WO2024055995A1 - 一种抗vegfa融合蛋白及其制备方法和应用 - Google Patents

一种抗vegfa融合蛋白及其制备方法和应用 Download PDF

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WO2024055995A1
WO2024055995A1 PCT/CN2023/118594 CN2023118594W WO2024055995A1 WO 2024055995 A1 WO2024055995 A1 WO 2024055995A1 CN 2023118594 W CN2023118594 W CN 2023118594W WO 2024055995 A1 WO2024055995 A1 WO 2024055995A1
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vegfa
fusion protein
seq
cells
antibody
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PCT/CN2023/118594
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English (en)
French (fr)
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黄金亮
黄文涛
陈蒙蒙
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寻济生物科技（北京）有限公司
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Publication of WO2024055995A1 publication Critical patent/WO2024055995A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine technology, and specifically relates to an anti-VEGFA fusion protein and its preparation method and application.
  • VEGFA vascular endothelial growth factor A
  • AMD wet age-related macular degeneration
  • the diffusion rate of drugs in the vitreous body directly affects the drug administration interval.
  • the diffusion rate of drugs is related to its charge, molecular weight, molecular shape and other properties. Drugs with larger molecular weights diffuse more slowly and have longer half-lives.
  • ranibizumab molecular weight: 48kDa
  • bevacizumab molecular weight: 149kDa
  • aflibercept molecular weight: 115kDa
  • Single domain antibodies or nanobodies are the variable region domains of heavy-chain antibodies that naturally lack light chains in camelids. They are the smallest stable antibody units with complete antigen-binding functions.
  • the molecular weight of a single domain antibody is about 13kDa, it has high thermal stability and good water solubility. The properties of the molecule itself have great potential to be developed into higher concentration protein preparations.
  • the present invention uses Nanobodies as the basic active unit for binding VEGFA, conducts bivalent design of Nanobodies, and fuses Fc fragments to prepare anti-VEGFA fusion proteins with high affinity.
  • the first aspect of the present invention provides an anti-VEGFA fusion protein.
  • the anti-VEGFA fusion protein includes two or more anti-VEGFA antibodies or antigen-binding fragments thereof, and an Fc fragment.
  • the anti-VEGFA antibody or Its antigen-binding fragment contains CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein,
  • the amino acid sequence of CDR-H1 contains SYTMG (SEQ ID NO: 1) or an amino acid sequence that is at least 80% identical to SYTMG (SEQ ID NO: 1);
  • the amino acid sequence of CDR-H2 includes AISKGGYKYX 1 X 2 VSLEA (SEQ ID NO: 2 ) or an amino acid sequence that is at least 80% identical to AISKGGYKYX 1
  • the amino acid sequence of CDR-H3 contains TRAYGSSRLX 3 LAX 4 TYEY (SEQ ID NO: 3) or is the same as TRAYGSSRLX 3 LAX 4 TYEY (SEQ ID NO: 3) has an amino acid sequence that is at least 80% identical.
  • the anti-VEGFA fusion protein includes at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and above) anti-VEGFA antibody or antigen-binding fragment thereof.
  • amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO: 1, 2, and 3 in sequence.
  • the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in the order from N-terminus to C-terminus.
  • the division of amino acids in the CDR region of the antibody adopts the Kabat numbering system.
  • the anti-VEGFA antibody or its antigen-binding fragment is directly connected or indirectly connected to the anti-VEGFA antibody or its antigen-binding fragment, and between the anti-VEGFA antibody or its antigen-binding fragment and the Fc fragment.
  • the direct or indirect connection includes direct or indirect connection to the N-terminal, C-terminal and/or Fc fragment of the anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies or its antigen-binding fragment and/or Fc fragment. or on internal residues.
  • the N-terminal, C-terminal and/or internal residues of the anti-VEGFA antibody or antigen-binding fragment thereof in the anti-VEGFA fusion protein are in contact with the N-terminal, C-terminal and/or internal residues of other anti-VEGFA antibodies or antigen-binding fragments thereof or Fc fragments. Internal residues are connected.
  • the N-terminus of the anti-VEGFA antibody or antigen-binding fragment thereof in the anti-VEGFA fusion protein is connected to the N-terminus or C-terminus of other anti-VEGFA antibodies or antigen-binding fragments thereof.
  • the C-terminus of the anti-VEGFA antibody or antigen-binding fragment thereof in the anti-VEGFA fusion protein is connected to the N-terminus or C-terminus of other anti-VEGFA antibodies or antigen-binding fragments thereof.
  • the C-terminus of the anti-VEGFA antibody or its antigen-binding fragment in the anti-VEGFA fusion protein or the C-terminus of other anti-VEGFA antibodies or its antigen-binding fragment is connected to the N-terminus of the Fc fragment.
  • the indirect connection is through a joint.
  • the linker is selected from connecting peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, aliphatic chains, biotin, streptavidin or avidin.
  • the connecting peptide can be a rigid linker, a flexible linker or a meaningless amino acid. It is further preferred that the amino acid sequence of the connecting peptide includes one or a combination of two or more of SEQ ID NOs: 77-91, 152-153 and 156.
  • two or more anti-VEGFA antibodies or antigen-binding fragments thereof are anti-VEGFA antibodies or antigen-binding fragments thereof with identical sequences, anti-VEGFA antibodies or antigen-binding fragments thereof with partially identical sequences, or anti-VEGFA antibodies with completely different sequences. Antibodies or antigen-binding fragments thereof.
  • the anti-VEGFA fusion protein also contains a secreted peptide.
  • the secreted peptide is connected to the N-terminus of the anti-VEGFA fusion protein.
  • the amino acid sequence of the secreted peptide can be SEQ ID NO: 151 or 155.
  • the Fc fragment includes SEQ ID NO: 134, 135 or 158.
  • the anti-VEGFA fusion protein includes the first anti-VEGFA antibody or its antigen-binding fragment, the connecting peptide, and the second anti-VEGFA antibody or its antigen-binding fragment in sequence from the N-terminus to the C-terminus. fragment.
  • the anti-VEGFA fusion protein includes secreted peptide, first anti-VEGFA antibody or antigen-binding fragment thereof, connecting peptide, second anti-VEGFA antibody or Its antigen-binding fragment.
  • the anti-VEGFA fusion protein includes secreted peptide, first anti-VEGFA antibody or antigen-binding fragment thereof, connecting peptide, second anti-VEGFA antibody or Its antigen-binding fragment and Fc fragment.
  • the anti-VEGFA fusion protein includes secreted peptide, first anti-VEGFA antibody or antigen-binding fragment thereof, connecting peptide, second anti-VEGFA antibody or Its antigen-binding fragment, tag (such as 6 ⁇ His).
  • X 1 X 2 in SEQ ID NO: 2 represents DS, DA, NT, DT, NA or NS
  • X 3 in SEQ ID NO: 3 represents R or K, and Represents D, N, E or K.
  • amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 include any of the following groups (see Table 1):
  • the antibody or its antigen-binding fragment is Nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fd' fragment, Fv fragment, dAb fragment, isolated CDR region, F(ab') 2 fragments, single domain antibodies, single chain antibody molecules or linear antibodies.
  • the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment includes any one of the amino acid sequences in SEQ ID NO: 13-32 and 35-61, or is the same as the amino acid sequence in SEQ ID NO: 13-32 and 35-61. Any amino acid sequence has at least 80% identity.
  • the specific sequence numbers are shown in Table 2.
  • the anti-VEGFA fusion protein binds to human or monkey VEGFA protein, wherein the human VEGFA protein has the same sequence as the monkey VEGFA protein.
  • the anti-VEGFA antibody or antigen-binding fragment thereof can be a humanized antibody or a fully human antibody.
  • the anti-VEGFA antibody or antigen-binding fragment thereof includes a humanized sequence, and the modification site of the humanized sequence is located in the non-CDR region. Further preferably, the humanized modification site is located in the non-CDR region. Framework and/or constant regions of antibodies.
  • the amino acid sequence of the anti-VEGFA fusion protein includes any one of the amino acid sequences of SEQ ID NO: 62-76, 92-133, 136-150 and 159, or is the same as SEQ ID NO : Any one of the amino acid sequences 62-76, 92-133, 136-150 and 159 has at least 80% identity.
  • a second aspect of the present invention provides a chimeric antigen receptor, the extracellular domain of which contains the above-mentioned anti-VEGFA fusion protein.
  • the chimeric antigen receptor further comprises any transmembrane region and/or intracellular signal transduction region conventional in the prior art.
  • the third aspect of the present invention provides a nucleic acid encoding the above-mentioned anti-VEGFA fusion protein or the above-mentioned chimeric antigen receptor.
  • a fourth aspect of the present invention provides a vector containing the above nucleic acid.
  • the vector can be expressed under in vivo or in vitro or ex vivo conditions.
  • the vector is a prokaryotic expression vector, a viral expression vector or a eukaryotic expression vector.
  • a fifth aspect of the present invention provides a cell containing the above-mentioned vector or the above-mentioned nucleic acid.
  • the cells may be eukaryotic cells or prokaryotic cells.
  • Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells or CHO cells, etc.
  • Prokaryotic cells such as E. coli, etc.
  • a sixth aspect of the present invention provides a method for preparing an anti-VEGFA fusion protein.
  • the preparation method includes introducing the above-mentioned nucleic acid into a host cell and inducing its expression.
  • the seventh aspect of the present invention provides an immune cell expressing the above-mentioned anti-VEGFA fusion protein or the above-mentioned chimeric antigen receptor.
  • the immune cells include but are not limited to lymphocytes (such as T cells, B cells, NK cells), dendritic cells, monocytes/macrophages, granulocytes, and mast cells.
  • the immune cells are CAR-immune cells.
  • An eighth aspect of the present invention provides a method for constructing immune cells.
  • the construction method includes transfecting the nucleic acid sequence encoding the chimeric antigen receptor of the present invention into immune cells for expression.
  • a ninth aspect of the present invention provides a product for treating and/or diagnosing diseases.
  • the product for treating and/or diagnosing diseases includes any of the following:
  • the products for treating and/or diagnosing diseases target cells expressing VEGFA, and the cells can be cardiomyocytes, proximal renal tubule cells, hepatocytes, vascular endothelial cells, granulosa cells, specialized epithelial cells, interstitial cells, Mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, Vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.
  • the product may be a kit, medicine, chip, antibody-drug conjugate, etc.
  • the disease is a disease related to the VEGFA signaling pathway. Further preferred examples include tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vasculopathy), and the like.
  • a tenth aspect of the present invention provides an antibody-drug conjugate (ADC), which contains the anti-VEGFA fusion protein of the present invention covalently bound to a drug.
  • ADC antibody-drug conjugate
  • An eleventh aspect of the present invention provides a method for detecting VEGFA.
  • the detection method includes contacting the sample to be tested with the above-mentioned anti-VEGFA fusion protein, and then detecting the content of the complex formed by VEGFA and the anti-VEGFA fusion protein.
  • the detection method is to detect the presence or content of VEGFA.
  • the presence means presence or absence, and the content can be expression amount or protein concentration, etc.
  • a twelfth aspect of the present invention provides a method for diagnosing diseases.
  • the method includes taking a sample and combining the sample with the anti-VEGFA fusion protein, the above chimeric antigen receptor, the above nucleic acid, and the above vector. , the above-mentioned cells, the above-mentioned immune cells or the above-mentioned products for treating and/or diagnosing diseases are contacted, and the content of the complex formed by VEGFA and anti-VEGFA fusion protein is detected.
  • the disease is a disease related to the VEGFA signaling pathway. Further preferred examples include tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vasculopathy), and the like.
  • the thirteenth aspect of the present invention provides an anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cells, and the above-mentioned immune cells in the preparation, treatment and/or prevention of VEGFA-related Application in products for diseases, or application in the preparation of products that block VEGFA-mediated vascular endothelial cell proliferation or inhibit angiogenesis, or application in the preparation of antibody drug conjugates or antibody diagnostic kits or tracers applications in.
  • the fourteenth aspect of the present invention provides a method for treating and/or preventing diseases, the method comprising applying to an individual the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, and the above-mentioned nucleic acid. vector, the above-mentioned cells, the above-mentioned immune cells or the above-mentioned products for treating and/or diagnosing diseases.
  • the disease is a disease related to the VEGFA signaling pathway. Further preferred ones include tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular diseases), and the like.
  • the fifteenth aspect of the present invention provides a method for blocking VEGFA-mediated proliferation of vascular endothelial cells or inhibiting angiogenesis.
  • the method includes combining vascular endothelial cells with the above-mentioned anti-VEGFA fusion protein and the above-mentioned chimeric Contact with the antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell, the above-mentioned immune cell or the above-mentioned product for treating and/or diagnosing diseases.
  • the method includes diluting the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cells, the above-mentioned immune cells or the above-mentioned immune cells with a culture medium containing vascular endothelial cells.
  • the product for treating and/or diagnosing disease is then incubated with an antigen (e.g. VEGFA165).
  • an antigen e.g. VEGFA165
  • the step of resuspending the vascular endothelial cells in culture medium is also included.
  • the resuspended vascular endothelial cells, antigen and the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cells, the above-mentioned immune cells or the above-mentioned treatment and/or Or products for diagnosing diseases are cultured on culture plates.
  • detection is performed after culture.
  • the incubation time is 0.5-5h, preferably 1-3h, such as 0.5, 1, 2, 3, 4, 5h.
  • the incubation temperature is room temperature-45°C, preferably 30-40°C, such as 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc.
  • the culture temperature is room temperature-45°C, preferably 30-40°C, such as 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc.
  • the culture is performed in an incubator with 5% CO2 .
  • the culture time is 1-5 days, preferably 2-4 days, such as 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, 5 days, etc.
  • the detection is to detect the number of viable vascular endothelial cells.
  • the sixteenth aspect of the present invention provides a method for treating and/or preventing diseases.
  • the method includes using the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, The above-mentioned cells, the above-mentioned immune cells or the above-mentioned products for treating and/or diagnosing diseases are in contact with the target cells.
  • the disease is a disease related to the VEGFA signaling pathway. Further preferred ones include tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular diseases), and the like.
  • the target cells are selected from VEGFA-expressing cells, such as cardiomyocytes, proximal renal tubule cells, hepatocytes, vascular endothelial cells, granulosa cells, specialized epithelial cells, mesenchymal cells, macrophages, Platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial Tube and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.
  • VEGFA-expressing cells such as cardiomyocytes, proximal renal tubule cells, hepatocytes, vascular endothelial cells, granulosa cells, specialized epithelial cells, mesenchymal cells, macrophages, Platelets, dendritic cells, activated T cells, retinal pigment epithelial cells,
  • the seventeenth aspect of the present invention provides the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cells, the above-mentioned immune cells, or the above-mentioned treatment and/or The application of disease diagnostic products in blocking the VEGFA signaling pathway.
  • the applications include:
  • angiogenesis such as fundus vascular diseases
  • AMD age-related macular degeneration
  • DME diabetic macular edema
  • Diabetic retinopathy central retinal vein occlusion
  • pathological myopia neovascular glaucoma, etc.
  • ophthalmic diseases involving angiogenesis such as fundus vascular diseases
  • fundus vascular diseases preferably age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retina lesions, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc.; and/or,
  • the anti-VEGFA fusion protein of the present invention has high affinity with VEGFA, can effectively block the binding of VEGFA to the receptor VEGFR, block the VEGFA signaling pathway, and effectively block pathological processes such as vascular endothelial cell proliferation and angiogenesis caused by VEGFA. Can potentially be used in the treatment of VEGFA-related diseases.
  • the "drugs" of the present invention can be used to treat humans or non-human animals, such as non-human mammals.
  • the medicine may contain pharmaceutically acceptable carriers, excipients or salts commonly found in the art.
  • the drugs may be administered via any suitable route of administration, such as gastrointestinal administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorganal, intranasal, Intraocular, instillation, intracerebral, intrathecal, transdermal, rectal, etc.) routes.
  • gastrointestinal administration e.g., oral
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorganal, intranasal, Intraocular, instillation, intracerebral, intrathecal, transdermal, rectal, etc.
  • the medicine can be in any suitable dosage form, such as gastrointestinal dosage form or parenteral dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, Syrup, liquid, emulsion, microemulsion, suspension, injection, spray, aerosol, powder mist, lotion, ointment, plaster, paste, patch, eye drops, nose drops, Sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc.
  • Various dosage forms of the medicine can be prepared according to conventional production methods in the pharmaceutical field.
  • the medicine may contain a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the anti-VEGFA fusion protein, the Nucleic acid, the vector, the cell, the immune cell, etc.
  • 0.01-99.5% specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%
  • the drug can be prepared with a protein concentration of 1-300mg/mL (such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 ,85,90,95,100,105,110,115,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290 , 300mg/mL) reagent.
  • 1-300mg/mL such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 ,85,90,95,100,105,110,115,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290 , 300mg/mL
  • the single dose of the drug can be 0.1-1000mg, such as 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80 , 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000mg.
  • “Pharmaceutically acceptable” as used herein means neither significantly stimulating the organism nor inhibiting the biological activity and properties of the active substance of the administered product.
  • the "...method” described in the present invention can be for the purpose of diagnosis and treatment of diseases, or it can also be for the purpose of diagnosis and treatment of non-diseases.
  • the "antigen-binding fragment" described in the present invention is a part of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody can specifically bind to the epitope on the target molecule of the antibody. It includes, for example, Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2 and variants of these fragments.
  • Fab is a monovalent fragment composed of VL, VH, CL and CH1 domains.
  • F(ab')2 a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region.
  • Fd Fd fragment consisting of VH and CH1 domains.
  • Fv Fv fragment consisting of the VL and VH domains of a single arm of an antibody.
  • Fab' is a Fab fragment with one or more cysteine residues at the C-terminus of the CH1 domain.
  • Fab'-SH is a Fab' with at least one free thiol group on the cysteine residue of the constant domain.
  • VH represents the heavy chain variable region
  • VL represents the light chain variable region
  • CL represents the light chain
  • CH1 is the CH1 that constitutes the heavy chain constant region.
  • the "Fc" region of the present invention contains two heavy chain fragments comprising the CH2 and CH3 domains of the antibody.
  • the two heavy chain segments form a dimer by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.
  • humanized antibody used in the present invention refers to the framework region and/or constant region part (such as CH region) of the antibody or all of the antibody being encoded by human antibody genes.
  • the CDR regions of the antibody are not engineered.
  • Treatment as used herein means to slow, interrupt, arrest, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily Involves the complete elimination of all disease-related signs, symptoms, conditions, or disorders.
  • Prevention as used in the present invention means a method implemented to prevent or delay the occurrence of a disease, illness or symptom in the body.
  • the "individual" mentioned in the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc.
  • the non-human mammals include but are not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, mink, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters , gerbils, chinchillas, squirrels) or monkeys, etc.
  • the word “comprising” or “includes” used in the present invention is an open-ended method.
  • the protein or nucleic acid may be composed of the sequence, or may be at one end of the protein or nucleic acid. Or it can have additional amino acids or nucleotides at both ends, but still have the same or similar activity as the original sequence.
  • a "tumor” as used herein may be any undesirable cell proliferation (or any disease manifesting itself as undesirable cell proliferation), neoplasia, or a predisposition or increased risk of undesirable cell proliferation, neoplasia, or neoplasia. It can be benign or malignant, primary or secondary (metastatic).
  • a neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, Endometrium, epithelial cells (e.g.
  • renal epithelial cells gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum , mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid , tongue, tonsils, trachea, uterus, vulva, white blood cells.
  • the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (such as glioma), intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, Skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma , medulloblastoma, meningioma, neurofibroma (such as neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myelogenous leukemia, acute Myeloid leuk
  • the "homology" mentioned in the present invention refers to the aspect of using protein sequences or nucleotide sequences. Those skilled in the art can adjust the sequences according to actual work needs, so that the used sequences are compared with the sequences obtained by the existing technology.
  • Figure 1 Stained gel image after purification of bivalent Nanobody (linker is 9GS).
  • Figure 2 ELISA test results of bivalent Nanobody (linker 9GS) binding to VEGFA.
  • FIG. 3 ELISA test results of bivalent Nanobody (linker is 9GS) competing for VEGFR2.
  • FIG. 4 Comparison of VEGFA-binding activities of bivalent Nanobodies and monovalent Nanobodies.
  • FIG. 5 Comparison of the activities of bivalent Nanobodies and monovalent Nanobodies competing for VEGFR2.
  • Figure 6 Stained gel image after purification of heterologous bivalent Nanobodies.
  • Figure 7 ELISA test of heterologous bivalent Nanobodies binding to VEGFA.
  • FIG. 8 ELISA test of heterologous bivalent Nanobodies competing for VEGFR2.
  • Figure 9 Stained gel after purification of bivalent Nanobodies with different linkers.
  • Figure 10 Test of VEGFA-binding activity of bivalent Nanobodies connected in series with different linkers.
  • FIG. 11 ELISA test of bivalent Nanobodies competing for VEGFR2.
  • Figure 12 Stained gel image of bivalent V1 with different linkers placed at 4°C for 30 days.
  • Figure 13 Absorbance of bivalent V1 with different linkers after being placed at 4°C for 30 days.
  • Figure 14 Stained gel image of bivalent V1 with different linkers placed at 40°C for 7 days.
  • Figure 15 The absorbance of bivalent V1 with different linkers after being placed at 40°C for 7 days and then at room temperature for 23 days.
  • Figure 16 Stained gel image after purification of bivalent humanized Nanobodies.
  • Figure 17 ELISA test of bivalent humanized Nanobodies binding to VEGFA.
  • Figure 18 Stained gel image after purification of monovalent Nanobody-Fc fusion protein.
  • Figure 19 ELISA test of monovalent Nanobody-Fc fusion protein or tandem bivalent Nanobody binding to VEGFA.
  • Figure 20 Binding dissociation curve (SPR) of monovalent Nanobody-Fc fusion protein (V1-SA1-Fc-m1) and VEGFA165.
  • Figure 21 Stained gel image after purification of bivalent Nanobody-Fc fusion protein.
  • Figure 22 ELISA test of bivalent Nanobody-Fc fusion protein binding to VEGFA.
  • Figure 23 Binding dissociation curve (SPR) of bivalent Nanobody-Fc fusion protein and VEGFA165.
  • Figure 24 Binding dissociation curve (SPR) of bivalent Nanobody-Fc fusion protein and Aflibercept with VEGFA165.
  • Figure 25 Activity test of bivalent Nanobody-Fc fusion protein 2V1-SA1-3GS-Fc binding to VEGFA of different species.
  • Figure 26 2V1-SA1-3GS-Fc and Aflibercept inhibit VEGFA-stimulated HUVEC proliferation.
  • Nanobodies binding to VEGFA In order to improve the efficiency of Nanobodies binding to VEGFA and improve the efficiency of Nanobodies in blocking the VEGFA/VEGFR interaction, two Nanobodies with the same sequence were fused in series to construct homologous bivalent Nanobodies, and biological activity studies were carried out. .
  • linker 9GS (linker) rich in glycine and serine (GS) with flexible spatial structure (amino acid sequence: GGGGSGGGS (SEQ ID NO: 78)) was selected to connect two Nanobodies with the same sequence in series.
  • the number of each Nanobody The coding gene was amplified from the expression plasmid of a monovalent Nanobody by PCR and constructed into the pVRC8400 expression vector.
  • the N-terminal of the first Nanobody was added with the nucleotide sequence of the secreted peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 151).
  • the nucleotide sequence encoding the linker 9GS is added to the C-terminus, followed by the nucleotide sequence encoding the second Nanobody, and the linker GGGGS (SEQ ID NO: 152) is sequentially added to the C-terminus of the second Nanobody. ) and the nucleotide sequence of the tag 6 ⁇ His.
  • the two nanobody nucleotide fragments were homologously recombined with the vector plasmid using a seamless cloning kit (purchased from: Beyotime, Cat. No.: D7010M) to form the correct expression plasmid.
  • the coding gene sequence of the positive control antibody BI-VEGF ab was synthesized (the antibody is a nanobody against VEGFA, and the antibody amino acid sequence comes from SEQ ID NO: 57 in patent US 9527925B2).
  • the C of the control antibody BI-VEGF ab was Add label 6 ⁇ His to the end.
  • the sequence names and numbers of the constructed exemplary bivalent Nanobodies are shown in Table 3.
  • the ELISA method was used to detect the VEGFA-binding activity of the purified bivalent Nanobody.
  • VEGFA antigen human VEGFA165
  • ELISA coating buffer a final concentration of 0.3 ⁇ g/mL, and add it to the enzyme plate. 100 ⁇ L/well, coated overnight at 4°C. After blocking with 5% skimmed milk powder, gradient dilutions of each bivalent Nanobody (0.001, 0.01, 0.1, 1, 3, 10, 100 nM) were added.
  • the solution used for antibody dilution is PBS (pH 7.4) containing 0.1% Tween-20, that is, PBST.
  • the EC50 of bivalent Nanobodies binding to VEGFA is shown in Table 4. It can be seen from the table that all nine candidate antibodies have high VEGFA binding activity.
  • VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from: Yiqiao Shenzhou, Cat.
  • VEGFR2-competing activity IC50 value
  • Table 5 The comparison of the VEGFR2-competing activity (IC50 value) of bivalent Nanobodies is shown in Table 5. It can be seen that the bivalent Nanobodies have strong competitive VEGFR2 activity, and the competitive activity is stronger than the positive control monovalent Nanobody BI-VEGF ab.
  • ELISA was used to compare the VEGFA-binding activities of bivalent Nanobodies (2V1-9GS, 2V15-9GS, 2V31-9GS, 2V43-9GS) and corresponding monovalent Nanobodies.
  • the experimental method is the same as "2. Detection of bivalent Nanobody-binding VEGFA activity" in Example 1.
  • the signal changes of monovalent and bivalent Nanobodies binding to VEGFA as a function of antibody concentration are shown in Figure 4.
  • the EC50 values of monovalent and bivalent Nanobodies binding to VEGFA are shown in Table 6. From the table, it can be seen that the activity of bivalent Nanobodies in binding VEGFA is stronger than that of monovalent Nanobodies.
  • SPR surface plasmon resonance
  • Biacore 8K instrument (Cytiva) was used, with the antigen VEGFA165 (unlabeled, purchased from: Yiqiao Shenzhou, product number: HPLC-10008-HNAH) as the ligand, and coupled to the CM5 chip through amino coupling reagents (EDC and NHS). , the coupling amount is about 400RU.
  • the mobile phase buffer was HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v Tween 20).
  • the analytes are monovalent Nanobodies and bivalent Nanobodies, and their dilution concentrations are: 20nM, 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, 0.3125nM.
  • the binding time of antigen and antibody was 240 seconds
  • the dissociation time was 1500 seconds
  • the flow rate was 30 ⁇ L/min.
  • the chip was regenerated with 100mM hydrochloric acid.
  • the binding and dissociation curves of the antibody and VEGFA165 were fitted using the "1:1 binding" model.
  • the obtained binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) are shown in Table 7.
  • the experimental method is the same as "3. Activity detection of bivalent Nanobody competition for VEGFR2" in Example 1.
  • the ELISA results of bivalent Nanobody 2V15-9GS and monovalent Nanobody V15 competing for VEGFR2 are shown in Figure 5.
  • Monovalent and bivalent Nanobodies compete for the activity of VEGFR2 (IC50 and maximum inhibition rate), see Table 8.
  • Monovalent Nanobodies with different amino acid sequences are connected in series through the linker 9GS to construct heterologous bivalent Nanobodies.
  • Nanobodies V1, V15, V30, and V40 with higher melting temperatures (Tm) were selected to construct heterologous bivalent Nanobodies.
  • Tm melting temperatures
  • a total of 6 heterologous bivalent Nanobodies were expressed. The names, sequence names and numbers are shown in Table 10.
  • the plasmid construction, expression and purification methods are the same as "1.
  • Bivalent Design of Nanobodies in Example 1.
  • the stained gel image of the antibody after Ni bead affinity purification is shown in Figure 6.
  • the molecular weight of the target protein is approximately 30kDa.
  • VEGFA-binding activity of the purified heterologous bivalent Nanobodies was evaluated.
  • the experimental method was the same as "2. Detection of VEGFA-binding activity of bivalent Nanobodies" in Example 1.
  • the ELISA results of heterologous bivalent Nanobodies binding to VEGFA are shown in Figure 7.
  • the EC50 of heterologous bivalent Nanobodies binding to VEGFA is shown in Table 11.
  • the results show that the six heterologous bivalent Nanobodies have similar activities in binding VEGFA (EC50 is 0.3-0.4nM), which is similar to the activity of homologous bivalent Nanobodies (two Nanobodies with the same sequence in series) (EC50 is about 0.24 nM, see Table 4 in Example 1).
  • the ELISA results of heterologous bivalent Nanobodies competing for VEGFR2 are shown in Figure 8.
  • the IC50 and inhibition rate of competition ELISA are shown in Table 11. The results show that heterologous bivalent Nanobodies have strong competition with VEGFR2 for binding to VEGFA.
  • the linkers connecting the two Nanobodies are mostly flexible 9GS, and the spatial orientations of the two Nanobodies are relatively independent and do not interfere with each other.
  • linkers with different properties may have an impact on the spatial conformation of the antibody and the antigen-binding efficiency. Therefore, different linkers were prepared and evaluated on the activity and stability of bivalent nanobodies. Influence.
  • linker properties on the biological activity and physical and chemical stability of bivalent nanobodies, 16 types of linkers were designed, including different linker lengths, possible secondary structures, hydrophilicity, etc.
  • the amino acid sequence of the linker is shown in Table 12.
  • Nanobodies V1 and V15 as model antibodies, bivalent Nanobodies with different linkers were constructed. Their expression vector construction, transient transformation and purification methods were the same as "1. Bivalent Design of Nanobodies" in Example 1. The sequence names and numbers of bivalent Nanobodies with different linkers are shown in Table 13.
  • the stained gel image of some antibodies after affinity purification with Ni beads is shown in Figure 9.
  • the molecular weight of the target protein is approximately 30kDa.
  • VEGFA-binding activity of the purified bivalent Nanobody was evaluated.
  • the experimental method was the same as "2. Detection of the VEGFA-binding activity of the bivalent Nanobody" in Example 1.
  • the ELISA results of some bivalent Nanobodies binding to VEGFA are shown in Figure 10, and the EC50 of binding to VEGFA is shown in Table 14.
  • bivalent Nanobody V1 For bivalent Nanobody V1, different linkers have little impact on the EC50 of bivalent V1 (EC50 is 0.2 ⁇ 0.3nM). Moreover, the activity of bivalent V1 without linker (2V1-NL) is different from that of bivalent V1 with linker added. Bivalent V1 activity is equivalent. For bivalent V15s connected in series with different linkers, their activities are not much different.
  • the ELISA results of bivalent Nanobodies competing for VEGFR2 are shown in Figure 11.
  • the IC50 and inhibition rate of competition ELISA are shown in Table 15. The results showed that for V15, the competitive activity of bivalent V15 (2V15-20S) formed by connecting 20S linkers in series was slightly worse than that of bivalent V15 formed by connecting other linkers in series.
  • the bivalent Nanobodies containing linkers 3GS and 9GS have better stability performance.
  • Nanobodies In the previous example, the original unhumanized antibody was bivalent and compared with linkers. Next, the bivalent human source will be constructed with linkers 3GS and 9GS or without linker (NL). Nanobodies.
  • Antibody humanization is to first compare the homology between the candidate antibody sequence and the human antibody sequence (through the database IGBLAST and IMGT), and then graft the CDR region of the candidate antibody into the human antibody framework region with high homology to form a complete
  • individual amino acids in the framework region of the human antibody need to be back mutated into the sequence corresponding to the position of the candidate antibody.
  • Bivalent humanized Nanobodies with different linkers were constructed using humanized antibodies of V1, V13, V15, V30, V40, and V43 as components. The amino acid sequence names and numbers of the constructed bivalent humanized Nanobodies are shown in Table 16.
  • the plasmid construction, expression and purification methods of the above bivalent Nanobodies are the same as "1. Bivalent Design of Nanobodies" in Example 1.
  • the stained gel image after purification of some proteins is shown in Figure 16.
  • the molecular weight of the target protein is approximately 30kDa.
  • the VEGFA-binding activity of the purified bivalent humanized Nanobody was evaluated.
  • the experimental method was the same as "2. Detection of VEGFA-binding activity of the bivalent Nanobody" in Example 1.
  • the ELISA results of bivalent humanized Nanobodies binding to VEGFA are shown in Figure 17.
  • the EC50 of ELISA is shown in Table 17. The results show that the vast majority of bivalent humanized Nanobodies can bind VEGFA efficiently.
  • dimerization domains or fragments can also be used, such as Fc of IgG1 antibodies (SEQ ID NO: 134), PPCP-Fc (SEQ ID NO: 158, that is, the remaining amino acid sequence after removing DKTHTC in SEQ ID NO: 134) or IgG1 Fc mutant Fc-m1 (SEQ ID NO: 135), connecting two Nanobodies in the same molecule.
  • IgG1 Fc amino acid sequence is:
  • amino acid sequence of PPCP-Fc is:
  • the mutant Fc-m1 of IgG1 Fc its amino acid sequence is:
  • the Nanobody and Fc were integrated into the vector pCDNA3.1(+).
  • the enzyme cutting sites used were: NheI and XbaI.
  • the secreted peptide of the fusion protein is: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 155), and then the target protein sequence is fused.
  • V1-SA1-Fc-m1 the linker amino acid sequence between V1-SA1 and Fc-m1 is: EPKSA (SEQ ID NO: 156).
  • the expression plasmid was constructed using the above method, but the C-terminus of Fc did not contain additional amino acids, that is, the fusion protein V1-SA1-Fc-m1 contained an Fc tag but no histidine tag.
  • Protein A beads were used for affinity purification. During protein elution, 0.1M citric acid (pH3.0) was used to elute the target protein and neutralize it quickly to obtain the target protein. protein.
  • the VEGFA binding activity test was carried out on the purified monovalent Nanobody-Fc fusion protein.
  • the ELISA experimental method was the same as "2. Activity detection of bivalent Nanobody binding to VEGFA" in Example 1.
  • the ELISA results are shown in Figure 19, and the EC50 is shown in Table 19.
  • V1-SA1-Fc-m1 monovalent Nanobody-Fc fusion protein
  • VEGFA VEGFA
  • Biacore 8K instrument (Cytiva) was used, with V1-SA1-Fc-m1 as the ligand, captured on the Protein A chip.
  • mobile phase buffer HBS-EP 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v Tween 20
  • Dilute V1-SA1-Fc-m1 Dilute V1-SA1-Fc-m1 to a protein concentration of 2 ⁇ g/mL, and its coupling amount is approximately 590RU.
  • the analyte was the antigen VEGFA165 (unlabeled, purchased from: GenScript, product number: Z03073), and its dilution concentrations were: 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78125nM, 0.390625nM .
  • the binding time of the fusion protein and the antigen VEGFA165 was 180 seconds, the dissociation time was 900 seconds, and the flow rate was 30 ⁇ L/min. After each association-dissociation cycle, the chip was regenerated with 10mM glycine hydrochloride (pH 1.5).
  • Example 7 the monovalent Nanobody-Fc fusion protein has high affinity with VEGFA.
  • the results of Example 2 show that the bivalent Nanobody binding activity to VEGFA is higher than that of the monovalent Nanobody. Therefore, a bivalent Nanobody-Fc fusion protein was constructed. , that is, the bivalent Nanobody in Table 16 is fused and expressed with IgG1 Fc (SEQ ID NO: 134).
  • the amino acid sequence names and numbers of some bivalent Nanobody-Fc fusion proteins are shown in Table 21.
  • An expression plasmid was constructed for the above bivalent Nanobody-Fc fusion protein, transiently transfected and purified.
  • the specific method is: based on the amino acid sequence of each fusion protein in the table, use gene synthesis or PCR methods to construct expression plasmids.
  • PCR method two identical Nanobody genes and Fc were integrated into the vector pCDNA3.1(+), and the enzyme cutting sites used were: NheI and XbaI.
  • PEI transfection reagent polyethylenimine
  • citric acid pH3.0
  • the stained gel image of an exemplary purified bivalent Nanobody-Fc fusion protein is shown in Figure 21. Under reducing SDS-PAGE conditions, the protein molecular weight is approximately 53 kDa.
  • ELISA was used to detect the binding activity of purified bivalent Nanobody-Fc fusion protein to VEGFA.
  • the ELISA method is: dilute human VEGFA165 (unlabeled, purchased from Yiqiao China, product number: HPLC-10008-HNAH) with ELISA coating buffer to a final concentration of 0.3ug/mL, coat overnight at 4°C, and After blocking with 5% skimmed milk powder (prepared with PBST), add different concentrations of fusion protein (the highest concentration is 100nM, the lowest concentration is 0.0001nM, 10 times concentration gradient, configured with PBST), incubate at 37°C for 1 hour, and wash the enzyme with PBST After standardizing the plate three times, add the detection antibody that recognizes the Fc tag: HRP-labeled Goat Anti-Human IgG antibody (purchased from: Abbkine, Cat.
  • VEGFA165 without label, purchased from: Yiqiao Shenzhou, product number: HPLC-10008-HNAH was coupled to the CM5 chip (due to the affinity of these proteins with VEGFA165 Higher, the dissociation is very slow, so reduce the coupling amount of VEGFA165).
  • Each fusion protein was diluted with mobile phase buffer HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v/v Tween 20).
  • the dilution concentrations are: 0.0244nM, 0.0977nM, 0.3906nM, 1.5625 nM and 6.25nM.
  • the binding time of antigen and antibody was 120 seconds
  • the dissociation time was 780 seconds
  • the flow rate was 40 ⁇ L/min.
  • the chip was regenerated with 100mM hydrochloric acid.
  • the binding and dissociation curve of the fusion protein and VEGFA165 was fitted using the "1:1 binding" model to obtain the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD).
  • the antigen human VEGFA165 from different reagent suppliers was replaced, and the KD of the fusion protein and VEGFA165 was repeatedly tested.
  • the KD values of the fusion protein (2V1-SA1-3GS-Fc) and the positive control drugs Aflibercept and VEGFA165 were measured.
  • Biacore 8K instrument (Cytiva) was used, using the "low fixed volume” mode, to couple VEGFA165 (unlabeled, purchased from: GenScript, product number: Z03073) on the CM5 chip, using mobile phase buffer HBS. -EP diluted protein.
  • the concentrations of 2V1-SA1-3GS-Fc dilution are: 0.091nM, 0.1828nM, 0.3656nM, 0.73125nM, 1.4625nM, 2.925nM, 5.85nM.
  • the concentrations of Aflibercept diluent are: 0.3125nM, 0.625nM, 1.25nM, 2.5nM, 5nM, 10nM, 20nM.
  • the binding time of antigen and antibody was 150 seconds
  • the dissociation time was 1000 seconds
  • the flow rate was 30 ⁇ L/min.
  • the binding and dissociation curve of the protein to be tested and VEGFA165 was fitted using the "1:1 binding" model to obtain the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD), as shown in Table 24.
  • the binding and dissociation curves (SPR) of bivalent Nanobody-Fc fusion protein (2V1-SA1-3GS-Fc) and Aflibercept with VEGFA165 are shown in Figure 24.
  • the fusion protein 2V1-SA1-3GS-Fc has extremely high affinity for human VEGFA165 from different reagent suppliers, the KD values are all at the pM level, and the KD values are significantly smaller than Aflibercept (about 1/12 of Aflibercept), which indicates that the affinity of the fusion protein 2V1-SA1-3GS-Fc to VEGFA165 is higher than that of Aflibercept.
  • the SPR test was carried out using a similar method (conjugated antigen VEGFA165) to compare the KD values of monovalent Nanobodies and bivalent Nanobodies with VEGFA (Table 7) and the KD values of the above bivalent Nanobody-Fc fusion protein with VEGFA. (Table 23, Table 24), the KD values of antibodies with different construction forms and VEGFA are summarized in Table 25.
  • bivalent Nanobodies and bivalent Nanobodies-Fc fusion proteins have 22.5 times and 105.5 times higher antigen affinity than monovalent Nanobodies. Therefore, as the number of antibodies or antigen-binding fragments in a protein molecule increases, the affinity of the protein molecule to the antigen will increase significantly.
  • the ELISA method was used to detect the cross-binding activity of 2V1-SA1-3GS-Fc and VEGFA proteins of different species.
  • mouse VEGFA164 was purchased from Yiqiao China with the product number 50159-MNAB
  • rat VEGFA164 was purchased from Yiqiao China with the product number 80006-RNAB.
  • Rabbit VEGFA121 is expressed in transiently transfected 293F cells and purified by Ni beads. After the nucleotide sequence of rabbit VEGFA121 was synthesized, it was cloned into the vector pCDNA3.1(+). The amino acid sequence of rabbit VEGFA121 is:
  • the N-terminal of the protein uses its own secreted peptide, and the C-terminal of the protein is sequentially added with a flexible linker (GGGGS (SEQ ID NO: 152)) and a 6-histidine tag (HHHHHH (SEQ ID NO: 154)).
  • GGGGS flexible linker
  • HHHHHH 6-histidine tag
  • the ELISA experimental method used is similar to "2. Activity detection of bivalent Nanobody binding to VEGFA" in Example 1. Different concentrations of 2V1-SA1-3GS-Fc are added to ELISA plates coated with different antigens and incubated for 1 hour. , add the detection antibody that recognizes the Fc tag: HRP-labeled Goat Anti-Human IgG antibody (purchased from: Abbkine, product number: A21050), incubate at room temperature for 45 minutes, wash the plate, develop TMB color and terminate the reaction, use a microplate reader Read OD450.
  • HRP-labeled Goat Anti-Human IgG antibody purchased from: Abbkine, product number: A21050
  • 2V1-SA1-3GS-Fc only has high binding activity with human VEGFA165, can weakly bind to rabbit VEGFA121, and cannot bind to mouse and rat VEGFA164.
  • the primary cell HUVEC proliferation inhibition experiment was used to verify the efficacy of the fusion protein 2V1-SA1-3GS-Fc at the cellular level, that is, blocking the activity of the VEGFA signaling pathway.
  • the positive control in the experiment was the marketed drug Aflibercept, and the negative control was the HIV virus-conjugated human IgG1 antibody (hlgG1control).
  • CellTiter-Glo luminescent cell viability assay was used to detect the number of viable HUVEC cells.
  • the experimental method is: normal passage of HUVEC (purchased from ScienCell, item number 8000) uses complete culture medium: ECM+5% FBS+1% ECGS (Purchased from ScienCell, the product numbers of the three reagents are 1001, 0010, and 5352 respectively).
  • ECM+1% FBS+0.05% ECGS dilute the protein to be tested with low serum medium (ECM+1% FBS+0.05% ECGS) to the required concentration: 1000nM to 0.0128nM, 5-fold concentration gradient (the final antibody concentration when incubating HUVEC cells is 250nM to 0.0032nM).
  • ECM+1% FBS+0.05% ECGS low serum medium
  • the required concentration 1000nM to 0.0128nM
  • 5-fold concentration gradient the final antibody concentration when incubating HUVEC cells is 250nM to 0.0032nM.
  • IC50 of fusion protein 2V1-SA1-3GS-Fc in inhibiting HUVEC proliferation is about half of the positive control Aflibercept, indicating that fusion protein 2V1-SA1-3GS-Fc can effectively block the downstream signaling pathway activated by VEGFA at the cellular level. , inhibits VEGFA-stimulated HUVEC proliferation, and the activity of the fusion protein 2V1-SA1-3GS-Fc at the cellular level is stronger than the positive control Aflibercept.

Abstract

提供了一种抗VEGFA融合蛋白及其制备方法和应用，该抗VEGFA融合蛋白与VEGFA具有高亲和力，能有效阻断VEGFA与受体VEGFR的结合，阻断VEGFA信号通路，有效阻断VEGFA导致的血管内皮细胞增殖以及血管新生等病理过程，能潜在应用于与VEGFA相关的疾病的治疗中。

Description

一种抗VEGFA融合蛋白及其制备方法和应用 技术领域
本发明涉及生物医药技术领域，具体涉及一种抗VEGFA融合蛋白及其制备方法和应用。
背景技术
血管异常增生与多种疾病的发生发展密切相关，如多种实体瘤和眼底血管疾病。研究表明，血管内皮生长因子A(VEGFA)是促进血管内皮细胞增殖和血管新生的重要诱因。因此，抑制VEGFA信号通路可以用于治疗血管异常增生相关的疾病。如湿性年龄相关黄斑变性(Age-related Macular Degeneration，AMD)，它是导致老年人群失明的最主要疾病，它是由于眼底VEGFA的过量表达，导致脉络膜血管新生，破坏视网膜正常功能。
目前，在湿性AMD的治疗中，已上市的抗VEGFA蛋白药物主要有4种，包括雷珠单抗(Ranibizumab)、阿柏西普(Aflibercept)、康柏西普(Conbercept)和Brolucizumab。此外，贝伐珠单抗(Bevacizumab)也被超标签(off-label)用于治疗湿性AMD。这些抗VEGF蛋白药物的给药途径是局部注射，如玻璃体腔注射，给药体积不超过100μL。Brolucizumab的最长给药间隔为3个月，其余药物的给药间隔是1～2个月注射一次。如在眼部注射，频繁的眼内注射会增加注射带来的眼压升高、眼内炎症、视网膜脱落等风险，因此临床上迫切需要长效抗VEGFA治疗药物，减少患者的注射次数。
提高药物和靶点的亲和力，即降低KD，可以降低药物的最低起效浓度。在湿性AMD治疗药物中，雷珠单抗(Ranibizumab)与VEGFA的亲和力较强(KD＝46pM)，而阿柏西普(Aflibercept)和康柏西普(Conbercept)与VEGFA的亲和力比雷珠单抗强100倍(KD＝0.5pM)。
药物在玻璃体中的扩散速率直接影响药物的给药间隔，药物的扩散速率与其电荷、分子量、分子形状等性质相关。大分子量的药物扩散较慢，半衰期也较长。在荷兰带兔(Dutch belted rabbit)玻璃体中开展的药代动力学研究中，雷珠单抗(分子量：48kDa)的半衰期是2.81天，贝伐珠单抗的(分子量：149kDa)的半衰期是4.22天，而阿柏西普(分子量：115kDa)的半衰期是4.58天。
因此，开发高亲和力、长半衰期的抗VEGFA融合蛋白对于提升湿性AMD患者依从性具有重大意义。
单域抗体或纳米抗体是骆驼类动物体内天然缺失轻链的重链抗体(heavy-chain antibody)的可变区结构域，是具有完整抗原结合功能的稳定的最小抗体单元。单域抗体的分子量约为13kDa，热稳定性高且水溶性好，分子本身的性质具有较大潜力可以开发为较高浓度的蛋白制剂。
因此，本发明以纳米抗体为结合VEGFA的基本活性单元，并进行纳米抗体的二价化设计，以及融合Fc片段，制备出具有高亲和力的抗VEGFA融合蛋白。
发明内容
本发明的第一方面，提供了一种抗VEGFA融合蛋白，所述的抗VEGFA融合蛋白包括两个或两个以上抗VEGFA抗体或其抗原结合片段，以及Fc片段，所述的抗VEGFA抗体或其抗原结合片段包含重链可变区的CDR-H1、CDR-H2和CDR-H3；其中，
CDR-H1的氨基酸序列包含SYTMG(SEQ ID NO：1)或与SYTMG(SEQ ID NO：1)具有至少80％同一性的氨基酸序列；
CDR-H2的氨基酸序列包含AISKGGYKYX1X2VSLEA(SEQ ID NO：2)或与AISKGGYKYX1X2VSLEA(SEQ ID NO：2)具有至少80％同一性的氨基酸序列；
CDR-H3的氨基酸序列包含TRAYGSSRLX3LAX4TYEY(SEQ ID NO：3)或与 TRAYGSSRLX3LAX4TYEY(SEQ ID NO：3)具有至少80％同一性的氨基酸序列。
优选的，所述的抗VEGFA融合蛋白包括至少两个(例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20以上)抗VEGFA抗体或其抗原结合片段。
在本发明的一个具体实施方式中，所述的CDR-H1、CDR-H2和CDR-H3的氨基酸序列依次如SEQ ID NO：1、2、3所示。
优选的，所述的CDR-H1、CDR-H2、CDR-H3的氨基酸序列以从N端到C端的顺序排列，本申请中抗体的CDR区氨基酸的划分采用Kabat编号系统。
优选的，抗VEGFA抗体或其抗原结合片段与抗VEGFA抗体或其抗原结合片段之间，抗VEGFA抗体或其抗原结合片段与Fc片段之间直接连接或者间接连接。
优选的，所述的其他抗VEGFA抗体或其抗原结合片段之间、其他抗VEGFA抗体或其抗原结合片段与抗VEGFA抗体或其抗原结合片段之间、其他抗VEGFA抗体或其抗原结合片段与Fc片段之间、Fc片段与抗VEGFA抗体或其抗原结合片段之间直接连接或间接连接。
优选的，所述的直接或者间接连接包括直接或者间接连接在所述的抗VEGFA抗体或其抗原结合片段、其他抗VEGFA抗体或其抗原结合片段和/或Fc片段的N端、C端和/或内部残基上。
优选的，抗VEGFA融合蛋白中抗VEGFA抗体或其抗原结合片段的N端、C端和/或内部残基与其他抗VEGFA抗体或其抗原结合片段或Fc片段的N端、C端和/或内部残基相连。
在本发明的一个具体实施方式中，抗VEGFA融合蛋白中抗VEGFA抗体或其抗原结合片段的N端与其他抗VEGFA抗体或其抗原结合片段的N端或C端相连。
在本发明的一个具体实施方式中，抗VEGFA融合蛋白中抗VEGFA抗体或其抗原结合片段的C端与其他抗VEGFA抗体或其抗原结合片段的N端或C端相连。
在本发明的一个具体实施方式中，抗VEGFA融合蛋白中抗VEGFA抗体或其抗原结合片段的C端或其他抗VEGFA抗体或其抗原结合片段的C端与Fc片段的N端相连。
优选的，所述的间接连接为通过接头连接。
优选的，所述的接头选自连接肽、寡肽、寡肽聚合物、多肽、多肽聚合物、PEG、核酸、多糖、脂肪链、生物素、链霉亲和素或亲和素。优选连接肽可以为刚性接头、柔性接头或无意义氨基酸，进一步优选连接肽的氨基酸序列包括SEQ ID NO：77-91、152-153和156中的一种或两种以上的组合。
优选的，两个或两个以上抗VEGFA抗体或其抗原结合片段为完全相同序列的抗VEGFA抗体或其抗原结合片段、部分相同序列的抗VEGFA抗体或其抗原结合片段或完全不同序列的抗VEGFA抗体或其抗原结合片段。
优选的，所述的抗VEGFA融合蛋白还包含分泌肽。所述的分泌肽连接在抗VEGFA融合蛋白的N端。进一步优选的，所述的分泌肽的氨基酸序列可以为SEQ ID NO：151或155。
在本发明的一个具体实施方式中，所述的Fc片段包括SEQ ID NO：134、135或158。
在本发明的一个具体实施方式中，所述的抗VEGFA融合蛋白从N端到C端依次包括第一个抗VEGFA抗体或其抗原结合片段、连接肽、第二个抗VEGFA抗体或其抗原结合片段。
在本发明的一个具体实施方式中，所述的抗VEGFA融合蛋白从N端到C端依次包括分泌肽、第一个抗VEGFA抗体或其抗原结合片段、连接肽、第二个抗VEGFA抗体或其抗原结合片段。
在本发明的一个具体实施方式中，所述的抗VEGFA融合蛋白从N端到C端依次包括分泌肽、第一个抗VEGFA抗体或其抗原结合片段、连接肽、第二个抗VEGFA抗体或其抗原结合片段、Fc片段。
在本发明的一个具体实施方式中，所述的抗VEGFA融合蛋白从N端到C端依次包括分泌肽、第一个抗VEGFA抗体或其抗原结合片段、连接肽、第二个抗VEGFA抗体或其抗原结合片段、标签(例如6×His)。
在本发明的一个具体实施方式中，SEQ ID NO：2中的X1X2代表DS、DA、NT、DT、NA或NS；SEQ ID NO：3中的X3代表R或K，X4代表D、N、E或K。
优选的，所述的CDR-H1、CDR-H2和CDR-H3的氨基酸序列包含下列任一组(见表1)：
A)SEQ ID NO：1、4、9；
B)SEQ ID NO：1、5、9；
C)SEQ ID NO：1、6、10；
D)SEQ ID NO：1、4、11；
E)SEQ ID NO：1、7、12；
F)SEQ ID NO：1、6、11；
G)SEQ ID NO：1、4、10；
H)SEQ ID NO：1、5、12；
I)SEQ ID NO：1、4、12；
J)SEQ ID NO：1、8、12；
K)SEQ ID NO：1、33、34。
表1 CDR-H1、CDR-H2和CDR-H3的氨基酸序列
优选的，所述的抗体或其抗原结合片段为纳米抗体、嵌合抗体、Fab片段、Fab'片段、Fd片段、Fd'片段、Fv片段、dAb片段、分离的CDR区、F(ab')2片段、单结构域抗体、单链抗体分子或线性抗体。
优选的，所述的抗VEGFA抗体或其抗原结合片段的氨基酸序列包含SEQ ID NO：13-32、35-61中的任一氨基酸序列，或与SEQ ID NO：13-32、35-61中的任一氨基酸序列具有至少80％同一性，具体序列编号见表2。
表2抗体氨基酸序列


优选的，所述的抗VEGFA融合蛋白结合人或猴VEGFA蛋白，其中，所述的人VEGFA蛋白与猴VEGFA蛋白序列相同。
所述的抗VEGFA抗体或其抗原结合片段可以为人源化抗体或全人源抗体。
优选的，所述的抗VEGFA抗体或其抗原结合片段包括人源化序列，所述的人源化序列的改造位点位于非CDR区，进一步优选的，所述的人源化改造位点位于抗体的框架区和/或恒定区。
在本发明的一个具体实施方式中，所述的抗VEGFA融合蛋白的氨基酸序列包含SEQ ID NO：62-76、92-133、136-150和159中的任一氨基酸序列，或与SEQ ID NO：62-76、92-133、136-150和159中的任一氨基酸序列具有至少80％同一性。
本发明的第二方面，提供了一种嵌合抗原受体，所述的嵌合抗原受体的胞外结构域包含上述的抗VEGFA融合蛋白。
优选的，所述的嵌合抗原受体还包含现有技术中常规的任何跨膜区和/或胞内信号传导区。
本发明的第三方面，提供了一种编码上述的抗VEGFA融合蛋白或上述的嵌合抗原受体的核酸。
本发明的第四方面，提供了一种包含上述核酸的载体。
所述的载体能够在体内或体外或离体条件下表达。优选的，所述的载体为原核表达载体、病毒表达载体或真核表达载体。例如大肠杆菌系列载体、噬菌体等等。
本发明的第五方面，提供了一种包含上述载体或上述核酸的细胞。
所述的细胞可以为真核细胞或原核细胞。
真核细胞包括动植物细胞，例如T细胞、酵母细胞、HEK293细胞或CHO细胞等等。
原核细胞例如大肠杆菌等。
本发明的第六方面，提供了一种抗VEGFA融合蛋白的制备方法，所述的制备方法包括将上述的核酸导入宿主细胞，诱导其表达。
本发明的第七方面，提供了一种免疫细胞，所述的免疫细胞表达上述的抗VEGFA融合蛋白或上述的嵌合抗原受体。
优选的，所述的免疫细胞包括但不限于淋巴细胞(例如T细胞、B细胞、NK细胞)、树突状细胞、单核/巨噬细胞、粒细胞、肥大细胞。
优选的，免疫细胞为CAR-免疫细胞。
本发明的第八方面，提供了一种免疫细胞的构建方法，所述的构建方法包括将编码本发明所述嵌合抗原受体的核酸序列转染至免疫细胞中表达获得。
本发明的第九方面，提供了一种治疗和/或诊断疾病的产品，所述的治疗和/或诊断疾病的产品包含下列任一种：
A)上述的抗VEGFA融合蛋白；
B)上述的嵌合抗原受体；
C)上述的核酸；
D)上述的载体；
E)上述的细胞；或，
F)上述的免疫细胞。
所述的治疗和/或诊断疾病的产品靶向表达VEGFA的细胞，所述的细胞可以为心肌细胞、近端肾小管细胞、肝细胞、血管内皮细胞、颗粒细胞、特化的上皮细胞、间充质细胞、巨噬细胞、血小板、树突状细胞、活化的T细胞、视网膜色素上皮细胞、视网膜中的Muller细胞、星形胶质细胞、成骨细胞、支气管与肺泡上皮细胞、周细胞、血管平滑肌细胞、肌成纤维细胞、角质形成细胞、肾系膜细胞或肿瘤细胞等。
优选的，所述的产品可以是试剂盒或药物或芯片或抗体药物偶联物等。
所述的疾病为与VEGFA信号通路相关的疾病。进一步优选可以为肿瘤、血管异常增生、眼科涉及血管新生的疾病(例如眼底血管病)等等。
本发明的第十方面，提供了一种抗体药物偶联物(antibody-drug conjugate,ADC)，其包含与药物共价结合的本发明所述的抗VEGFA融合蛋白。
本发明的第十一方面，提供了一种VEGFA的检测方法，所述的检测方法包括将待检样品与上述的抗VEGFA融合蛋白接触，然后检测VEGFA与抗VEGFA融合蛋白形成的复合物含量。
所述的检测方法为检测VEGFA的存在或含量。其中，所述的存在表示有无，所述的含量可以为表达量或蛋白浓度等。
本发明的第十二方面，提供了一种诊断疾病的方法，所述的方法包括取样，将样品与所述的抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞或上述的治疗和/或诊断疾病的产品接触，检测VEGFA与抗VEGFA融合蛋白形成的复合物的含量。
所述的疾病为与VEGFA信号通路相关的疾病。进一步优选可以为肿瘤、血管异常增生、眼科涉及血管新生的疾病(例如眼底血管病)等等。
本发明的第十三方面，提供一种抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞在制备治疗和/或预防与VEGFA相关的疾病的产品中的应用，或者，在制备阻断VEGFA介导的血管内皮细胞增殖或抑制血管新生的产品中的应用，或者，在制备抗体药物偶联物或抗体诊断试剂盒或示踪剂中的应用。
本发明的第十四方面，提供了一种治疗和/或预防疾病的方法，所述的方法包括向个体施加上述的抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞或上述的治疗和/或诊断疾病的产品。
所述的疾病为与VEGFA信号通路相关的疾病。进一步优选可以为肿瘤、血管异常增生、眼科涉及血管新生的疾病(例如眼底血管疾病)等等。
本发明的第十五方面，提供了一种阻断VEGFA介导的血管内皮细胞增殖或抑制血管新生的方法，所述的方法包括将血管内皮细胞与上述的抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞或上述的治疗和/或诊断疾病的产品接触。
优选的，所述的方法包括用包含血管内皮细胞的培养基稀释上述的抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞或上述的治疗和/或诊断疾病的产品，然后与抗原(例如VEGFA165)孵育。
优选的，孵育后还包括用培养基重悬血管内皮细胞的步骤。
优选的，将重悬的血管内皮细胞、抗原以及上述的抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞或上述的治疗和/或诊断疾病的产品在培养板上培养。
优选的，培养后进行检测。
优选的，所述的孵育时间为0.5-5h，优选1-3h，例如0.5、1、2、3、4、5h。
优选的，所述的孵育温度为室温-45℃，优选30-40℃，例如25、30、35、36、37、38、39、40、45℃等。
优选的，所述的培养温度为室温-45℃，优选30-40℃，例如25、30、35、36、37、38、39、40、45℃等。优选的，所述的培养在5％CO2的培养箱中进行。
优选的，所述的培养时间为1-5天，优选2-4天，例如1、1.5、2、2.5、3、2.5、4、4.5、5天等。
优选的，所述的检测为检测活的血管内皮细胞细胞数。
本发明的第十六方面，提供了一种治疗和/或预防疾病的方法，所述的方法包括使上述的抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞或上述的治疗和/或诊断疾病的产品与靶细胞接触。
所述的疾病为与VEGFA信号通路相关的疾病。进一步优选可以为肿瘤、血管异常增生、眼科涉及血管新生的疾病(例如眼底血管疾病)等等。
优选的，所述的靶细胞选自表达VEGFA的细胞，例如心肌细胞、近端肾小管细胞、肝细胞、血管内皮细胞、颗粒细胞、特化的上皮细胞、间充质细胞、巨噬细胞、血小板、树突状细胞、活化的T细胞、视网膜色素上皮细胞、视网膜中的Muller细胞、星形胶质细胞、成骨细胞、支气 管与肺泡上皮细胞、周细胞、血管平滑肌细胞、肌成纤维细胞、角质形成细胞、肾系膜细胞或肿瘤细胞等等。
本发明的第十七方面，提供了一种上述的抗VEGFA融合蛋白、上述的嵌合抗原受体、上述的核酸、上述的载体、上述的细胞、上述的免疫细胞或上述的治疗和/或诊断疾病的产品在阻断VEGFA信号通路中的应用。
优选的，所述的应用包括：
A)制备治疗和/或预防肿瘤的药物中的应用；
B)治疗和/或预防肿瘤中的应用；
C)制备治疗和/或预防眼科涉及血管新生的疾病(例如眼底血管疾病)的药物中的应用，所述的眼底血管疾病优选年龄相关性黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、糖尿病性视网膜病变、视网膜中央静脉阻塞、病理性近视、新生血管性青光眼等；
D)治疗和/或预防眼科涉及血管新生的疾病(例如眼底血管疾病)中的应用，所述的眼底血管疾病优选年龄相关性黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、糖尿病性视网膜病变、视网膜中央静脉阻塞、病理性近视、新生血管性青光眼等；和/或，
E)抑制血管异常增生中的应用，优选阻断血管内皮细胞增殖。
本发明所述的抗VEGFA融合蛋白与VEGFA具有高亲和力，能有效阻断VEGFA与受体VEGFR的结合，阻断VEGFA信号通路，有效阻断VEGFA导致的血管内皮细胞增殖以及血管新生等病理过程，能潜在应用于与VEGFA相关的疾病的治疗中。
本发明所述的“药物”可以用于治疗人或非人动物，例如非人哺乳动物。所述的药物可以包含现有技术常见的药学上可接受的载体、辅料或盐。所述的药物可以采用任何合适的给药途径，例如胃肠道给药(例如口服)或非胃肠道给药(例如，静脉内、肌内、皮下、皮内、器官内、鼻内、眼内、滴注、脑内、鞘内、透皮、直肠内等)途径。所述的药物可以为任何合适的剂型，例如经胃肠道给药剂型或非经胃肠道给药剂型，优选包括但不限于片剂、丸剂、粉剂、颗粒剂、胶囊剂、锭剂、糖浆剂、液体、乳剂、微乳剂、混悬剂、注射剂、喷雾剂、气雾剂、粉雾剂、洗剂、软膏剂、硬膏剂、糊剂、贴剂、滴眼剂、滴鼻剂、舌下片剂、栓剂、气雾剂、泡腾片、滴丸剂、凝胶剂等等。所述的药物的各种剂型可以按照药学领域的常规生产方法制备。所述的药物可以含有重量比为0.01-99.5％(具体如，0.01％、0.1％、0.5％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、99％、99.5％)的所述抗VEGFA融合蛋白、所述的核酸、所述的载体、所述的细胞、所述的免疫细胞等等。所述的药物可以制备为蛋白浓度为1-300mg/mL(例如1、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300mg/mL)的试剂。所述的药物的单次给药剂量可以为0.1-1000mg，例如0.1、0.2、0.5、0.75、1、1.25、1.5、1.75、2、2.25、2.5、3、5、10、20、50、80、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000mg。
本发明所述的“药学上可接受的”是指既不显著刺激生物体也不抑制所施用的产品的活性物质的生物学活性及特性。
本发明所述的“…方法”可以为疾病的诊断和治疗目的，也可以为非疾病的诊断和治疗目的。
本发明所述的“抗原结合片段”是保留抗体的特定结合活性的抗体的一部分，即抗体的任何部分能够与抗体的靶分子上的表位特异结合。它包括例如Fab、Fv、Fd、Fab'、Fab'-SH、F(ab')2和这些片段的变体。例如，抗体的重链和/或轻链，抗体的重链可变区和/或轻链可变区，或来自抗体的重链或轻链的单个或2个以上CDR。其中，Fab，即由VL、VH、CL和CH1结构域组成的单价片段。F(ab')2，即包含由铰链区的二硫桥连接的两个Fab片段的二价片段。Fd，由VH和CH1结构域组成的Fd片段。Fv，由抗体单臂的VL和VH结构域组成的Fv片段。Fab'，是在CH1结构域的C端具有一个或多个半胱氨酸残基的Fab片段。Fab'-SH是对其中恒定结构域的半胱氨酸残基带有至少一个游离硫醇基团的Fab'。其中，VH代表重链可变区，VL代表轻链可变区，CL代表轻链，CH1为组成重链恒定区的CH1。
本发明所述的“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由铰链区的两个或多个二硫键形成二聚体并通过CH3结构域的疏水作用保持在一起。
本发明所述的“人源化抗体”指抗体的框架区和/或恒定区部分(例如CH区)或抗体所有全部由人类抗体基因所编码。在本发明的一个具体实施方式中，抗体的CDR区未经过改造。
本发明所述的“治疗”表示在疾病已开始发展后减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性，但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。
本发明所述的“预防”表示为了阻止或延迟疾病或病症或症状在机体内的发生而实施的方式。
本发明所述的“个体”可以为人或非人哺乳动物，所述的非人哺乳动物可以为野生动物、动物园动物、经济动物、宠物、实验动物等等。优选的，所述的非人哺乳动物包括但不限于猪、牛、羊、马、驴、狐、貉、貂、骆驼、狗、猫、兔、鼠(例如大鼠、小鼠、豚鼠、仓鼠、沙鼠、龙猫、松鼠)或猴等等。
本发明所述的“包含”或“包括”为开放式写法，当用于描述蛋白质或核酸的序列时，所述蛋白质或核酸可以是由所述序列组成，或者在所述蛋白质或核酸的一端或两端可以具有额外的氨基酸或核苷酸，但仍然具有与原序列相同或相似的活性。
本发明所述的“肿瘤”可以是任何不良的细胞增殖(或本身表现为不良细胞增殖的任何疾病)、赘生物，或不良细胞增殖、赘生物或肿瘤的倾向性或风险增加。其可以是良性或恶性的，也可以是原发性或继发性(转移性)。赘生物可以是细胞的任何异常生长或增殖，并且可以位于任何组织中。组织的实例包括肾上腺、肾上腺髓质、肛门、阑尾、膀胱、血液、骨、骨髓、脑、乳腺、盲肠、中枢神经系统(包括或排除大脑)、小脑、子宫颈、结肠、十二指肠、子宫内膜、上皮细胞(例如肾上皮细胞)、胆囊、食道、神经胶质细胞、心脏、回肠、空肠、肾、泪腺、喉、肝、肺、淋巴、淋巴结、淋巴母细胞、上颌骨、纵隔、肠系膜、子宫肌层、鼻咽、网膜、口腔、卵巢、胰腺、腮腺、周围神经系统、腹膜、胸膜、前列腺、唾液腺、乙状结肠、皮肤、小肠、软组织、脾、胃、睾丸、胸腺、甲状腺、舌、扁桃体、气管、子宫、外阴、白细胞。进一步优选的，所述的肿瘤选自前列腺癌、乳腺癌、肝癌、胶质瘤(例如神经胶质瘤)、肠癌、宫颈癌、非小细胞肺癌、肺癌、胰腺癌、胃癌、膀胱癌、皮肤癌、横纹肌癌、舌鳞癌、鼻咽癌、卵巢癌、胎盘绒毛癌、淋巴瘤(例如非霍奇金淋巴瘤、霍奇金淋巴瘤、皮肤T细胞淋巴瘤)、白血病、直肠腺癌、成神经管细胞瘤、脑膜瘤、神经纤维瘤(例如神经纤维肉瘤)、室管膜瘤、神经鞘瘤、星形细胞瘤、黑色素瘤、间皮瘤、骨髓瘤、慢性粒细胞白血病、急性髓性白血病、骨髓增生异常综合征、慢性淋巴细胞白血病、表皮样癌、结肠癌、胸腺癌、血液癌、头颈癌或口咽癌。
本发明所述“同源性”，是指在使用蛋白序列或核苷酸序列的方面，本领域技术人员可以根据实际工作需要对序列进行调整，使使用序列与现有技术获得的序列相比，具有(包括但不限于)1％，2％，3％，4％，5％，6％，7％，8％，9％，10％，11％，12％，13％，14％，15％，16％，17％，18％，19％，20％，21％，22％，23％，24％，25％，26％，27％，28％，29％，30％，31％，32％，33％，34％，35％，36％，37％，38％，39％，40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，70％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.1％，99.2％，99.3％，99.4％，99.5％，99.6％，99.7％，99.8％，99.9％的同一性。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：二价纳米抗体(连接子为9GS)纯化后的考染胶图。
图2：二价纳米抗体(连接子为9GS)结合VEGFA的ELISA测试结果。
图3：二价纳米抗体(连接子为9GS)竞争VEGFR2的ELISA测试结果。
图4：二价纳米抗体与一价纳米抗体结合VEGFA活性比较。
图5：二价纳米抗体与一价纳米抗体竞争VEGFR2的活性比较。
图6：异源二价纳米抗体纯化后的考染胶图。
图7：异源二价纳米抗体结合VEGFA的ELISA测试。
图8：异源二价纳米抗体竞争VEGFR2的ELISA测试。
图9：不同连接子的二价纳米抗体纯化后的考染胶。
图10：不同连接子串联的二价纳米抗体结合VEGFA活性测试。
图11：二价纳米抗体竞争VEGFR2的ELISA测试。
图12：不同连接子的二价V1在4℃放置30天的考染胶图。
图13：不同连接子的二价V1在4℃放置30天后的吸光度。
图14：不同连接子的二价V1在40℃放置7天的考染胶图。
图15：不同连接子的二价V1在40℃放置7天后继续室温放置23天后的吸光度。
图16：二价人源化纳米抗体纯化后的考染胶图。
图17：二价人源化纳米抗体结合VEGFA的ELISA测试。
图18：单价纳米抗体-Fc融合蛋白纯化后的考染胶图。
图19：单价纳米抗体-Fc融合蛋白或串联的二价纳米抗体结合VEGFA的ELISA测试。
图20：单价纳米抗体-Fc融合蛋白(V1-SA1-Fc-m1)与VEGFA165的结合解离曲线(SPR)。
图21：二价纳米抗体-Fc融合蛋白纯化后的考染胶图。
图22：二价纳米抗体-Fc融合蛋白结合VEGFA的ELISA测试。
图23：二价纳米抗体-Fc融合蛋白与VEGFA165的结合解离曲线(SPR)。
图24：二价纳米抗体-Fc融合蛋白和Aflibercept与VEGFA165的结合解离曲线(SPR)。
图25：二价纳米抗体-Fc融合蛋白2V1-SA1-3GS-Fc结合不同物种VEGFA的活性测试。
图26：2V1-SA1-3GS-Fc和Aflibercept抑制VEGFA刺激的HUVEC增殖。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：二价纳米抗体的制备和活性研究
1、纳米抗体的二价化设计
为提高纳米抗体结合VEGFA的效率，并提升纳米抗体阻断VEGFA/VEGFR互作的效率，将两个序列相同的纳米抗体进行串联融合，即构建同源二价纳米抗体，并开展了生物活性研究。
首先，选择了空间结构灵活多变的富含甘氨酸和丝氨酸(GS)的连接子(linker)9GS(氨基酸序列为：GGGGSGGGS(SEQ ID NO：78))将两个序列相同的纳米抗体进行串联。将各纳米抗体的编 码基因通过PCR从一价纳米抗体的表达质粒中扩增，构建到pVRC8400表达载体上，其中第一个纳米抗体N端加入分泌肽：MDAMKRGLCCVLLLCGAVFVSPS(SEQ ID NO：151)的核苷酸序列，其C端添加编码连接子9GS的核苷酸序列，其后连接编码第二个纳米抗体的核苷酸序列，并在第二个纳米抗体的C端依次添加编码连接子GGGGS(SEQ ID NO：152)和标签6×His的核苷酸序列。两个纳米抗体核苷酸片段采用无缝克隆试剂盒(购自：碧云天，货号：D7010M)，与载体质粒同源重组，形成正确的表达质粒。与此同时，合成阳性对照抗体BI-VEGF ab的编码基因序列(该抗体是抗VEGFA的纳米抗体，抗体氨基酸序列来自专利US 9527925B2中的SEQ ID NO：57)，对照抗体BI-VEGF ab的C端添加标签6×His。构建的示例性的二价纳米抗体序列名称及编号见表3。
表3二价纳米抗体氨基酸序列名称及编号
二价纳米抗体的表达质粒测序正确后进行无内毒素提取。采用转染试剂聚乙烯亚胺(Polyethylenimine，简称PEI，linear，MW＝25kDa，购自：Polysciences，货号：23966-1)，对293F悬浮细胞进行瞬转，转染后第4天或第5天收集细胞上清，开展Ni珠亲和纯化。纯化后的蛋白经SDS-PAGE分析，分子量符合预期(～30kDa)。部分二价纳米抗体纯化后的考染胶图如图1所示。
2、二价纳米抗体结合VEGFA的活性检测
采用ELISA方法检测纯化后的二价纳米抗体结合VEGFA的活性。
用ELISA包被缓冲液稀释抗原人VEGFA165(无标签，购自：义翘神州，货号：HPLC-10008-HNAH，以下简称VEGFA)，使其终浓度为0.3μg/mL，加入酶标板中，100μL/孔，4℃包被过夜。经5％脱脂奶粉封闭后，加入各个二价纳米抗体的梯度稀释液(0.001，0.01，0.1，1，3，10，100nM)。抗体稀释所用溶液是含0.1％Tween-20的PBS(pH7.4)，即PBST。37℃孵育1小时，用PBST洗涤酶标板3次后，加入His-Tag鼠源单克隆抗体稀释液(购自：Proteintech，货号：66005-1-Ig)，37℃孵育1小时，洗板后加入HRP标记的山羊抗鼠IgG抗体稀释液(HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)，购自：Proteintech，货号：SA00001-1)，室温孵育45分钟。洗板后，每孔加入100μL TMB(购自：天根生化，货号：PA107-01)，37℃显色15分钟后，加入终止液50μL。用酶标仪测定在450nm处的吸光度(OD450)。部分二价纳米抗体结合VEGFA的信号随抗体浓度变化如图2所示。
二价纳米抗体结合VEGFA的EC50，见表4，从表中可知9个候选抗体均具有较高的VEGFA结合活性。
表4二价纳米抗体结合VEGFA的EC50值

3、二价纳米抗体竞争VEGFR2的活性检测
采用竞争ELISA进一步验证二价纳米抗体阻断VEGFA结合受体VEGFR2的活性。
用ELISA包被缓冲液稀释抗原VEGFA(终浓度为0.53μg/mL)，加入96孔酶标板中，100μL/孔，4℃包被过夜。经5％脱脂奶粉封闭，将终浓度为1nM的VEGFR2胞外区结构域VEGFR2-ECD-Fc(购自：义翘神州，货号：10012-H02H)与待测二价纳米抗体的梯度稀释液(0.01，0.1，1，3，10，100nM)浓度梯度，混合均匀，加入96孔板中，37℃孵育1小时，用PBST洗板3次后，加入HRP标记的山羊抗人IgG抗体稀释液(HRP-conjugated Goat Anti-Human IgG，购自：Proteintech，货号：A21050)，室温孵育45分钟。洗板后，每孔加入100μL TMB(购自：天根生化，货号：PA107-01)，37℃显色15分钟后，加入终止液50μL。用酶标仪测定在450nm处的吸光度(OD450)。竞争ELISA结果见图3。
二价纳米抗体竞争VEGFR2的活性(IC50值)比较见表5，可看出二价纳米抗体有较强的竞争VEGFR2活性，并且竞争活性强于阳性对照一价纳米抗体BI-VEGF ab。
表5二价纳米抗体竞争VEGFR2的活性(IC50值)
实施例2：二价纳米抗体与一价纳米抗体的VEGFA结合和阻断活性比较
1、二价纳米抗体与一价纳米抗体结合VEGFA活性比较
示例性地采用ELISA对比了二价纳米抗体(2V1-9GS、2V15-9GS、2V31-9GS、2V43-9GS)与相应的一价纳米抗体结合VEGFA的活性。实验方法与实施例1中“2、二价纳米抗体结合VEGFA的活性检测”相同。
一价和二价纳米抗体结合VEGFA的信号随抗体浓度变化如图4所示。一价和二价纳米抗体结合VEGFA的EC50值见表6，从表中可知二价纳米抗体结合VEGFA的活性要强于一价纳米抗体。
表6一价和二价纳米抗体结合VEGFA的EC50值
此外，利用表面等离子共振(SPR)技术示例性地分别测试一价纳米抗体与二价纳米抗体结合VEGFA的亲和力。采用Biacore 8K仪器(Cytiva)，以抗原VEGFA165(无标签，购自：义翘神州，货号：HPLC-10008-HNAH)为配体，通过氨基偶联试剂(EDC和NHS)偶联到CM5芯片上，偶联量约为400RU。实验过程中流动相缓冲液为HBS-EP(0.01M HEPES pH 7.4,0.15M NaCl,3mM EDTA,0.005％v/v Tween 20)。分析物为一价纳米抗体和二价纳米抗体，其稀释液浓度是：20nM、10nM、5nM、2.5nM、1.25nM、0.625nM、0.3125nM。动力学分析时，抗原与抗体的结合时间为240秒，解离时间为1500秒，流速是30μL/min。采用多循环模式，芯片经100mM盐酸再生。抗体与VEGFA165的结合和解离曲线采用“1：1 binding”模型拟合，获得的结合速率(ka)、解离速率(kd)和平衡解离常数(KD)见表7所示。
表7抗体与VEGFA165的结合速率(ka)、解离速率(kd)和平衡解离常数(KD)
从表7中，可以看出，二价纳米抗体与VEGFA有很高的亲和力(KD＝22pM)，一价纳米抗体与VEGFA的亲和力较高(KD＝116～719pM)，二者相差5.3～32.7倍。表明，一价抗体经二价化改造后，能显著提升结合靶点的亲和力。
2、二价纳米抗体与一价纳米抗体竞争VEGFR2活性比较
采用竞争ELISA比较一价和二价纳米抗体阻断VEGFA/VEGFR2互作的活性。
实验方法同实施例1中“3、二价纳米抗体竞争VEGFR2的活性检测”。二价纳米抗体2V15-9GS和一价纳米抗体V15分别竞争VEGFR2的ELISA结果见图5。
一价和二价纳米抗体竞争VEGFR2的活性(IC50和最大抑制率)，见表8。
最大抑制率计算公式是：
结果显示二价纳米抗体竞争VEGFR2的活性要强于一价纳米抗体。
表8一价和二价纳米抗体竞争VEGFR2的活性(IC50和最大抑制率)
实施例3：异源二价纳米抗体的设计和活性评估
1、异源二价纳米抗体的设计和制备
(1)单价抗体Tm值检测
选取8个单价抗体(V1、V15、V29、V30、V31、V36、V40、V43)，替换抗体所处缓冲液为：10mM His,pH＝6.5,40mM NaCl,5％sucrose,0.01％Tween-20，用UNCLE仪器(unchainedlabs)测定抗体的熔解温度(Tm)，结果见表9。结果表明这些候选抗体都具有一定的热稳定性。
表9单价抗体的熔解温度Tm
(2)异源二价纳米抗体的设计和制备
将不同氨基酸序列的单价纳米抗体通过连接子9GS串联，构建异源二价纳米抗体。选取熔解温度(Tm)较高的纳米抗体V1、V15、V30、V40构建异源二价纳米抗体，共表达6种异源二价纳米抗体，名称和序列名称及编号见表10。
表10异源二价纳米抗体的氨基酸序列名称及编号
对6种异源二价纳米抗体，其质粒构建和表达纯化方法与实施例1中“1、纳米抗体的二价化设计”相同。抗体经Ni珠亲和纯化后的考染胶图如图6所示。目标蛋白的分子量约为30kDa。
2、异源二价纳米抗体的活性研究
对纯化的异源二价纳米抗体开展VEGFA结合活性评估，实验方法与实施例1中“2、二价纳米抗体结合VEGFA的活性检测”相同。异源二价纳米抗体结合VEGFA的ELISA结果如图7所示。
异源二价纳米抗体结合VEGFA的EC50见表11。结果显示6种异源二价纳米抗体结合VEGFA的活性类似(EC50为0.3～0.4nM)，这与同源二价纳米抗体(串联两个序列相同的纳米抗体)的活性类似(EC50约为0.24nM，见实施例1中的表4)。
异源二价纳米抗体竞争VEGFR2的ELISA结果如图8所示。竞争ELISA的IC50和抑制率见表11。结果显示，异源二价纳米抗体有很强的与VEGFR2竞争结合VEGFA活性。
表11异源二价纳米抗体竞争VEGFR2的ELISA结果

实施例4：不同连接子对二价纳米抗体活性的影响
上述实施例中，连接两个纳米抗体的连接子多为柔性的9GS，两个纳米抗体的空间取向相对独立，互不干扰。但不同性质的连接子(如不同长度，亲疏水性，二级结构等)可能对抗体的空间构像以及抗原结合效率有影响，故制备并评估了不同连接子对二价纳米抗体活性和稳定性影响。
1、设计纳米抗体之间连接子和制备不同连接子的二价纳米抗体
为了解连接子性质对二价纳米抗体生物活性和物理化学稳定性的影响，设计了16种连接子，包括不同的连接子长度、可能形成的二级结构、亲水性等。连接子的氨基酸序列见表12。
表12连接子氨基酸序列

*WL代表Whitlow Linker。
以纳米抗体V1和V15为模型抗体，构建了不同连接子的二价纳米抗体，它们的表达载体构建、瞬转和纯化方法与实施例1中“1、纳米抗体的二价化设计”相同。不同连接子的二价纳米抗体序列名称及编号见表13。
表13二价纳米抗体氨基酸序列名称及编号
部分抗体经Ni珠亲和纯化后的考染胶图如图9所示。目标蛋白的分子量约为30kDa。
2、不同连接子串联的二价纳米抗体活性研究
对纯化的二价纳米抗体开展VEGFA结合活性评估，实验方法与实施例1中“2、二价纳米抗体结合VEGFA的活性检测”相同。部分二价纳米抗体结合VEGFA的ELISA结果如图10所示，结合VEGFA的EC50见表14。
对于二价的纳米抗体V1，不同连接子对二价V1的EC50影响较小(EC50为0.2～0.3nM)，而且，不含连接子的二价V1(2V1-NL)活性与添加连接子的二价V1活性相当。对于用不同连接子串联而成的二价V15，其活性也相差不大。
表14二价纳米抗体结合VEGFA的EC50值

二价纳米抗体竞争VEGFR2的ELISA结果如图11所示。竞争ELISA的IC50和抑制率见表15。结果显示，对于V15，连接子20S串联而成的二价V15(2V15-20S)的竞争活性稍差于其他连接子串联形成的二价V15。
表15二价纳米抗体竞争VEGFR2的ELISA的IC50和抑制率
实施例5：不同连接子对二价纳米抗体稳定性的影响
1、不同连接子在4℃下对二价纳米抗体稳定性的影响
将二价V1：2V1-3GS、2V1-(WL)2、2V1-EAAAK-Turn替换缓冲液至：10mM His,pH＝6.5,40mM NaCl,5％sucrose,0.01％Tween-20，蛋白浓度统一调整为0.5mg/mL，在0时刻，蛋白溶液澄清，4℃放置30天后进行SDS-PAGE和吸光度检测。蛋白的考染胶图见图12。结果显示，二价抗体2V1-EAAAK-Turn出现了分子量低于主条带的降解条带。而2V1-3GS和2V1-(WL)2泳道中未见明显降解片段，化学稳定性较好。
此外，测定了样品在600nm波长处的吸光度(OD600)，对比图见图13。可见，2V1-3GS的吸光度值最低，溶液最澄清，暗示相较于其他蛋白，2V1-3GS蛋白聚集程度相对较低。所以，3种连接子中，3GS更能维持二价V1的物理稳定性和化学稳定性。
2、不同连接子在高温下对二价纳米抗体稳定性的影响
将二价V1：2V1-9GS、2V1-30GS、2V1-39GS、2V1-(WL)2、2V1-(AP)5、2V1-(AP)10、2V1-EAAAK-Rigid替换缓冲液至：10mM His,pH＝6.5,40mM NaCl,5％sucrose,0.01％Tween-20，蛋白浓度统一调整为0.5mg/mL，在0时刻，蛋白溶液澄清，40℃放置7天后进行SDS-PAGE，蛋白的考染胶图见图14。结果显示，二价抗体2V1-EAAAK-Turn出现了分子量低于主条带的降解条带。而2V1-3GS和2V1-(WL)2泳道中未见明显降解片段，化学稳定性较好。
结果表明，在40℃加速条件下，7个二价V1中，只有2V1-9GS和2V1-(AP)10能维持大部分蛋白不发生降解。
将40℃放置7天后的样品继续放置在室温23天后，测定样品在600nm波长处的吸光度(OD600)，对比图见图15。可见，2V1-9GS的吸光度值最低，溶液最澄清，暗示2V1-9GS蛋白聚集程度相对较低。所以，7种连接子中，含9GS的二价V1有更好的物理稳定性和化学稳定性。
通过比较含不同连接子的二价纳米抗体在4℃和40℃下的化学稳定性和物理稳定性，含有连接子3GS和9GS的二价纳米抗体有更好的稳定性表现。
实施例6：二价人源化纳米抗体的活性研究
1、人源化纳米抗体的二价化设计
在前面的实施例中，对于原始的未做人源化改造的抗体进行了二价化以及连接子对比，接下来，将以连接子3GS和9GS或者不加连接子(NL)构建二价人源化纳米抗体。抗体人源化是先比对候选抗体序列与人的抗体序列同源性(通过数据库IGBLAST和IMGT)，再将候选抗体的CDR区嫁接到同源性高的人抗体框架区中，组成完整的人源化抗体，为了维持抗体的活性，需将人抗体框架区个别氨基酸恢复突变成候选抗体对应位置的序列。以V1、V13、V15、V30、V40、V43的人源化抗体为元件，构建不同连接子的二价人源化纳米抗体。构建的二价人源化纳米抗体氨基酸序列名称及编号见表16。
表16二价人源化纳米抗体氨基酸序列名称及编号
上述二价纳米抗体的质粒构建和表达纯化方法与实施例1中“1、纳米抗体的二价化设计”相同。部分蛋白纯化后的考染胶图如图16所示。目标蛋白的分子量约为30kDa。
2、人源化纳米抗体的二价化的活性测试
对纯化的二价人源化纳米抗体开展VEGFA结合活性评估，实验方法与实施例1中“2、二价纳米抗体结合VEGFA的活性检测”相同。二价人源化纳米抗体结合VEGFA的ELISA结果如图17所示。ELISA的EC50见表17。结果显示，绝大多数二价人源化纳米抗体能高效结合VEGFA。
表17二价人源化纳米抗体结合VEGFA的ELISA的EC50
实施例7：单价纳米抗体-Fc融合蛋白的制备和活性研究
1、单价纳米抗体-Fc融合蛋白的制备
实现纳米抗体的二价化，除了线性串联2个纳米抗体之外，还可以利用二聚化结构域或片段，如IgG1抗体的Fc(SEQ ID NO：134)，PPCP-Fc(SEQ ID NO：158，即将SEQ ID NO：134中的DKTHTC去除后的剩余氨基酸序列)或IgG1 Fc突变体Fc-m1(SEQ ID NO：135)，将2个纳米抗体连接在同一分子中。
其中，IgG1 Fc氨基酸序列为：
PPCP-Fc氨基酸序列为：
IgG1 Fc的突变体Fc-m1，其氨基酸序列为：
因此，开展了单价纳米抗体-Fc融合蛋白的表达。构建的纳米抗体-Fc融合蛋白氨基酸序列名称及编号见表18。
表18纳米抗体-Fc融合蛋白氨基酸序列名称及编号
为了便于对比线性串联的二价纳米抗体2V15-9GS(含有6个组氨酸的标签)，与融合蛋白V15-PPCP-Fc、V15-9GS-PPCP-Fc和V15-Fc在结合VEGFA方面的活性，在融合蛋白Fc段的C末端添加连接子(SGGS(SEQ ID NO：153))和6个组氨酸的标签(HHHHHH(SEQ ID NO：154))。单价纳米抗体-Fc融合蛋白的质粒构建过程中，用PCR方法分别扩增纳米抗体和含连接子和组氨酸标签的Fc，其中Fc片段的PCR模板来自于基因合成的Fc基因。采用两片段同源重组的方法，将纳米抗体和Fc整合到载体pCDNA3.1(+)上，所用酶切位点是：NheI和XbaI。融合蛋白的分泌肽为：METDTLLLWVLLLWVPGSTG(SEQ ID NO：155)，其后融合目的蛋白序列。这些融合蛋白质粒测序成功后，进行质粒无内毒素提取，采用转染试剂聚乙烯亚胺(Polyethylenimine，简称PEI，linear，MW＝25kDa，购自：Polysciences，货号：23966-1)，对293F悬浮细胞进行瞬转，转染后第4天或第5天收集细胞上清，开展Ni珠纯化。纯化后的蛋白经SDS-PAGE分析，还原条件下蛋白单体分子量约为39kDa，分子量符合预期。部分单价纳米抗体-Fc融合蛋白纯化后的考染胶图如图18所示。
对于人源化后的抗体V1-SA1与Fc的融合蛋白(V1-SA1-Fc-m1)，V1-SA1与Fc-m1之间的连接子氨基酸序列为：EPKSA(SEQ ID NO：156)。采用上述方法构建表达质粒，但Fc的C末端不含额外氨基酸，即融合蛋白V1-SA1-Fc-m1含Fc标签，不含组氨酸标签。V1-SA1-Fc-m1的表达质粒瞬转293F细胞后，采用Protein A珠亲和纯化，蛋白洗脱时使用0.1M柠檬酸(pH3.0)洗脱目标蛋白，并快速中和，获得目标蛋白。
2、单价纳米抗体-Fc融合蛋白的活性检测
对纯化的单价纳米抗体-Fc融合蛋白开展VEGFA结合活性测试，ELISA实验方法同实施例1中的“2、二价纳米抗体结合VEGFA的活性检测”。ELISA结果如图19所示，EC50如表19所示。
表19单价纳米抗体-Fc融合蛋白或串联的二价纳米抗体VEGFA结合活性测试

由表中数据可知，测试的单价纳米抗体-Fc融合蛋白与线性串联的二价纳米抗体均有很强的结合VEGFA活性。
利用表面等离子共振(SPR)技术测试单价纳米抗体-Fc融合蛋白(V1-SA1-Fc-m1)与VEGFA的亲和力。采用Biacore 8K仪器(Cytiva)，以V1-SA1-Fc-m1为配体，捕获在Protein A芯片上，固定时，用流动相缓冲液HBS-EP(0.01M HEPES pH 7.4,0.15M NaCl,3mM EDTA,0.005％v/v Tween 20)稀释V1-SA1-Fc-m1到蛋白浓度为2μg/mL，其偶联量约为590RU。实验过程中，分析物为抗原VEGFA165(无标签，购自：金斯瑞，货号：Z03073)，其稀释液浓度是：25nM、12.5nM、6.25nM、3.125nM、1.5625nM、0.78125nM、0.390625nM。动力学分析时，融合蛋白与抗原VEGFA165的结合时间为180秒，解离时间为900秒，流速是30μL/min。在每个“结合-解离”循环结束之后，芯片经再生处理，再生条件是10mM甘氨酸盐酸盐(pH 1.5)。融合蛋白V1-SA1-Fc-m1与抗原VEGFA165的结合解离曲线(图20)采用“1:1 binding”模型拟合获得结合速率(ka)、解离速率(kd)和平衡解离常数(KD)，见表20所示。
表20融合蛋白V1-SA1-Fc-m1与抗原VEGFA165的结合速率(ka)、解离速率(kd)和平衡解离常数(KD)
从SPR数据可知，单价纳米抗体-Fc融合蛋白V1-SA1-Fc-m1与VEGFA165具有很高的亲和力(KD＝0.148nM)。
实施例8：二价纳米抗体-Fc融合蛋白的制备和活性研究
1、二价纳米抗体-Fc融合蛋白的表达和纯化
实施例7中单价纳米抗体-Fc融合蛋白与VEGFA具有高亲和力，实施例2的结果表明二价纳米抗体结合VEGFA的活性要高于单价纳米抗体，因此，构建了二价纳米抗体-Fc融合蛋白，即将表16中的二价纳米抗体与IgG1 Fc(SEQ ID NO：134)融合表达，部分二价纳米抗体-Fc融合蛋白氨基酸序列名称及编号见表21。
表21二价纳米抗体Fc融合蛋白氨基酸序列名称及编号

对上述二价纳米抗体-Fc融合蛋白构建表达质粒，并瞬转和纯化。
具体方法是：根据表中各个融合蛋白的氨基酸序列，采用基因合成或PCR方法构建表达质粒。对于PCR的方法，将2个相同的纳米抗体基因和Fc整合到载体pCDNA3.1(+)上，所用酶切位点是：NheI和XbaI。融合蛋白的分泌肽为：METDTLLLWVLLLWVPGSTG(SEQ ID NO：155)，分泌肽后融合目的蛋白序列。经测序确认和无内毒素质粒提取，采用转染试剂聚乙烯亚胺(Polyethylenimine，简称PEI，linear，MW＝25kDa，购自：Polysciences，货号：23966-1)，对293F悬浮细胞进行瞬转，转染后第4天或第5天收集细胞上清，开展Protein A珠亲和纯化，蛋白洗脱时使用0.1M柠檬酸(pH3.0)洗脱目标蛋白，蛋白洗脱产物滴入0.5倍体积的1M柠檬酸钠(pH＝8.5)中进行快速中和，蛋白经浓缩和替换溶液后用于后续测试。
示例性的二价纳米抗体-Fc融合蛋白纯化后的考染胶图见图21，在还原型SDS-PAGE条件下，蛋白分子量约为53kDa。
2、二价纳米抗体-Fc融合蛋白的活性研究
用ELISA检测纯化的二价纳米抗体-Fc融合蛋白结合VEGFA的活性。ELISA方法为：用ELISA包被缓冲液稀释人VEGFA165(无标签，购自：义翘神州，货号：HPLC-10008-HNAH)，使其终浓度为0.3ug/mL,4℃包被过夜，经5％脱脂奶粉(用PBST配置)封闭后，加入不同浓度的融合蛋白(最高浓度为100nM，最低浓度为0.0001nM，10倍浓度梯度，用PBST配置)，37℃孵育1小时，用PBST洗涤酶标板3次后，加入识别Fc标签的检测抗体：HRP标记的Goat Anti-Human IgG抗体(购自：Abbkine，货号：A21050)，室温孵育45分钟，洗板后，每孔加入100μL TMB(购自：天根生化，货号：PA107-01)，37℃显色15分钟后，加入终止液50μL。用酶标仪测定在450nm处的吸光度(OD450)。
部分融合蛋白的ELISA结果见图22，其中Negative control为结合不相关靶点的IgG1 Fc融合蛋白。EC50见表22。
表22融合蛋白结合VEGFA的EC50
利用表面等离子共振(SPR)技术测定二价纳米抗体-Fc融合蛋白(2V1-SA1-3GS-Fc、2V1-SA1-NL-Fc、2V1-DP-3GS-Fc)与VEGFA165的亲和力。采用Biacore 8K仪器(Cytiva)，采用“低固定量”模式，将VEGFA165(无标签，购自：义翘神州，货号：HPLC-10008-HNAH)偶联在CM5芯片上(由于这些蛋白与VEGFA165亲和力较高，解离很慢，故减少VEGFA165偶联量)。用流动相缓冲液HBS-EP(0.01M HEPES pH 7.4,0.15M NaCl,3mM EDTA,0.005％v/v Tween 20)稀释各个融合蛋白，稀释液浓度是：0.0244nM，0.0977nM，0.3906nM，1.5625nM和6.25nM。动力学分析时，抗原与抗体的结合时间为120秒，解离时间为780秒，流速是40μL/min。采用多循环模式，芯片经100mM盐酸再生。融合蛋白与VEGFA165的结合解离曲线采用“1:1 binding”模型拟合获得结合速率(ka)、解离速率(kd)和平衡解离常数(KD)。
二价纳米抗体-Fc融合蛋白结合与VEGFA165的结合解离曲线见图23，拟合的KD见表23所示。
表23二价纳米抗体-Fc融合蛋白与VEGFA165的结合速率(ka)、解离速率(kd)和平衡解离常数(KD)
通过表23，可知3个二价纳米抗体-Fc融合蛋白与VEGFA165有很高的亲和力(KD为3.84～6.83pM)。
此外，更换不同试剂供应商来源的抗原人VEGFA165，重复测试融合蛋白与VEGFA165的KD。采用类似于上述SPR实验方法，测定了融合蛋白(2V1-SA1-3GS-Fc)和阳性对照药物Aflibercept(阿柏西普)与VEGFA165的KD值。具体来说，采用Biacore 8K仪器(Cytiva)，采用“低固定量”模式，将VEGFA165(无标签，购自：金斯瑞，货号：Z03073)偶联在CM5芯片上，用流动相缓冲液HBS-EP稀释蛋白。2V1-SA1-3GS-Fc稀释液浓度是：0.091nM、0.1828nM、0.3656nM、0.73125nM、1.4625nM、2.925nM、5.85nM。Aflibercept稀释液浓度是：0.3125nM、0.625nM、1.25nM、2.5nM、5nM、10nM、20nM。动力学分析时，抗原与抗体的结合时间为150秒，解离时间为1000秒，流速是30μL/min。采用多循环模式，芯片经10mM甘氨酸盐酸盐(pH＝1.5)再生。待测蛋白与VEGFA165的结合解离曲线采用“1:1 binding”模型拟合获得结合速率(ka)、解离速率(kd)和平衡解离常数(KD)，见表24所示。二价纳米抗体-Fc融合蛋白(2V1-SA1-3GS-Fc)和Aflibercept与VEGFA165的结合解离曲线(SPR)如图24所示。
表24 2V1-SA1-3GS-Fc和Aflibercept与VEGFA165的结合速率(ka)、解离速率(kd)和平衡解离常数(KD)
对比表23和表24中的KD值可知，融合蛋白2V1-SA1-3GS-Fc与不同试剂供应商来源的人VEGFA165均有极高的亲和力，KD均为pM级别，且KD值明显小于Aflibercept(约为Aflibercept的1/12)，这表明，融合蛋白2V1-SA1-3GS-Fc与VEGFA165的亲和力高于Aflibercept。
以上采用近似的方法(偶联抗原VEGFA165)开展了SPR测试，对比一价纳米抗体和二价纳米抗体与VEGFA的KD值(表7)以及上述二价纳米抗体-Fc融合蛋白与VEGFA的KD值(表23、表24)，不同构建形式的抗体与VEGFA的KD值汇总如表25所示。
表25不同构建形式的抗体与VEGFA的KD值汇总
从表25可以看出，二价纳米抗体和二价纳米抗体-Fc融合蛋白相比一价纳米抗体，与抗原亲和力分别提升22.5倍和105.5倍。所以，随着一个蛋白分子中抗体或抗原结合片段数目的增加，蛋白分子与抗原的亲和力将显著增加。
采用ELISA方法检测2V1-SA1-3GS-Fc与不同物种VEGFA蛋白的交叉结合活性。其中，小鼠VEGFA164购自义翘神州，货号为50159-MNAB，大鼠VEGFA164购自义翘神州，货号为80006-RNAB。兔VEGFA121为瞬转293F细胞表达，经Ni珠纯化而得。兔VEGFA121的核苷酸序列合成后，克隆至载体pCDNA3.1(+)中。兔VEGFA121的氨基酸序列是：
蛋白N端采用其自身分泌肽，蛋白C端依次添加柔性连接子(GGGGS(SEQ ID NO：152))和6个组氨酸的标签(HHHHHH(SEQ ID NO：154))。
所用的ELISA实验方法与实施例1中“2、二价纳米抗体结合VEGFA的活性检测”近似，将不同浓度的2V1-SA1-3GS-Fc加入包被有不同抗原的ELISA板中，孵育1h后，加入识别Fc标签的检测抗体：HRP标记的Goat Anti-Human IgG抗体(购自：Abbkine，货号：A21050)，室温孵育45分钟，经洗板、TMB显色以及终止反应后，用酶标仪读取OD450。
结合VEGFA的ELISA数据如图25所示，EC50见表26。
表26 2V1-SA1-3GS-Fc与不同物种VEGFA结合的EC50或最大结合值
由上可知，2V1-SA1-3GS-Fc仅与人VEGFA165有较高结合活性，能弱结合兔VEGFA121，且不能结合小鼠、大鼠的VEGFA164。
利用原代细胞HUVEC增殖抑制实验，验证融合蛋白2V1-SA1-3GS-Fc在细胞水平的药效，即阻断VEGFA信号通路的活性，实验中阳性对照是上市药物Aflibercept，阴性对照是结合HIV病毒的人IgG1抗体(hIgG1control)。采用CellTiter-Glo发光型细胞活力实验检测活的HUVEC细胞数目。实验方法是：HUVEC(购自ScienCell，货号8000)的正常传代采用完全培养基：ECM+5％FBS+1％ECGS (购自ScienCell，3个试剂的货号分别是1001、0010、5352)。实验开始时，将待测蛋白用低血清培养基(ECM+1％FBS+0.05％ECGS)稀释到所需浓度：1000nM到0.0128nM，5倍浓度梯度(最终HUVEC细胞孵育时的抗体浓度为250nM到0.0032nM)。将抗体稀释液与200ng/mL的VEGFA165溶液等体积混合，放置在37℃的细胞培养箱中孵育2小时。将混合物加入到96孔板中(50μL/孔)，然后将HUVEC细胞用低血清培养基重悬，并以50μL/孔的体积转移到96孔板中(确保每孔种6×103个HUVEC细胞)。培养板中加入细胞、VEGF和蛋白稀释液的记为Cell+VEGF+Protein。同时设置对照组：仅有细胞且不加VEGF和蛋白(Cell only，预期细胞增殖最慢)，以及有细胞和VEGF且无蛋白(Cell and VEGF only，预期细胞增殖最快)。将96孔板放置在37℃、5％CO2的培养箱中培养3天。培养结束后，向每个孔中加入CellTiter-Glo试剂(购自Promega，货号：G7573)，并充分混合。通过酶标仪(Envision model，PerkinElmer)测量发光强度。IC50值是使用GraphPad Prism 9软件通过四参数非线性回归分析计算的。
实验结果如图26所示。拟合的IC50和最大抑制率见表27。抑制率计算公式为：Inhibition(％)＝{1-[(Cell+VEGF+Protein)-(Cell only)/(Cell and VEGF only)-(Cell only)]}*100
表27 HUVEC增殖抑制的IC50和最大抑制率
由此可知融合蛋白2V1-SA1-3GS-Fc抑制HUVEC增殖的IC50约为阳性对照Aflibercept的一半，表明，融合蛋白2V1-SA1-3GS-Fc能在细胞水平高效阻断VEGFA所激活的下游信号通路，抑制VEGFA刺激的HUVEC增殖，并且，融合蛋白2V1-SA1-3GS-Fc在细胞水平的活性强于阳性对照Aflibercept。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (22)

  1. 一种抗VEGFA融合蛋白，其特征在于，所述的抗VEGFA融合蛋白包括两个或两个以上抗VEGFA抗体或其抗原结合片段，以及Fc片段，所述的抗VEGFA抗体或其抗原结合片段包含重链可变区的CDR-H1、CDR-H2和CDR-H3；其中，
    CDR-H1的氨基酸序列包含SYTMG(SEQ ID NO：1)或与SYTMG(SEQ ID NO：1)具有至少80％同一性的氨基酸序列；
    CDR-H2的氨基酸序列包含AISKGGYKYX1X2VSLEA(SEQ ID NO：2)或与AISKGGYKYX1X2VSLEA(SEQ ID NO：2)具有至少80％同一性的氨基酸序列；
    CDR-H3的氨基酸序列包含TRAYGSSRLX3LAX4TYEY(SEQ ID NO：3)或与TRAYGSSRLX3LAX4TYEY(SEQ ID NO：3)具有至少80％同一性的氨基酸序列。
  2. 根据权利要求1所述的抗VEGFA融合蛋白，其特征在于，抗VEGFA抗体或其抗原结合片段与抗VEGFA抗体或其抗原结合片段之间，抗VEGFA抗体或其抗原结合片段与Fc片段之间直接连接或者间接连接。
  3. 根据权利要求2所述的抗VEGFA融合蛋白，其特征在于，抗VEGFA融合蛋白中抗VEGFA抗体或其抗原结合片段的N端、C端和/或内部残基与其他抗VEGFA抗体或其抗原结合片段或Fc片段的N端、C端和/或内部残基相连。
  4. 根据权利要求1-3任一所述的抗VEGFA融合蛋白，其特征在于，两个或两个以上抗VEGFA抗体或其抗原结合片段为完全相同序列的抗VEGFA抗体或其抗原结合片段、部分相同序列的抗VEGFA抗体或其抗原结合片段或完全不同序列的抗VEGFA抗体或其抗原结合片段。
  5. 根据权利要求1-4任一所述的抗VEGFA融合蛋白，其特征在于，SEQ ID NO：2中的X1X2代表DS、DA、NT、DT、NA或NS；SEQ ID NO：3中的X3代表R或K，X4代表D、N、E或K。
  6. 根据权利要求1-5任一所述的抗VEGFA融合蛋白，其特征在于，所述的CDR-H1、CDR-H2和CDR-H3的氨基酸序列包含下列任一组：
    A)SEQ ID NO：1、4、9；
    B)SEQ ID NO：1、5、9；
    C)SEQ ID NO：1、6、10；
    D)SEQ ID NO：1、4、11；
    E)SEQ ID NO：1、7、12；
    F)SEQ ID NO：1、6、11；
    G)SEQ ID NO：1、4、10；
    H)SEQ ID NO：1、5、12；
    I)SEQ ID NO：1、4、12；
    J)SEQ ID NO：1、8、12；
    K)SEQ ID NO：1、33、34。
  7. 根据权利要求1-6任一所述的抗VEGFA融合蛋白，其特征在于，所述的抗VEGFA抗体或其抗原结合片段为纳米抗体、嵌合抗体、Fab片段、Fab'片段、Fd片段、Fd'片段、Fv片段、dAb片段、分离的CDR区、F(ab')2片段、单结构域抗体、单链抗体分子或线性抗体。
  8. 根据权利要求1-7任一所述的抗VEGFA融合蛋白，其特征在于，所述的抗VEGFA抗体或其抗原结合片段的氨基酸序列包含SEQ ID NO：13-32、35-61中的任一氨基酸序列，或与SEQ ID NO：13-32、35-61中的任一氨基酸序列具有至少80％同一性。
  9. 根据权利要求1-8任一所述的抗VEGFA融合蛋白，其特征在于，所述的抗VEGFA融合蛋白的氨基酸序列包含SEQ ID NO：62-76、92-133、136-150和159中的任一氨基酸序列，或与SEQ ID NO：62-76、92-133、136-150和159中的任一氨基酸序列具有至少80％同一性。
  10. 一种嵌合抗原受体，其特征在于，所述的嵌合抗原受体的胞外结构域包含权利要求1-9任一所述的抗VEGFA融合蛋白。
  11. 一种编码权利要求1-9任一所述的抗VEGFA融合蛋白或权利要求10所述的嵌合抗原受体的核酸。
  12. 一种包含权利要求11所述的核酸的载体。
  13. 一种包含权利要求11所述的核酸或权利要求12所述的载体的细胞。
  14. 一种免疫细胞，其特征在于，所述的免疫细胞表达权利要求1-9任一所述的抗VEGFA融合蛋白或权利要求10所述的嵌合抗原受体。
  15. 一种抗VEGFA融合蛋白的制备方法，其特征在于，所述的制备方法包括将权利要求11所述的核酸导入宿主细胞，诱导其表达。
  16. 一种治疗和/或诊断疾病的产品，其特征在于，所述的治疗和/或诊断疾病的产品包含下列任一种：
    A)权利要求1-9任一所述的抗VEGFA融合蛋白；
    B)权利要求10所述的嵌合抗原受体；
    C)权利要求11所述的核酸；
    D)权利要求12所述的载体；
    E)权利要求13所述的细胞；或，
    F)权利要求14所述免疫细胞。
  17. 一种权利要求1-9任一所述的抗VEGFA融合蛋白、权利要求10所述的嵌合抗原受体、权利要求11所述的核酸、权利要求12所述的载体、权利要求13所述的细胞或权利要求14所述免疫细胞或权利要求16所述的治疗和/或诊断疾病的产品在阻断VEGFA信号通路中的应用；
    优选的，所述的应用包括：
    A)制备治疗和/或预防肿瘤的药物中的应用；
    B)制备治疗和/或预防眼科涉及血管新生的疾病(例如眼底血管疾病)的药物中的应用，所述的眼底血管疾病优选年龄相关性黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、糖尿病性视网膜病变、视网膜中央静脉阻塞、病理性近视、新生血管性青光眼等；和/或，
    C)抑制血管异常增生中的应用，优选阻断血管内皮细胞增殖。
  18. 一种治疗和/或预防疾病的方法，其特征在于，所述的方法包括向个体施加权利要求1-9任一所述的抗VEGFA融合蛋白、权利要求10所述的嵌合抗原受体、权利要求11所述的核酸、权利要求12所述的载体、权利要求13所述的细胞、权利要求14所述免疫细胞或权利要求16所述的治疗和/或诊断疾病的产品。
  19. 一种治疗和/或预防疾病的方法，所述的方法包括使权利要求1-9任一所述的抗VEGFA融合蛋白、权利要求10所述的嵌合抗原受体、权利要求11所述的核酸、权利要求12所述的载体、权利要求13所述的细胞、权利要求14所述免疫细胞或权利要求16所述的治疗和/或诊断疾病的产品与靶细胞接触。
  20. 根据权利要求19所述的方法，其特征在于，所述的靶细胞选自表达VEGFA的细胞，例如心肌细胞、近端肾小管细胞、肝细胞、血管内皮细胞、颗粒细胞、特化的上皮细胞、间充质细胞、巨噬细胞、血小板、树突状细胞、活化的T细胞、视网膜色素上皮细胞、视网膜中的Muller细胞、星形胶质细胞、成骨细胞、支气管与肺泡上皮细胞、周细胞、血管平滑肌细胞、肌成纤维细胞、角质形成细胞、肾系膜细胞或肿瘤细胞等等。
  21. 根据权利要求18-20任一所述的方法，其特征在于，所述的疾病为与VEGFA信号通路相关的疾病，优选可以为肿瘤、血管异常增生、眼科涉及血管新生的疾病。
  22. 一种阻断VEGFA介导的血管内皮细胞增殖或抑制血管新生的方法，其特征在于，所述的方法包括将血管内皮细胞与权利要求1-9任一所述的抗VEGFA融合蛋白、权利要求10所述的嵌合抗原受体、权利要求11所述的核酸、权利要求12所述的载体、权利要求13所述的细胞、权利要求14所述免疫细胞或权利要求16所述的治疗和/或诊断疾病的产品接触。
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