WO2022089108A1 - 抗il5纳米抗体及其应用 - Google Patents

抗il5纳米抗体及其应用 Download PDF

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WO2022089108A1
WO2022089108A1 PCT/CN2021/119969 CN2021119969W WO2022089108A1 WO 2022089108 A1 WO2022089108 A1 WO 2022089108A1 CN 2021119969 W CN2021119969 W CN 2021119969W WO 2022089108 A1 WO2022089108 A1 WO 2022089108A1
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seq
nanobody
antibody
fusion protein
present
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PCT/CN2021/119969
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French (fr)
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万亚坤
朱敏
盖军伟
李光辉
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 EP21884838.0A priority Critical patent/EP4238989A1/en
Priority to KR1020237018299A priority patent/KR20230098629A/ko
Priority to US18/251,303 priority patent/US20230399395A1/en
Priority to JP2023526251A priority patent/JP2023549701A/ja
Publication of WO2022089108A1 publication Critical patent/WO2022089108A1/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]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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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
    • G01N33/563—Immunoassay; Biospecific binding assay; Materials therefor involving antibody fragments
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    • 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
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    • 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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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
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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
    • C07K2317/565—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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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]
    • G01N2333/5409—IL-5

Definitions

  • the present invention relates to the technical field of biomedicine or biopharmaceuticals, and more particularly to an anti-IL5 nanobody and application thereof.
  • Eosinophils play an important role in protecting the body from infection. But in some individuals, elevated levels of eosinophils may lead to inflammation and play a role in the development of certain inflammatory diseases.
  • IL5 is the most selective for eosinophils among the known cytokines, and can regulate the growth, differentiation, recruitment, activation and survival of eosinophils. Its receptor IL5R ⁇ is highly expressed on eosinophils and plays a key role in eosinophils to clear allergens in blood and tissues. IL5 is currently considered to be one of the key drivers of the Th2 pathway, and the binding of IL5 to its receptor will further activate the downstream JAK-STAT signaling pathway.
  • IL5 has played an important role in the field of immunotherapy.
  • Three monoclonal antibodies targeting IL5/IL5R ⁇ have been approved for marketing worldwide, two of which are IL5 antibodies. (GSK's Mepolizumab and Teva's Reslizumab), one is an IL5R ⁇ antibody (AstraZeneca's Benralizumab), bringing new treatment options to patients.
  • the IL5 humanized monoclonal antibody injection (610) of Sunshine Guojian, a local Chinese pharmaceutical company, and the IL5 antibody (SHR-1703) of Hengrui Medicine have also been approved by the State Drug Administration for clinical trials.
  • GSK's Mepolizumab The world's first approved IL5 monoclonal antibody, GSK's Mepolizumab, has been approved for multiple clinical studies, targeting moderate asthma, eosinophilic granulomatosis with polyangiitis (EGPA), chronic obstructive pulmonary disease (COPD), Chronic rhinosinusitis with nasal polyps (CRSwNP), severe eosinophilic syndrome, severe atopic dermatitis, severe bilateral nasal polyps and other indications.
  • EGPA eosinophilic granulomatosis with polyangiitis
  • COPD chronic obstructive pulmonary disease
  • COSwNP Chronic rhinosinusitis with nasal polyps
  • severe eosinophilic syndrome severe atopic dermatitis, severe bilateral nasal polyps and other indications.
  • Nanobody namely heavy chain nanobody VHH (variable domain of heavy chain of heavy-chain antibody) - there is a natural The heavy-chain antibody (HCAb) lacking the light chain, and the nanobody composed of only one heavy chain variable region obtained by cloning its variable region is a stable and binding with complete function currently available.
  • Nanobodies have the characteristics of high stability, good water solubility, simple humanization, high targeting, and strong penetrability. Nanobodies are gradually becoming an emerging force in the new generation of antibody diagnosis and treatment.
  • the purpose of the present invention is to provide an anti-IL5 nanobody and its application.
  • the first aspect of the present invention provides an anti-IL5 Nanobody, the Nanobody can specifically bind to IL5, and the CDR of the VHH chain in the Nanobody is one or more selected from the group kind:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains the ability to bind to IL5.
  • the VHH chain of the anti-IL5 Nanobody further includes a framework region FR, and the framework region FR is one or more selected from the group consisting of:
  • the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.
  • amino acid sequence of the VHH chain of the anti-IL5 Nanobody is selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, or a combination thereof.
  • the anti-IL5 Nanobody includes monomer, bivalent (bivalent antibody), tetravalent (tetravalent antibody), and/or multivalent (multivalent antibody).
  • the anti-IL5 Nanobody comprises two VHH chains having the amino acid sequences shown in SEQ ID NO: 41 and/or SEQ ID NO: 47, preferably, one of the VHH chains connected by a linker peptide.
  • sequence of the connecting peptide is (G 4 S) 4 .
  • the anti-IL5 Nanobody can recognize two different IL5 epitopes.
  • the nanobodies include humanized antibodies, camel-derived antibodies, and chimeric antibodies.
  • the second aspect of the present invention provides an anti-IL5 antibody, which is an antibody against the interleukin 5 (IL5) epitope and has the anti-IL5 nanobody according to the first aspect of the present invention.
  • IL5 interleukin 5
  • the anti-IL5 antibody includes a monomer, a bivalent (divalent antibody), a tetravalent (tetravalent antibody), and/or a multivalent (multivalent antibody).
  • the anti-IL5 antibody comprises one or more antibodies with SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 41 or SEQ ID NO: 17
  • SEQ ID NO: 8 SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 41 or SEQ ID NO: 17
  • the anti-IL5 antibody comprises two VHH chains having the amino acid sequence shown in SEQ ID NO:41 and/or SEQ ID NO:47.
  • the structure of the anti-IL5 antibody from the N-terminus to the C-terminus is shown in formula I:
  • A1 and A2 are each independently any one of the anti-IL5 Nanobodies described in the first aspect of the present invention.
  • B is the Fc fragment of IgG.
  • L is no or flexible joint.
  • the A1 and A2 are each independently a VHH chain having the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:47.
  • the A1 is a VHH chain having the amino acid sequence shown in SEQ ID NO: 41
  • the A2 is a VHH chain having the amino acid sequence shown in SEQ ID NO: 47.
  • the A1 is a VHH chain with the amino acid sequence shown in SEQ ID NO:47
  • the A2 is a VHH chain with the amino acid sequence shown in SEQ ID NO:41.
  • the flexible linker is a linking peptide.
  • VHH chains are connected through a linking peptide.
  • sequence of the connecting peptide is (G 4 S) 4 .
  • amino acid sequence of the antibody is shown in SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:59.
  • the antibody can specifically bind to the IL5 protein with correct spatial structure.
  • the antibody can effectively block the interaction between IL5 and IL5R.
  • the antibody can recognize IL5 of human and cynomolgus monkey, but not IL5 of mouse.
  • the affinity of the antibody to IL5 is less than 1 nM.
  • the antibody has good IL5/IL5R blocking activity, and the blocking activity is significantly better than that of the control antibody Nucala.
  • the control antibody Nucala is GlaxoSmithKline's marketed drug Mepolizumab, that is, mepolizumab.
  • the antibody can effectively inhibit the proliferation of TF-1 cells induced by IL5, and its inhibitory activity is better than that of the control antibody Nucala.
  • the antibody is a nanobody.
  • an anti-IL5 Nanobody Fc fusion protein is provided, and the structure of the fusion protein from the N-terminus to the C-terminus is as shown in formula Ia or Ib:
  • A is one or more anti-IL5 Nanobodies according to the first aspect of the present invention.
  • B is the Fc fragment of IgG.
  • L is no or flexible joint.
  • the flexible linker is a linking peptide.
  • the Fc fragment of IgG includes the Fc fragment of human IgG.
  • the Fc fragment of IgG is selected from the group consisting of Fc fragments of IgG1, IgG2, IgG3, IgG4, or a combination thereof.
  • the Fc fragment of the IgG is IgG4.
  • amino acid sequence of the Fc fragment is shown in SEQ ID NO:63.
  • amino acid sequence of the fusion protein is such as SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO: 59 shown.
  • the fusion protein is a Nanobody Fc fusion protein against the IL5 epitope.
  • the fourth aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of the anti-IL5 nanobody according to the first aspect of the present invention, the anti-IL5 nanobody according to the second aspect of the present invention The anti-IL5 antibody, or the anti-IL5 nanobody Fc fusion protein of the third aspect of the present invention.
  • the polynucleotide sequence is in a combined form, preferably, the polynucleotide sequence comprises SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36 , SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, or SEQ ID NO:54 One or more of ID NO: 62.
  • the polynucleotide includes DNA or RNA.
  • the fifth aspect of the present invention provides an expression vector containing the polynucleotide according to the fourth 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 a combination thereof.
  • the expression vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a viral vector such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • the sixth aspect of the present invention provides a host cell, the host cell contains the expression vector of the fifth aspect of the present invention, or the polynucleotide of the fourth aspect of the present invention is integrated into 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, bacteriophage, or a combination thereof.
  • the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or a combination thereof.
  • the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, Trichoderma, or a combination thereof.
  • the host cell is Pichia pastoris.
  • the seventh aspect of the present invention provides a method for producing an anti-IL5 nanobody or an Fc fusion protein thereof, comprising the steps of:
  • step (c) Optionally, purify and/or modify the anti-IL5 Nanobody or its Fc fusion protein obtained in step (b).
  • the eighth aspect of the present invention provides an immunoconjugate, the immunoconjugate contains:
  • a conjugation 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 radionuclide includes:
  • 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
  • a therapeutic isotope 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 group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, 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 reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatin ( auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4) ), taxanes, benzodiazepines, or benzodiazepine containing drugs (eg, pyrrolo[1,4]benzodiazepines (PBDs), indole indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • auristatin auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4)
  • taxanes eg, benzodiazepines
  • the toxin is selected from the group consisting of: auristatins (eg, auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansinoid, gatetoxin, grate Cannatoxin A-chain, combretastatin, docarmicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, Tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthraxdione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, acacia Toxin, abrin A chain, capsular root toxin A chain, ⁇ -sarcinus, gelonin, mitogellin,
  • the coupling moiety is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing Enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles that can detect products , prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or nanoparticles in any form.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the ninth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition contains:
  • the coupling part of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
  • the pharmaceutical composition also contains other drugs for the treatment of asthma, atopic dermatitis, arthritis, allergic rhinitis and/or eczema, such as corticosteroids (TCS), nedocromil sodium, Sodium cromoglycate, theophylline, a leukotriene receptor antagonist, or a combination thereof.
  • TCS corticosteroids
  • nedocromil sodium nedocromil sodium
  • Sodium cromoglycate theophylline
  • leukotriene receptor antagonist a combination thereof.
  • the pharmaceutical composition is used to prepare a medicament for preventing and/or treating diseases or conditions related to IL5/IL5R signaling.
  • the tenth aspect of the present invention provides the anti-IL5 Nanobody according to the first aspect of the present invention, the anti-IL5 Nanobody according to the second aspect of the present invention, and the anti-IL5 Nanobody according to the third aspect of the present invention Fc fusion Use of the protein, or the immunoconjugate according to the eighth aspect of the present invention; (a) for the preparation of a medicament for the prevention and/or treatment of diseases or conditions related to IL5/IL5R signaling; (b) for the preparation of Reagents, assay plates or kits for the detection of IL5.
  • the diseases or conditions include but are not limited to: asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, sinusitis, nasal polyps, chronic obstructive pulmonary disease, eosinophilic granulomatous Polyangiitis, hypereosinophilic syndrome, or a combination thereof.
  • the IL5 is human IL5.
  • the reagents shown are diagnostic reagents.
  • the diagnostic reagent shown is a contrast agent
  • the reagent is used to detect IL5 protein or its fragment in the sample.
  • a multispecific antibody comprises: the anti-IL5 nanobody according to the first aspect of the present invention, or the second invention of the present invention. of anti-IL5 antibodies.
  • the multispecific antibody further comprises a second antigen binding region targeting a target selected from the group consisting of IL-4R, IL-4R ⁇ , IL-13, IL-13R, IL-11, IL -11R, or a combination thereof.
  • the second antigen-binding region is a nanobody.
  • the multispecific antibody includes one or more second antigen binding regions.
  • the multispecific antibody further comprises the Fc segment of the antibody.
  • the twelfth aspect of the present invention provides a recombinant protein, the recombinant protein has:
  • the tag sequence includes Fc tag, HA tag and 6His tag.
  • the recombinant protein specifically binds to IL5 protein.
  • the anti-IL5 nanobody according to the first aspect of the present invention or the anti-IL5 antibody according to the second aspect of the present invention, or the anti-IL5 antibody according to the third aspect of the present invention
  • the diseases or conditions include but are not limited to: asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, sinusitis, nasal polyps, chronic obstructive pulmonary disease, eosinophilic granulomatous Polyangiitis, hypereosinophilic syndrome, or a combination thereof.
  • the detection includes flow detection and cellular immunofluorescence detection.
  • the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
  • a fourteenth aspect of the present invention provides a method for detecting IL5 protein in a sample, the method comprising the steps of:
  • the method is a non-diagnostic and non-therapeutic method.
  • an IL5 protein detection reagent comprises:
  • the coupling part of the immunoconjugate is a diagnostic isotope.
  • the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the group consisting of isotope 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 (eg, water preparation, injection, freeze-dried powder, tablet, buccal preparation, aerosol preparation).
  • the sixteenth aspect of the present invention provides a kit for detecting IL5 protein, the kit contains the immunoconjugate according to the eighth aspect of the present invention or the detection reagent according to the fifteenth aspect of the present invention, and manual.
  • the instructions describe that the kit is used to non-invasively detect the IL5 expression of the test object.
  • the seventeenth aspect of the present invention provides a use of the immunoconjugate according to the eighth aspect of the present invention for preparing a contrast agent for detecting IL5 protein in vivo.
  • the detection is used for asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema, sinusitis, nasal polyps, chronic obstructive pulmonary disease, eosinophilic granulomatosis with polyangiitis, high Diagnosis or prognosis of hypereosinophilic syndrome, etc.
  • Figure 1 shows the IL5 Nanobody screening enrichment process. After five rounds of panning, IL5-specific Nanobody phages were 120-fold and 14.6-fold enriched in the two libraries, respectively.
  • Figure 2 shows the results of a preliminary flow cytometry screening for Nanobodies that block the interaction of IL5 with IL5R. The results showed that 11 of the 96 nanobodies had blocking activity.
  • Figure 3 shows the results of species cross-activity of 11 blocking IL5 nanobodies detected by ELISA. The results showed that all 11 nanobodies could recognize human and cynomolgus monkey IL5, but could not recognize mouse IL5.
  • Figure 4 shows the affinity results of nanobodies detected by Fortebio using biofilm layer interference technology. The results showed that the affinity of 11 nanobodies to IL5 was less than 1 nM.
  • Figure 5 shows the results of the binding activity of Nanobodies detected by ELISA. The results showed that the binding activities of the four nanobodies were better than those of the control antibody Nucala.
  • Figure 6 shows the blocking activity of candidate IL5 Nanobodies identified by flow cytometry. The results showed that the four nanobodies all had good IL5/IL5R blocking activities, and the blocking activities of Nb21 and Nb66 were significantly better than those of the control antibody Nucala.
  • Figure 7 shows the results of ELISA detection of the antigen recognition epitope consistency of blocking nanobodies. The results showed that Nb21 and Nb66 were different antigen recognition epitopes.
  • Figure 8 shows the blocking activity of humanized bivalent Nanobodies detected by flow cytometry. The results showed that the activity of humanized bivalent antibodies was significantly higher than that of monovalent antibodies, and the blocking activity of HuNb21-HuNb66-Fc and HuNb66-HuNb21-Fc bivalent bi-epitope antibodies was the best, and was significantly better than the control antibody Nucala .
  • Figure 9 shows the results of Fortebio's detection of the bi-epitope bivalent IL5 Nanobody in the fermentation supernatant of Pichia pastoris. The results showed that under the fermentation conditions, the expression of bivalent bi-epitope antibodies increased continuously with the extension of culture time, and the antibody yield of 202 hours of fermentation could reach 13 g/L.
  • Figure 10 shows the results of SDS-PAGE detection of the bi-epitope bivalent IL5 Nanobody Pichia fermentation supernatant. The results showed that the double-epitope IL5 nanobody HuNb66-HuNb21 was expressed in Pichia pastoris with a yield of up to 13 g/L, and the target protein expression supernatant had high purity.
  • Figure 11 shows the results of flow cytometry detection of the blocking activity of bivalent bi-epitopic IL5 Nanobodies.
  • Figure 12 shows the detection results of the inhibitory effect of bivalent bi-epitope IL5 Nanobody on the proliferation of TF1 cells.
  • the inventors unexpectedly discovered a type of IL5 nanobody for the first time.
  • the experimental results show that the nanobody of the present invention can specifically recognize human and cynomolgus IL5, but not mouse IL5. , has good specificity and binding activity; the nanobody of the present invention has good IL5/IL5R blocking activity, and the blocking activity is significantly better than that of the control antibody Nucala; the nanobody of the present invention can effectively inhibit IL5-induced TF-1 cells. Proliferation, its inhibitory activity is better than that of the control antibody Nucala; the expression yield of the nanobody of the present invention in Pichia pastoris can reach 13 g/L, and the target protein expression supernatant has high purity.
  • Nanobodies of the invention As used herein, the terms “Nanobodies of the invention”, “Nanobodies of the invention”, “anti-IL5 Nanobodies of the invention”, “IL5 Nanobodies of the invention”, “anti-IL5 Nanobodies”, “IL5 Nanobodies” Having the same meaning and being used interchangeably, both refer to Nanobodies that specifically recognize and bind to IL5 (including human IL5).
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a 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. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) composed of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete function.
  • VHH nanobody
  • CH1 light chain and heavy chain constant region 1
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. 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 parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable domains of native heavy and light chains each contain four FR regions, which are generally in a b-sheet configuration, connected by three CDRs that form linking loops, and in some cases may form part of the b-sheet structure.
  • the CDRs in each chain are tightly packed together by 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. 1, 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, such as involvement in 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 the conjugate.
  • the present invention also includes cell surface markers or antigens that bind to the anti-IL5 antibodies or fragments thereof.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • 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 a polypeptide that specifically binds to IL5 protein, such as a protein or polypeptide having a heavy chain variable region . They may or may not contain the starting methionine.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a variable region-containing heavy chain, as long as the variable region is associated with the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), four FR amino acids
  • FRs framework regions
  • FRs framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the 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. Accordingly, the present invention includes those molecules having CDR-bearing antibody heavy chain variable regions, as long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues 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 with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region having IL5-binding activity.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , 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-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
  • the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
  • the present invention also provides other polypeptides, such as fusion proteins comprising Nanobodies or fragments thereof.
  • the present invention also includes fragments of the Nanobodies of the present 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” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best 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.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • Polynucleotides encoding the mature polypeptides of the invention include: coding sequences encoding only the mature polypeptide; coding sequences and various additional coding sequences for the mature polypeptide; coding sequences (and optional additional coding sequences) for the mature polypeptide and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is more than 95%. Furthermore, 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 method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by 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 suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can 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 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.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various isolation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
  • Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include, but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • Interleukin-5 IL5
  • Interleukin-5 has the highest selectivity for eosinophils among the known cytokines, and can regulate the growth, activation, survival and migration of eosinophils. Interleukin-5 exerts its proliferation and differentiation effects through receptors comprising interleukin-5-specific alpha and common beta-subunits. IL5 plays a key role in the migration of eosinophils from the bone marrow to the lungs and other organs. IL5 signaling through JAK-STAT, Btk and Ras/Raf-ERK signaling maintains B cell and eosinophil survival and function.
  • IL5 is currently recognized as one of the key drivers of the Th2 pathway.
  • Interleukin-5 receptor alpha (IL5R ⁇ )
  • the receptor of IL5, IL5R ⁇ is highly expressed on eosinophils and plays a key role in eosinophils clearing allergens from blood and tissues.
  • the IL5 receptor consists of ⁇ and ⁇ c chains, the ⁇ subunit is specific for the IL5 molecule, and the ⁇ c subunit is also recognized by interleukin 3 (IL3) and granulocyte macrophage colony stimulating factor (GM-CSF).
  • IL3 interleukin 3
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the expression of IL5R ⁇ in activated B cells is regulated by a variety of transcription factors, including E12, E47, Sp1, c/EBP ⁇ and Oct2.
  • 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, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions 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 IL5 protein molecules, and thus can be used to treat asthma, atopic dermatitis, arthritis, allergic rhinitis, eczema and the like.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above-mentioned Nanobody (or its conjugate) of the present invention and a pharmaceutically acceptable amount. acceptable carrier or excipient.
  • acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention may 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 generally 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 specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • amino acid sequence of the VHH chain of the anti-IL5 Nanobody is selected from SEQ ID NO:8, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:41, SEQ ID One or more of NO:47.
  • the anti-IL5 Nanobody includes a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and/or a multivalent (multivalent antibody).
  • the anti-IL5 Nanobody comprises two VHH chains having amino acid sequences as shown in SEQ ID NO:41 and/or SEQ ID NO:47.
  • VHH chains are connected through a linking peptide.
  • sequence of the connecting peptide is (G 4 S) 4 .
  • 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 IL5 is labeled with colloidal gold to obtain a nanobody labeled with colloidal gold.
  • the present invention also relates to methods of detecting IL5 protein.
  • the method steps are roughly as follows: obtaining a cell and/or tissue sample; lysing the sample in a medium; detecting the level of IL5 protein in the lysed sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample existing in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the present invention also provides a detection kit for detecting IL5 level, the kit includes an antibody that recognizes IL5 protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, such as various buffers, detection Labeling, detection substrates, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the nanobody of the present invention has a wide range of biological application value and clinical application value, and its application involves diagnosis and treatment of IL5-related diseases, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy against IL5.
  • the nanobody of the present invention can effectively block the interaction between IL5 and IL5R.
  • the nanobody of the present invention can recognize IL5 of human and cynomolgus monkey, but not IL5 of mouse.
  • the nanobody of the present invention has stronger binding activity and blocking activity.
  • the nanobody of the present invention can be expressed in Pichia pastoris, the expression yield can reach 13 g/L, and the target protein expression supernatant has high purity.
  • the nanobody of the present invention can effectively inhibit the proliferation of TF-1 cells induced by IL5, and the inhibitory effect is better than that of the control antibody Nucala.
  • Example 1 Screening of IL5-specific Nanobodies by Phage Display Technology
  • the IL5 nanobody clones with different sequences were inoculated into 1 mL of TB medium containing appropriate concentration of ampicillin, and cultured at 37 °C in a constant temperature shaker to the logarithmic growth phase. IPTG inducer was added, and induced at 28 °C for 16 h; after 16 h, the osmotic The cells were broken by pressure impact method to obtain the crude nanobody extract; 1E6 HEK293F/IL5Ra cells were taken from each sample and resuspended in 0.5% BSA-PBS buffer, and 200 ⁇ L of the above IL5 nanobody crude extract was added respectively, and a negative control was set at the same time.
  • ELISA was used to detect whether the 11 nanobodies obtained in Example 2 could cross-react with IL5 of other species.
  • 1 ⁇ g/mL human IL5, mouse IL5, cynomolgus monkey IL5 and IgG1 proteins were added to the ELISA plate for overnight coating, 4°C, 100uL/well; after washing with PBST for 5 times, 300uL of 1% BSA was added to each well at room temperature Blocked for 2 hours; washed 5 times with PBST, added 100uL of 2 ⁇ g/mL prokaryotically expressed Nanobodies (Nb6, Nb13, Nb20, Nb21, Nb25, Nb26, Nb50, Nb66, Nb71, Nb85, Nb92) and incubated at 37°C 1 hour; washed 5 times with PBST, added 100uL diluted mouse anti-HA antibody (1:2000 dilution) and incubated at 37°C for 1 hour; washed 5 times with PBST,
  • the binding kinetics of the 11-strain Nanobodies obtained in Example 2 to human IL5 were measured by Bio-layer interferometry BLI using a Fortebio Red96 instrument.
  • IL5 antigen protein was diluted to 1.5 ⁇ g/mL with PBST buffer;
  • 11 strains of IL5 Nanobodies were diluted 2-fold with PBST buffer for six concentration gradients (30 nM, 15 nM, 7.5 nM, 3.75 nM, 1.88 nM , 0.94nM), set the operating conditions of the instrument: temperature 30°C, Shake speed 1000rpm.
  • the antibody was captured using a probe coated with Protein A, the capture time was 180s; the antigen was combined with the serial dilution, the binding time was 300s; the dissociation time was 360s; 10mM glycine (pH1.7) was regenerated for 3 times, 5s each time.
  • the analysis was performed using FortebioAnalysis 9.0 version, the 1:1 binding model Global model was fitted, and the association rate (Kon), the dissociation rate (Kdis) and the dissociation constant KD were calculated. The results are shown in Figure 4, the affinity of 11 nanobodies to IL5 is less than 1 nM.
  • Example 5 ELISA to detect the binding activity of IL5 Nanobodies
  • ELISA was used to detect and compare the binding activity of 4 nanobodies (Nb21, Nb25, Nb66 and Nb92) with different sequences to IL5. All IL5 Nanobodies were diluted to 1 ⁇ g/mL, and 100 ⁇ L of each well was coated, overnight at 4°C. After washing 5 times with PBST, 300 uL of 1% BSA was added to each well and placed at 37°C for 2 hours. Wash 5 times with PBST, add 100uL of serially diluted IL5-biotin, with a concentration gradient of 2 ⁇ g/mL starting with 2-fold serial dilution for 12 points, add the sample and incubate at 37°C for 1 hour.
  • Example 6 Detection of blocking activity of IL5 antibody by flow cytometry
  • the HEK293F stably transfected cells with high IL5R expression were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Wash the cells once with 5 mL of PBS and then add 2 mL of PBS to resuspend the cells. After counting, the cells are distributed into 96-well plates with 3 ⁇ 10 5 cells per well.
  • the 4 strains of nanobodies (Nb21, Nb25, Nb66 and Nb92) and the control antibody Nucala were serially diluted 2-fold (20 ⁇ g/ml, 10 ⁇ g/ml, 5 ⁇ g/ml, 2.5 ⁇ g/ml, 1.25 ⁇ g/ml, 0.625 ⁇ g/ml).
  • the diluted antibodies were mixed with equal volumes of 2.5 ⁇ g/mL IL5-biotin respectively Then resuspend the cells in a 96-well plate, incubate at 4°C for 20 minutes; centrifuge at 3000rpm at 4°C, add 200uL PBS/well, resuspend and then centrifuge at 3000rpm at 4°C for 4min; add diluted SA- PE antibody (used at 0.3:100 dilution), incubate at 4°C for 20min; centrifuge at 3000rpm for 4min at 4°C, discard the supernatant, add 200uL PBS/well to wash the cells, wash twice, then add 200uL PBS to resuspend the cells, and transfer to flow cytometry Tube, flow cytometer to detect PE signal in
  • ELISA method was used to detect whether the two nanobodies with better blocking activity (Nb21 and Nb66) in Example 6 recognized different IL5 epitopes. Specifically, 1 ⁇ g/mL IL5 antigen protein was coated at 4°C overnight; the ELISA plate was washed 5 times with PBST, and then 1% BSA was added for blocking at room temperature for 2 hours; the ELISA plate was washed 5 times with PBST, and 100 uL of diluted antibody was added to mix Incubate at 37°C for 1 hour (the working concentration of the nanobody is 20 ⁇ g/mL, the working concentration of the biotinylated nanobody is 3 ⁇ g/mL, and the three groups of samples are: 3 ⁇ g/mL Nb21-biotin, 20 ⁇ g/mL Nb21+3 ⁇ g/mL Nb21-biotin, 20 ⁇ g/mL Nb66+3 ⁇ g/mL Nb21-biotin); then wash the EL
  • the humanized antibodies were combined in pairs, fused with Fc and expressed in the pCDNA3.1+ vector.
  • the fused sequences are shown in Table 3.
  • the constructed plasmid was transfected into HEK293F cells, and for the method of expression, see Example 3 of Patent CN2018101517526.
  • Example 9 Detection of blocking activity of bivalent humanized IL5 Nanobodies by flow cytometry
  • the purified humanized bivalent antibody obtained in Example 8 was compared with the monovalent Nanobody and the positive control Nucala for blocking activity at the cellular level. Specifically: the HEK293F stably transfected cells with high IL5R expression were centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Wash the cells once with 5 mL of PBS and then add 2 mL of PBS to resuspend the cells. After counting, the cells are distributed into 96-well plates with 3 ⁇ 10 5 cells per well.
  • the antibodies to be tested were diluted by 2-fold gradient (80 ⁇ g/ml, 40 ⁇ g/ml, 20 ⁇ g/ml, 10 ⁇ g/ml, 5 ⁇ g/ml, 2.5 ⁇ g/ml, 1.25 ⁇ g/ml, 0.625 ⁇ g/ml, 0.31 ⁇ g/ml) , 0.16 ⁇ g/ml, 0.08 ⁇ g/ml, 0.04 ⁇ g/ml), mix the diluted antibodies with IL5-biotin, resuspend the cells in 96-well plates, and incubate at 4°C for 20 minutes; 3000rpm, After centrifugation at 4°C, add 200uL PBS/well, resuspend at 3000rpm, centrifuge at 4°C for 4min; add diluted SA-PE antibody (0.3:100 dilution), incubate at 4°C for 20min; centrifuge at 3000rpm for 4min at 4°C, Discard the supernatant, add 200uL PBS/well to
  • Example 10 Expression of bivalent bi-epitopic IL5 Nanobodies in Pichia pastoris
  • the above humanized Nb66 and Nb21 are combined to form a bivalent bi-epitope Nanobody, and the antibody amino acid sequence is shown in SEQ ID NO: 61, and the sequence is subjected to Pichia pastoris codon optimization.
  • the base sequence is as shown in SEQ ID NO: 62, cloned into the pPICZaA vector and subsequently expressed using Pichia pastoris.
  • the expression method is as follows: pPICZaA-HuNb66-HuNb21 was linearized with Sac I restriction endonuclease and then electrotransformed into X-33 competent cells;
  • pPICZaA vector provided by Invitrogen
  • the single clones above were placed in BMGY medium.
  • the OD value of BMGY medium reached about 20, the cells were collected and then replaced in BMMY medium, and cultured at 28°C and 250 rpm; after that, samples were taken every 24 hours, and the final volume was 1%.
  • the samples were centrifuged at 12,000 rpm for 5 min, and the supernatant was taken and stored at -20°C; after continuous induction for 5 days, the culture was terminated, and the supernatant was taken to determine the content of the target protein. Then, a high-yielding clone was selected and cultured in a 7L fermenter.
  • the fermentation conditions were induction culture at 24°C, pH 6.5, and methanol feed rate of 8.5mL/L/h. The supernatant was taken at different time points in the fermentation process to detect the content of the target protein.
  • the bivalent bi-epitope antibody expressed by the above yeast was purified by Protein A affinity chromatography, and the blocking activity was detected after obtaining a relatively pure antibody.
  • the detection method was the same as that in Example 9.
  • Example 12 Inhibitory effect of bivalent bi-epitope IL5 Nanobody on proliferation of TF1 cells

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Abstract

提供了IL5纳米抗体及其应用。具体地提供了一种IL5纳米抗体，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纳米抗体的生产方法。该纳米抗体能够特异性识别人和食蟹猴的IL5，不识别小鼠的IL5，具有良好的结合活性；具有良好的IL5/IL5R阻断活性，且阻断活性显著优于对照抗体Nucala；能够有效抑制IL5诱导的TF-1细胞的增殖，其抑制活性优于对照抗体Nucala；在毕赤酵母中的表达产量可达13g/L，目的蛋白表达上清纯度高。

Description

抗IL5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，更具体地涉及一种抗IL5纳米抗体及其应用。
背景技术
嗜酸性粒细胞在保护机体免受感染方面起着重要作用。但在一些个体中，嗜酸性粒细胞水平升高可能导致炎症，并在某些炎症性疾病的发展中发挥作用。IL5是已知细胞因子中对嗜酸性粒细胞选择性最高的，可以调控嗜酸性粒细胞的生长、分化、招募、活化以及存活。而其受体IL5Rα在嗜酸性粒细胞上高表达，对于嗜酸性细胞清除血液及组织中的过敏原起到关键作用。IL5目前被认为是Th2通路的关键驱动因子之一，IL5与其受体结合后将进一步激活下游JAK-STAT信号通路。
近年来，作为免疫调节药物研发领域的重要靶点，IL5已经在免疫治疗领域发挥了重要作用，全球范围内已有三款靶向IL5/IL5Rα的单抗药物获批上市，其中两款为IL5抗体(GSK公司的Mepolizumab和Teva公司的Reslizumab)，1款为IL5Rα抗体(AstraZeneca公司的Benralizumab)，为患者带来了新的治疗选择。而中国本土药企三生国健的IL5人源化单克隆抗体注射液(610)、恒瑞医药的IL5抗体(SHR-1703)也已都获得国家药品监督管理局临床试验批准。全球首个获批上市的IL5单抗药物即GSK公司的Mepolizumab已经获批多项临床研究，针对中度哮喘、嗜酸性肉芽肿性多血管炎(EGPA)、慢性阻塞性肺疾病(COPD)、伴有鼻息肉的慢性鼻窦炎(CRSwNP)、重度嗜酸性粒细胞增多综合征、重度特异性皮炎、重度双侧鼻息肉等适应症。
截至目前，市场上尚未有针对IL5靶点的纳米抗体药物公布，而纳米抗体(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诊断及治疗中的新兴力量。
因此，开发一种新型抗IL5纳米抗体，具有较好的临床应用前景，并且有望填补IL5纳米抗体药物的市场空白，造福患者。
发明内容
本发明的目的在于提供一种抗IL5纳米抗体及其应用。
本发明的第一方面，提供了一种抗IL5纳米抗体，所述纳米抗体能够特异性结合IL5，且所述纳米抗体中的VHH链的互补决定区CDR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:10所示的CDR1、SEQ ID NO:11所示的CDR2、和SEQ ID NO:12所示的CDR3；
(3)SEQ ID NO:19所示的CDR1、SEQ ID NO:20所示的CDR2、和SEQ ID NO:21所示的CDR3；和
(4)SEQ ID NO:28所示的CDR1、SEQ ID NO:29所示的CDR2、和SEQ ID NO:30所示的CDR3。
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IL5结合能力的衍生序列。
在另一优选例中，所述抗IL5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:13所示的FR1、SEQ ID NO:14所示的FR2、SEQ ID NO:15所示的FR3、和SEQ ID NO:16所示的FR4；
(3)SEQ ID NO:22所示的FR1、SEQ ID NO:23所示的FR2、SEQ ID NO:24所示的FR3、和SEQ ID NO:25所示的FR4；
(4)SEQ ID NO:31所示的FR1、SEQ ID NO:32所示的FR2、SEQ ID NO:33所示的FR3、和SEQ ID NO:34所示的FR4；
(5)SEQ ID NO:37所示的FR1、SEQ ID NO:38所示的FR2、SEQ ID NO:39所示的FR3、和SEQ ID NO:40所示的FR4；和
(6)SEQ ID NO:43所示的FR1、SEQ ID NO:44所示的FR2、SEQ ID NO:45所示的FR3、和SEQ ID NO:46所示的FR4。
在另一优选例中，所述的CDR1、CDR2和CDR3被框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述抗IL5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、SEQ ID NO:26、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:47、或其组合。
在另一优选例中，所述抗IL5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IL5纳米抗体包括两个具有如SEQ ID NO:41和/或SEQ ID NO:47中所示的氨基酸序列的VHH链，较佳地，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—(G mS n) y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
在另一优选例中，所述连接肽的序列为(G 4S) 4。
在另一优选例中，所述抗IL5纳米抗体能够识别两种不同的IL5表位。
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。
本发明的第二方面，提供了一种抗IL5抗体，它是针对白细胞介素5(IL5)表位的抗体，并且具有本发明的第一方面所述的抗IL5纳米抗体。
在另一优选例中，所述抗IL5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IL5抗体包括一条或多条具有如SEQ ID NO:8、SEQ ID NO:17、SEQ ID NO:26、SEQ ID NO:35、SEQ ID NO:41或SEQ ID NO:47所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL5抗体包括两条具有如SEQ ID NO:41和/或SEQ ID NO:47所示的氨基酸序列的VHH链。
在另一优选例中，所述抗IL5抗体从N端到C端的结构如式I所示：
A1-A2-L-B(I)；
其中，
“-”为肽键或连接肽；
A1和A2各自独立地为本发明第一方面所述的抗IL5纳米抗体的任一种；
B为IgG的Fc片段；和
L为无或柔性接头。
在另一优选例中，所述的A1和A2各自独立地为具有如SEQ ID NO:41或SEQ ID NO:47所示的氨基酸序列的VHH链。
在另一优选例中，所述的A1为具有如SEQ ID NO:41所示的氨基酸序列的VHH链，所述的A2为具有如SEQ ID NO:47所示的氨基酸序列的VHH链。
在另一优选例中，所述的A1为具有如SEQ ID NO:47所示的氨基酸序列的VHH链，所述的A2为具有如SEQ ID NO:41所示的氨基酸序列的VHH链。
在另一优选例中，所述的柔性接头为连接肽。
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—(G mS n) y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
在另一优选例中，所述连接肽的序列为(G 4S) 4。
在另一优选例中，所述抗体的氨基酸序列如SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、或SEQ ID NO:59所示。
在另一优选例中，所述的抗体能够特异性结合具有正确空间结构的IL5蛋白。
在另一优选例中，所述的抗体能够有效阻断IL5与IL5R的相互作用。
在另一优选例中，所述的抗体能够识别人、食蟹猴的IL5，不识别小鼠的IL5。
在另一优选例中，所述的抗体对IL5的亲和力小于1nM。
在另一优选例中，所述的抗体具有良好的IL5/IL5R阻断活性，且阻断活性显著优于对照抗体Nucala。其中，对照抗体Nucala为葛兰素史克公司的上市药品Mepolizumab，即美泊利单抗。
在另一优选例中，所述的抗体能够有效抑制IL5诱导的TF-1细胞的增殖，其抑制活性优于对照抗体Nucala。
在另一优选例中，所述抗体为纳米抗体。
本发明第三方面，提供了一种抗IL5纳米抗体Fc融合蛋白，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
A-L-B(Ia)；
B-L-A(Ib)；
其中，
“-”为肽键；
A为一个或多个本发明第一方面所述的抗IL5纳米抗体；
B为IgG的Fc片段；和
L为无或柔性接头。
在另一优选例中，所述的柔性接头为连接肽。
在另一优选例中，所述IgG的Fc片段包括人的IgG的Fc片段。
在另一优选例中，所述IgG的Fc片段选自下组：IgG1、IgG2、IgG3、IgG4的Fc片段、或其组合。
在另一优选例中，所述IgG的Fc片段为IgG4。
在另一优选例中，所述Fc片段的氨基酸序列如SEQ ID NO:63所示。
在另一优选例中，所述融合蛋白的氨基酸序列如SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、或SEQ ID NO:59所示。
在另一优选例中，所述融合蛋白为针对IL5表位的纳米抗体Fc融合蛋白。
本发明的第四方面，提供了一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：本发明的第一方面所述的抗IL5纳米抗体、本发明的第二方面所述的抗IL5抗体、或本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白。
在另一优选例中，所述多核苷酸序列为组合形式，优选地，所述多核苷酸序列包含SEQ ID NO:9、SEQ ID NO:18、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:42、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60或SEQ ID NO:62中的一种或多种。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
本发明的第五方面，提供了一种表达载体，所述表达载体含有本发明第四方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移系统、或其组合。
优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
本发明的第六方面，提供了一种宿主细胞，所述宿主细胞含有本发明的第五方面所述的表达载体，或其基因组中整合有本发明的第四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。
本发明的第七方面，提供了一种产生抗IL5纳米抗体或其Fc融合蛋白的方法，包括步骤：
(a)在适合产生纳米抗体或其Fc融合蛋白的条件下，培养本发明的第六方面所述的宿主细胞，从而获得含所述抗IL5纳米抗体或其Fc融合蛋白的培养物；
(b)从所述培养物中分离或回收所述的抗IL5纳米抗体或其Fc融合蛋白；以及
(c)任选地，纯化和/或修饰得步骤(b)中获得的抗IL5纳米抗体或其Fc融合蛋白。
本发明的第八方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第一方面所述的抗IL5纳米抗体、或如本发明第二发明所述的抗IL5抗体、或如本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177、或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。
在另一优选例中，特别有用的细胞毒性药物的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))、长春花生物碱(vinca alkaloids)、或其组合。
在另一优选例中，所述的毒素选自下组：耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、 相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素、或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联部分选自下组：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))或任何形式的纳米颗粒。
本发明的第九方面，提供了一种药物组合物，所述药物组合物含有：
(i)如本发明第一方面所述的抗IL5纳米抗体、或如本发明第二发明所述的抗IL5抗体、或如本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白、或如本发明的第八方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为药物、毒素、和/或治疗用同位素。
在另一优选例中，所述的药物组合物中还含有治疗哮喘、特应性皮炎、关节炎、过敏性鼻炎和/或湿疹的其他药物，如皮质类固醇(TCS)、奈多罗米钠、色甘酸钠、茶碱、白三烯受体拮抗剂、或其组合。
在另一优选例中，所述的药物组合物用于制备预防和/或治疗与IL5/IL5R信号传导相关的疾病或病症的药物。
本发明的第十方面，提供了本发明第一方面所述的抗IL5纳米抗体、如本发明第二发明所述的抗IL5抗体、如本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白、或如本发明第八方面所述的免疫偶联物的用途；(a)用于制备预防和/或治疗与IL5/IL5R信号传导相关的疾病或病症的药物；(b)用于制备检测IL5的试剂、检测板或试剂盒。
在另一优选例中，所述疾病或病症包括但不限于：哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹、鼻窦炎、鼻息肉、慢性阻塞性肺疾病、嗜酸性肉芽肿性多血管炎、高嗜酸性粒细胞增多综合征、或其组合。
在另一优选例中，所述IL5为人IL5。
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IL5蛋白或其片段。
在本发明的第十一方面，提供了一种多特异性抗体，所述的多特异性抗体包含：如本发明第一方面所述的抗IL5纳米抗体，或如本发明第二发明所述的抗IL5抗体。
在另一优选例中，多特异性抗体还包括靶向选自下组的靶点的第二抗原结合区：IL-4R、IL-4Rα、IL-13、IL-13R、IL-11、IL-11R、或其组合。
在另一优选例中，所述的第二抗原结合区为纳米抗体。
在另一优选例中，所述多特异性抗体包括一个或多个第二抗原结合区。
在另一优选例中，所述多特异性抗体还包含抗体的Fc段。
本发明的第十二方面，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IL5纳米抗体、或如本发明第二发明所述的抗IL5抗体、或如本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括Fc标签、HA标签和6His标签。
在另一优选例中，所述的重组蛋白特异性结合于IL5蛋白。
在本发明的第十三方面，提供了本发明第一方面所述的抗IL5纳米抗体、或如本发明第二发明所述的抗IL5抗体、或如本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白、或如本发明的第八方面所述的免疫偶联物的用途，用于检测样品中IL5蛋白，或用于治疗和/或预防与IL5/IL5R信号传导相关的疾病或病症。
在另一优选例中，所述疾病或病症包括但不限于：哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹、鼻窦炎、鼻息肉、慢性阻塞性肺疾病、嗜酸性肉芽肿性多血管炎、高嗜酸性粒细胞增多综合征、或其组合。
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。
本发明的第十四方面，提供了一种检测样品中IL5蛋白的方法，所述方法包括步骤：
(1)将样品本发明第一方面所述的抗IL5纳米抗体、或如本发明第二发明所述的抗IL5抗体、或如本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白、或本发明的第八方面所述的免疫偶联物接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在IL5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。
在本发明的第十五方面，提供了一种IL5蛋白检测试剂，所述的检测试剂包含：
(i)本本发明第一方面所述的抗IL5纳米抗体、或如本发明第二发明所述的抗IL5抗体、或如本发明第三方面所述的抗IL5纳米抗体Fc融合蛋白、或本发明的第八方面所述的免疫偶联物；以及
(ii)检测学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。
在另一优选例中，所述的检测学上可接受的载体为无毒的、惰性的水性载体介质。
在另一优选例中，所述的检测试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述的检测试剂用于体内检测。
在另一优选例中，所述的检测试剂的剂型为液态或粉状(如水剂，针剂，冻干粉，片剂，含服剂，吸雾剂)。
本发明的第十六方面，提供一种检测IL5蛋白的试剂盒，所述试剂盒含有本发明的第八方面所述的免疫偶联物或本发明的第十五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IL5表达。
本发明的第十七方面，提供了一种本发明的第八方面所述的免疫偶联物的用途，用于制备体内检测IL5蛋白的造影剂。
在另一优选例中，所述检测用于哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹、鼻窦炎、鼻息肉、慢性阻塞性肺疾病、嗜酸性肉芽肿性多血管炎、高嗜酸性粒细胞增多综合征等的诊断或预后。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了IL5纳米抗体筛选富集过程。经过五轮淘选，两个文库中IL5特异性的纳米抗体噬菌体分别出现120倍和14.6倍富集。
图2显示了流式细胞术初步筛选具有阻断IL5与IL5R相互作用的纳米抗体的结果。结果表明：96株纳米抗体中有11株抗体具有阻断活性。
图3显示了ELISA检测11株阻断型IL5纳米抗体的种属交叉活性结果。结果表明：11株纳米抗体均能识别人和食蟹猴IL5，而无法识别小鼠IL5。
图4显示了利用生物膜层干涉技术Fortebio检测纳米抗体的亲和力结果。结果表明：11株纳米抗体对IL5的亲和力均小于1nM。
图5显示了ELISA检测纳米抗体的结合活性结果。结果表明：4株纳米抗体的结合活性均优于对照抗体Nucala。
图6显示了流式细胞术鉴定候选IL5纳米抗体的阻断活性。结果表明：4株纳米抗体均具有良好的IL5/IL5R阻断活性，其中Nb21、Nb66的阻断活性显著优于对照抗体Nucala。
图7显示了ELISA检测阻断型纳米抗体的抗原识别表位一致性结果。结果表明：Nb21与Nb66为不同抗原识别表位。
图8显示了流式细胞术检测人源化二价纳米抗体的阻断活性。结果表明：人源化二价抗体较单价抗体的活性显著升高，其中HuNb21-HuNb66-Fc及HuNb66-HuNb21-Fc二价双表位抗体的阻断活性最优，且显著优于对照抗体Nucala。
图9显示了Fortebio检测双表位二价IL5纳米抗体毕赤酵母发酵上清液中的含量结果。结果表明：在该发酵条件下，二价双表位抗体的表达量随培养时间的延长不断增加，发酵培养202小时的抗体产量可达13g/L。
图10显示了双表位二价IL5纳米抗体毕赤酵母发酵上清液的SDS-PAGE检测结果。结果表明：双表位IL5纳米抗体HuNb66-HuNb21在毕赤酵母中的表达产量可达13g/L，目的蛋白表达上清纯度高。
图11显示了流式细胞术检测二价双表位IL5纳米抗体的阻断活性结果。结果表明：酵母表达的二价双表位抗体HuNb66-HuNb21的阻断活性(IC 50＝0.841μg/mL)优于对照抗体Nucala(IC 50＝2.035μg/mL)。
图12显示了二价双表位IL5纳米抗体对TF1细胞的增殖抑制作用检测结果。结果表明：酵母表达的二价双表位抗体能够有效抑制IL5诱导的TF-1细胞的增殖，其抑制活性(IC 50HuNb66-HuNb21＝0.0512nM)优于对照抗体对TF-1细胞的增殖抑制作用(IC 50Nucala＝0.1782nM)。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的筛选，首次意外地发现一类IL5纳米抗体，实验结果表明，本发明纳米抗体能够特异性识别人和食蟹猴的IL5，不识别小鼠的IL5，具有良好的特异性和结合活性；本发明纳米抗体具有良好的IL5/IL5R阻断活性，且阻断活性显著优于对照抗体Nucala；本发明纳米抗体能够有效抑制IL5诱导的TF-1细胞的增殖，其抑制活性优于对照抗体Nucala；本发明纳米抗体在毕赤酵母中的表达产量可达13g/L，目的蛋白表达上清纯度高。
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IL5纳米抗体”、“本发明IL5纳米抗体”、“抗IL5纳米抗体”、“IL5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IL5(包括人IL5)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IL5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IL5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IL5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。
白细胞介素-5(IL5)
白细胞介素-5(IL5)是已知细胞因子中对嗜酸性粒细胞选择性最高的，可以调控嗜酸性粒细胞的生长、活化、存活及迁移。白细胞介素-5通过包含白介素-5特异性α和共同β-亚单位的受体来发挥其增殖和分化的作用。IL5在嗜酸性粒细胞从骨髓迁移至肺部及其他器官时发挥关键作用。IL5信号通过JAK-STAT、Btk和Ras/Raf-ERK信号传导，维持B细胞和嗜酸性粒细胞的存活和功能。在体内过度表达IL5可显著增加嗜酸性粒细胞和B细胞数量，而缺乏IL5或IL5受体功能基因的小鼠在B细胞和嗜酸性粒细胞系中显示出许多发育和功能损伤。在人类中，IL5的生物学效应是嗜酸性粒细胞的最佳特征。IL5目前被认为是Th2通路的关键驱动因子之一。
白细胞介素-5受体α(IL5Rα)
IL5的受体IL5Rα在嗜酸性粒细胞上高表达，对于嗜酸性细胞清除血液及组织中的过敏原起到关键作用。IL5受体由α和βc链组成，α亚基对IL5分子具有特异性，而βc亚基也被白介素3(IL3)和粒细胞巨噬细胞集落刺激因子(GM-CSF)识别。IL5Rα在活化B细胞中的表达受多种转录因子的调控，包括E12、E47、Sp1、c/EBPβ和Oct2。
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IL5蛋白分子，因而可用于治疗哮喘、特应性皮炎、关节炎、过敏性鼻炎、湿疹等。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的纳米抗体(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IL5纳米抗体
本发明中，所述抗IL5纳米抗体的VHH链的氨基酸序列选自SEQ ID NO:8、SEQ ID NO:17、SEQ ID NO:26、SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:47中的一种或多种。
在本发明的一个优选例中，所述抗IL5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IL5纳米抗体包括两条具有如SEQ ID NO:41和/或SEQ ID NO:47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—(G mS n) y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
在另一优选例中，所述连接肽的序列为(G 4S) 4。
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，IL5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IL5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IL5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IL5水平的检测试剂盒，该试剂盒包括识别IL5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IL5相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对IL5的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明纳米抗体能够有效阻断IL5与IL5R的相互作用。
(b)本发明纳米抗体能够识别人、食蟹猴的IL5，不识别小鼠的IL5。
(c)本发明纳米抗体相较于对照抗体Nucala具有更强的结合活性和阻断活性。
(d)本发明纳米抗体可在毕赤酵母中表达，表达产量可达13g/L，且目的蛋白表达上清纯度高。
(e)本发明纳米抗体能够有效抑制IL5诱导的TF-1细胞的增殖，且抑制作用优于对照抗体Nucala。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：噬菌体展示技术筛选IL5特异性的纳米抗体
前期将高纯度的人IL5蛋白与免疫佐剂混合，免疫2只新疆双峰驼，7次免疫后取外周血，随后从中提取淋巴细胞分离RNA。将RNA反转录后通过巢式PCR扩增分离出VHH基因，再将VHH片段克隆至pMECS载体，再电转化至TG1感受态细胞中，建立抗IL5纳米抗体噬菌体展示文库。经测定，两个文库的库容分别为6.4×10 8CFU和1.3×10 8CFU，文库目的片段VHH的插入率分别达91.7％和100％。随后利用噬菌体展示技术筛选IL5特异性的纳米抗体，具体筛选方法参见专利CN110144011B实施例3中的方案。经过5轮“结合-洗涤-洗脱”的富集过程，最终分别获得120倍和14.6倍特异性噬菌体富集(图1)。再经PE-ELISA鉴定及测序分析，最终获得96株序列不同的IL5特异性纳米抗体。
实施例2：阻断型IL5纳米抗体筛选
将不同序列的IL5纳米抗体克隆株接种于1mL含有合适浓度的氨苄青霉素的 TB培养基中，37℃恒温摇床培养至对数生长期时加入IPTG诱导剂，28℃诱导16h；16h之后利用渗透压冲击法破碎菌体，获得纳米抗体粗提液；每个样品取1E6个HEK293F/IL5Ra细胞重悬于0.5％BSA-PBS buffer中，分别加入200μL上述IL5纳米抗体粗提液，同时设置阴性对照(裂解液)，所有样本加入合适浓度的IL5-Biotin，4℃孵育20分钟；再用1×PBS洗涤2次，加SA-PE，4℃避光孵育20分钟，PBS洗涤2次细胞后用流式细胞仪(BD Caliber)检测，结果如图2所示：初步筛选到11株具有良好阻断效果的IL5纳米抗体，其中阳性细胞百分率数值越低代表抗体阻断效果越佳，其中，11株纳米抗体的编号分别为Nb6、Nb13、Nb20、Nb21、Nb25、Nb26、Nb50、Nb66、Nb71、Nb85、Nb92。经序列分析，11株阻断型IL5纳米抗体序列可分为4个家族，后续重点研究对象如表1所示。
表1 4株阻断型纳米抗体氨基酸及碱基序列表
Figure PCTCN2021119969-appb-000001
实施例3：IL5纳米抗体的种属特异性检测
利用ELISA检测实施例2获得的11株纳米抗体能否与其他种属IL5发生交叉反应。将1μg/mL人IL5、小鼠IL5、食蟹猴IL5及IgG1蛋白分别加入酶标板中包被过夜，4℃，100uL/孔；用PBST洗涤5次后，每孔加入300uL 1％BSA室温封闭2小时；用PBST洗涤5次后，分别加入100uL 2μg/mL原核表达的纳米抗体(Nb6、Nb13、Nb20、Nb21、Nb25、Nb26、Nb50、Nb66、Nb71、Nb85、Nb92)于37℃下孵育1小时；再用PBST洗涤5次，加入100uL稀释的鼠抗HA抗体(1:2000稀释)于37℃下孵育1小时；用PBST洗涤5次，加入100uL稀释的碱性磷酸酶修饰的抗鼠抗体(1:2000稀释)于37℃下孵育1小时；再用PBST洗涤5次，加入显色液，酶标仪405nm波长下测定吸收值。结果如图3所示：11株纳米抗体均能识别人和食蟹猴IL5，而无法识别小鼠IL5。
实施例4：IL5纳米抗体的亲和力测定
将实施例2获得的11株纳米抗体针对人IL5的结合动力学通过生物膜层干涉 技术(Bio-layer interferometry BLI)，使用Fortebio Red96仪器测量。对于动力学测量，将IL5抗原蛋白用PBST缓冲液稀释至1.5μg/mL；11株IL5纳米抗体用PBST缓冲液2倍梯度稀释六个浓度梯度(30nM，15nM，7.5nM，3.75nM，1.88nM，0.94nM)，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180s；结合梯度稀释的抗原，结合时间300s；解离时间360s；10mM甘氨酸(PH1.7)再生3次，每次5s。使用FortebioAnalysis9.0版本进行分析，1：1结合模型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4所示，11株纳米抗体对IL5的亲和力均小于1nM。
实施例5：ELISA检测IL5纳米抗体的结合活性
利用ELISA方法检测比较4株序列不同的纳米抗体(Nb21、Nb25、Nb66和Nb92)与IL5的结合活性。将所有IL5纳米抗体稀释至1μg/mL，取100uL每孔包被，4℃过夜。PBST洗5遍，然后每孔加入300uL的1％BSA置于37℃封闭2小时。PBST洗5遍，加入100uL梯度稀释的IL5-biotin，浓度梯度以2μg/mL起始2倍梯度稀释12个点，加入样品37℃孵育1小时。PBST洗5遍，再加入100uL SA-HRP(用PBS 1:5000稀释使用)37℃孵育1小时。再用PBST洗5遍，每孔加100uL TMB显色液，室温避光反应5min，再加入50uL 2M的硫酸终止反应，酶标仪在450nm处测吸光值。结果如图5所示：4株纳米抗体的结合活性均优于对照抗体Nucala。
实施例6：流式细胞术检测IL5抗体的阻断活性
将高表达IL5R的HEK293F稳转细胞用1000rpm离心5min，弃上清。用5mL PBS洗涤细胞一次后再加入2mL PBS重悬细胞，计数后将细胞分装至96孔板中，每孔3×10 5个细胞。分别将4株纳米抗体(Nb21、Nb25、Nb66和Nb92)及对照抗体Nucala进行2倍梯度稀释(20μg/ml、10μg/ml、5μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml、0.31μg/ml、0.16μg/ml、0.08μg/ml、0.04μg/ml、0.02μg/ml、0.01μg/ml)，用稀释好的抗体分别与2.5μg/mL IL5-biotin等体积混合后重悬分装在96孔板中的细胞，于4℃孵育20分钟；3000rpm，4℃离心后加入200uL PBS/孔，重悬后再于3000rpm，4℃离心4min；加入稀释好的SA-PE抗体(0.3:100稀释使用)，4℃孵育20min；3000rpm，4℃离心4min，弃上清，加入200uL PBS/孔洗涤细胞，洗涤2次，再加入200uL PBS重悬细胞，转移至流式管中，流式细胞仪检测每个样本PE信号。结果如图6所示，4株纳米抗体均具有良好的IL5/IL5R阻断活性，其中Nb21、Nb66的阻断活性显著优于对照抗体Nucala。
实施例7：IL5纳米抗体的抗原识别表位分析
利用ELISA方法检测实施例6中2株阻断活性较好的纳米抗体(Nb21和Nb66)是否识别不同的IL5抗原表位。具体地，于4℃包被1μg/mL IL5抗原蛋白过夜；PBST洗涤酶标版5次后加入1％BSA室温封闭2小时；再用PBST洗涤酶标版5 次，加入100uL稀释好的抗体混合液37℃孵育1小时(纳米抗体工作浓度为20μg/mL，生物素化的纳米抗体工作浓度为3μg/mL，3组样品分别为：3μg/mL Nb21-biotin、20μg/mL Nb21+3μg/mL Nb21-biotin、20μg/mL Nb66+3μg/mL Nb21-biotin)；再用PBST洗涤酶标版5次，加入100uL SA-HRP(1:5000稀释使用)置于37℃孵育1小时；再用PBST洗5遍，每孔加100uL TMB显色液，室温避光反应5min，再加入50uL 2M的硫酸终止反应，酶标仪在450nm处测吸光值。结果如图7所示：Nb21与Nb66具有不同抗原识别表位。
实施例8：二价人源化纳米抗体的构建及表达
将上述实施例6中2株具有较好阻断活性的纳米抗体Nb21、Nb66分别进行骨架区人源化改造，改造方案参见专利CN110144010B实施例3。人源化后抗体序列如表2所示。
表2 IL5纳米抗体人源化序列表
Figure PCTCN2021119969-appb-000002
将人源化后的抗体分别两两组合，并与Fc融合表达，构建于pCDNA3.1+载体。融合后的序列如表3所示。
表3人源化IL5纳米抗体序列表
抗体结构 氨基酸序列 碱基序列
HuNb21-Fc SEQ ID NO:49 SEQ ID NO:50
HuNb66-Fc SEQ ID NO:51 SEQ ID NO:52
HuNb21-HuNb21-Fc SEQ ID NO:53 SEQ ID NO:54
HuNb21-HuNb66-Fc SEQ ID NO:55 SEQ ID NO:56
HuNb66-HuNb66-Fc SEQ ID NO:57 SEQ ID NO:58
HuNb66-HuNb21-Fc SEQ ID NO:59 SEQ ID NO:60
将构建好的质粒转染HEK293F细胞，表达的方法参见专利CN2018101517526实施例3。
实施例9：流式细胞术检测二价人源化IL5纳米抗体的阻断活性
将实施例8获得的纯化的人源化二价抗体与单价纳米抗体及阳性对照Nucala进行细胞水平阻断活性比较。具体地：将高表达IL5R的HEK293F稳转细胞用1000rpm离心5min，弃上清。用5mL PBS洗涤细胞一次后再加入2mL PBS重悬细胞，计数后将细胞分装至96孔板中，每孔3×10 5个细胞。分别将待测抗体进行 2倍梯度稀释(80μg/ml、40μg/ml、20μg/ml、10μg/ml、5μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml、0.31μg/ml、0.16μg/ml、0.08μg/ml、0.04μg/ml)，用稀释好的抗体分别与IL5-biotin混合后重悬分装在96孔板中的细胞，于4℃孵育20分钟；3000rpm，4℃离心后加入200uL PBS/孔，重悬后再于3000rpm，4℃离心4min；加入稀释好的SA-PE抗体(0.3:100稀释使用)，4℃孵育20min；3000rpm，4℃离心4min，弃上清，加入200uL PBS/孔洗涤细胞，洗涤2次，再加入200uL PBS重悬细胞，转移至流式管中，流式细胞仪检测每个样本PE信号。
结果如图8所示：人源化二价抗体较单价抗体的活性显著升高，其中HuNb21-HuNb66-Fc及HuNb66-HuNb21-Fc异源二价抗体的阻断活性更好，且显著优于对照抗体Nucala。
实施例10：二价双表位IL5纳米抗体在毕赤酵母中的表达
优选以上人源化的Nb66和Nb21组合形成二价双表位纳米抗体，抗体氨基酸序列如SEQ ID NO:61所示，将该序列进行毕赤酵母密码子优化后碱基序列如SEQ ID NO:62所示，克隆至pPICZaA载体上，随后利用毕赤酵母进行表达。简要地，表达方法如下：用Sac I限制性内切酶线性化pPICZaA-HuNb66-HuNb21后电转化至X-33感受态细胞中；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，具体实施方案可参见Invitrogen公司提供的pPICZaA载体说明书；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；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，取上清液测定其目的蛋白含量。随后挑选一株高产克隆进行7L发酵罐培养，发酵条件为24℃诱导培养，pH 6.5，甲醇补料速率为8.5mL/L/h。在发酵过程不同时间点取上清液进行目的蛋白含量检测，结果如图9所示：双表位IL5纳米抗体HuNb66-HuNb21在毕赤酵母中的表达产量可达约13g/L。将取到的样品稀释13倍进行SDS-PAGE检测，结果如图10所示：目的蛋白清晰，表达上清纯度较高。
实施例11：二价双表位IL5纳米抗体的阻断活性检测
将以上酵母表达的二价双表位抗体利用Protein A亲和层析纯化，获得较高纯度的抗体后进行阻断活性检测，检测方法同实施例9。结果如图11所示，酵母表达的二价双表位抗体HuNb66-HuNb21的阻断活性(IC 50＝0.841μg/mL)显著优于对照抗体Nucala(IC 50＝2.035μg/mL)。
实施例12：二价双表位IL5纳米抗体对TF1细胞的增殖抑制作用
将复苏培养的TF-1细胞(IL5诱导处理)于1000rpm离心5min，弃上清；用5mL PBS重悬，1000rpm再离心5min；20mL 1640培养基重悬细胞计数，稀释 细胞溶液浓度至6×10 5/mL，分装60uL/孔到96孔板中；分别将50uL梯度稀释的IL5抗体均与50uL 25ng/mL的IL5蛋白混合后，取40uL混合液加到细胞溶液中；同时向所有细胞周边孔补加200uL PBS，以防止有细胞孔溶液蒸发，置于37℃，5％CO 2培养箱培养72小时；取出细胞培养板，加入CCK8溶液10uL/孔，置于37℃，显色4小时；显色结束后，用酶标仪读取各孔的OD450值。
结果如图12所示：酵母表达的二价双表位抗体能够有效抑制IL5诱导的TF-1细胞的增殖，其抑制活性(IC 50 HuNb66-HuNb21＝0.0512nM)优于对照抗体对TF-1细胞的增殖抑制作用(IC 50 Nucala＝0.1782nM)。
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IL5纳米抗体，其特征在于，所述纳米抗体能够特异性结合IL5，且所述纳米抗体中的VHH链的互补决定区CDR为选自下组的一种或多种：
    (1)SEQ ID NO:19所示的CDR1、SEQ ID NO:20所示的CDR2和SEQ ID NO:21所示的CDR3；
    (2)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；
    (3)SEQ ID NO:10所示的CDR1、SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3；和
    (4)SEQ ID NO:28所示的CDR1、SEQ ID NO:29所示的CDR2和SEQ ID NO:30所示的CDR3。
  2. 如权利要求1所述的抗IL5纳米抗体，其特征在于，所述抗IL5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:13所示的FR1、SEQ ID NO:14所示的FR2、SEQ ID NO:15所示的FR3和SEQ ID NO:16所示的FR4；
    (3)SEQ ID NO:22所示的FR1、SEQ ID NO:23所示的FR2、SEQ ID NO:24所示的FR3和SEQ ID NO:25所示的FR4；
    (4)SEQ ID NO:31所示的FR1、SEQ ID NO:32所示的FR2、SEQ ID NO:33所示的FR3和SEQ ID NO:34所示的FR4；
    (5)SEQ ID NO:37所示的FR1、SEQ ID NO:38所示的FR2、SEQ ID NO:39所示的FR3和SEQ ID NO:40所示的FR4；和
    (6)SEQ ID NO:43所示的FR1、SEQ ID NO:44所示的FR2、SEQ ID NO:45所示的FR3和SEQ ID NO:46所示的FR4。
  3. 如权利要求1所述的抗IL5纳米抗体，其特征在于，所述抗IL5纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:26、SEQ ID NO:47、SEQ ID NO:8、SEQ ID NO:41、SEQ ID NO:17、SEQ ID NO:35或其组合。
  4. 一种抗IL5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IL5纳米抗体。
  5. 如权利要求4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。
  6. 如权利要求4所述的抗体，其特征在于，所述抗IL5抗体包括一条或多条具有如SEQ ID NO:26、SEQ ID NO:47、SEQ ID NO:8、SEQ ID NO:41、SEQ ID  NO:17、或SEQ ID NO:35所示的氨基酸序列的VHH链。
  7. 一种抗IL5纳米抗体Fc融合蛋白，其特征在于，所述融合蛋白从N端到C端的结构如式Ia或Ib所示：
    A-L-B(Ia)；
    B-L-A(Ib)；
    其中，
    A为一个或多个如权利要求1所述的抗IL5纳米抗体；
    B为IgG的Fc片段；和
    L为无或柔性接头。
  8. 如权利要求7所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列如SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、或SEQ ID NO:59所示。
  9. 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IL5纳米抗体、权利要求4所述的抗IL5抗体、或权利要求7所述的抗IL5纳米抗体Fc融合蛋白。
  10. 一种表达载体，其特征在于，所述表达载体含有权利要求9所述的多核苷酸。
  11. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求10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。
  12. 一种产生抗IL5纳米抗体、抗IL5抗体或其Fc融合蛋白的方法，其特征在于，包括步骤：
    (a)在适合产生纳米抗体或其Fc融合蛋白的条件下，培养权利要求11所述的宿主细胞，从而获得含所述抗IL5纳米抗体或其Fc融合蛋白的培养物；
    (b)从所述培养物中分离或回收所述的抗IL5纳米抗体或其Fc融合蛋白；以及
    (c)任选地，纯化和/或修饰得步骤(b)中获得的抗IL5纳米抗体或其Fc融合蛋白。
  13. 一种免疫偶联物，其特征在于，所述免疫偶联物含有：
    (a)如权利要求1所述的抗IL5纳米抗体、或如权利要求4所述的抗IL5抗体、或如权利要求7所述的抗IL5纳米抗体Fc融合蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
  14. 一种药物组合物，其特征在于，所述药物组合物含有：
    (i)如权利要求1所述的抗IL5纳米抗体、或如权利要求4所述的抗IL5抗体、或如权利要求7所述的抗IL5纳米抗体Fc融合蛋白、或如权利要求13所述的免疫偶联物；以及
    (ii)药学上可接受的载体。
  15. 如权利要求1所述的抗IL5纳米抗体、如权利要求4所述的抗IL5抗体、 如权利要求7所述的抗IL5纳米抗体Fc融合蛋白、或如权利要求13所述的免疫偶联物的用途；(a)用于制备预防和/或治疗与IL5/IL5R信号传导相关的疾病或病症的药物；(b)用于制备检测IL5的试剂、检测板或试剂盒。
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