Anti-VEGFA (vascular endothelial growth factor A) antibody or antigen binding fragment thereof and application thereof

Abstract

The invention relates to an anti-VEGFA antibody or an antigen binding fragment thereof and application thereof, the anti-VEGFA antibody or the antigen binding fragment thereof has the advantages of high thermal stability, high water solubility, small molecular weight, simple expression and purification process and high affinity with VEGFA, and has the effects of effectively blocking a VEGFA signal channel, effectively blocking pathological processes such as vascular endothelial cell proliferation and angiogenesis caused by VEGFA, and improving the anti-VEGFA antibody or the antigen binding fragment thereof. The compound can be potentially applied to treatment of diseases related to VEGFA (vascular endothelial growth factor A).

Description

An anti-VEGFA antibody or its antigen-binding fragment and its application

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-VEGFA antibody or an antigen-binding fragment thereof and applications thereof.

[0002] Vascular endothelial growth factor A (VEGFA) is a cytokine closely related to angiogenesis. VEGFA binds to its receptors VEGFR1 and VEGFR2, activates the downstream signaling pathways of the two receptors, and promotes endothelial cell proliferation and angiogenesis.

[0003] Pathological angiogenesis occurs in a variety of solid tumors, inflammatory reactions, and retinal vascular diseases. In the later stages of tumor cell proliferation, new blood vessels are required to provide sufficient nutrients to support tumor growth and metastasis. Therefore, inhibiting angiogenesis is an important means of treating cancer. The first anti-VEGFA monoclonal antibody, Bevacizumab, was approved in 2004 for the treatment of metastatic colorectal cancer. Overexpression of VEGFA is also closely related to retinal vascular proliferation, such as wet age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy (DR). Therefore, anti-VEGFA drugs have become the first choice for the treatment of retinal vascular proliferation.

[0004] Currently, there are four main anti-VEGFA protein drugs on the market for the treatment of wet AMD, including Ranibizumab, Aflibercept, Conbercept, and Brolucizumab. In addition, Bevacizumab is also used off-label to treat wet AMD. These anti-VEGFA protein drugs include traditional monoclonal antibodies, antibody fragments Fab and scFv, and receptor-Fc fusion proteins. There are no single-domain antibodies (variable domain of heavy-chain antibody, VHH) or nanobody (Nb) drugs on the market.

[0005] 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 unit with complete antigen-binding function. The molecular weight of single-domain antibodies is about 13kDa, with high thermal stability and good water solubility. As a new type of antibody, the development of nanobodies has received more and more attention.

[0006] Therefore, the present invention provides a novel anti-VEGFA antibody or an antigen-binding fragment thereof, which is used to block the binding of VEGF to VEGFR1 and VEGR2, thereby inhibiting the VEGF downstream signaling pathway and inhibiting the cell proliferation stimulated by VEGF.

[0007]

[0008] In a first aspect, the present invention provides an anti-VEGFA antibody or an antigen-binding fragment thereof, wherein the anti-VEGFA antibody or the antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and / or CDR-H3 of the heavy chain variable region.

[0009] The amino acid sequence of CDR-H1 comprises SYTMG (SEQ ID NO: 1) or an amino acid sequence that is at least 80% identical to SYTMG (SEQ ID NO: 1);

[0010] The amino acid sequence of CDR-H2 comprises AISKGGYKYX1X2VSLEA (SEQ ID NO: 2) or an amino acid sequence that is at least 80% identical to AISKGGYKYX1X2VSLEA (SEQ ID NO: 2);

[0011] The amino acid sequence of CDR-H3 comprises TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3) or an amino acid sequence that is at least 80% identical to TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3).

[0012] Wherein, X in SEQ ID NO: 2 or SEQ ID NO: 3 can be any natural amino acid residue, such as alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), valine (V).

[0013] In a specific embodiment of the present invention, X1X2 in SEQ ID NO: 2 represents DS, DA, NT, DT, NA or NS; X3 in SEQ ID NO: 3 represents R or K, and X4 represents D, N, E or K.

[0014] In a specific embodiment of the present invention, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 include any of the following groups (see Table 1 for details):

[0015] A) SEQ ID NO: 1, 4, 9;

[0016] B) SEQ ID NO: 1, 5, 9;

[0017] C) SEQ ID NO: 1, 6, 10;

[0018] D) SEQ ID NO: 1, 4, 11;

[0019] E) SEQ ID NO: 1, 7, 12;

[0020] F) SEQ ID NO: 1, 6, 11;

[0021] G) SEQ ID NO: 1, 4, 10;

[0022] H) SEQ ID NO: 1, 5, 12;

[0023] I) SEQ ID NO: 1, 4, 12;

[0024] J) SEQ ID NO: 1, 8, 12;

[0025] K) SEQ ID NO: 1, 33, 34.

[0026] Table 1 Amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of candidate antibodies

[0027] Preferably, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are arranged in order from N-terminus to C-terminus. The amino acids in the antibody CDR region are divided according to the Kabat numbering system in the present application.

[0028] The anti-VEGFA antibody or antigen-binding fragment thereof comprises a heavy chain variable region, and a CH2 and / or CH3 region.

[0029] The structure of the anti-VEGFA antibody or its antigen-binding fragment is a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a F(ab')2 fragment, a single domain antibody, a single chain antibody molecule or a linear antibody.

[0030] The anti-VEGFA antibody or antigen-binding fragment thereof can be a dual-target specific antibody or an antibody with three or more targets (more than three, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more targets, etc.).

[0031] The anti-VEGFA antibody or antigen-binding fragment thereof may be a linear antibody.

[0032] The anti-VEGFA antibody or antigen-binding fragment thereof may be a single domain antibody or a nanobody.

[0033] The anti-VEGFA antibody or antigen-binding fragment thereof may be a humanized antibody or a fully human antibody.

[0034] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof comprises a humanized sequence, and the modification site of the humanized sequence is located in a non-CDR region. Further preferably, the humanized modification site is located in the framework region and / or constant region of the antibody.

[0035] In a specific embodiment of the present invention, the anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody. Compared with full-length IgG antibody, Fab, and scFv, at the same mass, the nanobody has a higher molar concentration and can bind to more antigen molecules.

[0036] The anti-VEGFA antibody or antigen-binding fragment thereof can bind to human or monkey VEGFA protein, wherein the human VEGFA protein has the same sequence as the monkey VEGFA protein.

[0037] Preferably, the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment comprises any one of SEQ ID NOs: 13-32, 35-61, 64-67, or has at least 80% identity with any one of SEQ ID NOs: 13-32, 35-61, 64-67.

[0038] In a specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof is shown in any one of SEQ ID NOs: 13-32, 35-61, 64-67.

[0039] The anti-VEGFA antibody or antigen-binding fragment thereof can inhibit or compete for the binding of other antibodies (preferably antibodies that bind to the same or overlapping epitopes as the anti-VEGFA antibody of the present invention) to human or monkey VEGFA protein.

[0040] The anti-VEGFA antibody or antigen-binding fragment thereof can be constructed by any conventional method in the prior art, such as artificial synthesis or eukaryotic expression or prokaryotic expression.

[0041] In a second aspect, the present invention provides an anti-VEGFA antibody or an antigen-binding fragment thereof.

[0042] The anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody.

[0043] The anti-VEGFA antibody or antigen-binding fragment thereof comprises any amino acid sequence of SEQ ID NO: 13-32, 35-61, 64-67, or has at least 80% identity with any amino acid sequence of SEQ ID NO: 13-32, 35-61, 64-67.

[0044] In a specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof is shown in any one of SEQ ID NOs: 13-32, 35-61, 64-67.

[0045] The third aspect of the present invention provides a method for screening anti-VEGFA antibodies or antigen-binding fragments thereof, wherein the method comprises immunizing alpacas with human or monkey VEGFA protein.

[0046] The fourth aspect of the present invention provides a fusion protein, wherein the fusion protein comprises the above-mentioned anti-VEGFA antibody or antigen-binding fragment thereof.

[0047] Preferably, the fusion protein further comprises other anti-VEGFA antibodies or other target antibodies or their antigen-binding fragments or other functional components in addition to the above-mentioned anti-VEGFA antibodies or their antigen-binding fragments.

[0048] Preferably, the functional components include, but are not limited to, serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and their markers, PEGylated components or Fc fragments, or a combination of two or more thereof.

[0049] Preferably, the other targets are selected from IgG, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Endoglin (CD105), TGF, Integrin, Integrin receptor, interleukin (such as IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptor (such as IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R , IL12R, IL15Rα, IL17R, IL23R, etc.), PCSK9, TNF-α, TNFR, RANKL, complement protein C3, complement protein C5, G protein-coupled receptor (GPCR), GLP1R, CD3, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, CD111, CD112, CD123, CD133, CD138, CD155, CD171, Claudin 18.2, OX40, ICOS, CTLA4, 4-1BB, TCR, B7-1, B7-2, BTLA, TIM-3, LAG3, Galectin-9, PD-L1, PD-L2, PD-1, TIGIT, EGFR, Her2, PSCA, CEA, F AP, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL 3 or 5T4.

[0050] The structure of the antibody or its antigen-binding fragment is a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a F(ab')2 fragment, a single domain antibody, a single chain antibody molecule or a linear antibody.

[0051] Preferably, the fusion protein comprises at least one anti-VEGFA antibody or an antigen-binding fragment thereof.

[0052] Preferably, the fusion protein comprises at least one other anti-VEGFA antibody or an antigen-binding fragment thereof.

[0053] Preferably, the fusion protein contains at least one antibody or antigen-binding fragment thereof or other functional components targeting other targets.

[0054] Among them, the anti-VEGFA antibodies or antigen-binding fragments thereof, between the anti-VEGFA antibodies or antigen-binding fragments thereof and other anti-VEGFA antibodies or antigen-binding fragments thereof, between the anti-VEGFA antibodies or antigen-binding fragments thereof and other target antibodies or antigen-binding fragments or other functional components, between other anti-VEGFA antibodies and other target antibodies or antigen-binding fragments or other functional components, between antibodies or antigen-binding fragments or other functional components of other targets, and between other anti-VEGFA antibodies or antigen-binding fragments thereof.

[0055] The indirect connection can be through a linker, a functional domain and / or a linker for coupling. Wherein, the linker is selected from a connecting peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, a nucleic acid, a polysaccharide, a fatty chain, biotin, streptavidin or avidin.

[0056] The functional domain is one or a combination of two or more of an Fc fragment, serum albumin, cytokine, transferrin, scaffold protein, VEGFA or an antibody or antigen-binding fragment thereof of other targets.

[0057] The linker used for coupling includes one or a combination of two or more of toxins, drugs, nucleic acids, PEG, radionuclides and markers thereof.

[0058] Preferably, the direct or indirect connection includes direct or indirect connection to the N-terminus, C-terminus and / or internal residues of the anti-VEGFA antibody or antigen-binding fragment thereof, other anti-VEGFA antibodies and / or other target antibodies.

[0059] The anti-VEGFA antibody or antigen-binding fragment thereof, other anti-VEGFA antibodies or other target antibodies contained in the fusion protein may be connected in the order that the N-terminus, C-terminus and / or internal residues of one antibody are connected to the N-terminus, C-terminus and / or internal residues of another antibody.

[0060] In a specific embodiment of the present invention, the anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies or other target antibodies contained in the fusion protein can be connected in the order of the N-terminus of one antibody to the N-terminus or C-terminus of another antibody.

[0061] In a specific embodiment of the present invention, the connection order of the anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies or other target antibodies contained in the fusion protein can be that the C-terminus of one antibody is connected to the N-terminus or C-terminus of another antibody.

[0062] In a specific embodiment, the fusion protein comprises an anti-VEGFA antibody and an Fc fragment.

[0063] Preferably, the fusion protein comprises any one of the amino acid sequences in SEQ ID NOs: 64-67, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NOs: 64-67.

[0064] Preferably, the fusion protein further comprises a tag.

[0065] Preferably, the tag is connected to the C-terminus of the fusion protein.

[0066] In a fifth aspect, the present invention provides a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises the above-mentioned anti-VEGFA antibody or an antigen-binding fragment thereof.

[0067] Preferably, the chimeric antigen receptor further comprises any transmembrane region and / or intracellular signal transduction region conventional in the prior art.

[0068] In a sixth aspect, the present invention provides a nucleic acid encoding the above-mentioned anti-VEGFA antibody or antigen-binding fragment thereof, the above-mentioned fusion protein or the above-mentioned chimeric antigen receptor.

[0069] The seventh aspect of the present invention provides a vector, wherein the vector comprises the above-mentioned nucleic acid.

[0070] The vector can be expressed in vivo, in vitro or in vitro. Preferably, the vector is a prokaryotic expression vector, a viral expression vector or a eukaryotic expression vector, such as an E. coli series vector, a bacteriophage, etc.

[0071] The eighth aspect of the present invention provides a host cell, wherein the host cell comprises the above-mentioned nucleic acid or the above-mentioned vector.

[0072] The host cell may be a eukaryotic cell or a prokaryotic cell.

[0073] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells or CHO cells, etc.

[0074] Prokaryotic cells such as Escherichia coli.

[0075] The ninth aspect of the present invention provides a method for preparing a host cell, which comprises introducing the above-mentioned nucleic acid or vector into the host cell and then inducing its expression.

[0076] The tenth aspect of the present invention provides a method for preparing an anti-VEGFA antibody or an antigen-binding fragment thereof, the preparation method comprising introducing a nucleic acid or a vector encoding the anti-VEGFA antibody or an antigen-binding fragment thereof into a host cell, and then inducing its expression.

[0077] In an eleventh aspect of the present invention, an immune cell is provided, wherein the immune cell expresses the above-mentioned anti-VEGFA antibody or antigen-binding fragment thereof or the above-mentioned chimeric antigen receptor.

[0078] Preferably, the immune cells include but are not limited to lymphocytes (eg, T cells, B cells, NK cells), dendritic cells, monocytes / macrophages, granulocytes, and mast cells.

[0079] Preferably, the immune cells are CAR-immune cells.

[0080] The twelfth aspect of the present invention provides a method for constructing an immune cell, the method comprising transfecting a nucleic acid sequence encoding the chimeric antigen receptor of the present invention into an immune cell for expression.

[0081] In a thirteenth aspect of the present invention, there is provided a product for treating and / or diagnosing a disease, wherein the product for treating and / or diagnosing a disease comprises any one of the following:

[0082] A) the above-mentioned anti-VEGFA antibody or antigen-binding fragment thereof;

[0083] B) the above fusion protein;

[0084] C) the chimeric antigen receptor described above;

[0085] D) Immune cells mentioned above

[0086] E) the above nucleic acid;

[0087] F) the vector described above; or,

[0088] G) The host cell described above.

[0089] The product for treating and / or diagnosing the disease targets cells expressing VEGFA, and the cells may be cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular 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 and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

[0090] Preferably, the product can be a kit, a drug, a chip, or an antibody-drug conjugate, etc. The disease is a disease related to the VEGFA signaling pathway. Further preferably, it can be a tumor, abnormal vascular proliferation, angiogenesis-related ophthalmic disease (such as fundus vascular disease), etc.

[0091] In a fourteenth aspect, the present invention provides an antibody-drug conjugate (ADC), which comprises the anti-VEGFA antibody or antigen-binding fragment thereof of the present invention covalently bound to a drug.

[0092] In a fifteenth aspect, the present invention provides a method for detecting VEGFA, which comprises contacting a sample to be tested with the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, and then detecting the content of a complex formed by VEGFA and the anti-VEGFA antibody or its antigen-binding fragment.

[0093] The detection method is to detect the presence or content of VEGFA, wherein the presence refers to the presence or absence, and the content can be the expression level or protein concentration, etc.

[0094] In the sixteenth aspect of the present invention, a method for diagnosing a disease is provided, which comprises taking a sample, contacting the sample with the anti-VEGFA antibody or its antigen-binding fragment, the chimeric antigen receptor, the fusion protein, the nucleic acid, the vector, the host cell, the immune cell or the product for treating and / or diagnosing the disease, and detecting the content of the complex formed by VEGFA and the anti-VEGFA antibody or its antigen-binding fragment.

[0095] The disease is a disease related to the VEGFA signaling pathway, and more preferably may be a tumor, abnormal vascular proliferation, angiogenesis-related ophthalmic disease (eg, fundus vascular disease), and the like.

[0096] In the seventeenth aspect of the present invention, a method for treating and / or preventing a disease is provided, which comprises applying the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease to an individual.

[0097] The disease is a disease related to the VEGFA signaling pathway, and more preferably may be a tumor, abnormal vascular proliferation, angiogenesis-related ophthalmic disease (eg, fundus vascular disease), and the like.

[0098] In the eighteenth aspect of the present invention, a method for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis is provided, the method comprising contacting vascular endothelial cells with the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease.

[0099] Preferably, the method comprises diluting the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease with a serum-free culture medium of vascular endothelial cells, and then incubating with the antigen (e.g., VEGFA165).

[0100] Preferably, after incubation, the method comprises a step of discarding the complete culture medium of the vascular endothelial cells in the culture plate.

[0101] Preferably, the antigen and the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease are added to a vascular endothelial cell culture plate and cultured on the culture plate.

[0102] Preferably, the detection is performed after culturing.

[0103] Preferably, the incubation time is 0.5-5 h, preferably 1-3 h, for example 0.5, 1, 2, 3, 4, 5 h.

[0104] Preferably, the incubation temperature is room temperature-45°C, preferably 30-40°C, for example 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc.

[0105] Preferably, 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. Preferably, the culture is carried out in an incubator with 5% CO2.

[0106] Preferably, the culture time is 1-5 days, preferably 2-4 days, for example 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, 5 days, etc.

[0107] Preferably, the detection is the detection of the number of live vascular endothelial cells.

[0108] In the nineteenth aspect of the present invention, a method for treating and / or preventing a disease is provided, which method comprises using the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease to contact with target cells.

[0109] The disease is a disease related to the VEGFA signaling pathway, and more preferably may be a tumor, abnormal vascular proliferation, angiogenesis-related ophthalmic disease (eg, fundus vascular disease), and the like.

[0110] Preferably, the target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular 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 and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

[0111] The twentieth aspect of the present invention provides an application of the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned immune cell in the preparation of a product for treating and / or preventing a disease associated with VEGFA, or in the preparation of a product for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, or in the preparation of an antibody-drug conjugate or an antibody diagnostic kit or a tracer.

[0112] The disease may be a tumor, abnormal vascular proliferation, an ophthalmic disease involving angiogenesis (eg, fundus vascular disease), and the like.

[0113] The product can be a kit, a drug, a chip, an antibody-drug conjugate, etc.

[0114] The present invention provides a new anti-VEGFA antibody or antigen-binding fragment thereof, which has high thermal stability, high water solubility, small molecular weight, simple expression and purification process, high affinity with VEGFA, and can effectively block the VEGFA signaling pathway, effectively block the pathological processes such as vascular endothelial cell proliferation and angiogenesis caused by VEGFA, and can be potentially used in the treatment of diseases related to VEGFA. It may also bring greater clinical value, such as better tissue penetration, lower production costs, and more convenient drug administration methods.

[0115] The "medicine" of the present invention can be used to treat humans or non-human animals, such as non-human mammals. The medicine can contain pharmaceutically acceptable carriers, excipients or salts common in the prior art. The medicine can be administered by any suitable route, such as gastrointestinal administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, rectal, etc.). The medicine can be in any suitable dosage form, such as a gastrointestinal dosage form or a parenteral dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, drop pills, gels, etc. The various dosage forms of the medicine can be prepared according to conventional production methods in the pharmaceutical field. The drug may contain 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 antibody or its antigen-binding fragment, the nucleic acid, the vector, the host cell, the immune cell, etc. by weight. The drug can be prepared as a reagent with a protein concentration of 1-300 mg / mL (e.g., 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, 300 mg / mL). The single dose of the drug can be 0.1-1000 mg, for example 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, 1000 mg.

[0116] The term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0117] The "method" described in the present invention can be used for the purpose of diagnosis and treatment of diseases, or for the purpose of diagnosis and treatment of non-diseases.

[0118] 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, Fab', F(ab')2, Fv, Fd and variants of these fragments. For example, the heavy chain and / or light chain of an antibody, the heavy chain variable region and / or light chain variable region of an antibody, or a single or more than two CDRs from the heavy chain or light chain of an antibody. Among them, Fab, that is, a monovalent fragment consisting of VL, VH, CL and CH1 domains. F(ab')2, that is, a divalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region. Fd, an Fd fragment consisting of VH and CH1 domains. Fv, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody. Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH is a Fab' in which the cysteine ​​residue of the constant domain carries at least one free thiol group. Among them, VH represents the heavy chain variable region, VL represents the light chain variable region, and CL represents the light chain.

[0119] The "CH2" or "CH3" described in the present invention refers to the CH2 or CH3 of the heavy chain constant region. The complete heavy chain constant region is composed of three domains: CH1, CH2 and CH3. Specifically, the CH2 domain refers to the antibody heavy chain polypeptide portion extending from about EU position 231 to EU position 340 (according to Kabat's EU numbering system). The uniqueness of the CH2 domain is that it is not closely paired with another domain. The CH3 domain refers to the antibody heavy chain polypeptide portion extending from about EU position 341 to EU position 446.

[0120] The "Fc" region of the present invention contains two heavy chain fragments including the CH2 and CH3 domains of an antibody. The two heavy chain fragments form dimers by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.

[0121] The "linear antibody" described in the present invention comprises a pair of tandem Fd segments (VH-CH1-VH-CH1).

[0122] The words “comprising” or “including” described in the present invention are open-ended expressions. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0123] The "homology" mentioned in the present invention refers to that in terms 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 have (including but not limited to) 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%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 9%, 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% homology.

[0124] The "humanized antibody" of the present invention refers to the framework region and / or constant region portion (such as CH region) of the antibody or the entirety of the antibody encoded by human antibody genes. In a specific embodiment of the present invention, the CDR region of the antibody has not been modified.

[0125] The "individual" described 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. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0126] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0127] The term "prevention" as used herein refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in an organism.

[0128] The term "diagnosis" as used herein refers to finding out whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression of a disease or its possible future progression.

[0129] The tumor of the present invention can be any undesirable cell proliferation (or any disease that manifests itself as undesirable cell proliferation), neoplasm, or increased tendency or risk of undesirable cell proliferation, neoplasm or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). Neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblastic cells, 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, leukocytes. Further preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., 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 carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, neurilemoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0130] The "retinal vascular disease" mentioned in the present invention refers to a general term for diseases occurring in the retinal arteries or veins, or diseases related to choroidal angiogenesis, including but not limited to age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc.

[0131] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0132] Figure 1: Gel staining of candidate antibodies purified from different batches.

[0133] Figure 2: Signals of different candidate antibodies binding to VEGFA change with antibody concentration.

[0134] Figure 3: Competition ELISA results of different candidate antibodies.

[0135] Figure 4: Inhibition rate of HUVEC cell proliferation by different candidate antibodies.

[0136] Figure 5: ELISA detection of the activity of antibody V1 binding to human VEGFA121.

[0137] Figure 6: ELISA detection of antibody V1 binding activity to mouse VEGFA164.

[0138] Figure 7: ELISA detection of antibody V1 binding activity to rat VEGFA164.

[0139] Figure 8: Cootistained gel image after purification of humanized antibodies.

[0140] Figure 9: ELISA test of humanized antibody binding to VEGFA.

[0141] Figure 10: ELISA test of humanized antibodies competing for VEGFR2.

[0142] FIG. 11 : ELISA test of humanized antibodies V30 and V43 competing for VEGFR2.

[0143] FIG. 12 : Binding and dissociation curves (SPR) of V1-SA1 and VEGFA165.

[0144] Figure 13: Melting temperatures (Tm) of different humanized antibodies.

[0145] Figure 14: SDS-PAGE gel image of humanized antibody V1-SA1 and positive control antibody BI-VEGF ab after purification.

[0146] Figure 15: Cow staining gel image after purification of monovalent nanobody-Fc fusion protein.

[0147] Figure 16: ELISA test of monovalent Nanobody-Fc fusion protein binding to VEGFA.

[0148] FIG. 17 : Binding and dissociation curves (SPR) of V1-SA1-Fc-m1 and VEGFA165.

[0149] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0150] Example 1: Alpaca immunization and antibody screening

[0151] 1. Alpaca Immunity

[0152] Human VEGFA165 (unlabeled, purchased from Sino Biological, catalog number: HPLC-10008-HNAH, hereinafter referred to as VEGFA) was emulsified and immunized to alpacas. Two alpacas were selected, and each animal was immunized with 1 mg of VEGFA protein each time, once every 2 weeks. Starting from the second immunization, serum titer was tested. Among them, alpaca 1# was immunized with antigen 4 times, and alpaca 2# was immunized with antigen 3 times. After serum titer detection, both reached the library construction standard.

[0153] 2. Competitive activity detection of post-immunization serum blocking the binding of VEGFA and VEGFR2

[0154] The VEGF receptor extracellular region VEGFR2-ECD was biotin labeled (named VEGFR2-biotin), VEGFA was diluted to 0.5 μg / mL with CBS buffer, coated with ELISA plate, and incubated at 4°C overnight. After blocking with 3% skim milk powder, the plate was washed, and the alpaca serum and negative serum (alpaca serum before antigen immunization) were diluted 5 times, 15 times, 45 times, 135 times, 405 times, 1215 times, and 3645 times, respectively, and diluted to a final concentration of 0.5 μg / mL VEGFR2-biotin solution, and then 100 μL was added to the blocked wells, placed at room temperature for 1 hour, PBST (PBS containing 0.1% Tween20, pH7.4) was used to wash the plate 3 times, streptavidin-HRP was added, placed at room temperature for 1 hour, PBST was used to wash the plate 3 times, TMB was added for color development for 15 minutes, stop solution was added, and the absorbance at a wavelength of 450 nm was read with an ELISA reader. The results of the serum competition activity test of the two alpacas are shown in Table 2-3.

[0155] Table 2 Competitive activity test results of 1# alpaca serum

[0156] Table 3 Competitive activity test results of 2# alpaca serum

[0157] The results showed that antibodies that blocked the binding of VEGFA and VEGFR2 were detected in both 1# and 2# alpacas, and could be used for library construction.

[0158] 3. Antibody library construction

[0159] 40 mL of alpaca peripheral blood was drawn using a blood collection needle. After lysing the red blood cells according to the instructions of the red blood cell lysis solution, the white blood cells were collected and added to trizol for freezing. Before building the library, trizol was used to extract total RNA and reverse transcription of cDNA from the separated alpaca PBMC, and the variable region VH fragment was amplified using nanoantibody-specific primers. The above-obtained fragments and the pcomb3X vector were digested with SfiI enzyme, and after digestion, they were mixed in appropriate proportions and connected with T4 ligase. After connection, it was used for electroporation of XL1-Blue competent cells. According to the growth of the colonies, the transformation library capacity of the two alpacas was calculated to be 6.52×10 8 and 4.12×10 8 The clones were randomly picked from the transformant colonies and sent for sequencing verification, and the sequencing results showed the correct nanobody sequence.

[0160] 4. Antibody library screening

[0161] The phage display method was used to screen phages that could bind to VEGFA.

[0162] For the 1# alpaca library, after 4 rounds of enrichment screening, a single clone from 1 plate of 96 wells was picked (the screening method was recorded as VEGF-4 th At the same time, the fourth round of phages were subjected to competitive elution: 10 μg / mL VEGFR2 extracellular domain VEGFR2-ECD-His (prepared in-house) was used to competitively elute the phages that bound to VEGF, and it was expected that antibodies that could compete with VEGFR2 for binding to VEGFA were obtained. Five 96-well plates were picked for the obtained phages to detect the binding activity of phages to VEGFA (the screening method was recorded as VEGF-5 th In addition, the phages obtained from the VEGFR2 competitive screening were further subjected to competitive elution screening with the control monoclonal antibody Bevacizumab (Avastin), that is, 10 μg / mL Bevacizumab antibody was used to competitively elute the phages that bind to VEGFA, and a 96-well plate was picked for the obtained phages to detect the binding activity of the phages to VEGFA (the screening method was recorded as VEGF-6 th Bevacizumab competition), the phage was detected by using an antibody that recognizes the phage coat protein labeled with HRP, labeled as anti-M13 (HRP). The detection results of positive clones with VEGFA binding activity among the above monoclonal clones are shown in Table 4.

[0163] Table 4 Detection results of positive clones binding to VEGFA activity after screening of 1# alpaca library

[0164] In addition, biotin-labeled VEGFA was used to screen phages that bind to more epitopes. The method was as follows: Avidin was first coated, and then biotinylated VEGFA was incubated. After merging the 1# alpaca library with the 2# alpaca library, the phages that bind to VEGFA were obtained by incubating and eluting the library with biotinylated VEGFA, and the process was repeated. After multiple rounds of enrichment, a 96-well plate monoclonal colony was screened. The phages in the supernatant were tested for VEGFA binding activity by ELISA, and the results of positive clones with binding activity are shown in Table 5.

[0165] Table 5 Results of screening positive clones for binding to VEGFA activity after library merging

[0166] 5. Preliminary screening of antibody sequences

[0167] After plasmid sequencing and codon translation of the above-mentioned phage with VEGFA binding activity, the amino acid sequence of the candidate nanoantibody (CDR region + framework region) was obtained. Antibodies with the same sequence were combined, and candidate antibodies with different CDR sequences are shown in Table 6.

[0168] Table 6 Amino acid sequences of candidate antibodies

[0169] 6. Expression and purification of candidate antibodies

[0170] Some candidate antibodies in the above table were selected, expressed in mammalian cells, purified, and then subjected to ELISA testing to repeatedly verify the antibody's VEGFA binding activity.

[0171] The coding gene of each nanobody was amplified from the original phage plasmid by PCR and constructed into the pVRC8400 expression vector, wherein the secretory peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 68) was added to the N-terminus of the antibody, and the flexible linker GGGGS (SEQ ID NO: 69) and the label 6×His were added to the C-terminus of the antibody in sequence. At the same time, the coding gene sequence of the positive control antibody BI-VEGF ab was synthesized (the antibody is an anti-VEGFA nanobody, and the antibody amino acid sequence is from SEQ ID NO: 57 in patent US 9527925B2), and the label 6×His was added to the C-terminus of the control antibody BI-VEGF ab. After the expression plasmids of each antibody were sequenced correctly, a large amount of endotoxin-free extraction was performed. The 293F suspension cells were transiently transfected with the transfection reagent polyethylenimine (PEI, linear, MW = 25 kDa, purchased from Polysciences, catalog number: 23966-1). The cell supernatant was collected on the 4th or 5th day after transfection and Ni bead affinity purification was performed. The purified protein was analyzed by SDS-PAGE, and the molecular weight was in line with expectations (~15 kDa). The positive control antibody BI-VEGF ab and some candidate antibodies after purification were shown in Figure 1.

[0172] Example 2: Antibody activity test

[0173] 1. Activity test of candidate antibodies binding to VEGFA

[0174] The antigen VEGFA was diluted with ELISA coating buffer (final concentration was 0.3 μg / mL), added to the ELISA plate, 100 μL / well, and coated at 4°C overnight. After blocking with 5% skim milk powder, the positive control antibody BI-VEGF ab (the antibody is an anti-VEGFA nanobody, the antibody amino acid sequence is from SEQ ID NO: 57 in patent US 9527925B2, and the construction method of its expression plasmid and the protein purification method are the same as the step "6. Expression and purification of candidate antibodies" in Example 1) and the gradient dilution of the candidate antibody to be tested (0.001, 0.01, 0.1, 1, 3, 10, 100 nM) were added, and incubated at 37° C. for 1 hour. After washing the plate, His-Tag monoclonal antibody dilution (purchased from: Proteintech, item number: 66005-1-Ig) was added, and incubated at 37° C. for 1 hour. After washing the plate, HRP-conjugated Affinipure Goat Anti-Mouse IgG antibody dilution (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), purchased from Proteintech, catalog number: SA00001-1), incubated at room temperature for 45 minutes. After washing, 100 μL TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using an enzyme-labeled instrument.

[0175] The changes in signals of some antibodies binding to VEGFA as a function of antibody concentration are shown in FIG2 .

[0176] The VEGFA binding data in the ELISA test were fitted to obtain the EC50 of the antibody binding to VEGFA, as shown in Table 7. It can be seen that all the 10 candidate antibodies in the table have high VEGFA binding activity.

[0177] Table 7 VEGFA binding activity detection of candidate antibodies

[0178] The amino acid residues in the amino acid sequences of the above-mentioned antibody CDR regions are partially different, and the antibody activity fluctuates within an acceptable range.

[0179] In addition, the affinity of the nanobody to VEGFA was tested using surface plasmon resonance (SPR) technology. The Biacore 8K instrument (Cytiva) was used, with the antigen VEGFA165 (unlabeled, purchased from Sino-Biological, catalog number: HPLC-10008-HNAH) as the ligand, coupled to the CM5 chip through amino coupling reagents (EDC and NHS), and the coupling amount was about 400RU. During the experiment, 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 were the candidate antibodies and the positive control antibody BI-VEGF ab, and the dilution concentrations were: 80nM, 40nM, 20nM, 10nM, 5nM, 2.5nM. In the kinetic analysis, the binding time of the antigen and the antibody was 240 seconds, the dissociation time was 1500 seconds, and the flow rate was 30 μL / min. The multi-cycle mode was adopted, and the chip was regenerated by 100 mM hydrochloric acid. The binding and dissociation curves of the candidate antibody and VEGFA165 were fitted with the "1:1 binding" model to obtain the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD), as shown in Table 8.

[0180] Table 8 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of candidate antibodies binding to VEGFA165

[0181] As can be seen from Table 8, the candidate antibody has a high affinity to VEGFA165, and the KD value is within 3 times of the positive control, so it is believed that the affinity of the candidate antibody is comparable to that of the positive control protein.

[0182] 2. Competitive VEGFR2 activity test

[0183] To further verify the activity of candidate antibodies in blocking VEGFA binding to receptor VEGFR2, a competitive ELISA was performed.

[0184] Antigen VEGFA was diluted with ELISA coating buffer (final concentration of 0.53 μg / mL) and added to a 96-well ELISA plate at 100 μL / well for overnight coating at 4°C. After blocking with 5% skim milk powder, VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from Sino Biological, catalog number: 10012-H02H) with a final concentration of 1 nM and gradient dilutions of the antibody to be tested (0.01, 0.1, 1, 3, 10, 100 nM) were mixed evenly and added to a 96-well plate, incubated at 37°C for 1 hour, and after washing, HRP-conjugated Goat Anti-Human IgG antibody dilution (HRP-conjugated Goat Anti-Human IgG, purchased from Abbkine, catalog number: A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 μL TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well, and after color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using an ELISA reader. The competitive ELISA results are shown in Figure 3.

[0185] The comparison of antibody competition activity for VEGFR2 (IC50 value and maximum inhibition rate) is shown in Table 9. It can be seen that the 12 candidate antibodies in the table can compete with VEGFR2 for binding to VEGFA. The maximum inhibition rate calculation formula is:

[0186] Table 9 Activity of candidate antibodies in competing for VEGFR2 (IC50 values ​​and maximum inhibition rates)

[0187] 3. Activity of candidate antibodies in inhibiting HUVEC cell proliferation

[0188] Candidate antibodies V1, V13, V15 and V30 were validated to block VEGFA-stimulated proliferation of human umbilical vein endothelial cells (HUVEC).

[0189] Primary HUVEC cells (derived from China Center for Type Culture Collection (CCTCC)) were digested and diluted to 5×10^4 cell / mL, inoculated in 96-well plates at 100 μL / well in complete culture medium (F-12K+0.1 mg / mL heparin+ECGS+10% FBS), and treated with drugs after 5 hours of adherent culture.

[0190] A mixture of VEGFA and candidate antibody dilutions was prepared using DMEM / F12 (HAM) 1:1 culture medium (serum-free, supplemented with penicillin and streptomycin, containing glutamine, purchased from Biological Industries, catalog number: 01-172-1ACS), wherein the final concentration of VEGFA in each dilution was 35 ng / mL, and the concentration gradient of the candidate antibody V1 was 1000000 pM, 33333.33 pM, 111111.11 pM, 37037.04 pM, 12345.68 pM, 4115.23 pM, 1371.74 pM, 457.25 pM, 152.42 pM, 50.81 pM, 16.94 pM, and 5.65 pM. The concentration gradients of V13, V15, and V30 are 1000000pM, 33333.33pM, 111111.11pM, 37037.04pM, 12345.68pM, 4115.23pM, 1371.74pM, 457.25pM, 152.42pM, and 5.65pM.

[0191] The negative control antibody (Negative control) is a nanobody that binds to an irrelevant antigen, and its concentration gradient is (3-fold decreasing): 1000000 pM, 33333.33 pM, 111111.11 pM, 37037.04 pM, 12345.68 pM, 4115.23 pM, 1371.74 pM, 457.25 pM, and 152.42 pM.

[0192] At the same time, controls without VEGFA (minimum cell proliferation) and without any antibody containing only VEGFA (maximum cell proliferation) were set up. The mixture of VEGFA and each antibody dilution was incubated at 37°C for 1 hour. After the original culture medium in the 96-well cell plate was aspirated, the mixture of VEGFA and each antibody dilution was added to the cell plate. Two cell wells were set for each concentration. Cultured in a 37°C cell culture incubator for 72 hours. CCK-8 solution (purchased from: Solebao, catalog number: CA1210) was added to the 96-well plate, the culture plate was incubated in the incubator for 3 hours, and the absorbance (OD450) at 450nm was measured with an enzyme reader.

[0193] According to the difference in readings of the control wells (without VEGFA and only containing VEGFA without any antibody), the inhibition rate of cell proliferation by the candidate antibody at different concentrations was calculated. The calculation formula of the inhibition rate is:

[0194] The inhibition rate curves of each antibody on HUVEC cell proliferation, as well as the maximum inhibition rate and IC50 are shown in FIG4 and Table 10.

[0195] Table 10 Maximum inhibition rate and IC50 value of each antibody on HUVEC cell proliferation

[0196] Therefore, all four candidate antibodies can inhibit the VEGFA signaling pathway at the cellular level, that is, inhibit the proliferation of HUVEC cells stimulated by VEGFA. At the highest antibody concentration (1 μM), candidate antibody V1 can effectively inhibit HUVEC proliferation (the maximum inhibition rate is close to 100%). From the perspective of IC50, candidate antibody V30 is relatively more active.

[0197] ELISA tests for binding to VEGFA and ELISA tests for competing with VEGFR2, as well as activity detection for inhibiting HUVEC proliferation at the cellular level, showed that the candidate antibody can efficiently bind to VEGFA and block VEGFA from binding to VEGFR2, thereby inhibiting the VEGFA-VEGFR signaling pathway and inhibiting the proliferation of vascular endothelial cells.

[0198] 4. Activity detection of candidate antibodies binding to human VEGFA121 and mouse VEGFA164

[0199] An ELISA experiment was performed using the same method as in "1. Activity test of candidate antibodies binding to VEGFA" in this example, i.e., 0.3 μg / mL of human VEGFA121 (purchased from Sino Biological, Catalog No.: 10008-HNAH) or mouse VEGFA164 (purchased from Sino Biological, Catalog No.: 50159-MNAB), or rat VEGFA164 (purchased from Sino Biological, Catalog No.: 80006-RNAB) was coated to detect the activity of His-tagged candidate antibody V1 binding to human VEGFA121, mouse VEGFA164 and rat VEGFA164.

[0200] The activity of antibody V1 in binding to human VEGFA121 was detected by ELISA, as shown in FIG5 and Table 11.

[0201] Table 11 Activity of antibody V1 binding to human VEGFA121

[0202] The binding activity of antibody V1 to mouse VEGFA164 is shown in FIG6 .

[0203] The binding activity of antibody V1 to rat VEGFA164 is shown in FIG7 .

[0204] In summary, the candidate antibody V1 has a high activity in binding to human VEGFA121.

[0205] 5. Tm value detection of candidate antibodies

[0206] Eight candidate antibodies (V1, V15, V29, V30, V31, V36, V40, V43) were selected, and the buffer in which the antibodies were placed was replaced with: 10 mM His, pH=6.5, 40 mM NaCl, 5% sucrose, 0.01% Tween-20, and the melting temperature (Tm) of the antibodies was measured using a UNCLE instrument (unchainedlabs). The results are shown in Table 12. The results show that these candidate antibodies all have a certain degree of thermal stability.

[0207] Table 12 Melting temperature Tm of candidate antibodies

[0208] Example 3: Humanization of anti-VEGFA nanobody

[0209] The candidate antibody V1 was used as the starting antibody for humanization. The candidate antibody V1 sequence was first compared with the human antibody sequence homology through the antibody databases IGBLAST and IMGT, and then the human antibody framework region with high homology was combined with the CDR region of the candidate antibody to construct a complete humanized antibody. The method for constructing the expression plasmid of the humanized antibody and the protein purification method are the same as the step "6. Expression and purification of candidate antibodies" in Example 1.

[0210] The purified antibodies were tested for their binding activity to VEGFA using the ELISA method. After the initial humanization of V1, the antibody's binding activity to VEGFA was greatly reduced, and a few amino acids in the framework region were restored to mutation to obtain 9 optimized V1 humanized antibodies, whose amino acid sequences are shown in Table 13.

[0211] Table 13 Amino acid sequences of humanized candidate antibodies

[0212] Transient expression and purification were performed on these restored mutant humanized antibodies. The experimental method of the first step of purification was the same as the step "6. Expression and purification of candidate antibodies" in Example 1. Among them, the positive control BI-VEGF ab and humanized antibodies V1-DP and V1-SA1 were purified by Ni bead affinity purification and then purified by cation exchange chromatography (S column). The SDS-PAGE stained gel after partial protein purification is shown in Figure 8 (A), and the molecular weight of the target protein is in line with expectations (~15kDa).

[0213] The CDR regions of candidate antibodies V13, V15, V30, V40, and V43 were transplanted to the framework region after the humanization restoration mutation of V1, and these candidate antibodies were humanized, expressed, and purified. The partially purified humanized antibodies are shown in Figure 8 (B) (C) (D), and the molecular weight of the target protein is consistent with the expectation (~15 kDa).

[0214] Example 4: Activity detection of humanized anti-VEGFA nanobody

[0215] The obtained humanized antibodies were tested for VEGFA binding activity using the ELISA method, and the experimental method was the same as "1. Activity test of candidate antibodies binding to VEGFA" in Example 2. The ELISA test results of some humanized antibodies binding to VEGFA are shown in FIG9 .

[0216] The VEGFA binding data in the ELISA test were fitted to obtain the EC50 of the humanized antibody binding to VEGFA, as shown in Table 14. The data showed that after humanization, the activity of the candidate antibody in binding to VEGFA was similar to that before humanization (V40).

[0217] Table 14 EC50 values ​​of humanized antibodies binding to VEGFA

[0218] To further verify the activity of humanized antibodies in blocking VEGFA binding to receptor VEGFR2, competitive ELISA was performed. The experimental method is similar to "2. Competitive VEGFR2 activity test" in Example 2, except that for humanized antibodies of V1, V13, V15 and V43, the final concentration of VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from: Sino Biological, Cat. No.: 10012-H02H) is 2 nM. The concentration gradient of V1 humanized antibody is: 0.01, 0.1, 1, 10, 30, 100, 1000 nM. The concentration gradient of V13, V15 and V43 humanized antibodies is: 0.03, 0.3, 3, 10, 30, 300 nM.

[0219] The results showed that the humanized antibodies of V1 and V15 competed with VEGFR2 for binding to VEGFA, respectively, and restored to the activity before humanization (i.e., V1 and V15), and the remaining humanized antibodies showed similar competitive VEGFR2 activity. The competition ELISA results of some humanized antibodies are shown in Figure 10, and the IC50 and maximum inhibition rate are shown in Table 15. The maximum inhibition rate calculation formula is:

[0220] Table 15 Competitive ELISA results and maximum inhibition rate of humanized antibodies

[0221] In addition, ELISA tests for blocking VEGFR2 were performed on the humanized antibodies of V30 and the rest of the humanized antibodies of V43. The experimental method is similar to "2. Competitive VEGFR2 activity test" in Example 2, except that the final concentration of the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from Sino Biological, Cat. No.: 10012-H02H) is 1 nM. The concentration gradient of the humanized antibodies is: 0.03, 0.3, 3, 10, 30, 300 nM.

[0222] The ELISA test results of humanized antibodies V30 and V43 competing for VEGFR2 are shown in FIG11 .

[0223] The activity of the antibody in competing with VEGFR2 (IC50 value and maximum inhibition rate) is shown in Table 16. It can be seen that the humanized antibody can compete with VEGFR2 for binding to VEGFA, and the activity is similar to that before humanization (V30). The maximum inhibition rate calculation formula is:

[0224] Table 16 Antibody Competition for VEGFR2 Activity (IC50 Value and Maximum Inhibition Rate)

[0225] Using surface plasmon resonance (SPR) technology, coupled with antigen VEGFA165, the affinity of the nanobody to VEGFA before and after humanization was tested. The experimental method and data fitting method are the same as "1. Activity test of candidate antibodies binding to VEGFA" in Example 2. The obtained KD is shown in Table 17.

[0226] Table 17 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of humanized antibodies to VEGFA

[0227] In addition, the affinity of the humanized antibody V1-SA1 to VEGFA was tested by coupling the antibody using surface plasmon resonance (SPR) technology. The antibody V1-SA1 was used as a ligand and coupled to the CM5 chip through amino coupling reagents (EDC and NHS) using a Biacore 8K instrument (Cytiva): the coupling conditions were 10mM sodium acetate (pH4.0) and the V1-SA1 protein concentration was 20μg / mL. The coupling amount of the ligand V1-SA1 was 371.8RU. The mobile phase buffer during the experiment was HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v / v Tween 20). The analyte was the antigen VEGFA165 (unlabeled, purchased from: GenScript, catalog number: Z03073), and its dilution concentrations were: 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM. In the kinetic analysis, the binding time of the antigen and the antibody was 180 seconds, the dissociation time was 200 seconds, and the flow rate was 30μL / min. The multi-cycle mode was adopted, and the chip was regenerated with 10mM glycine hydrochloride (pH=1.5). The binding curve of the antigen and antibody was fitted with the "Two state reaction" model to obtain the binding rate, dissociation rate and KD.

[0228] The binding and dissociation curves of V1-SA1 and VEGFA165 are shown in FIG12 , and the fitted KDs are shown in Table 18 .

[0229] Table 18 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of humanized antibody V1-SA1 and VEGFA

[0230] From Tables 17 and 18, it can be seen that antibody V1 maintained a high affinity for VEGFA165 after humanization.

[0231] Example 5: Stability study of humanized anti-VEGFA nanobody

[0232] 1. Comparison of Tm values ​​of V1 humanized antibodies

[0233] Antibodies with high thermal stability are more conducive to antibody production and long-term storage, so the melting temperatures (Tm) of different humanized antibodies were compared.

[0234] For the 9 humanized antibodies against V1, the buffer was replaced with PBS (pH=7.4) and the Tm was measured using the UNCLE instrument (unchainedlabs). The ranking of Tm is shown in Figure 13. The Tm values ​​of the humanized antibodies fluctuated within an acceptable range, among which V1-DP and V1-SA1 had higher Tm values, indicating that the thermal stability of these two antibodies is likely to be better than that of other humanized antibodies.

[0235] 2. Study on the colloidal stability of humanized antibody V1-SA1

[0236] The coding genes of humanized antibody V1-SA1 and positive control antibody BI-VEGF ab were constructed into expression vector pCDNA3.1(+), wherein the secretory peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 68) was added to the N-terminus of the antibody, and the flexible linker GGS and the label 6×His were added to the C-terminus of the antibody in sequence. The two antibodies were transiently expressed and Ni-bead affinity purified, and the experimental method was the same as the step "6. Expression and purification of candidate antibodies" in Example 1. After purification, they were separated by SDS-PAGE, and the gel staining results are shown in Figure 14. It can be seen from the figure that the molecular weight of the two proteins is in line with expectations (~15kDa), and the protein expression amount and SDS-PAGE staining purity are shown in Table 19. It can be seen that the expression amount of V1-SA is much higher than that of BI-VEGF ab.

[0237] Table 19 Transient expression of humanized antibody V1-SA1 and positive control antibody BI-VEGF ab and protein purity on SDS-PAGE gel

[0238] The V1-SA1 and BI-VEGF ab proteins were exchanged into 1xPBS (pH=7.4), the protein concentration was adjusted to 10 mg / mL, and the proteins were analyzed by HPLC-SEC (column: Zenix-C SEC-300, Sepax). The percentage of protein monomers, aggregates and fragments is shown in Table 20. It can be seen that the percentage of aggregates of V1-SA is much less than that of BI-VEGF ab, indicating that V1-SA1 is less prone to aggregation.

[0239] Table 20 Proportion of each component of antibody V1-SA1 and BI-VEGF ab in SEC-HPLC analysis

[0240] The antibodies V1-SA1 and BI-VEGF ab (protein concentration was 10 mg / mL) were exchanged into PBS and placed at 37°C for a period of time (0 day and 2 days) after sterile filtration. The properties of the protein solutions were observed and the colloidal stabilities of the two were compared. The results are shown in Table 21.

[0241] Table 21 Comparison of properties, average particle size and PDI of antibodies V1-SA1 and BI-VEGF ab placed at 37°C for 0 and 2 days

[0242] According to the principle of dynamic light scattering, the antibody V1-SA1 and BI-VEGF ab solutions (diluted 4 times with PBS) placed at 37°C for a period of time (0 days and 2 days) were measured for protein particle size using a nanoparticle size and potential analyzer (NS-90Z, OMEK), and the results of protein average particle size (Z-Average) and polydispersity index (PDI) are shown in Table 21. For the sample at time 0, the average hydration kinetic diameter and PDI of V1-SA1 are smaller than those of BI-VEGF ab. Considering that the molecular weights of the two are similar, it means that BI-VEGF ab has a certain proportion of high molecular weight protein aggregates in the buffer solution. Under the same conditions, the particle size distribution of V1-SA1 is more uniform than that of BI-VEGF ab, and the proportion of high molecular weight protein aggregates is significantly lower.

[0243] As can be seen from Table 21, for the samples placed at 37°C for 2 days, V1-SA1 remained clear, while the BI-VEGF ab protein solution precipitated, indicating that the colloidal stability of V1-SA1 was better than that of BI-VEGF ab. At this time, the average hydration kinetic diameter of V1-SA1 was much smaller than that of BI-VEGF ab, and changed less than that at time 0; while the average hydration kinetic diameter of BI-VEGF ab was much larger than that of V1-SA1, and increased significantly compared to the BI-VEGF ab solution at time 0, indicating that BI-VEGF ab further formed high molecular weight protein aggregates under high temperature conditions.

[0244] Example 6: Preparation and activity study of monovalent nanobody-Fc fusion protein

[0245] 1. Preparation of monovalent nanobody-Fc fusion protein

[0246] This example carries out the fusion expression of monovalent nanobody and IgG1 Fc (SEQ ID NO: 62) or PPCP-Fc (SEQ ID NO: 75, the remaining amino acid sequence after removing DKTHTC in SEQ ID NO: 62) or IgG1 Fc mutant Fc-m1 (SEQ ID NO: 63).

[0247] Among them, the IgG1 Fc amino acid sequence is:

[0248] The amino acid sequence of PPCP-Fc is:

[0249] The amino acid sequence of the mutant Fc-m1 of IgG1 Fc is:

[0250] The amino acid sequence of the constructed Nanobody-Fc fusion protein is shown in Table 22.

[0251] Table 22 Nanobody-Fc fusion protein amino acid sequence

[0252] In order to compare the linear tandem bivalent nanobody 2V15-9GS (SEQ ID NO: 74) (containing a 6-histidine tag based on 2V15-9GS) with the fusion proteins V15-PPCP-Fc, V15-9GS-PPCP-Fc and V15-Fc in binding to VEGFA, a linker (SGGS (SEQ ID NO: 70)) and a 6-histidine tag (HHHHHH (SEQ ID NO: 71)) were added to the C-terminus of the Fc segment of the fusion protein. During the plasmid construction of the monovalent nanobody-Fc fusion protein, the nanobody and the Fc containing the linker and the histidine tag were amplified by PCR, respectively, wherein the PCR template of the Fc fragment came from the gene synthesized Fc gene. The nanobody and Fc were integrated into the vector pCDNA3.1(+) by the method of homologous recombination of the two fragments, and the restriction sites used were: NheI and XbaI. The secretory peptide of the fusion protein is: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 72), which is then fused to the target protein sequence. After the plasmids of these fusion proteins were sequenced successfully, the plasmids were extracted without endotoxin, and the transfection reagent polyethyleneimine (Polyethylenimine, referred to as PEI, linear, MW = 25kDa, purchased from: Polysciences, item number: 23966-1) was used to transiently transfect 293F suspension cells. The cell supernatant was collected on the 4th or 5th day after transfection, and Ni bead purification was carried out. The purified protein was analyzed by SDS-PAGE. The molecular weight of the protein monomer under reducing conditions was about 39kDa, which was in line with expectations. The stained gel image of some monovalent nanobody-Fc fusion proteins after purification is shown in Figure 15.

[0253] 2V15-9GS (SEQ ID NO: 74)

[0254] For the fusion protein of the humanized antibody V1-SA1 and Fc (V1-SA1-Fc-m1), the amino acid sequence of the linker between V1-SA1 and Fc-m1 is: EPKSA (SEQ ID NO: 73). The expression plasmid was constructed by 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 did not contain a histidine tag. After the expression plasmid of V1-SA1-Fc-m1 was transiently transfected into 293F cells, Protein A beads were used for affinity purification. When eluting the protein, 0.1M citric acid (pH 3.0) was used to elute the target protein, and it was quickly neutralized to obtain the target protein.

[0255] 2. Activity detection of monovalent nanobody-Fc fusion protein

[0256] The purified monovalent nanobody-Fc fusion protein was tested for VEGFA binding activity, and the ELISA experimental method was the same as "1. Activity test of candidate antibody binding to VEGFA" in Example 2. The ELISA results are shown in Figure 16, and the EC50 is shown in Table 23.

[0257] Table 23 VEGFA binding activity test of monovalent nanobody-Fc fusion protein

[0258] It can be seen from the data in the table that the tested monovalent nanobody-Fc fusion protein and the linear tandem bivalent nanobody both have strong VEGFA binding activity.

[0259] The affinity of monovalent nanobody-Fc fusion protein (V1-SA1-Fc-m1) to VEGFA was tested by surface plasmon resonance (SPR) technology. Using Biacore 8K instrument (Cytiva), V1-SA1-Fc-m1 was used as ligand and captured on Protein A chip. When fixed, V1-SA1-Fc-m1 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) to a protein concentration of 2μg / mL, and its coupling amount was about 590RU. During the experiment, the analyte was the antigen VEGFA165 (unlabeled, purchased from: GenScript, catalog number: Z03073), and its dilution concentrations were: 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78125nM, 0.390625nM. During the kinetic analysis, 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 30μL / min. After each "binding-dissociation" cycle, the chip was regenerated, and the regeneration condition was 10mM glycine hydrochloride (pH 1.5). The binding and dissociation curves of the fusion protein and the antigen VEGFA165 are shown in Figure 17. The "1:1 binding" model was used to fit the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD), as shown in Table 24.

[0260] Table 24 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of monovalent nanobody-Fc fusion protein to VEGFA165

[0261] From the SPR data, it can be seen that the monovalent nanobody-Fc fusion protein V1-SA1-Fc-m1 has a very high affinity with VEGFA165 (KD = 0.148nM). In Table 18 and Figure 12 of "6. Activity detection of humanized antibodies" in Example 2 above, it can be seen that the KD of the monovalent nanobody V1-SA1 and VEGFA165 is 4.38nM. Therefore, after the monovalent nanobody is fused with Fc, the affinity with VEGFA165 is increased by nearly 30 times compared to the unfused Fc, indicating that the anti-VEGFA antibody-Fc fusion protein has a stronger target affinity and potentially better efficacy in blocking the VEGFA signaling pathway.

[0262] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0263] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

An anti-VEGFA antibody or an antigen-binding fragment thereof, characterized in that: The anti-VEGFA antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein the amino acid sequence of CDR-H1 comprises 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 comprises AISKGGYKYX1X2VSLEA (SEQ ID NO: 2) or an amino acid sequence that is at least 80% identical to AISKGGYKYX1X2VSLEA (SEQ ID NO: 2); the amino acid sequence of CDR-H3 comprises TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3) or an amino acid sequence that is at least 80% identical to TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3). The anti-VEGFA antibody or antigen-binding fragment thereof according to claim 1, characterized in that X1 and X2 in SEQ ID NO: 2 represent DS, DA, NT, DT, NA or NS; X3 in SEQ ID NO: 3 represents R or K, and X4 represents D, N, E or K. The anti-VEGFA antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 include any of the following groups: 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. The anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The anti-VEGFA antibody or antigen-binding fragment thereof comprises a humanized sequence, wherein the modification site of the humanized sequence is located in a non-CDR region, and preferably, the modification site of the humanized sequence is located in the framework region and / or constant region of the antibody. The anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that The anti-VEGFA antibody or its antigen-binding fragment is a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a F(ab')2 fragment, a single domain antibody, a single chain antibody molecule or a linear antibody. The anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that The amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment comprises any one of SEQ ID NOs: 13-32, 35-61, 64-67, or has at least 80% identity with any one of SEQ ID NOs: 13-32, 35-61, 64-67. The anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that The anti-VEGFA antibody or antigen-binding fragment thereof binds to human or monkey VEGFA protein. A fusion protein, characterized in that The fusion protein comprises the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-7. The fusion protein according to claim 8, characterized in that The fusion protein further comprises other anti-VEGFA antibodies or antigen-binding fragments thereof or other target antibodies or antigen-binding fragments thereof or other functional components in addition to the anti-VEGFA antibody or antigen-binding fragment thereof described in any one of claims 1 to 7. Preferably, the other functional components include but are not limited to serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and their markers, PEGylated components or Fc fragments, one or a combination of two or more thereof; preferably, the other targets are selected from IgG, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Endoglin (CD105), TGF, Integrin, Integrin receptor, interleukin-1β, leukocyte antigen 2, leukocyte antigen 3, leukocyte antigen 4, leukocyte antigen 5, leukocyte antigen 6, leukocyte antigen 7, leukocyte antigen 8, leukocyte antigen 9, leukocyte antigen 10, leukocyte antigen 11, leukocyte antigen 12, leukocyte antigen 13, leukocyte antigen 14, leukocyte antigen 15, leukocyte antigen 16, leukocyte antigen 17, leukocyte antigen 18, leukocyte antigen 29, leukocyte antigen 21, leukocyte antigen 22, leukocyte antigen 23, leukocyte antigen 24, leukocyte antigen 19, leukocyte antigen 25, leukocyte antigen 27, leukocyte antigen 28, leukocyte antigen 29 Interleukin receptors (such as IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptors (such as IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), PCSK9, TNF-α, TNFR, RANKL, complement proteins C3, complement protein C5, G protein-coupled receptor (GPCR), GLP1R, CD3, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, CD111, CD112, CD123, CD133, CD138, CD155, CD171, Claudin 18.2, OX40, ICOS, CTLA4, 4-1BB, TCR, B7-1, B7-2, BTLA, TIM-3, LAG3, Galectin-9, PD-L1, PD-L2, PD-1, TIGIT, EGFR, Her2, PSCA, CEA, FAP, EGFRVIII, BC MA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3 or 5T4. A chimeric antigen receptor, characterized in that The extracellular domain of the chimeric antigen receptor comprises the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7. An immune cell, characterized in that The immune cell expresses the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 or the chimeric antigen receptor according to claim 10. A nucleic acid, characterized in that The nucleic acid encodes the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-7 or encodes the fusion protein according to any one of claims 8-9. A carrier, characterized in that The vector comprises the nucleic acid according to claim 12. A host cell, characterized in that The host cell comprises the nucleic acid of claim 12 or the vector of claim 13. A method for preparing an anti-VEGFA antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The preparation method comprises introducing nucleic acid or vector encoding anti-VEGFA antibody or antigen-binding fragment thereof into host cells, and then inducing expression thereof. A method for screening an anti-VEGFA antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The method comprises immunizing alpacas with human or monkey VEGFA protein. A product for treating and / or diagnosing a disease, characterized in that The product for treating and / or diagnosing a disease comprises any one of the following: A) an anti-VEGFA antibody or an antigen-binding fragment thereof as described in any one of claims 1-7; B) a fusion protein as described in any one of claims 8-9; C) a chimeric antigen receptor as described in claim 10; D) an immune cell as described in claim 11; E) a nucleic acid as described in claim 12; F) a vector as described in claim 13; or, G) a host cell as described in claim 14. A use of the anti-VEGFA antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 7, the fusion protein as claimed in any one of claims 8 to 9, the chimeric antigen receptor as claimed in claim 10, the immune cell as claimed in claim 11, the nucleic acid as claimed in claim 12, the vector as claimed in claim 13, or the host cell as claimed in claim 14 in the preparation of a product for treating and / or preventing a disease associated with VEGFA, or in the preparation of a product for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, or in the preparation of an antibody-drug conjugate or an antibody diagnostic kit or a tracer. A method for detecting VEGFA, characterized in that: The detection method comprises contacting the sample to be tested with the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and then detecting the content of the complex formed by VEGFA and the anti-VEGFA antibody or antigen-binding fragment thereof. A method for treating and / or preventing a disease, characterized in that The method comprises applying to an individual an anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, a fusion protein according to any one of claims 8 to 9, a chimeric antigen receptor according to claim 10, an immune cell according to claim 11, a nucleic acid according to claim 12, a vector according to claim 13, a host cell according to claim 14, or a product for treating and / or diagnosing a disease according to claim 17. A method for treating and / or preventing a disease, characterized in that The method comprises contacting the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the fusion protein according to any one of claims 8 to 9, the chimeric antigen receptor according to claim 10, the immune cell according to claim 11, the nucleic acid according to claim 12, the vector according to claim 13, the host cell according to claim 14, or the product for treating and / or diagnosing the disease according to claim 17 with a target cell. The method according to claim 21, characterized in that The target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular 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 and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc. The method according to any one of claims 20 to 22, characterized in that: The disease is a disease associated with the VEGFA signaling pathway; preferably, the disease is selected from tumors, abnormal vascular proliferation, or ophthalmic diseases involving angiogenesis (such as fundus vascular disease). A method for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, characterized in that: The method comprises contacting vascular endothelial cells with the anti-VEGFA antibody or antigen-binding fragment thereof described in any one of claims 1-7, the fusion protein described in any one of claims 8-9, the chimeric antigen receptor described in claim 10, the immune cell described in claim 11, the nucleic acid described in claim 12, the vector described in claim 13, the host cell described in claim 14, or the product for treating and / or diagnosing the disease described in claim 17.

Images