WO2021042694A1 - 抗vegf单域抗体及其应用 - Google Patents

抗vegf单域抗体及其应用 Download PDF

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WO2021042694A1
WO2021042694A1 PCT/CN2020/078595 CN2020078595W WO2021042694A1 WO 2021042694 A1 WO2021042694 A1 WO 2021042694A1 CN 2020078595 W CN2020078595 W CN 2020078595W WO 2021042694 A1 WO2021042694 A1 WO 2021042694A1
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antibody
vegf
single domain
seq
present
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PCT/CN2020/078595
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English (en)
French (fr)
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万亚坤
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 EP20861119.4A priority Critical patent/EP4032905A4/en
Priority to US17/640,083 priority patent/US20220324954A1/en
Priority to KR1020227010990A priority patent/KR20220058592A/ko
Priority to JP2022514739A priority patent/JP7336122B2/ja
Publication of WO2021042694A1 publication Critical patent/WO2021042694A1/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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/35—Valency
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    • 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
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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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07—ORGANIC CHEMISTRY
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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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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
    • G01N2333/475—Assays involving growth factors
    • 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
    • G01N2333/475—Assays involving growth factors
    • G01N2333/515—Angiogenesic factors; Angiogenin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/164—Retinal disorders, e.g. retinopathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/16—Ophthalmology
    • G01N2800/168—Glaucoma

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-VEGF single domain antibody and its application.
  • vascular endothelial growth factor drugs include Bevacizumab (trade name Avastin), Conbercept, Aflibercept and the like.
  • CFDA China Food and Drug Administration
  • Aliya aflibercept intraocular injection solution
  • RMB 1998 per bottle of 4ml containing 100mg of bevacizumab
  • the recommended dose is 5mg/kg, and it is administered every 14 days.
  • aflibercept is 5850 yuan per 0.1ml bottle (including 4mg aflibercept), and its dosing regimen is once every two months, 6 times a year, which is 35,100 yuan a month. year.
  • Single domain antibody (nanobody, Nb), namely heavy-chain single-domain antibody VHH (variable domain of heavy chain of heavy-chain antibody)-there are heavy-chain antibodies (HCAb) that naturally lack light chains in camels
  • VHH variable domain of heavy chain of heavy-chain antibody
  • HCAb heavy-chain antibodies
  • the single domain antibody consisting of only one heavy chain variable region obtained by cloning its variable region is the smallest unit with complete function and stable antigen binding that is currently available.
  • Single-domain antibodies have the characteristics of high stability, good water solubility, simple humanization, high targeting, and strong penetration. They play a huge function beyond imagination in immunological experiments, diagnosis and treatment. Single-domain antibodies are gradually becoming an emerging force in the diagnosis and treatment of a new generation of antibodies.
  • the purpose of the present invention is to provide a new type of anti-VEGF single domain antibody and its application.
  • the purpose of the present invention is to provide a method that can effectively block the interaction between VEGFA and VEGFR2, VEGFA and VEGFR1, has a good inhibitory effect on neovascularization, has a good inhibitory effect on solid tumors, and has good Specific single domain antibody.
  • the first aspect of the present invention provides a complementarity determining region CDR region of an anti-VEGF single domain antibody single domain antibody VHH chain.
  • the complementarity determining region CDR of the VHH chain includes CDR1 and SEQ shown in SEQ ID NO.:1 CDR2 shown in ID NO.: 2 and CDR3 shown in SEQ ID NO.: 3.
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4.
  • the second aspect of the present invention provides a VHH chain of an anti-VEGF single domain antibody.
  • the VHH chain includes a framework region FR and the complementarity determining region CDR described in the first aspect of the present invention.
  • the framework region FR includes:
  • VHH chain of the anti-VEGF single domain antibody is shown in SEQ ID NO.: 8 or 14.
  • the heavy chain variable region includes CDR1 shown in SEQ ID NO.:1 and CDR2 shown in SEQ ID NO.:2. And CDR3 shown in SEQ ID NO.: 3.
  • the third aspect of the present invention provides an anti-VEGF antibody, the anti-VEGF antibody having the VHH chain described in the second aspect of the present invention.
  • the anti-VEGF antibody includes a double-chain antibody, a single-chain antibody, and a single-domain antibody.
  • the anti-VEGF antibody is selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.
  • the anti-VEGF antibody can be an antibody fragment, such as: Fab, Fab', (Fab')2 or other antibody derivatives known in the field, etc., as well as IgA, IgD, IgE, IgG And any one or more of IgM antibodies or antibodies of other subtypes.
  • the anti-VEGF antibody is an anti-VEGF single domain antibody.
  • the anti-VEGF antibody includes monomer, bivalent (bivalent antibody), and/or multivalent (multivalent antibody).
  • the anti-VEGF antibody includes one or more VHH chains having the amino acid sequence shown in SEQ ID NO.: 8 or SEQ ID NO.: 14.
  • VHH chain sequence of the anti-VEGF antibody is shown in SEQ ID NO.: 8 and/or SEQ ID NO.: 14.
  • the anti-VEGF antibody includes two VHH chains having the amino acid sequence shown in SEQ ID NO.: 8 or SEQ ID NO.: 14.
  • the anti-VEGF antibody has a VHH chain with an amino acid sequence as shown in SEQ ID NO. 8 and/or SEQ ID NO. 14.
  • the two VHH chains having the amino acid sequence shown in SEQ ID NO. 14 are connected by a linker.
  • the linker is selected from the following group: GGGGSGGGS (SEQ ID NO. 18), GS (SEQ ID NO. 19), GGGGS (SEQ ID NO. 20).
  • amino acid sequence of the anti-VEGF antibody is shown in SEQ ID NO.16.
  • the bivalent anti-VEGF antibody is hubi-Nb24(Y).
  • the fourth aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of: the CDR region of the anti-VEGF single domain antibody VHH chain of the first aspect of the present invention, The VHH chain of the anti-VEGF single domain antibody described in the second aspect of the present invention, or the anti-VEGF single domain antibody described in the third aspect of the present invention.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.: 9 or 15.
  • the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.17.
  • 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 following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.
  • the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a viral vector such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • the sixth aspect of the present invention provides a host cell containing the expression vector of the fifth aspect of the present invention, or the polynucleotide of the fourth aspect of the present invention integrated into its genome.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.
  • the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.
  • the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, fission yeast, Trichoderma, or a combination thereof.
  • the eukaryotic cells are selected from the group consisting of insect cells such as armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.
  • the host cell is preferably a mammalian cell, more preferably HEK293 cell, CHO cell, BHK cell, NSO cell or COS cell.
  • the host cell is Pichia pastoris.
  • the seventh aspect of the present invention provides a method for producing an anti-VEGF single domain antibody, which includes the steps:
  • step (c) Optionally, purify and/or modify the VEGF single domain antibody of step (b).
  • the anti-VEGF single domain antibody has an amino acid sequence as shown in SEQ ID NO.: 8 or 14.
  • the anti-VEGF single domain antibody has an amino acid sequence as shown in SEQ ID NO.16.
  • the eighth aspect of the present invention provides an immunoconjugate, which contains:
  • a coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof .
  • the radionuclide includes:
  • Diagnostic isotopes said diagnostic isotopes are selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • Therapeutic isotope said therapeutic isotope is selected from the following group: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133 Yb-169, Yb-177 , Or a combination thereof.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatin ( auristatins), camptothecins, docarmycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 DM1 and DM4
  • benzodiazepines or benzodiazepine containing drugs such as pyrrolo[1,4] benzodiazepines (PBDs), indole Indolinobenzodiazepines (indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PBDs pyrrolo[1,4] benzodiazepines
  • indole Indolinobenzodiazepines indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • the toxin is selected from the following group: auristatin (for example, auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, mettancilol, a toxin, and Ephedrine A-chain, combstatin, docarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, Tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthracisin dione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia Toxin, acacia toxin A chain, capsulatoxin A chain, ⁇ -sarcin, white tree toxin, mitogellin, retstrictocin
  • the coupling portion is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or capable of producing Detectable enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, magnetic nanoparticles , Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutics (for example, cisplatin) or any form of nanoparticles.
  • MRI magnetic resonance imaging
  • CT electro computed tomography technology
  • the immunoconjugate contains: a multivalent (such as bivalent) VHH chain of the anti-VEGF single domain antibody according to the second aspect of the present invention, as described in the third aspect of the present invention The described anti-VEGF single domain antibody.
  • the multivalent means that the amino acid sequence of the immunoconjugate includes a plurality of repeated VHH chains of the anti-VEGF single-domain antibody according to the second aspect of the present invention, such as those of the present invention.
  • the third aspect of the anti-VEGF single domain antibody is also preferred.
  • the ninth aspect of the present invention provides the use of the VHH chain of the anti-VEGF single domain antibody described in the second aspect of the present invention and the anti-VEGF single domain antibody described in the third aspect of the present invention for preparing:
  • the tenth aspect of the present invention provides a pharmaceutical composition, which contains:
  • the coupling part of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
  • the pharmaceutical composition also contains other drugs for treating tumors, such as cytotoxic drugs.
  • the pharmaceutical composition is used to block the interaction between VEGFA and VEGFR2, VEGFA and VEGFR1.
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare a medicament for the treatment of VEGF-related diseases or conditions, and the diseases or conditions include tumors or cancers or eye diseases.
  • the tumor or cancer includes but is not limited to one or more of breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, and nasopharyngeal cancer.
  • the ocular diseases include but are not limited to age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma and other ophthalmic diseases involving neovascularization.
  • the eleventh aspect of the present invention provides one or more uses of the anti-VEGF single domain antibody of the third aspect of the present invention; for preparing
  • the use is diagnostic and/or non-diagnostic, and/or therapeutic and/or non-therapeutic.
  • the twelfth aspect of the present invention provides an antibody comprising one or more VHH chains of the anti-VEGF single domain antibody according to the second aspect of the present invention.
  • the antibody includes two VHH chains of the anti-VEGF single domain antibody described in the second aspect of the present invention.
  • the antibody has the heavy chain variable region VHH as described in the second aspect of the present invention.
  • the antibody can specifically target the VEGFA protein with the correct spatial structure.
  • the antibody can recognize human, mouse, rabbit, and monkey VEGFA.
  • the antibody does not cross-react with human VEGFB, VEGFC, and VEGFD.
  • the antibody can block the interaction between VEGFA and VEGFR2, VEGFA and VEGFR1.
  • the antibody can inhibit the formation of new blood vessels.
  • the antibody is a single domain antibody.
  • the thirteenth aspect of the present invention provides a recombinant protein, the recombinant protein having:
  • the tag sequence includes Fc tag, HA tag and 6His tag.
  • the recombinant protein specifically binds to the VEGFA protein.
  • the VHH chain of the anti-VEGF single domain antibody according to the second aspect of the present invention is provided the anti-VEGF single domain antibody according to the third aspect of the present invention, or the anti-VEGF single domain antibody according to the present invention.
  • the use of the immunoconjugate described in the eighth aspect of the invention is used to prepare medicaments, reagents, detection plates or kits;
  • the reagent, detection plate or kit is used to: detect VEGF protein in the sample;
  • the medicament is used to treat or prevent diseases or disorders related to VEGF.
  • the detection includes flow cytometry and cellular immunofluorescence detection.
  • the disease or condition includes tumor or cancer or eye disease.
  • the tumor or cancer includes but is not limited to one or more of breast cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, thyroid cancer, and nasopharyngeal cancer.
  • the ocular diseases include but are not limited to age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma and other ophthalmic diseases involving neovascularization.
  • the fifteenth aspect of the present invention provides a method of treating diseases, the method comprising administering the single domain antibody according to the third aspect of the present invention or the eighth aspect of the present invention to a subject in need Immunoconjugates.
  • the subject includes mammals, such as humans, mice, rabbits, and monkeys.
  • the sixteenth aspect of the present invention provides a method for detecting VEGFA protein in a sample, the method comprising the steps:
  • the method is a non-diagnostic and non-therapeutic method.
  • a VEGFA protein detection reagent comprises:
  • the coupling part of the immunoconjugate is an isotope for diagnosis.
  • the detection-acceptable carrier is a non-toxic and inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the following group: isotope tracer, contrast agent, flow detection reagent, cellular immunofluorescence detection reagent, nano magnetic particle and imaging Agent.
  • the detection reagent is used for in vivo detection.
  • the dosage form of the detection reagent is liquid or powder (such as liquid, injection, lyophilized powder, tablet, buccal, or mist).
  • the eighteenth aspect of the present invention provides a kit for detecting VEGFA protein, the kit containing the immunoconjugate according to the eighth aspect of the present invention or the detection reagent according to the seventeenth aspect of the present invention, And instructions.
  • the instructions stated that the kit is used to non-invasively detect the expression of VEGFA in the test subject.
  • the nineteenth aspect of the present invention provides a use of the immunoconjugate of the eighth aspect of the present invention for preparing a contrast agent for detecting VEGFA protein in vivo.
  • the detection is used for the diagnosis or prognosis of cancer.
  • the twentieth aspect of the present invention provides a framework region FR of an anti-VEGF single domain antibody VHH chain.
  • the framework region FR of the VHH chain is represented by FR1 shown in SEQ ID NO.: 4, SEQ ID NO.: FR2 shown in 5, FR3 shown in SEQ ID NO.: 6, FR4 shown in SEQ ID NO.: 7; or FR1 shown in SEQ ID NO.: 10, SEQ ID NO.: 11 Composition of FR2, FR3 shown in SEQ ID NO.: 12, and FR4 shown in SEQ ID NO.: 13.
  • the twenty-first aspect of the present invention provides a method for treating VEGF-related diseases or disorders by administering the pharmaceutical composition according to the tenth aspect of the present invention to a subject in need.
  • the subject includes mammals, such as humans.
  • Figure 1 is the result of ELISA identification of single domain antibodies capable of blocking the interaction between human VEGFA and VEGFR2.
  • the blocking activity of the identified candidate antibody Nb24 was significantly better than that of the control antibody Avastin.
  • Avastin is Bevacizumab.
  • Figure 2 is the test result of the inhibitory effect of the candidate antibody on the proliferation of HUVEC cells.
  • the identified candidate antibody Nb24 has a stronger inhibitory effect on the proliferation of HUVEC cells than the control antibody Avastin.
  • Figure 3 shows the results of SEC-HPLC detection of huNb24 bivalent expressed in yeast.
  • the sample purified by ammonium sulfate precipitation was identified by SEC-HPLC, and the purity of the sample could reach 94.11%.
  • Figure 4 shows the blocking activity of candidate antibodies after humanization by ELISA.
  • Nb24 is a pre-humanized antibody
  • huNb24 is a post-humanized antibody.
  • Figure 5 shows the blocking activity of humanized bivalent antibodies expressed in yeast by ELISA.
  • hu bi-Nb24(Y) is a humanized bivalent antibody expressed in yeast.
  • Figure 6 compares the blocking activity of humanized bivalent antibodies with similar products on the market using ELISA.
  • Eylea is Aflibercept and Conbercept is Conbercept.
  • Fig. 7 is the detection result of the proliferation inhibitory effect of the humanized bivalent antibody expressed by yeast on HUVEC cells.
  • Figure 8 is the result of ELISA testing whether the candidate antibody can cross-react with the VEGF family protein.
  • the results show that the humanized bivalent antibody can react with human VEGFA, but does not cross-react with other proteins of the same family, such as VEGFB, VEGFC, VEGFD, and has good specificity.
  • Figure 9 is the result of ELISA testing whether the candidate antibody can cross-react with other species of VEGF. The results show that the humanized bivalent antibody can simultaneously recognize human, mouse, and rabbit VEGFA.
  • Figure 10 is the result of ELISA detecting the blocking of the interaction between human VEGFA and VEGFR1 by the humanized bivalent antibody expressed by yeast.
  • Figure 11 is the statistical results of the non-perfusion area of the retina in OIR model mice. Different concentrations of yeast-expressed humanized bivalent antibodies were administered to mice. The area of the non-perfusion area of the retina of the experimental group was smaller than that of the positive control group.
  • Figure 12 shows the statistical results of retinal neovascularization clusters in OIR model mice. Compared with Aflibercept Eylea (positive control), humanized bivalent antibodies expressed by yeast at different concentrations have a more obvious inhibitory effect on mouse retinal neovascularization clusters, and there are statistical differences.
  • Figure 13 shows the results of SEC-HPLC testing the stability of candidate antibodies at different temperatures.
  • Figure 13A shows the stability results of the candidate antibody placed at 4°C for 1 month. The results show that the antibody did not show significant purity changes when placed at 4°C for 1 month under non-prepared conditions, showing better stability.
  • Figure 13B shows the stability results of the candidate antibody stored at 25°C for 1 month. The results show that the antibody does not show significant purity changes after being stored at 25°C for 1 month under non-prepared conditions, showing better stability.
  • Figure 13C shows the stability results of the candidate antibody stored at 40°C for 15 days. The results show that the antibody does not show significant purity changes when stored at 40°C for 15 days under non-prepared conditions, showing better stability.
  • Figure 13D shows the stability results of the candidate antibody at -20°C for 5 times of repeated freezing and thawing. The results show that the antibody was placed at -20°C for 5 times of repeated freezing and thawing under non-preparation conditions without significant changes in purity. Good stability.
  • the inventors unexpectedly discovered a class of anti-VEGF single domain antibodies for the first time.
  • the experimental results show that the single domain antibodies of the present invention can specifically recognize VEGFA and do not interact with VEGFB, VEGFC, and VEGFD. Cross-reaction, with good specificity; can effectively block the interaction of VEGFA and VEGFR2, VEGFA and VEGFR1; and has a good inhibitory effect on angiogenesis.
  • the single domain antibody of the present invention is easy to produce. This completes the present invention.
  • the present invention uses human-derived VEGFA protein to immunize camels to obtain a high-quality immune single domain antibody gene library. Then the VEGFR protein molecule is coupled to the ELISA plate to display the correct spatial structure of the VEGFR protein.
  • the antigen in this form is used to screen the immune single domain antibody gene library (camel heavy chain antibody phage display gene library) using phage display technology to obtain VEGFA specific single domain antibody gene. Then the gene was transferred to mammalian cells, and a single domain antibody strain with high specificity that can be expressed in mammalian cells was obtained. Then by ELISA, flow cytometry, luciferase reporter gene detection system and other methods to identify anti-VEGF single domain antibodies with blocking activity.
  • the terms “single domain antibody of the present invention”, “single domain antibody of the present invention”, “anti-VEGF antibody of the present invention”, “VEGF single domain antibody of the present invention”, and “anti-VEGF single domain antibody” have the same Meaning, used interchangeably, both refer to single domain antibodies that specifically recognize and bind to VEGFA (including human VEGFA).
  • the variable region of the single domain antibody of the present invention has CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.:2, and CDR3 shown in SEQ ID NO.:3.
  • the framework region of the single-domain antibody of the present invention has (a) FR1 shown in SEQ ID NO.: 4, FR2 shown in SEQ ID NO.: 5, FR3 shown in SEQ ID NO.: 6, And FR4 shown in SEQ ID NO.: 7, or (b) FR1 shown in SEQ ID NO. 10, FR1 shown in SEQ ID NO. 11, FR2 shown in SEQ ID NO. 12, and FR3 shown in SEQ ID NO. 12, and SEQ ID NO. FR4 shown in .13.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.
  • single domain antibody As used herein, the terms “single domain antibody”, “VHH”, “nanobody”, “single domain antibody” (single domain antibody, sdAb, or nanobody) have the same meaning and can be used interchangeably, Refers to the cloning of the variable region of the heavy chain of an antibody to construct a single domain antibody (VHH) consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete functions. Usually, after obtaining an antibody with naturally missing light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • variable means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments of the present invention to form ⁇ conjugate.
  • the present invention also includes cell surface markers or antigens that bind to the anti-VEGF antibody or fragments thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the heavy chain variable region and the heavy chain constant region described above.
  • the terms "antibody of the present invention”, “protein of the present invention”, or “polypeptide of the present invention” are used interchangeably, and all refer to a polypeptide that specifically binds to the VEGF protein, such as a protein or polypeptide having a heavy chain variable region. . They may or may not contain the starting methionine.
  • variable regions which are divided into 4 framework regions (FR), 4 FR amino acids
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ sheets formed by the FRs between them are close to each other in space structure, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody.
  • the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified here. Sex.
  • the present invention includes not only complete antibodies, but also fragments, derivatives, and analogs of the antibodies.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogues of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or proprotein sequence, or with Fusion protein formed by 6His tag
  • the antibody of the present invention refers to a polypeptide having VEGFA binding activity and including the above-mentioned CDR regions.
  • the term also includes variant forms of polypeptides containing the above-mentioned CDR regions that have the same functions as the antibodies of the present invention. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the present invention also provides other polypeptides.
  • the present invention also includes fragments of the single domain antibody of the present invention.
  • the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 compared to the amino acid sequence of the antibody of the present invention. Two amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table 1 through amino acid substitutions.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence or fragments of the antibody of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be carried out by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the present invention can be used alone, or can be combined or coupled with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or those capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nano magnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutics ( For example, cisplatin) or any form of nanoparticles.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • VEGF Vascular Endothelial Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • VEGFR Vascular Endothelial Growth Factor Receptor
  • MAPK mitogen-activated protein kinase
  • VEGF can restore tissue oxygen supply when blood circulation is insufficient.
  • VEGF can cause disease symptoms.
  • overexpression of VEGF can lead to vascular diseases on the retina, such as diabetic retinopathy.
  • solid tumors cannot grow beyond a certain limited size without sufficient blood vessel supply to obtain the nutrients needed for growth. Therefore, in order to overcome this limitation, solid tumors will express VEGF to enable their growth and metastasis.
  • VEGF-A is the most important factor, which can regulate normal and pathological angiogenesis (ie, angiogenesis).
  • the biological effects of VEGF-A are mediated by binding to its specific receptors, which are mainly the specific receptor vascular endothelial growth factor receptor 1 (VEGFR-1) and the specific receptor vascular endothelial growth factor Receptor 1 (VEGFR-2).
  • VEGFR-2 is considered to be the main VEGFR, which has an important effect on the proliferation of vascular endothelial cells.
  • VEGFR-2 induces VEGF to bind dimers and receptors that require autophosphorylation through intracellular kinases, thereby enhancing cell mitosis.
  • VEGF-C and VEGF-D can regulate the production of lymphatic vessels.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind VEGFA protein molecules, and thus can be used to treat tumors.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned single domain antibody (or conjugate thereof) of the present invention and a pharmaceutical Acceptable carrier or excipient.
  • a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention can
  • 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 not more than about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the anti-VEGF single domain antibody includes monomer, bivalent (bivalent antibody), and/or multivalent (multivalent antibody).
  • the anti-VEGF single domain antibody includes one, two or more VHH chains having the amino acid sequence shown in SEQ ID NO. 8 and/or SEQ ID NO. 14.
  • the anti-VEGF single domain antibody includes two VHH chains having the amino acid sequence shown in SEQ ID NO. 8 and/or SEQ ID NO. 14.
  • the anti-VEGF single domain antibody has a VHH chain with the amino acid sequence shown in SEQ ID NO. 8 and/or SEQ ID NO. 14.
  • the anti-VEGF single domain antibody includes two VHH chains having the amino acid sequence shown in SEQ ID NO.14.
  • the two VHH chains having the amino acid sequence shown in SEQ ID NO. 8 are connected by a linker.
  • the two VHH chains having the amino acid sequence shown in SEQ ID NO. 14 are connected by a linker.
  • the linker is selected from the following group: GGGGSGGGS (SEQ ID NO. 18), GS (SEQ ID NO. 19), GGGGS (SEQ ID NO. 20).
  • amino acid sequence of the anti-VEGF single domain antibody is shown in SEQ ID NO.16.
  • the bivalent anti-VEGF single domain antibody is hubi-Nb24(Y).
  • the single domain antibody carries a detectable label. More preferably, the label is selected from the following group: isotope, colloidal gold label, colored label or fluorescent label.
  • Colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the anti-VEGF single domain antibody is labeled with colloidal gold to obtain the colloidal gold labeled single domain antibody.
  • novel anti-VEGF single domain antibody of the present invention has good specificity and high titer.
  • the present invention also relates to a method for detecting VEGF protein.
  • the method steps are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; and detect the level of VEGF protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a sample containing cells in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention.
  • the kit further includes a container, instructions for use, buffers, and the like.
  • the present invention also provides a detection kit for detecting the level of VEGF.
  • the kit includes an antibody that recognizes the VEGF protein, a lysis medium used to dissolve the sample, and general reagents and buffers required for detection, such as various buffers, detection Labeling, detecting substrates, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the single domain antibody of the present invention has a wide range of biological and clinical application value, and its application involves many fields such as diagnosis and treatment of VEGF-related diseases, basic medical research, and biological research.
  • a preferred application is for clinical diagnosis and targeted therapy for VEGF.
  • the single domain antibody of the present invention specifically targets the VEGF protein with the correct spatial structure.
  • the single domain antibody of the present invention can recognize human, mouse, rabbit, and monkey VEGF.
  • the single domain antibody of the present invention only recognizes human VEGFA, does not cross-react with VEGFB, VEGFC, and VEGFD, and has good specificity.
  • the single domain antibody of the present invention can block the interaction between VEGFA and VEGFR2, VEGFA and VEGFR1, and is higher than the blocking activity of aflibercept.
  • the single domain antibody of the present invention has a good inhibitory effect on neovascularization, and the effect is better than the marketed product aflibercept.
  • the single domain antibody of the present invention has good anti-tumor activity, and the effect is better than the marketed product Avastin.
  • the single domain antibody of the present invention exhibits better stability under non-preparation conditions.
  • the constructed library volume was 6.4 ⁇ 10 8 CFU and 5.5 ⁇ 10 8 CFU, respectively, and the insertion rates were 91.7% and 95.8%, respectively.
  • the library was screened, and the two libraries were screened for 6 and 5 rounds respectively to obtain the enrichment of phage containing antibody genes.
  • the ELISA method was used to screen single domain antibodies that can block the interaction between human VEGFA and VEGFR2.
  • (1) Coat the VEGFR2 protein onto an enzyme-labeled plate (1ug/mL, 100uL/well) and incubate overnight at 4°C; (2) After washing 5 times with PBST, add 300uL 1% BSA blocking solution and incubate at 37°C 2 (3) After washing 5 times with PBST, add 50uL of the antibody sample (2-fold dilution from 40ug/mL), then add 50uL 0.08ug/mL biotinylated VEGFA protein to each well, 37°C Incubate for 1 hour; (4) After washing 5 times with PBST, add 100uL of SA-HRP (1:100,000 dilution) and incubate at 37°C for 1 hour; (5) After washing 5 times with PBST, add 100uL of TMB color developing solution, 37 Develop the color at °C for 10min, add 2M H 2 SO 4
  • Example 3 Inhibition of proliferation of HUVEC cells by candidate antibody
  • the method is as follows: (1) Trypsinize the well-grown HUVEC cells, neutralize the complete medium, wash it with PBS, resuspend at a concentration of 3E4/mL, and aliquot 100uL/well into 96-well plates, 37 °C, 5% CO 2 , culture for 20h. (2) Dilute VEGFA to 100ng/mL with 2% FBS DMEM on the second day, and dilute the antibody to 10000ng/mL, 5000ng/mL, 2500ng/mL, 1250ng/mL, 312.50ng/mL, 78.13ng/mL, 39.06 ng/mL, 9.77ng/mL, 2ng/mL.
  • the candidate antibody is humanized and modified, keeping the variable regions unchanged, and humanized design is performed for the four framework region sequences.
  • modification method please refer to the method of Example 4 in the patent CN2018101517526.
  • humanized antibody huNb24 sequence was constructed on the pFUSE vector, the humanized single domain antibody was fused with the Fc sequence, and expressed using the HEK293F system. The expressed protein can be used for subsequent verification.
  • the modified sequence is shown in Table 2 below:
  • the above humanized antibody was constructed into a bivalent form, and the linker GGGGSGGGS (SEQ ID NO.18) was used to connect.
  • the amino acid sequence after linking is shown in SEQ ID NO.16 (the corresponding coding nucleotide sequence is shown in SEQ ID No. 17), and then expressed using Pichia pastoris. Briefly, the expression method is as follows: (1) The single domain antibody bivalent sequence shown in SEQ ID NO.
  • Example 6 Detection of blocking activity of humanized antibodies and bivalent antibodies by ELISA
  • the detection method is the same as in Example 2, and the results are shown in Figure 4:
  • Example 7 Humanized bivalent antibody inhibits the proliferation of HUVEC cells
  • the detection method is the same as in Example 3, and the results are shown in Figure 7:
  • the humanized bivalent antibody hu bi-Nb24(Y) expressed by yeast has better proliferation inhibitory effects on HUVEC cells than similar marketed control products (IC 50 hu bi-Nb24).
  • (Y) 53.59 ng/mL
  • IC 50 Eylea 65.96 ng/mL
  • IC 50 Conbercept 129.7 ng/mL
  • IC 50 Avastin 254.7 ng/mL).
  • Example 8 ELISA to detect the specificity of candidate antibodies
  • ELISA is used to test whether the candidate antibody can cross-react with other species of VEGF.
  • (1) Add 1ug/mL of the antibody to be tested to the ELISA plate and coat overnight at 4°C, 100uL/well; (2) After washing 5 times with PBST, add 300uL 1% BSA to each well and block for 2 hours at room temperature; (3) ) After washing 5 times with PBST, add 100uL 1ug/mL Biotin-hVEGFA (human), Biotin-mVEGFA (mouse), Biotin-rVEGFA (rabbit) and incubate at 37°C for 1 hour; (4) After washing 5 times with PBST , Add 100uL diluted SA-HRP (diluted 1:5000) and incubate for 1 hour at 37; (5) After washing 5 times with PBST, add 100uL TMB color developing solution, develop color at 37°C for 10min, add 2M H 2 SO 4 The reaction was terminated with 50uL/well, and the absorbance
  • the humanized bivalent antibody hu bi-Nb24(Y) can simultaneously recognize human, mouse, and rabbit VEGFA.
  • this candidate antibody can also recognize VEGFA of cynomolgus monkey.
  • Example 9 Detection of the blocking effect of humanized bivalent antibody on VEGFR1/VEGFA by ELISA
  • Example 10 Inhibitory effect of candidate antibody on the growth of blood vessels in the eye of neonatal mouse OIR model
  • the area of the non-perfusion area of the retina of the drug hubi-Nb24(Y) at three concentrations (1.5mg/mL, 1.0mg/mL, 0.5mg/mL) is smaller than that of Eylea, but there is no statistical difference; in Figure 12, the comparison with OIR In comparison, the three concentrations of the drug hubi-Nb24(Y) (1.5mg/mL, 1.0mg/mL, 0.5mg/mL) and Eylea can reduce the new blood vessel clusters, and all have statistical significance; compared with Eylea ,
  • the drug hubi-Nb24(Y) has three concentrations (1.5mg/mL, 1.0mg/mL, 0.5mg/mL) of the percentage of retinal neovascularization clusters are all less than it, and there is a statistical difference; with the drug hubi -The increase in the concentration of Nb24(Y) reduces the percentage of retinal neovascular clusters at P17
  • the candidate antibody hubi-Nb24(Y) with a concentration of 1mg/mL was placed in a 10mM PB solution, and placed at -20°C (repeated freezing and thawing), 4°C, 25°C, and 40°C. After different time points Sampling for SEC-HPLC detection. Using Adcance Bio SEC 130A 2.7um 7.8*300mm column, the detection wavelength is 280nm, tassel 0.5mL/min at room temperature, and 200mM pH7.0PB solution is used as the flow isocratic elution for 30min.
  • the test results are shown in Figure 13.
  • the candidate antibody was stored at 4°C for 1 month (Figure 13A), 25°C for 1 month (Figure 13B), 40°C for 15 days (Figure 13C), and -20°C repeated freezing and thawing 5 Under the second ( Figure 13D) conditions, the purity did not change significantly, indicating that the antibody exhibited better stability under non-prepared conditions.

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Abstract

本发明提供了抗VEGF单域抗体及其应用。具体地，本发明提供了一种抗VEGF单域抗体及其VHH链。本发明还提供了编码上述单域抗体或其VHH链的编码序列、相应的表达载体和能够表达该单域抗体的宿主细胞，以及本发明单域抗体的生产方法。本发明单域抗体能够特异性识别人VEGFA，不与VEGFB、VEGFC、VEGFD发生交叉反应，具有良好的特异性；本发明单域抗体能同时识别人、鼠、兔、猴子的VEGFA，能够有效阻断VEGFA与VEGFR2、VEGFA与VEGFR1的相互作用；且对新生血管生成有极好的抑制作用；本发明单域抗体在非制剂条件下表现出较佳的稳定性。

Description

抗VEGF单域抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及一种抗VEGF单域抗体及其应用。
背景技术
现有的血管内皮生长因子药物包括贝伐珠单抗(Bevacizumab，商品名为Avastin)、康柏西普(Conbercept)、阿柏西普(Aflibercept)等。2018年2月13日，国家食品药品监督管理总局(CFDA)已批准艾力雅(阿柏西普眼内注射溶液)用于治疗成人糖尿病性黄斑水肿。用于抗肿瘤治疗的贝伐珠单抗(Bevacizumab，商品名为Avastin)的价格是1998元每瓶4ml(含100mg贝伐珠单抗)，推荐剂量是5mg/kg，每14天给药1次，假如一位患者的体重为60kg，每次应用300mg贝伐珠单抗，也就是三瓶，一个月用药2次，也就是11988元一个月。用于眼科疾病治疗的康柏西普的价格是5550元每支0.2ml(含10mg康柏西普)，它的给药方案是前三月每月注射一次，后续三月注射一次，一年共6次，也就是33300元一年。此外，阿柏西普的价格是5850元每瓶0.1ml(含4mg阿柏西普)，它的给药方案是每两个月给药1次，一年共6次，也就是35100元一年。
单域抗体(nanobody，Nb)，即重链单域抗体VHH(variable domain of heavy chain of heavy-chain antibody)—骆驼体内存在着天然缺失轻链的重链抗体(heavy-chain antibody，HCAb)，克隆其可变区而得到的只由一个重链可变区组成的单域抗体，是目前可以得到的具有完整功能的稳定的可结合抗原的最小单位。单域抗体具有稳定性高、水溶性好、人源化简单、靶向性高、穿透性强等特点，在免疫实验、诊断与治疗中，发挥着超乎想象的巨大功能。单域抗体正逐渐成为新一代抗体诊断及治疗中的新兴力量。
开发一种新型抗VEGF单域抗体，使其具有较好的特异性、阻断活性，更佳的临床药效，并且生产简便，能够降低生产成本，减轻患者用药负担，已经成为亟待解决的问题。
发明内容
本发明的目的在于提供一种新型抗VEGF单域抗体及其应用。
具体地，本发明的目的在于提供一种能够有效阻断VEGFA与VEGFR2、VEGFA与VEGFR1的相互作用、对新生血管生成有良好的抑制作用，对实体瘤具有良好的 抑制作用，且具有较好的特异性的单域抗体。
本发明的第一方面，提供了一种抗VEGF单域抗体单域抗体VHH链的互补决定区CDR区，所述VHH链的互补决定区CDR包括SEQ ID NO.:1所示的CDR1、SEQ ID NO.:2所示的CDR2、和SEQ ID NO.:3所示的CDR3。
在另一优选例中，所述的CDR1、CDR2和CDR3被框架区FR1、FR2、FR3和FR4所隔开。
本发明的第二方面，提供了一种抗VEGF单域抗体的VHH链，所述的VHH链包括框架区FR和本发明的第一方面所述的互补决定区CDR。
在另一优选例中，所述的框架区FR包括：
(a)SEQ ID NO.:4所示的FR1、SEQ ID NO.:5所示的FR2、SEQ ID NO.:6所示的FR3、和SEQ ID NO.:7所示的FR4；或
(b)SEQ ID NO.:10所示的FR1、SEQ ID NO.:11所示的FR2、SEQ ID NO.:12所示的FR3、和SEQ ID NO.:13所示的FR4。
在另一优选例中，所述的抗VEGF单域抗体的VHH链如SEQ ID NO.:8或14所示。
此外，还提供一种新型抗VEGF单域抗体的重链可变区，所述的重链可变区包括SEQ ID NO.:1所示的CDR1、SEQ ID NO.:2所示的CDR2、和SEQ ID NO.:3所示的CDR3。
本发明的第三方面，提供了一种抗VEGF抗体，所述的抗VEGF抗体具有本发明的第二方面所述的VHH链。
在另一优选例中，所述抗VEGF抗体包括双链抗体、单链抗体、单域抗体。
在另一优选例中，所述抗VEGF抗体选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
在另一优选例中，所述抗VEGF抗体可以是抗体片段，如：Fab、Fab'、(Fab')2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
在另一优选例中，所述抗VEGF抗体为抗VEGF单域抗体。
在另一优选例中，所述抗VEGF抗体包括单体、二价体(二价抗体)、和/或多价体(多价抗体)。
在另一优选例中，所述抗VEGF抗体包括一条或多条具有如SEQ ID NO.:8或SEQ ID NO.:14所示的氨基酸序列的VHH链。
在另一优选例中，所述抗VEGF抗体的VHH链序列如SEQ ID NO.:8和/ 或SEQ ID NO.:14所示。
在另一优选例中，所述抗VEGF抗体包括两条具有如SEQ ID NO.:8或SEQ ID NO.:14中所示的氨基酸序列的VHH链。
在另一优选例中，所述抗VEGF抗体具有如SEQ ID NO.8和/或SEQ ID NO.14中所示的氨基酸序列的VHH链。
在另一优选例中，所述两条具有如SEQ ID NO.14中所示的氨基酸序列的VHH链之间通过连接子进行连接。
在本发明的一个优选例中，所述连接子选自以下序列：(G aS b) x—(G mS n) y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
在本发明的一个优选例中，所述连接子选自下组：GGGGSGGGS(SEQ ID NO.18)、GS(SEQ ID NO.19)、GGGGS(SEQ ID NO.20)。
在另一优选例中，所述抗VEGF抗体的氨基酸序列如SEQ ID NO.16所示。
在本发明的一个优选例中，所述二价抗VEGF抗体为hu bi-Nb24(Y)。
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VEGF单域抗体VHH链的CDR区、本发明的第二方面所述的抗VEGF单域抗体的VHH链、或本发明的第三方面所述的抗VEGF单域抗体。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.:9或15所示的核苷酸序列。
在另一优选例中，所述多核苷酸具有如SEQ ID NO.17所示的核苷酸序列。
在另一优选例中，所述的多核苷酸包括DNA或RNA。
本发明的第五方面，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：巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉、或其组合。
在另一优选例中，所示真核细胞选自下组：草地粘虫等昆虫细胞、烟草等植物细胞、BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞、或其组合。
在另一优选例中，所述宿主细胞优选为哺乳动物细胞，更优选HEK293细胞、CHO细胞、BHK细胞、NSO细胞或COS细胞。
在另一优选例中，所述的宿主细胞为巴斯德毕赤酵母。
本发明的第七方面，提供了一种产生抗VEGF单域抗体的方法，包括步骤：
(a)在适合产生单域抗体的条件下，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VEGF单域抗体的培养物；以及
(b)从所述培养物中分离或回收所述的抗VEGF单域抗体；以及
(c)任选地，纯化和/或修饰得步骤(b)的VEGF单域抗体。
在另一优选例中，所述的抗VEGF单域抗体具有如SEQ ID NO.:8或14所示的氨基酸序列。
在另一优选例中，所述的抗VEGF单域抗体具有如SEQ ID NO.16所示的氨基酸序列。
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)如本发明第二方面所述的抗VEGF单域抗体的VHH链、或如本发明第三方面所述的抗VEGF单域抗体；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))、化疗剂(例如，顺铂)或任何形式的纳米颗粒。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明的第二方面所述的抗VEGF单域抗体的VHH链、如本发明的第三方面所述的抗VEGF单域抗体。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多 个重复的如本发明的第二方面所述抗VEGF单域抗体的VHH链、如本发明的第三方面所述的抗VEGF单域抗体。
本发明的第九方面，提供了本发明的第二方面所述的抗VEGF单域抗体的VHH链、本发明的第三方面所述的抗VEGF单域抗体的用途，用于制备：
(a)用于抑制血管生成的药物；
(b)用于治疗与VEGF相关的疾病或病症的药物。
本发明的第十方面，提供了一种药物组合物，所述药物组合物含有：
(i)如本发明的第一方面所述的抗VEGF单域抗体VHH链的互补决定区CDR区、如本发明的第二方面所述的抗VEGF单域抗体的VHH链、如本发明的第三方面所述的抗VEGF单域抗体、或如本发明的第九方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为药物、毒素、和/或治疗用同位素。
在另一优选例中，所述的药物组合物中还含有治疗肿瘤的其他药物，如细胞毒性药物。
在另一优选例中，所述的药物组合物用于阻断VEGFA与VEGFR2、VEGFA与VEGFR1的相互作用。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备治疗与VEGF相关的疾病或病症的药物，所述疾病或病症包括肿瘤或癌症或眼部疾病。
在另一优选例中，所述肿瘤或癌症包括但不限于：乳腺癌、肺癌、食管癌、胃癌、大肠癌、肺癌、甲状腺癌、鼻咽癌中的一种或多种。
在另一优选例中，所述眼部疾病包括但不限于年龄相关性黄斑变性、糖尿病性视网膜病变、视网膜静脉阻塞、病理性近视、新生血管性青光眼以及其它涉及新生血管的眼科疾病。
本发明的第十一方面，提供了本发明的第三方面所述的抗VEGF单域抗体的一种或多种的用途；用于制备
(a)用于抑制血管生成的药物；
(b)用于治疗与VEGF相关的疾病或病症的药物；
(c)用于检测人VEGF分子；
(d)用于流式检测；
(e)用于细胞免疫荧光检测；
(f)用于治疗肿瘤；
(g)用于肿瘤诊断。
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。
本发明的第十二方面，提供了一种抗体，所述抗体包括一条或多条如本发明的第二方面所述的抗VEGF单域抗体的VHH链。
在另一优选例中，所述抗体包括两条本发明的第二方面所述的抗VEGF单域抗体的VHH链。
在另一优选例中，所述抗体具有如本发明的第二方面所述的重链可变区VHH。
在另一优选例中，所述的抗体能够特异性针对具有正确空间结构的VEGFA蛋白。
在另一优选例中，所述的抗体能够识别人、小鼠、兔子、猴子的VEGFA。
在另一优选例中，所述的抗体不与人的VEGFB、VEGFC、VEGFD发生交叉反应。
在另一优选例中，所述的抗体能够阻断VEGFA与VEGFR2、VEGFA与VEGFR1的相互作用。
在另一优选例中，所述的抗体能够抑制新生血管的生成。
在另一优选例中，所述抗体为单域抗体。
本发明的第十三方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明的第二方面所述的VHH链或如本发明的第三方面所述的抗VEGF单域抗体；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VEGFA蛋白。
在本发明的第十四方面，提供了如本发明的第二方面所述的抗VEGF单域抗体的VHH链、如本发明的第三方面所述的抗VEGF单域抗体、或如本发明的第八方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；
其中，所述试剂、检测板或试剂盒用于：检测样品中VEGF蛋白；
其中，所述药剂用于治疗或预防与VEGF相关的疾病或病症。
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，所述疾病或病症包括肿瘤或癌症或眼部疾病。
在另一优选例中，所述肿瘤或癌症包括但不限于：乳腺癌、肺癌、食管癌、胃癌、大肠癌、肺癌、甲状腺癌、鼻咽癌中的一种或多种。
在另一优选例中，所述眼部疾病包括但不限于年龄相关性黄斑变性、糖尿病性视网膜病变、视网膜静脉阻塞、病理性近视、新生血管性青光眼以及其它涉及新生血管的眼科疾病。
本发明的第十五方面，提供了一种治疗疾病的方法，所述方法包括，给需要的对象施用本发明的第三方面所述的单域抗体、或本发明的第八方面所述的免疫偶联物。
在另一优选例中，所述的对象包括哺乳动物，如人、小鼠、兔子、猴子。
本发明的第十六方面，提供了一种检测样品中VEGFA蛋白的方法，所述方法包括步骤：
(1)将样品与本发明的第二方面所述的VHH链、本发明的第三方面所述的单域抗体、或本发明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VEGFA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。
在本发明的第十七方面，提供了一种VEGFA蛋白检测试剂，所述的检测试剂包含：
(i)本发明的第二方面所述的VHH链、本发明的第三方面所述的单域抗体、或本发明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)。
本发明的第十八方面，提供一种检测VEGFA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十七方面所述的检测试 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VEGFA表达。
本发明的第十九方面，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VEGFA蛋白的造影剂。
在另一优选例中，所述检测用于癌症的诊断或预后。
本发明的第二十方面，提供了一种抗VEGF单域抗体VHH链的框架区FR，所述的VHH链的框架区FR由SEQ ID NO.:4所示的FR1，SEQ ID NO.:5所示的FR2，SEQ ID NO.:6所示的FR3，SEQ ID NO.:7所示的FR4组成；或由SEQ ID NO.:10所示的FR1、SEQ ID NO.:11所示的FR2、SEQ ID NO.:12所示的FR3、和SEQ ID NO.:13所示的FR4组成。
本发明第二十一方面，提供了一种治疗与VEGF相关的疾病或病症的方法，给所需要的对象施用本发明第十方面所述的药物组合物。
在另一优选例中，所述的对象包括哺乳动物，如人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是ELISA鉴定能够阻断人VEGFA与VEGFR2相互作用的单域抗体的结果。经鉴定候选抗体Nb24的阻断活性显著优于对照抗体Avastin。其中，安维汀(Avastin)为贝伐珠单抗(Bevacizumab)。
图2是候选抗体对HUVEC细胞的增殖抑制作用检测结果。经鉴定候选抗体Nb24对HUVEC细胞的增殖抑制作用强于对照抗体Avastin。
图3是酵母表达的huNb24二价体的SEC-HPLC检测结果。经硫酸铵沉淀纯化后的样品用SEC-HPLC鉴定，其样品纯度可到达94.11％。
图4是ELISA检测候选抗体人源化后的阻断活性。结果表明，人源化前与人源化后抗体的阻断活性相当(IC 50 Nb24＝0.045ug/mL，IC 50 huNb24＝0.038ug/mL)，因此人源化成功。其中，Nb24为人源化前抗体，huNb24为人源化后抗体。
图5是ELISA检测酵母表达的人源化二价抗体的阻断活性。结果表明，酵母表达的单域抗体二价体的阻断活性显著提高(IC 50 huNb24＝0.044ug/mL，IC 50 hu bi-Nb24(Y)＝0.013ug/mL)，且显著高于对照抗体Avastin的阻断活性(IC 50  Avastin＝0.331ug/mL)。其中，hu bi-Nb24(Y)为酵母表达的人源化二价抗体。
图6是利用ELISA检测比较人源化二价抗体与同类上市产品的阻断活性。结果表明，酵母表达的人源化二聚体抗体的阻断活性具有明显优势，均优于几种上市产品的阻断活性(IC 50 hu bi-Nb24(Y)＝0.022ug/mL，IC 50 Eylea＝0.085ug/mL，IC 50 Conbercept＝0.088ug/mL，IC 50 Avastin＝0.439ug/mL)。其中，Eylea为阿柏西普，Conbercept为康柏西普。
图7是酵母表达的人源化二价抗体对HUVEC细胞的增殖抑制作用检测结果。结果表明，酵母表达的人源化二价抗体的效果优于同类上市对照产品(IC 50  hu bi-Nb24(Y)＝53.59ng/mL，IC 50 Eylea＝65.96ng/mL，IC 50 Conbercept＝129.7ng/mL，IC 50 Avastin＝254.7ng/mL)。
图8是ELISA检测候选抗体能否与VEGF同家族蛋白发生交叉反应的结果。结果表明，人源化二价抗体能够与人VEGFA反应，而不与同家族的其他蛋白如VEGFB、VEGFC、VEGFD发生交叉反应，其特异性良好。
图9是ELISA检测候选抗体能否与与其他种属VEGF发生交叉反应的结果。结果表明，人源化二价抗体能够同时识别人、鼠、兔的VEGFA。
图10是ELISA检测酵母表达的人源化二价抗体对人VEGFA与VEGFR1相互作用阻断的结果。结果表明，候选抗体能够阻断人VGEFA与VEGFR1的相互作用(IC 50 hu bi-Nb24(Y)＝0.168ug/mL)，且阻断活性优于对照抗体Avastin(IC 50  Avastin＝0.967ug/mL)。
图11是OIR模型小鼠视网膜无灌注区面积统计结果。对小鼠进行不同浓度的酵母表达的人源化二价抗体用药，实验组小鼠视网膜无灌注区面积均小于阳性对照组。
图12是OIR模型小鼠视网膜新生血管簇统计结果。与阿柏西普Eylea(阳性对照)相比，不同浓度的酵母表达的人源化二价抗体对小鼠的视网膜新生血管簇抑制效果更加明显，且具有统计学差异。
图13是SEC-HPLC检测候选抗体在不同温度下的稳定性结果。图13A显示了候选抗体在4℃放置1个月的稳定性结果，结果表明，该抗体在非制剂条件下放置于4℃1个月未出现明显的纯度变化，表现出较佳的稳定性。图13B显示了候选抗体在25℃放置1个月的稳定性结果，结果表明，该抗体在非制剂条件下放置于25℃1个月未出现明显的纯度变化，表现出较佳的稳定性。图13C显示了候选抗体在40℃放置15天的稳定性结果，结果表明，该抗体在非制剂条件下放置于40℃15天未出现明显的纯度变化，表现出较佳的稳定性。图13D显示了候选抗体在-20℃反复冻融5次的稳定性结果，结果表明，该抗体在非制剂条件下放置于-20℃反复冻融5次未出现明显的纯度变化，表现出较佳的稳 定性。
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抗VEGF单域抗体，实验结果表明，本发明单域抗体能够特异性识别VEGFA，不与VEGFB、VEGFC、VEGFD发生交叉反应，具有良好的特异性；能够有效阻断VEGFA与VEGFR2、VEGFA与VEGFR1的相互作用；且对新生血管生成有良好的抑制作用。本发明单域抗体生成简便。由此完成了本发明。
具体地，本发明利用人源的VEGFA蛋白免疫骆驼，获得高质量的免疫单域抗体基因文库。然后将VEGFR蛋白分子偶联在酶标板上，展示VEGFR蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫单域抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了VEGFA特异性的单域抗体基因。再将此基因转至哺乳动物细胞中，从而获得了能在哺乳动物细胞中高效表达的、且特异性高的的单域抗体株。然后通过ELISA、流式细胞术、荧光素酶报告基因检测系统等方法鉴定出具有阻断活性的抗VEGF单域抗体。
术语
如本文所用，术语“本发明单域抗体”、“本发明的单域抗体”、“本发明的抗VEGF抗体”、“本发明VEGF单域抗体”、“抗VEGF单域抗体”具有相同的含义，可互换使用，均指特异性识别和结合于VEGFA(包括人VEGFA)的单域抗体。优选地，本发明的单域抗体的可变区具有SEQ ID NO.1所示的CDR1、SEQ ID NO.:2所示的CDR2、和SEQ ID NO.:3所示的CDR3。更优选地，本发明的单域抗体的框架区具有(a)SEQ ID NO.:4所示的FR1、SEQ ID NO.:5所示的FR2、SEQ ID NO.:6所示的FR3、和SEQ ID NO.:7所示的FR4，或(b)SEQ ID NO.10所示的FR1、SEQ ID NO.11所示的FR2、SEQ ID NO.12所示的FR3、和SEQ ID NO.13所示的FR4。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体”、“VHH”、“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb，或纳米抗体nanobody)具有相同的 含义并可互换使用，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-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β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VEGF抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合VEGF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。
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VEGFA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，除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段的多核苷酸分子。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))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
血管内皮生长因子(Vascular Endothelial Growth Factor，VEGF)
血管内皮生长因子(Vascular Endothelial Growth Factor，VEGF)，是一种高度特异性的促血管内皮细胞生长因子，VEGF与内皮细胞膜上其受体，血管内皮细胞生长因子受体(Vascular Endothelial Growth Factor Receptor，VEGFR)结合，引起受体的自身磷酸化，从而激活有丝分裂原活化蛋白激酶(MAPK)，实现有丝分裂原特性，诱导内皮细胞增生。因其具有血管新生的特性，VEGF可以于血液循环不足时恢复组织的氧气供应。当组织中的VEGF过度表达时，可导致疾病症状。例如，VEGF过度表达会导致视网膜上的血管疾病，如糖尿病性视网膜病。此外，实体瘤在没有足够的血管供应以获得生长所需的营养情况下，无法生长超过一定限制的大小，因此为了克服该限制，实体瘤会表达VEGF以使得其生长及转移。
VEGF家族成员包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、VEGF-F和胎盘生长因子(Placenta Growth Factor，PGF)。其中VEGF-A是最重要的因子，其可调控正常和病理性的血管新体(即血管生成)。VEGF-A的生物学效应是依靠与其特异性受体结合而介导的，与其结合的主要是特异性受体血管内皮生长因子受体1(VEGFR-1)和特异性受体血管内皮生长因子受体1(VEGFR-2)。其中，VEGFR-2被认为是主要的VEGFR，其对血管内皮细胞的增生有重要影响。VEGFR-2通过细胞内激酶诱导VEGF绑定二聚体和需要自身磷酸化的受体，从而加强细胞的有丝分裂。VEGF-C和VEGF-D则可调控淋巴管的生成。
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，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)：瘤内、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VEGFA蛋白分子，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
抗VEGF单域抗体
在本发明中，所述抗VEGF单域抗体包括单体、二价体(二价抗体)、和/或多价体(多价抗体)。
在本发明的一个优选例中，所述抗VEGF单域抗体包括一条、两条或多条具有如SEQ ID NO.8和/或SEQ ID NO.14所示的氨基酸序列的VHH链。
典型的，所述抗VEGF单域抗体包括两条具有如SEQ ID NO.8和/或SEQ ID NO.14中所示的氨基酸序列的VHH链。
典型的，所述抗VEGF单域抗体具有如SEQ ID NO.8和/或SEQ ID NO.14中所示的氨基酸序列的VHH链。
典型的，所述抗VEGF单域抗体包括两条具有如SEQ ID NO.14中所示的氨基酸序列的VHH链。
在本发明的一个优选例中，所述两条具有如SEQ ID NO.8中所示的氨基酸序列的VHH链之间通过连接子进行连接。
在本发明的一个优选例中，所述两条具有如SEQ ID NO.14中所示的氨基酸 序列的VHH链之间通过连接子进行连接。
在本发明的一个优选例中，所述连接子选自以下序列：(G aS b) x—(G mS n) y，其中a，b，m，n，x，y＝0或1或2或3或4或5或6或7或8或9或10(较佳地，a＝4而b＝1，m＝3而n＝1)。
在本发明的一个优选例中，所述连接子选自下组：GGGGSGGGS(SEQ ID NO.18)、GS(SEQ ID NO.19)、GGGGS(SEQ ID NO.20)。
在本发明的一个优选例中，所述抗VEGF单域抗体的氨基酸序列如SEQ ID NO.16所示。
在本发明的一个优选例中，所述二价抗VEGF单域抗体为hu bi-Nb24(Y)。
标记的单域抗体
在本发明的一个优选例中，所述单域抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，抗VEGF的单域抗体用胶体金标记，得到胶体金标记的单域抗体。
本发明的新型抗VEGF单域抗体具有很好的特异性，很高的效价。
检测方法
本发明还涉及检测VEGF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VEGF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VEGF水平的检测试剂盒，该试剂盒包括识别VEGF蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的单域抗体有广泛生物应用价值和临床应用价值，其应用涉及到与VEGF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VEGF的临床诊断和靶向治疗。
本发明的主要优点包括：
(a)本发明单域抗体特异性针对具有正确空间结构的VEGF蛋白。
(b)本发明单域抗体能够识别人、小鼠、兔子、猴子的VEGF。
(c)本发明单域抗体只识别人的VEGFA，不与VEGFB、VEGFC、VEGFD发生交叉反应，具有较好的特异性。
(d)本发明单域抗体能够阻断VEGFA与VEGFR2、VEGFA与VEGFR1的相互作用，且高于阿柏西普的阻断活性。
(e)本发明单域抗体对新生血管生成有良好的抑制作用，且效果优于上市产品阿柏西普。
(f)本发明单域抗体具有良好的抗肿瘤活性，且效果优于上市产品Avastin。
(g)本发明单域抗体的生产简便。
(h)本发明单域抗体在非制剂条件下表现出较佳的稳定性。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1：抗VEGF单域抗体的筛选及表达
为了获得特异性针对人VEGF的单域抗体，首先利用哺乳动物细胞HEK293F瞬转表达人VEGFA蛋白，经亲和纯化后用于骆驼免疫。具体方法可参见专利CN2018101517526实施例1和实施例2中的方法描述。简要地，将纯化的VEGFA蛋白免疫两只新疆双峰驼，7次免疫结束后从骆驼外周血中分离total RNA，随后反转录和PCR扩增出VHH基因，再将VHH基因克隆至噬菌体载体pMECS上，转化至TG1中构建成噬菌体展示文库。构建的文库库容分别为6.4×10 8CFU和5.5×10 8CFU，插入率分别为91.7％和95.8％。随后进行文库筛选，两个文库分别经6轮及5轮筛选获得含抗体基因的噬菌体富集。从每个文库挑选300颗克隆进行PE-ELISA鉴定，将获得的阳性克隆进行测序，再将序列不同的单域抗体与Fc融合表达，利用HEK293F系统瞬时表达抗体，表达方法参见专利CN2018101517526实施例3中的方法描述。
实施例2：阻断型抗VEGF单域抗体的筛选
利用ELISA法筛选能够阻断人VEGFA与VEGFR2相互作用的单域抗体。(1)将VEGFR2蛋白包被至酶标板上(1ug/mL，100uL/孔)，4℃孵育过夜；(2)用PBST洗涤5次后，加入300uL 1％BSA封闭液，37℃孵育2小时；(3)用PBST洗涤5次后，加入50uL梯度稀释好的抗体样品(从40ug/mL开始2倍梯度稀释)，再每孔加入50uL 0.08ug/mL生物素化的VEGFA蛋白，37℃孵育1小时；(4)用PBST洗涤5次后，加入SA-HRP(1:100000稀释)100uL，37℃孵育1小时；(5)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1所示：Nb24的阻断活性显著优于对照抗体Avastin(IC 50 Nb24＝0.0149ug/mL，IC 50  Avastin＝0.2172ug/mL)。
实施例3：候选抗体对HUVEC细胞的增殖抑制作用
简要地，方法如下：(1)将生长良好的HUVEC细胞胰酶消化，完全培养基中和，PBS洗一遍，按照3E4/mL的浓度重悬，100uL/孔分装到96孔板中，37℃，5％CO 2，培养20h。(2)第二天用2％FBS的DMEM稀释VEGFA至100ng/mL，梯度稀释抗体至10000ng/mL、5000ng/mL、2500ng/mL、1250ng/mL、312.50ng/mL、78.13ng/mL、39.06ng/mL、9.77ng/mL、2ng/mL。(3)在另一块96孔板中加入60uL VEGFA和等体积的稀释好的抗体混匀，放置于37℃共同孵育2h；每种混合液3个复孔。(4)从培养箱中取出细胞培养板，吸去上清，然后分别在对应的孔中加入100ul(3)中的混合液，置于37℃孵育72h。(5)72h后，加入CCK8溶液10ul/孔，显色2h，显色结束后，用酶标仪读取OD450波长下的吸光值。结果如图2所示：候选抗体Nb24对HUVEC细胞的增殖抑制作用强于对照抗体Avastin(IC 50 Nb24＝29.58ng/mL，IC 50 Avastin＝72.28ng/mL)。
实施例4：抗体Nb24人源化及表达
将候选抗体进行人源化改造，保持可变区不变，针对四个骨架区序列进行人源化设计，改造方法参见专利CN2018101517526中实施例4的方法。再将人源化后的抗体huNb24序列构建在pFUSE载体上，使人源化的单域抗体与Fc序列融合，利用HEK293F系统表达，表达后的蛋白可用于后续验证。改造后的序列如下表2所示：
表2
Figure PCTCN2020078595-appb-000001
实施例5：人源化二价抗体的构建及表达
将以上人源化的抗体构建成二价形式，利用连接子GGGGSGGGS(SEQ ID NO.18)连接，连接后氨基酸序列如SEQ ID NO.16所示(其对应的编码核苷酸序列如SEQ ID NO.17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SEQ ID NO.16所示的单域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pPICZaA-Nb24-Nb24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，具体实施方案可参见Invitrogen公司提供的pPICZaA载体说明书；(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)将取到的样品进行SDS-PAGE检测，同时收集样品用硫酸铵沉淀法纯化目的抗体。结果如图3所示：从表达上清中进行硫酸铵沉淀纯化获得的单域抗体二价体抗体hu bi-Nb24(Y)，经SEC-HPLC检测其纯度为94.11％，可用于后续研究。
实施例6：ELISA检测人源化抗体及二价抗体的阻断活性
检测方法同实施例2，结果如图4所示：人源化抗体的阻断活性与人源化前抗体的阻断活性相当(IC 50 Nb24＝0.045ug/mL，IC 50 huNb24＝0.038ug/mL)，因此表明，该人源化改造成功。再将人源化后的单域抗体与酵母表达的二价抗体进行 阻断活性比较，重复以上实验，结果如图5所示：酵母表达的单域抗体二价体hu bi-Nb24(Y)的阻断活性提高3倍以上(IC 50 huNb24＝0.044ug/mL，IC 50 hu  bi-Nb24(Y)＝0.013ug/mL)，且显著高于对照抗体Avastin的阻断活性(IC 50  Avastin＝0.331ug/mL)。再次利用以上实验方法检测比较候选抗体与同类上市产品的阻断活性，结果如图6所示：酵母表达的人源化二聚体抗体hu bi-Nb24(Y)的阻断活性具有明显优势，IC 50 hu bi-Nb24(Y)＝0.022ug/mL，IC 50 Eylea＝0.085ug/mL，IC 50 Conbercept＝0.088ug/mL，IC 50 Avastin＝0.439ug/mL。由此表明，酵母表达的人源化二聚体较现有上市产品具有显著优异的阻断活性。
实施例7：人源化二价抗体对HUVEC细胞的增殖抑制作用
检测方法同实施例3，结果如图7所示：酵母表达的人源化二价抗体hu bi-Nb24(Y)对HUVEC细胞的增殖抑制作用优于同类上市对照产品(IC 50 hu  bi-Nb24(Y)＝53.59ng/mL，IC 50 Eylea＝65.96ng/mL，IC 50 Conbercept＝129.7ng/mL，IC 50  Avastin＝254.7ng/mL)。
实施例8：ELISA检测候选抗体的特异性
利用ELISA验证候选抗体能否与VEGF同家族蛋白发生交叉反应。(1)将1ug/mL待测抗体加入酶标板中包被过夜，4℃，100uL/孔；(2)用PBST洗涤5次后，每孔加入300uL 1％BSA室温封闭2小时；(3)用PBST洗涤5次后，加入100uL 1ug/mL Biotin-hVEGFA、Biotin-hVEGFB、Biotin-hVEGFC、Biotin-hVEGFD于37℃下孵育1小时；(4)用PBST洗涤5次后，加入100uL稀释的SA-HRP(1:5000稀释)于37下孵育1小时；(5)用PBST洗涤5次后，加入加入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8所示：人源化二价抗体hu bi-Nb24(Y)能够与人VEGFA反应，而不与同家族的其他蛋白如VEGFB、VEGFC、VEGFD发生交叉反应。
同样地，利用ELISA检测候选抗体能否与其他种属VEGF发生交叉反应。(1)将1ug/mL待测抗体加入酶标板中包被过夜，4℃，100uL/孔；(2)用PBST洗涤5次后，每孔加入300uL 1％BSA室温封闭2小时；(3)用PBST洗涤5次后，加入100uL 1ug/mL Biotin-hVEGFA(人)、Biotin-mVEGFA(鼠)、Biotin-rVEGFA(兔)于37℃下孵育1小时；(4)用PBST洗涤5次后，加入100uL稀释的SA-HRP(1:5000稀释)于37下孵育1小时；(5)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9所示：人源化二价抗体hu bi-Nb24(Y)能够同时识别人、鼠、兔的VEGFA。此外，由于人VEGF121与食蟹猴对应位置的序列完全相 同，因此，可以认为该候选抗体也能够识别食蟹猴的VEGFA。
实施例9：ELISA检测人源化二价抗体对VEGFR1/VEGFA的阻断作用
(1)将VEGFR1蛋白包被至酶标板上(1ug/mL，100uL/孔)，4℃孵育过夜；(2)用PBST洗涤5次后，加入300uL 1％BSA封闭液，37℃孵育2小时；(3)用PBST洗涤5次后，加入50uL梯度稀释好的抗体样品(从20ug/mL开始2倍梯度稀释)，再每孔加入50uL 0.08ug/mL生物素化的VEGFA蛋白，37℃孵育1小时；(4)用PBST洗涤5次后，加入SA-HRP(1:100000稀释)100uL，37℃孵育1小时；(5)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10所示：候选抗体hu bi-Nb24(Y)能够阻断人VGEFA与VEGFR1的相互作用(IC 50 hu  bi-Nb24(Y)＝0.168ug/mL)，且阻断活性优于对照抗体Avastin(IC 50  Avastin＝0.967ug/mL)。
实施例10：候选抗体对新生小鼠OIR模型眼内血管生长的抑制作用
(1)采用经典的研究新生血管的小鼠模型—OIR模型。将7日龄的雄性C57BL/6J小鼠在75％氧浓度密闭容器中饲养至12日，期间定时监测氧箱内氧气浓度，使其维持在75％，后移至正常空气环境饲养5天。P7(7日龄)-P12(12日龄)对应视网膜血管闭塞期，P12-P17(17日龄)对应缺氧-异常血管增殖期，P17-P21(21日龄)对应异常增殖血管恢复期。选用P17为异常新生血管的观察点，于P12时，于OIR造模组的小鼠玻璃体注射1uL不同浓度的抗体药物hu bi-Nb24(Y)(1.5mg/mL、1.0mg/mL、0.5mg/mL)，对照抗体Eylea浓度为40mg/mL，后将小鼠移至正常空气中饲养至P17。
(2)FITC-IB4视网膜血管内皮细胞染色进行视网膜铺片，观察OIR模型中P17时异常新生血管簇及视网膜无灌注区：处死C57BL/6J小鼠后，立即摘除小鼠眼球，4％多聚甲醛固定1-2h，在手术显微镜下，沿角膜缘剪开球壁，去除晶状体、玻璃体，将视网膜神经纤维层和色素上皮层分离，去除巩膜、脉络膜和视网膜色素上皮层，将视网膜神经纤维层在PBS中漂洗，清除残存玻璃体；用含1mg/mL BSA及0.3％TX-100的PBS封闭1-2h；加入FITC-IB4溶液中(1:50稀释)，4℃48h；取出后PBS冲洗15分钟×3次，视网膜神经纤维层向上平铺在载玻片上，以视乳头为中心放射状切开，封片剂封片，荧光显微镜和共聚焦显微镜下观察拍照并统计。
结果如图11和图12所示：图11中，与OIR阴性对照组相比，药物hu bi-Nb24(Y)三种浓度(1.5mg/mL、1.0mg/mL、0.5mg/mL)均可减少视网膜无灌注区的面积，且具有统计学意义(p<0.05)；阳性对照药阿柏西普Eylea(40mg/mL)亦 可减少视网膜无灌注区面积。药物hu bi-Nb24(Y)三种浓度(1.5mg/mL、1.0mg/mL、0.5mg/mL)的视网膜无灌注区面积均小于Eylea，但无统计学差异；图12中，与OIR对照相比，药物hu bi-Nb24(Y)三种浓度(1.5mg/mL、1.0mg/mL、0.5mg/mL)与Eylea均可减少新生血管簇，且均具有统计学意义；与Eylea相比，药物hu bi-Nb24(Y)三种浓度(1.5mg/mL、1.0mg/mL、0.5mg/mL)的视网膜新生血管簇的百分比均小于它，且具有统计学差异；随着药物hu bi-Nb24(Y)浓度的增加，P17时视网膜新生血管簇的百分比减少(0.5VS 1，p＝0.0027；0.5vs 1.5，p＝0.0177；1vs 1.5，p＝0.2417)。
实施例11：候选抗体的稳定性研究
将浓度为1mg/mL的候选抗体hu bi-Nb24(Y)置于10mM PB溶液中，分别放置在-20℃(反复冻融)、4℃、25℃、40℃条件下，经不同时间点取样进行SEC-HPLC检测。使用Adcance Bio SEC 130A 2.7um 7.8*300mm column，检测波长为280nm，室温下流苏0.5mL/min，以200mM pH7.0PB溶液为流动相等度洗脱30min。
检测结果如图13所示，候选抗体在4℃放置1个月(图13A)、25℃放置1个月(图13B)、40℃放置15天(图13C)、-20℃反复冻融5次(图13D)条件下，其纯度均未发生明显变化，表明该抗体在非制剂条件下表现出较佳的稳定性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明序列信息：
Figure PCTCN2020078595-appb-000002
Figure PCTCN2020078595-appb-000003

Claims (16)

  1. 一种抗VEGF单域抗体VHH链的互补决定区CDR区，其特征在于，所述VHH链的互补决定区CDR包括SEQ ID NO.:1所示的CDR1、SEQ ID NO.:2所示的CDR2、和SEQ ID NO.:3所示的CDR3。
  2. 一种抗VEGF单域抗体的VHH链，其特征在于，所述的VHH链包括框架区FR和权利要求1所述的互补决定区CDR，优选地，所述的框架区FR包括：
    (a)SEQ ID NO.:4所示的FR1、SEQ ID NO.:5所示的FR2、SEQ ID NO.:6所示的FR3、和SEQ ID NO.:7所示的FR4；或
    (b)SEQ ID NO.:10所示的FR1、SEQ ID NO.:11所示的FR2、SEQ ID NO.:12所示的FR3、和SEQ ID NO.:13所示的FR4。
  3. 一种抗VEGF抗体，其特征在于，所述的抗VEGF抗体具有权利要求2所述的VHH链。
  4. 如权利要求3所述的抗VEGF抗体，其特征在于，所述抗VEGF抗体包含一条或多条具有如SEQ ID NO.:8或SEQ ID NO.:14所示的氨基酸序列的VHH链。
  5. 如权利要求3所述的抗VEGF抗体，其特征在于，所述抗VEGF抗体的氨基酸序列如SEQ ID NO.16所示。
  6. 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白质：权利要求1所述的抗VEGF单域抗体VHH链的CDR区、权利要求2所述的抗VEGF单域抗体的VHH链、权利要求3所述的抗VEGF抗体。
  7. 一种表达载体，其特征在于，所述表达载体含有权利要求6所述的多核苷酸。
  8. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的表达载体，或其基因组中整合有权利要求6所述的多核苷酸。
  9. 一种产生抗VEGF单域抗体的方法，其特征在于，包括步骤：
    (a)在适合产生单域抗体的条件下，培养权利要求8所述的宿主细胞，从而获得含所述抗VEGF单域抗体的培养物；以及
    (b)从所述培养物中分离或回收所述的抗VEGF单域抗体；以及
    (c)任选地，纯化和/或修饰得步骤(b)的VEGF单域抗体。
  10. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求2所述的抗VEGF单域抗体的VHH链、或如权利要求3所述的 抗VEGF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。
  11. 权利要求2所述的抗VEGF单域抗体的VHH链、权利要求3所述的抗VEGF抗体的用途，其特征在于，用于制备
    (a)用于抑制血管生成的药物；
    (b)用于治疗与VEGF相关的疾病或病症的药物。
  12. 一种药物组合物，其特征在于，所述药物组合物含有：
    (a)权利要求1所述的抗VEGF单域抗体VHH链的互补决定区CDR区、权利要求2所述的抗VEGF单域抗体的VHH链、或权利要求3所述的抗VEGF抗体、或权利要求10所述的免疫偶联物；以及
    (b)药学上可接受的载体。
  13. 一种抗体，其特征在于，所述抗体包括一条或多条权利要求2所述的抗VEGF单域抗体的VHH链。
  14. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)权利要求2所述的VHH链或权利要求3所述的抗VEGF抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  15. 一种检测样品中VEGFA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2所述的VHH链、权利要求3所述的抗体或权利要求10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VEGFA蛋白。
  16. 一种VEGFA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2所述的VHH链、权利要求3所述的抗体、或权利要求10所述的免疫偶联物；以及
    (ii)检测学上可接受的载体。
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