿瘤免受死亡。因此TSLP对于肿瘤的存活也是至关重要的。
胸腺基质淋巴生成素受体(thymic stromal lymphopoietin receptor，TSLPR)
TSLPR受体为I型跨膜蛋白质，属于造血细胞因子受体家族成员。功能性TSLPR复合物是由TSLPR和IL-7Ra组成。TSLPR也称为细胞因子受体样分子2或I型细胞因子受体σ1。TSLP通过结合其高亲和力异二聚体受体复合物启动细胞内2型信号传导，异二聚体受体复合物由其特异性受体TSLPR组成，其与IL-2、IL-4、IL-9和IL-15中的共同受体γ链具有24％的同源性，并且不包含δ-共同链IL-2家族，以及共同表达TSLPR和IL-7Rα的细胞中的IL-7Rα亚单位(CD127)。TSLP最初与TSLPR结合，随后招募IL-7Rα链。
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TSLP蛋白分子，因而可用于治疗与TSLP相关的疾病或病症(包括免疫系统疾病或肿瘤疾病)。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％)的本发明上述的纳米抗体(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TSLP纳米抗体
本发明中，所述抗TSLP纳米抗体的VHH链的氨基酸序列选自SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34或SEQ ID NO:38中的一种或多种。
在本发明的一个优选例中，所述抗TSLP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TSLP纳米抗体包括两条具有如SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34或SEQ ID NO:38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：(G aS b) 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。
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，TSLP的纳米抗体用胶体金标记，得到胶体金标记的纳米抗体。
检测方法
本发明还涉及检测TSLP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TSLP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TSLP水平的检测试剂盒，该试剂盒包括识别TSLP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TSLP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TSLP的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明的纳米抗体能够有效阻断TSLP与TSLPR的相互作用。
(b)本发明的纳米抗体相较于对照抗体Tezepelumab具有更强的阻断活性。
(c)本发明的纳米抗体可在毕赤酵母中表达，摇瓶表达产量较高，且发酵罐产量可达17-23g/L。
(d)本发明的纳米抗体对BaF3/TSLPR-IL7R细胞中pSTAT5信号通路的抑制作用显著优于对照抗体Tezepelumab。
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 TSLP特异性纳米抗体筛选
将弗林位点突变的人TSLP氨基酸序列按照人密码子优化后，将其碱基序列克隆至pFUSE载体上，转染HEK293F细胞后纯化出人TSLP蛋白。将高纯度的蛋白与免疫佐剂混合后分别免疫四只新疆双峰驼，每周一次，七次免疫后取骆驼外周血，分离RNA并扩增VHH基因片段，随后将其克隆至pMECS载体，电转化至TG1感受态细胞中建立高质量噬菌体展示纳米抗体文库。经检测，四个文库的库容均达到1x10 9CFU以上，片段插入率在80％以上。
利用噬菌体展示技术筛选TSLP特异性纳米抗体。经过三轮“吸附-洗涤-富集”的过程，各文库均出现特异性纳米抗体噬菌体富集。从中随机挑选1200个克隆进行ELISA检测及测序分析，最终获得312株差异序列抗体。
实施例2流式细胞术筛选阻断型TSLP纳米抗体
将以上序列不同的纳米抗体克隆接种于TB培养基中培养并用IPTG诱导过夜，裂解细胞收集上清用于阻断活性检测。将培养好的CHOZEN/TSLPR稳转细胞分装至96孔板中，每孔3E5个细胞，3000rpm离心3min去掉上清液，加入各抗体的裂解液及TSLP-Biotin蛋白孵育20min。离心弃上清，加入稀释的SA-PE抗体，4℃孵育20min。再次离心弃上清，每孔加入200uL PBS重悬细胞，流式细胞仪检测样本PE信号。结果表明：共有64株候选抗体具有阻断TSLP与TSLPR相互作用的功能。结合序列分析，挑选出31株不同家族的抗体进行表达纯化。纯化方法参见专利CN110144011A中实施例4。
将以上31株纯化的抗体再次进行流式细胞术阻断活性检测。将培养好的HEK293F/TSLPR瞬转细胞分装至96孔板中，每孔3E5个细胞，3000rpm离心3min去掉上清液，加入梯度稀释好的各抗体(从20ug/mL开始2倍梯度稀释)及TSLP-Biotin蛋白孵育20min。本次实验以Tezepelumab为对照抗体。随后离心弃上清，加入稀释的SA-PE抗体，4℃孵育20min。再次离心弃上清，每孔加入200uL PBS重悬细胞，流式细胞仪检测样本PE信号。结果如图1A和1B所示，其中14株抗体的阻断活性显著优于对照抗体Tezepelumab，14株抗体的编号分别为Nb1-41、Nb1-51、Nb1-59、Nb3-18、Nb3-43、Nb5-31、Nb6-29、Nb7-54、Nb10-55、Nb10-63、Nb10-87、Nb11-6、Nb11-72、Nb11-75。
实施例3抗体亲和力测定
通过生物膜层干涉技术(Bio-layer interferometry BLI)对14株阻断型TSLP纳米抗体的结合动力学进行检测。对于动力学测量，将候选抗体用PBST缓冲液稀释至5ug/mL，TSLP-Fc抗原用PBST缓冲液2倍梯度稀释六个浓度梯度(从20nM开始2倍梯度稀释)，设置仪器运行条件：温度30℃，Shake speed 1000rpm。使用已包被Protein A的探针捕获抗体，捕获时间60s；结合梯度稀释的抗原，结合时间240s；解离时间300s；10mM甘氨酸(pH1.7)再生2次，每次5s。使用Fortebio Analysis 9.0版本进行分析，Global模式进行拟合，计算结合速率(Kon)，解离速率(Kdis)和解离常数KD。结果如图2所示。
实施例4毕赤酵母表达人源化二价抗体
对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。人源化后抗体的氨基酸序列如表1所示。
Figure PCTCN2022093166-appb-000001
将以上人源化的抗体构建成二价形式，利用连接子(G 4S)4连接，连接后的序列如SEQ ID NO.:40、SEQ ID NO.:41和SEQ ID NO.:42所示，随后利用毕赤酵母进行表达。简要地，表达方法如下：(1)将以上TSLP纳米抗体二价体序列构建至pPICZaA载体；(2)用Sac I限制性内切酶线性化后电转化至X-33感受态细胞中；(3)将电转的样品分别涂布在含有不同浓度博来霉素抗性的YPD平板培养基上，置于30℃培养箱中培养3-4天；(4)待平板培养基上长出单克隆后，挑取不同浓度平板上的单克隆置于BMGY培养基中，当BMGY培养液OD值达到20左右，收集菌体后更换至BMMY培养基中，28℃250rpm培养；(5)之后每24h取样，并补加终体积为1％的甲醇并取样；样品12000rpm离心5min，取上清，-20℃保存；连续诱导5天，结束培养；(6)将取到的上清样品进行SDS-PAGE检测。二价单域抗体的摇瓶表达量如图3所示。
实施例5 ELISA检测人源化二价抗体阻断活性
将以上纯化的二价人源化抗体进行ELISA检测阻断活性。将人TSLP抗原蛋白分装至96孔板中，4℃过夜孵育；用PBST洗涤5次后，加入300uL 1％BSA封闭液，37℃孵育2小时；用PBST洗涤5次后，加入50uL梯度稀释好的抗体样品，再每孔加入50uL 0.02ug/mL生物素化的TSLPR蛋白，37℃孵育1小时；用PBST洗涤5次后，加入SA-HRP(1:100000稀释)100uL，37℃孵育1小时；用PBST洗涤5次后，加入TMB显色液100uL，37℃显色10min，加入2M H 2SO 4 50uL/孔终止反应，酶标仪450nm波长下测定吸收值。结果如图4所示：三株人源化二价抗体的阻断活性显著优于对照抗体Tezepelumab。
实施例6人源化二价抗体对BaF3/TSLPR-IL7R细胞中pSTAT5信号通路的抑制作用
将生长良好的BaF3/TSLPR-IL7R细胞于1000rpm离心5min，PBS重悬细胞，以每孔1x10 5个细胞孔分装到96孔板中。将25uL稀释好的人TSLP因子和梯度稀释的二价人源化抗体或者Tezepelumab混合，37度孵育30分钟后将混合物加入到细胞中37℃，5％CO 2放置20分钟。PBS洗涤细胞后，加入甲醛固定，再加入预冷的甲醇孵育10分钟。最后加入抗磷酸化STAT5-PE标记抗体孵育30分钟，洗涤细胞后上流式细胞仪检测PE信号值。结果如图5所示，其中两株人源化抗体对BaF3/TSLPR-IL7R细胞中pSTAT5信号通路的抑制作用显著优于对照抗体Tezepelumab。
实施例7人源化二价抗体在7L发酵罐中的产量评估
将以上3个抗体的表达菌株甘油菌分别按1:100扩增培养一级种子，随后转入新鲜培养基中进行二级种子培养，待二级种子培养合格后接入7L发酵罐中进行发酵培养，自动流加氨水调节发酵培养pH为6.0。培养过程中，定时取样检测发酵液pH、菌体湿重、菌液OD值等，并根据溶氧变化调节搅拌转速、罐压和氧气通气量。根据发酵菌体湿重和溶氧变化，在发酵培养的不同阶段分别流加甘油补料培养基和甲醇补料培养基。甲醇诱导培养160h～200h，停止发酵。经检测上述三个人源化二价抗体的产量分别为17g/L，19g/L和23g/L。该抗体产量远远高于已报到的所有类型抗体发酵表达产量，达到行业领先水平。
序列信息：
Figure PCTCN2022093166-appb-000002
Figure PCTCN2022093166-appb-000003
Figure PCTCN2022093166-appb-000004
Figure PCTCN2022093166-appb-000005
Figure PCTCN2022093166-appb-000006
Figure PCTCN2022093166-appb-000007
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 (19)

  1. 一种抗TSLP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:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3；
    (2)SEQ ID NO:14所示的CDR1、SEQ ID NO:15所示的CDR2、和SEQ ID NO:16所示的CDR3；和
    (3)SEQ ID NO:27所示的CDR1、SEQ ID NO:28所示的CDR2、和SEQ ID NO:29所示的CDR3。
  2. 如权利要求1所述的抗TSLP纳米抗体，其特征在于，所述抗TSLP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: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；
    (2)SEQ ID NO:10所示的FR1、SEQ ID NO:5所示的FR2、SEQ ID NO:6所示的FR3、和SEQ ID NO:11所示的FR4；
    (3)SEQ ID NO:17所示的FR1、SEQ ID NO:18所示的FR2、SEQ ID NO:19所示的FR3、和SEQ ID NO:20所示的FR4；
    (4)SEQ ID NO:23所示的FR1、SEQ ID NO:18所示的FR2、SEQ ID NO:19所示的FR3、和SEQ ID NO:24所示的FR4；
    (5)SEQ ID NO:30所示的FR1、SEQ ID NO:31所示的FR2、SEQ ID NO:32所示的FR3、和SEQ ID NO:33所示的FR4；和
    (6)SEQ ID NO:36所示的FR1、SEQ ID NO:31所示的FR2、SEQ ID NO:32所示的FR3、和SEQ ID NO:37所示的FR4。
  3. 如权利要求1所述的抗TSLP纳米抗体，其特征在于，所述抗TSLP纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:34、SEQ ID NO:38、或其组合。
  4. 一种抗TSLP抗体，其特征在于，它是针对TSLP表位的抗体，并且具有权利要求1所述的抗TSLP纳米抗体。
  5. 一种抗TSLP抗体，其特征在于，所述抗体包括一个或多个如权利要求 1所述的抗TSLP纳米抗体。
  6. 如权利要求4或5所述的抗体，其特征在于，所述抗体包括单体、二价抗体、和/或多价抗体。
  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TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP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TSLP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TSLP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TSLP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TSLP纳米抗体。
  11. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)权利要求1所述的抗TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP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，其特征在于，所述的多特异性抗体包含：权利要求1所述的抗TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP抗体。
  13. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的抗TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP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，其特征在于，所述药物组合物含有：
    (i)权利要求1所述的抗TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP抗体、或权利要求11所述的免疫偶联物或权利要求12所述的多特异性抗体或权利要求13所述的重组蛋白；以及
    (ii)药学上可接受的载体。
  15. 权利要求1所述的抗TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP抗体、如权利要求11所述的免疫偶联物或权利要求12所述的多特异性抗体或权利要求13所述的重组蛋白或权利要求14所述的药物 组合物的用途；其特征在于，(a)用于制备预防和/或治疗与TSLP相关的疾病的药物；和/或(b)用于制备检测TSLP的试剂、检测板或试剂盒。
  16. 一种检测样品中TSLP蛋白的方法，其特征在于，所述方法包括步骤：
    (1)将样品与权利要求1所述的抗TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP抗体、或权利要求11所述的免疫偶联物或权利要求12所述的多特异性抗体或权利要求13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TSLP蛋白。
  17. 一种TSLP蛋白检测试剂，其特征在于，所述的检测试剂包含：
    (i)权利要求1所述的抗TSLP纳米抗体、权利要求4所述的抗TSLP抗体或权利要求5所述的抗TSLP抗体、或权利要求11所述的免疫偶联物或权利要求12所述的多特异性抗体或权利要求13所述的重组蛋白；以及
    (ii)检测学上可接受的载体。
  18. 一种检测TSLP蛋白的试剂盒，其特征在于，所述试剂盒含有权利要求7所述的免疫偶联物或权利要求17所述的检测试剂，以及说明书。
  19. 一种权利要求11所述的免疫偶联物的用途，其特征在于，用于制备体内检测TSLP蛋白的造影剂。
PCT/CN2022/093166 2021-02-05 2022-05-16 抗tslp纳米抗体及其应用 WO2023142309A1 (zh)

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