Anti-VEGFA antibody or its antigen-binding fragment, and its use

Abstract

The present invention provides an anti-VEGFA antibody or antigen-binding fragment thereof, and uses thereof. The anti-VEGFA antibody or antigen-binding fragment thereof has high thermostability, high water solubility, small molecular weight, simple expression and purification processes, and high affinity for VEGFA. The anti-VEGFA antibody or antigen-binding fragment thereof can effectively inhibit the VEGFA signaling pathway and pathological processes caused by VEGFA, such as vascular endothelial cell proliferation and angiogenesis, and is expected to be used in the treatment of VEGFA-related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and specifically relates to an anti-VEGFA antibody or its antigen-binding fragment, and its use.

Background Art

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

[0003] Pathological angiogenesis occurs in diseases such as various solid tumors, inflammatory reactions, and fundus vascular diseases. In the late stage of tumor cell growth, it depends on the provision of sufficient nutrients from new blood vessels to support tumor growth and metastasis. Therefore, inhibition of angiogenesis is an important means for treating cancer. Bevacizumab, the first anti-VEGFA monoclonal antibody, was approved for the treatment of metastatic colorectal cancer in 2004. Overexpression of VEGFA is also closely related to fundus vascular proliferation such as exudative 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 fundus vascular proliferation.

[0004] Currently, four major anti-VEGFA protein agents are commercially available for the treatment of exudative AMD, including ranibizumab, aflibercept, convert, and brolucizumab. In addition, bevacizumab is also used off-label for the treatment of exudative AMD. These anti-VEGFA protein agents include conventional monoclonal antibodies, antibody fragments Fab and scFv, and receptor-Fc fusion proteins, but currently, single-domain antibodies (VHH: variable domain of heavy-chain antibody) or nanobody (Nb) agents are not commercially available.

[0005] Single-domain antibodies, or nanobodies, are variable region domains of heavy-chain antibodies that naturally lack light chains in the bodies of camelids. They are the smallest, most stable antibody units with complete antigen-binding function. Single-domain antibodies have a molecular weight of approximately 13 kDa, exhibit high thermal stability and excellent water solubility. As a novel antibody form, the development of nanobodies is attracting increasing attention.

[0006] Accordingly, the present invention provides a novel anti-VEGFA antibody or its antigen-binding fragment, which is used to inhibit the binding of VEGFR1 and VEGR2 to VEGF, thereby inhibiting the downstream signaling pathway of VEGF and inhibiting VEGF-stimulated cell proliferation. [Overview of the Initiative]

[0007] A first aspect of the present invention provides an anti-VEGFA antibody or an antigen-binding fragment thereof, wherein the anti-VEGFA antibody or the antigen-binding fragment comprises heavy chain variable regions CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 includes 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 an amino acid sequence that is at least 80% identical to AISKGGYKYX1X2VSLEA(SEQ ID NO:2) or AISKGGYKYX1X2VSLEA(SEQ ID NO:2). The amino acid sequence of CDR-H3 includes TRAYGSSRLX3LAX4TYEY(SEQ ID NO:3) or an amino acid sequence that is at least 80% identical to TRAYGSSRLX3LAX4TYEY(SEQ ID NO:3).

[0008] In SEQ ID NO:2 or SEQ ID NO:3, X may 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), or valine (V).

[0009] In a specific embodiment of the present invention, 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.

[0010] In specific embodiments of the present invention, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 include any of the following groups (specifically shown in Table 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

[0011] Table 1: Amino acid sequences of antibody candidates CDR-H1, CDR-H2, and CDR-H3 [Table 1]

[0012] Preferably, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in order from the N-terminus to the C-terminus, and in this application, the amino acids of the CDR region of the antibody are partitioned using the Kabat numbering system.

[0013] The anti-VEGFA antibody or its antigen-binding fragment comprises a heavy chain variable region and CH2 and / or CH3 regions.

[0014] The structure of the anti-VEGFA antibody or its antigen-binding fragment may be a nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fd' fragment, Fv fragment, dAb fragment, isolated CDR region, F(ab')2 fragment, single-domain antibody, single-chain antibody molecule, or linear antibody.

[0015] The anti-VEGFA antibody or its antigen-binding fragment may be a dual-target specific antibody or a specific antibody having three or more targets (for example, 3, 4, 5, 6, 7, 8, 9, 10 or more targets).

[0016] The anti-VEGFA antibody or its antigen-binding fragment may be a linear antibody.

[0017] The anti-VEGFA antibody or its antigen-binding fragment may be a single-domain antibody or a nanobody.

[0018] The anti-VEGFA antibody or its antigen-binding fragment may be a humanized antibody or a fully human antibody.

[0019] Preferably, the anti-VEGFA antibody or its antigen-binding fragment contains a humanized sequence, and the modified site of the humanized sequence is located in a non-CDR region. More preferably, the humanized modification site is located in the framework region and / or constant region of the antibody.

[0020] In one specific embodiment of the present invention, the anti-VEGFA antibody or its antigen-binding fragment is a nanobody. Compared with a full-length IgG antibody, Fab, or scFv, at the same mass, the nanobody has a higher molar concentration and can bind more antigen molecules.

[0021] The anti-VEGFA antibody or its antigen-binding fragment can bind to human or monkey VEGFA protein, where the human VEGFA protein and monkey VEGFA protein have the same sequence.

[0022] Preferably, the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment includes any one of the amino acid sequences of SEQ ID NO: 13-32, 35-61, 64-67, or has at least 8*% identity with any one of the amino acid sequences of SEQ ID NO: 13-32, 35-61, 64-67.

[0023] In one specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment is represented by any one of the amino acid sequences of SEQ ID NO: 13-32, 35-61, 64-67.

[0024] The anti-VEGFA antibody or its antigen-binding fragment can inhibit or compete with the binding of other antibodies (preferably those that bind to the same or overlapping epitopes as the anti-VEGFA antibodies described in the present invention) to human or monkey VEGFA protein.

[0025] The anti-VEGFA antibody or its antigen-binding fragment may be constructed by any conventional method in the prior art. Examples include artificial synthesis, eukaryotic expression, or prokaryotic expression.

[0026] A second aspect of the present invention provides an anti-VEGFA antibody or its antigen-binding fragment.

[0027] The anti-VEGFA antibody or its antigen-binding fragment is a nanobody.

[0028] The anti-VEGFA antibody or its antigen-binding fragment contains any amino acid sequence among SEQ ID NO: 13-32, 35-61, or 64-67, or has at least 80% identity with any amino acid sequence among SEQ ID NO: 13-32, 35-61, or 64-67.

[0029] In one particular embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment is represented by SEQ ID NO: 13-32, 35-61, or 64-67.

[0030] A third aspect of the present invention provides a method for screening anti-VEGFA antibodies or antigen-binding fragments thereof, comprising immunizing alpacas with human or monkey VEGFA proteins.

[0031] A fourth aspect of the present invention provides a fusion protein comprising the above-mentioned anti-VEGFA antibody or its antigen-binding fragment.

[0032] Preferably, the fusion protein further comprises other anti-VEGFA antibodies other than the anti-VEGFA antibody or its antigen-binding fragment, an antibody against another target or its antigen-binding fragment, or another functional component.

[0033] Preferably, the functional components include, but are not limited to, one or more combinations of 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.

[0034] Preferably, the other targets are IgG, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Endoglin (CD105), TGF, Integrin, Integrin receptor, interleukins (e.g., IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptors (e.g., 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 Selected from 3 or 5T4.

[0035] The structure of the antibody or its antigen-binding fragment may be a nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fd' fragment, Fv fragment, dAb fragment, isolated CDR region, F(ab')2 fragment, single-domain antibody, single-chain antibody molecule, or linear antibody.

[0036] Preferably, the fusion protein comprises at least one anti-VEGFA antibody or its antigen-binding fragment.

[0037] Preferably, the fusion protein comprises at least one other anti-VEGFA antibody or its antigen-binding fragment.

[0038] Preferably, the fusion protein comprises at least an antibody or its antigen-binding fragment against one other target, or another functional component.

[0039] Of these, the following connections are made directly or indirectly: between the anti-VEGFA antibody or its antigen-binding fragment; between the anti-VEGFA antibody or its antigen-binding fragment and other anti-VEGFA antibodies or their antigen-binding fragments; between the anti-VEGFA antibody or its antigen-binding fragment and antibodies against other targets or their antigen-binding fragments, or other functional components; between other anti-VEGFA antibodies and antibodies against other targets or their antigen-binding fragments, or other functional components; between antibodies against other targets or their antigen-binding fragments, or other functional components; and between other anti-VEGFA antibodies or their antigen-binding fragments.

[0040] The aforementioned indirect linking may be linked by a linker, a functional domain and / or a linker for coupling. Here, the linker is selected from linking peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEGs, nucleic acids, polysaccharides, fatty acid chains, biotin, streptavidin, or avidin.

[0041] The functional domain is one or more combinations of Fc fragments, serum albumin, cytokines, transferrin, scaffold proteins, VEGFA, or antibodies against other targets or their antigen-binding fragments.

[0042] The linker for coupling includes one or more combinations of toxins, drugs, nucleic acids, PEGs, radionuclides, and their markers.

[0043] Preferably, direct or indirect ligation means direct or indirect ligation to the N-terminus, C-terminus, and / or internal residues of the anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies, and / or antibodies against other targets.

[0044] The linking order of the anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies, or antibodies against other targets contained in the fusion protein may be such that the N-terminus, C-terminus, and / or internal residues of one antibody are linked to the N-terminus, C-terminus, and / or internal residues of another antibody.

[0045] In one particular embodiment of the present invention, the linkage order of the anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies or antibodies against other targets contained in the fusion protein may be such that the N-terminus of one antibody is linked to the N-terminus or C-terminus of another antibody.

[0046] In one particular embodiment of the present invention, the linkage order of the anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies or antibodies against other targets contained in the fusion protein may be such that the C-terminus of one antibody is linked to the N-terminus or C-terminus of another antibody.

[0047] In one particular embodiment, the fusion protein comprises an anti-VEGFA antibody and an Fc fragment.

[0048] Preferably, the fusion protein contains any amino acid sequence from SEQ ID NO: 64 to 67, or has at least 80% identity with any amino acid sequence from SEQ ID NO: 64 to 67.

[0049] Preferably, the fusion protein further includes a tag.

[0050] Preferably, the tag is ligated to the C-terminus of the fusion protein.

[0051] A fifth aspect of the present invention provides a chimeric antigen receptor in which the extracellular domain comprises the above-mentioned anti-VEGFA antibody or its antigen-binding fragment.

[0052] Preferably, the chimeric antigen receptor further comprises any conventional transmembrane region and / or intracellular signaling region in the prior art.

[0053] A sixth aspect of the present invention provides nucleic acids encoding the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, the above-mentioned fusion protein, or the above-mentioned chimeric antigen receptor.

[0054] A seventh aspect of the present invention provides a vector containing the above-mentioned nucleic acid.

[0055] The vector can be expressed under in vivo, in vitro, or ex vivo conditions. Preferably, the vector is a prokaryotic expression vector, a viral expression vector, or a eukaryotic expression vector, such as an E. coli vector or a phage.

[0056] An eighth aspect of the present invention provides a host cell containing the above-mentioned nucleic acid or vector.

[0057] The host cell may be a eukaryotic cell or a prokaryotic cell.

[0058] Eukaryotic cells include plant and animal cells, such as T cells, yeast cells, HEK293 cells, or CHO cells.

[0059] Prokaryotic cells include, for example, E. coli.

[0060] A ninth aspect of the present invention provides a method for producing host cells, which includes introducing the above-mentioned nucleic acid or vector into host cells and subsequently inducing its expression.

[0061] A tenth aspect of the present invention provides a method for producing an anti-VEGFA antibody or an antigen-binding fragment thereof, comprising introducing a nucleic acid or vector encoding an anti-VEGFA antibody or its antigen-binding fragment into a host cell and subsequently inducing its expression.

[0062] An eleventh aspect of the present invention provides immune cells that express the above-mentioned anti-VEGFA antibody or its antigen-binding fragment, or the above-mentioned chimeric antigen receptor.

[0063] Preferably, the immune cells include, but are not limited to, lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, monocytes / macrophages, granulocytes, and mast cells.

[0064] Preferably, the immune cells are CAR-immune cells.

[0065] A twelfth aspect of the present invention provides a method for constructing immune cells, which includes obtaining immune cells by transfecting and expressing a nucleic acid sequence encoding a chimeric antigen receptor described in the present invention.

[0066] In the thirteenth aspect of the present invention, A) The above-mentioned anti-VEGFA antibody or its antigen-binding fragment, B) The above fusion protein, C) The above-mentioned chimeric antigen receptor, D) The above immune cells E) The above nucleic acids, F) The above vector, or G) Provide a product for treating and / or diagnosing a disease, comprising any one of the host cells described above.

[0067] Products for treating and / or diagnosing the aforementioned diseases target and express VEGFA in cells, which may include cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granule cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, retinal Müller cells, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells, or tumor cells.

[0068] Preferably, the product may be a kit, drug, chip, or antibody-drug conjugate. The disease is a disease related to the VEGFA signaling pathway. More preferably, it may be an eye disease involving tumors, abnormal angiogenesis, or neovascularization (e.g., retinal vascular disease).

[0069] A fourteenth aspect of the present invention provides an antibody-drug conjugate (ADC) comprising the anti-VEGFA antibody of the present invention or its antigen-binding fragment, which is covalently bound to a drug.

[0070] A 15th aspect of the present invention provides a method for detecting VEGFA, which includes contacting a test sample 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.

[0071] The detection method described above detects the presence or content of VEGFA. The presence indicates the presence or absence of VEGFA, and the content may be the expression level or protein concentration, etc.

[0072] A sixteenth aspect of the present invention provides a method for diagnosing a disease, comprising 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 a product for treating and / or diagnosing the disease, and detecting the content of a complex formed by VEGFA and the anti-VEGFA antibody or its antigen-binding fragment.

[0073] The aforementioned disease is a disease related to the VEGFA signaling pathway. More preferably, it may be an eye disease involving tumors, abnormal angiogenesis, or neovascularization (e.g., retinal vascular disease).

[0074] A 17th aspect of the present invention provides a method for treating and / or preventing a disease, comprising administering to an individual 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 a product for treating and / or diagnosing the above-mentioned disease.

[0075] The aforementioned disease is a disease related to the VEGFA signaling pathway. More preferably, it may be an eye disease involving tumors, abnormal angiogenesis, or neovascularization (e.g., retinal vascular disease).

[0076] In an 18th aspect of the present invention, a method is provided for inhibiting VEGFA-mediated vascular endothelial cell proliferation or angiogenesis, 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 a product for treating and / or diagnosing the above-mentioned disease.

[0077] 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 a product for treating and / or diagnosing the above-mentioned disease in serum-free medium for vascular endothelial cells, and then incubating it with an antigen (e.g., VEGFA165).

[0078] Preferably, the process includes the step of discarding the complete medium of vascular endothelial cells in the culture plate after incubation.

[0079] 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 cells, the above-mentioned immune cells, or the above-mentioned product for treating and / or diagnosing the disease are added to a vascular endothelial cell culture plate and cultured in the culture plate.

[0080] Preferably, detection is performed after culturing.

[0081] Preferably, the incubation time is 0.5 to 5 hours, preferably 1 to 3 hours, for example, 0.5, 1, 2, 3, 4, or 5 hours.

[0082] Preferably, the incubation temperature is room temperature to 45°C, preferably 30 to 40°C, for example 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc.

[0083] Preferably, the culture temperature is room temperature to 45°C, preferably 30 to 40°C, for example 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc. Preferably, the culture is carried out in a 5% CO2 incubator.

[0084] Preferably, the culture time is 1 to 5 days, preferably 2 to 4 days, for example, 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, 5 days, etc.

[0085] Preferably, the detection involves detecting the number of living vascular endothelial cells.

[0086] A 19th aspect of the present invention provides a method for treating and / or preventing a disease, comprising contacting target 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 a product for treating and / or diagnosing the above-mentioned disease.

[0087] The aforementioned disease is a disease related to the VEGFA signaling pathway. More preferably, it may be an eye disease involving tumors, abnormal angiogenesis, or neovascularization (e.g., retinal vascular disease).

[0088] Preferably, the target cells are selected from cells that express VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granule cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, retinal Müller cells, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells, or tumor cells.

[0089] A 20th aspect of the present invention provides the use 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 manufacture of a product for treating and / or preventing a VEGFA-related disease, a product for inhibiting VEGFA-mediated vascular endothelial cell proliferation or angiogenesis, or an antibody-drug conjugate, antibody diagnostic kit or tracer.

[0090] The aforementioned disease may also include tumors, abnormal blood vessel proliferation, and eye diseases accompanied by neovascularization (e.g., retinal vascular diseases).

[0091] The aforementioned product may be a kit, drug, chip, or antibody-drug conjugate, etc.

[0092] This invention provides a novel anti-VEGFA antibody or its antigen-binding fragment, which exhibits high thermal stability, high water solubility, low molecular weight, simple expression and purification processes, high affinity for VEGFA, effectively inhibits the VEGFA signaling pathway, effectively inhibits pathological processes such as vascular endothelial cell proliferation and angiogenesis caused by VEGFA, and is expected to be used for the treatment of VEGFA-related diseases. Furthermore, it may also bring greater clinical value, such as superior tissue penetration, lower production costs, and easier administration.

[0093] The “drug” of the present invention can be used to treat humans or non-human animals such as non-human mammals. The drug may contain pharmaceutically acceptable carriers, excipients, or salts commonly found in the prior art. The drug may be administered by any suitable route, such as gastrointestinal (e.g., orally) or parenteral (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorganic, intranasal, intraocular, intrainfusion, intracerebral, subarachnoid, percutaneous, rectal, etc.) routes. The drug may be in any suitable dosage form, such as gastrointestinal or parenteral dosage form, and preferably includes, but is not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder aerosols, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, drops, gels, etc. The various dosage forms of the drug can be manufactured according to conventional manufacturing methods in the pharmaceutical field. The drug may contain the anti-VEGFA antibody or its antigen-binding fragment, the nucleic acid, the vector, the host cell, the immune cell, etc., in a weight ratio of 0.01 to 99.5% (specifically, for example, 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 may be manufactured as a reagent with a protein concentration of 1 to 300 mg / mL (for example, 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 may be 0.1 to 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, or 1000 mg.

[0094] In this invention, "pharmaceutically acceptable" means that it does not significantly stimulate the organism or inhibit the biological activity and properties of the active substance of the administered product.

[0095] The "...method" of the present invention may be for the purpose of diagnosing or treating a disease, or it may be for a purpose other than diagnosing or treating a disease.

[0096] The "antigen-binding fragment" of the present invention refers to a part of an antibody that retains the specific binding activity of the antibody, i.e., any part of the antibody that specifically binds to an epitope on the antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, Fv, Fd, and variants of these fragments. Examples include the heavy and / or light chain of an antibody, the heavy chain variable region and / or light chain variable region of an antibody, or one or more CDRs derived from the heavy or light chain of an antibody. Of these, Fab is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. F(ab')2 is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region. Fd is an Fd fragment consisting of a VH domain and a CH1 domain. Fv is an Fv fragment consisting of a VL domain and a VH domain 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' molecule that has at least one free thiol at a cysteine ​​residue in its constant domain. Of these, VH represents the heavy chain variable region, VL represents the light chain variable region, and CL represents the light chain.

[0097] In this invention, "CH2" or "CH3" refers to the CH2 or CH3 domain of the heavy chain constant region. The complete heavy chain constant region consists of three domains: CH1, CH2, and CH3. Specifically, the CH2 domain refers to the portion of the antibody heavy chain polypeptide extending from approximately EU position 231 to EU position 340 (according to Kabat's EU numbering system). The CH2 domain is unique in that it is not tightly paired with another domain. The CH3 domain refers to the portion of the antibody heavy chain polypeptide extending from approximately EU position 341 to EU position 446.

[0098] The "Fc" region of the present invention comprises two heavy chain fragments, each containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments form a dimer via two or more disulfide bonds within the hinge region and are linked by hydrophobic interactions of the CH3 domains.

[0099] The "linear antibody" of the present invention includes a pair of tandem Fd segments (VH-CH1-VH-CH1).

[0100] The terms "contains" or "includes" in this invention are open expressions and, when used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence or have the same or similar activity as the original sequence, even if additional amino acids or nucleotides are present at one or both ends of the protein or nucleic acid.

[0101] The "homology" of the present invention means that, with respect to the protein sequence or nucleotide sequence used, a person skilled in the art would know that the sequence used is 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%, 4 This means that the sequences can be adjusted as needed for the actual work to have homology of 7%, 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%, and 99.9% (including, but not limited to, these percentages).

[0102] In this invention, the term "humanized antibody" refers to an antibody whose framework region and / or constant region portion (e.g., the CH region), or the entire antibody, is encoded by a human antibody gene. In certain embodiments of this invention, the CDR region of the antibody is not modified.

[0103] The term "individual" in this invention may refer to a human or a non-human mammal, and non-human mammals may include wild animals, zoo animals, economic animals, pets, laboratory animals, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cows, sheep, horses, donkeys, foxes, raccoons, minks, camels, dogs, cats, rabbits, rodents (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys.

[0104] The term "treatment" in this invention means slowing, interrupting, preventing, controlling, stopping, reducing, or reversing the signs, symptoms, impairments, conditions, or progression or severity of a disease after it has begun to develop, but does not necessarily mean completely eliminating all signs, symptoms, conditions, or impairments associated with the disease.

[0105] In this invention, "prevention" means a method implemented to prevent or delay the onset of a disease, condition, or symptom in the body.

[0106] In this invention, "diagnosis" means determining whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or determining the progression or potential future progression of a disease.

[0107] The tumor of the present invention may be any undesirable cell proliferation (or any disease that manifests as undesirable cell proliferation), neoplasm, or an increased tendency or risk of undesirable cell proliferation, neoplasm, or tumor. It may be benign or malignant, primary or secondary (metastatic). A neoplasm may be any abnormal growth or proliferation of cells and may be present in any tissue. Examples of tissues include the adrenal glands, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, mammary glands, cecum, central nervous system (with or without brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidneys, lacrimal glands, larynx, liver, lungs, lymph, lymph nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, retina, oral cavity, ovaries, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary glands, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testes, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, and leukocytes. More 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, squamous cell carcinoma of the tongue, nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermal carcinoma, colon cancer, thymic carcinoma, hematological cancer, head and neck cancer, oropharyngeal cancer, and the like.

[0108] In this invention, the term "fundus vascular disease" refers to a general term for diseases occurring in the retinal arteries and veins, or diseases related to choroidal neovascularization. This includes, but is not limited to, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, and neovascular glaucoma. [Brief explanation of the drawing]

[0109] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. [Figure 1] These are Coomassie brilliant blue stained gel images of purified antibody candidates from various batches. [Figure 2] This figure shows the results of how the binding signal to VEGFA of various antibody candidates changes with antibody concentration. [Figure 3] This is a diagram showing the results of a competitive ELISA for various antibody candidates. [Figure 4] This shows the inhibition rate of HUVEC cell proliferation by various antibody candidates. [Figure 5] This is an ELISA detection of the combined activity of antibody V1 with human VEGFA121. [Figure 6] This is an ELISA detection of the mouse VEGFA164 binding activity of antibody V1. [Figure 7] This is an ELISA detection of the rat VEGFA164 binding activity of antibody V1. [Figure 8] This is a Coomassie brilliant blue stained gel image after purification of the humanized antibody. [Figure 9] This is an ELISA test for the binding of humanized antibodies to VEGFA. [Figure 10] This is an ELISA test for VEGFR2 competition among humanized antibodies. [Figure 11] This is an ELISA test for VEGFR2 competition between humanized antibodies against V30 and V43. [Figure 12] This is the binding-dissociation curve (SPR) between V1-SA1 and VEGFA165. [Figure 13] This shows the melting temperatures (Tm) of various humanized antibodies. [Figure 14] These are SDS-PAGE gel images of the humanized antibody V1-SA1 and the positive control antibody BI-VEGF ab after purification. [Figure 15] This is a Coomassie brilliant blue stained gel image of a monovalent nanobody-Fc fusion protein after purification. [Figure 16] This is an ELISA test for VEGFA binding of monovalent nanobody-Fc fusion proteins. [Figure 17]This is the binding-dissociation curve (SPR) for V1-SA1-Fc-m1 and VEGFA165. [Modes for carrying out the invention]

[0110] The following describes the technical solutions in the embodiments of the present invention clearly and completely with reference to the drawings of the embodiments of the present invention, and it is clear that the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments that a person skilled in the art could obtain without creative work based on the embodiments of the present invention are all within the scope of the protection of the present invention.

[0111] Example 1: Preliminary screening of alpaca immunities and antibodies

[0112] 1. Alpaca immunity Human VEGFA165 (untagged, purchased from Yiqiao Shenzhou Co., Ltd., catalog number: HPLC-10008-HNAH, hereinafter simply referred to as VEGFA) was emulsified and used to immunize alpacas. Two alpacas were selected, and each animal was immunized with 1 mg of VEGFA protein every two weeks. Serum was collected from the second immunization onwards, and the titer was detected. Of these, alpaca #1 was immunized with the antigen four times, and alpaca #2 was immunized with the antigen three times. Serum titer was detected, and both met the library construction criteria.

[0113] 2. Detection of competitive activity of post-immunization serum that inhibits the binding of VEGFR2 to VEGFA. The extracellular domain of the VEGF receptor, VEGFR2-ECD, was labeled with biotin (named VEGFR2-biotin), VEGFA was diluted to 0.5 μg / mL with CBS buffer, coated onto an ELISA plate, and left overnight at 4°C. After blocking with 3% skim milk powder, the plate was washed, and alpaca serum and negative serum (alpaca serum before antigen immunization) were diluted 5-fold, 15-fold, 45-fold, 135-fold, 405-fold, 1215-fold, and 3645-fold. 100 μL of a 0.5 μg / mL diluted VEGFR2-biotin solution was taken from each well, added to blocked wells, left at room temperature for 1 hour, washed the plate three times with PBST (PBS containing 0.1% Tween20, pH 7.4), streptavidin-HRP was added, left at room temperature for 1 hour, washed the plate three times with PBST, TMB was added and allowed to develop for 15 minutes, then stop solution was added and the absorbance at a wavelength of 450 nm was read using a microplate reader. The results of detecting competitive activity in the serum of two alpacas are shown in Tables 2 and 3.

[0114] Table 2:1#Results of detection of competitive activity in alpaca serum [Table 2]

[0115] Table 3:2#Results of detection of competitive activity in alpaca serum [Table 3]

[0116] The results indicate that antibodies inhibiting the binding of VEGFR2 to VEGFA were detected in both alpaca 1# and 2#, suggesting they can be used for library construction.

[0117] 3. Construction of an antibody library 40 mL of alpaca peripheral blood was collected using a blood collection needle. After lysing the red blood cells according to the instructions for the red blood cell lysate, white blood cells were collected and cryopreserved with trizol. Before constructing the library, total RNA was extracted from alpaca PBMCs isolated using trizol, and cDNA reverse transcription was performed. Variable region VH fragments were amplified using nanobody-specific primers. The resulting fragments and the pcomb3X vector were digested with SfiI enzyme, mixed in appropriate proportions, and ligated with T4 ligase. After ligation, the cells were subjected to electrochemical transformation of XL1-Blue competent cells. Based on colony growth, the transformed library volume of two alpacas was calculated to be 6.52 × 10⁶ each. 8 and 4.12 × 10 8 Clones were randomly selected from the transformed colonies and subjected to sequencing verification, and the sequencing results showed the correct nanobody sequence.

[0118] 4. Screening of antibody libraries We used phage display to screen for phages that can bind to VEGFA.

[0119] For the 1# alpaca library, after four concentration screenings, a single clone was selected from 96 wells on one plate (the screening method was VEGF-4 th (Recorded as concentrated), the binding activity of phages to VEGFA was detected. Competitive elution was also performed on the fourth phage sample. Using the 10 μg / mL VEGFR2 extracellular domain VEGFR2-ECD-His (manufactured in-house), phages binding to VEGF were competitively eluted. It is expected that antibodies capable of competing with VEGFR2 for binding to VEGFA will be obtained. For the obtained phages, five 96-well plates were selected, and the binding activity of the phages to VEGFA was detected (screening method: VEGF-5 th(Recorded as VEGFR2 competition). Furthermore, competitive elution screening was performed on the phages obtained by VEGFR2 competitive screening using the reference monoclonal antibody bevacizumab (Avastin), i.e., competitive elution of phages that bind to VEGFA was performed using 10 μg / mL bevacizumab antibody. One 96-well plate was selected for the obtained phages, and the phage binding activity to VEGFA was detected (screening method was VEGF-6 th Recorded as (competition with bevacizumab). The detected phage is an HRP-labeled antibody that recognizes the phage coat protein and is labeled as anti-M13(HRP). Table 4 shows the detection results of positive clones with VEGFA-binding activity among the above monoclones.

[0120] Table 4:1# Detection results of positive clones with VEGFA binding activity after screening of the alpaca library [Table 4]

[0121] Furthermore, biotin-labeled VEGFA was used to screen for phages that bind to a wider range of epitopes. The method was as follows: First, alpaca libraries 1 and 2 were coated with avidin and then incubated with biotinylated VEGFA. The libraries were merged, and VEGFA-binding phages were obtained through incubation and elution of the libraries and biotinylated VEGFA. This process was repeated. After multiple enrichments, single-clonal colonies in 96-well plates were screened. Phages in the supernatant were subjected to ELISA to detect VEGFA-binding activity. The results for positive clones with binding activity are shown in Table 5.

[0122] Table 5: Detection results after screening for positive clones with VEGFA binding activity after merging libraries. [Table 5]

[0123] 5. Preliminary screening of antibody sequences Plasmid sequencing and codon translation of the above phages exhibiting VEGFA-binding activity were performed to obtain the amino acid sequences of nanobody candidates (CDR region + framework region), and antibodies with the same sequence were merged. Antibody candidates with different CDR sequences are shown in Table 6.

[0124] Table 6: Amino acid sequences of antibody candidates [Table 6] TIFF0007897657000007.tif249170TIFF0007897657000008.tif19170

[0125] 6. Expression and purification of antibody candidates Several antibody candidates were selected from the table above, expressed in mammalian cells, purified, and subjected to ELISA testing to repeatedly verify the VEGFA-binding activity of the antibodies.

[0126] The coding genes for each nanobody were amplified from the original phage plasmid by PCR and constructed in a pVRC8400 expression vector. Here, the secreted peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 68) was added to the N-terminus of the antibody, followed by the flexible linker GGGGS (SEQ ID NO: 69) and the tag 6×His to the C-terminus. Simultaneously, the coding gene sequence for the positive control antibody BI-VEGF ab was synthesized (this antibody is an anti-VEGFA nanobody, and its amino acid sequence is derived from SEQ ID NO: 57 of U.S. Patent No. 9527925B2). The tag 6×His was added to the C-terminus of the control antibody BI-VEGF ab. After determining the sequence of the expression plasmid for each antibody, if accurate, endotoxin-free large-scale extraction was performed. Using the transfection reagent polyethyleneimine (PEI, linear, MW=25kDa, purchased from Polysciences, catalog number: 23966-1), 293F suspension cells were transiently transfected. Cell supernatant was collected 4 or 5 days post-transfection and purified using Ni bead affinity. Analysis of the purified proteins by SDS-PAGE revealed the molecular weight to be as expected (approximately 15kDa). Figure 1 shows images of the Coomassie brilliant blue stained gels after purification of the positive control antibody BI-VEGF ab and some antibody candidates. Example 2: Testing of antibody activity

[0127] 1. Testing the VEGFA binding activity of antibody candidates. The antigen VEGFA was diluted in ELISA coating buffer (final concentration 0.3 μg / mL), added to an ELISA plate at a rate of 100 μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder, the positive control antibody BI-VEGF ab (this antibody is an anti-VEGFA nanobody, and the amino acid sequence of the antibody is derived from SEQ ID NO: 57 described in U.S. Patent No. 9527925 B2; the method for constructing its expression plasmid and purifying the protein is the same as in step 6, "Expression and Purification of Antibody Candidate" of Example 1) and gradient dilutions of the antibody candidate under test (0.001, 0.01, 0.1, 1, 3, 10, 100 nM) were added, and the plates were incubated at 37°C for 1 hour. After washing the plates, His-Tag monoclonal antibody diluent (purchased from Proteintech, catalog number: 66005-1-Ig) was added, and the plates were incubated at 37°C for 1 hour. After washing the plates, HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), purchased from Proteintech, catalog number: SA00001-1, was added and incubated at room temperature for 45 minutes. After washing the plates again, 100 μL of TMB (purchased from Tiangen, catalog number: PA107-01) was added to each well and incubated at 37°C for 15 minutes, followed by the addition of 50 μL of stop solution. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0128] The changes in the VEGFA binding signal of some antibodies with antibody concentration are shown in Figure 2.

[0129] The EC50 of the antibodies' binding to VEGFA was obtained by fitting the VEGFA binding data from the ELISA test, and is shown in Table 7. All 10 antibody candidates in the table were found to have high VEGFA binding activity.

[0130] Table 7: Detection of VEGFA-binding activity of candidate antibodies [Table 7]

[0131] The amino acid sequences in the CDR region of the antibodies described above differ in some amino acid residues, and the antibody activity varies within an acceptable range.

[0132] Furthermore, the affinity of nanobodies to VEGFA was tested using surface plasmon resonance (SPR) technology. Using a Biacore 8K instrument (Cytiva), the antigen VEGFA165 (untagged, purchased from Yiqiao Shenzhou, catalog number: HPLC-10008-HNAH) was coupled to a CM5 chip via amino coupling reagents (EDC and NHS) using an amino coupling reagent, with a coupling volume of approximately 400 RU. During the experiment, the mobile phase buffer was HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20). The analytes were each antibody candidate and the positive control antibody BI-VEGF ab, at dilutions of 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, and 2.5 nM. Kinetic analysis showed an antigen-antibody binding time of 240 seconds, a dissociation time of 1500 seconds, and a flow rate of 30 μL / min. The tip was regenerated with 100 mM hydrochloric acid using multicycle mode. The binding and dissociation curves of the antibody candidate and VEGFA165 were fitted using a "1:1 binding" model to obtain the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD), as shown in Table 8.

[0133] Table 8: Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of antibody candidates to VEGFA165. [Table 8]

[0134] Table 8 shows that the antibody candidate has high affinity for VEGFA165, and the KD value is within 3 times the difference from the positive control, indicating that the affinity of the antibody candidate is equivalent to that of the positive control protein.

[0135] 2. Testing of competitive activity of VEGFR2 To further validate the activity of candidate antibodies that inhibit the VEGFA-binding receptor VEGFR2, competitive ELISA was performed.

[0136] The antigen VEGFA was diluted in ELISA coating buffer (final concentration 0.53 μg / mL), added to a 96-well ELISA plate at a rate of 100 μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder, the VEGFR2-ECD-Fc extracellular domain of VEGFR2 (purchased from Yiqiao Shenzhou, catalog number 10012-H02H) at a final concentration of 1 nM was uniformly mixed in a concentration gradient with gradient dilutions of the antibody under test (0.01, 0.1, 1, 3, 10, 100 nM), added to the 96-well plate, and incubated at 37°C for 1 hour. After washing the plate, HRP-conjugated Goat Anti-Human IgG (purchased from Abbkine, catalog number A21050) was added and incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from Tiangen, catalog number: PA107-01) was added to each well, and the mixture was allowed to develop color at 37°C for 15 minutes, after which 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader. The results of the competitive ELISA are shown in Figure 3.

[0137] Table 9 shows a comparison of the competitive activity (IC50 value and maximum inhibition rate) of antibodies against VEGFR2. The 12 antibody candidates in the table were found to be able to compete with VEGFR2 for binding to VEGFA. The formula for calculating the maximum inhibition rate is as follows:

number

[0138] Table 9: Competitive activity of antibody candidates in VEGFR2 (IC50 value and maximum inhibition rate) [Table 9] 3. Inhibitory activity of candidate antibodies against HUVEC cell proliferation

[0139] We verified that antibody candidates V1, V13, V15, and V30 inhibit the proliferation of human umbilical vein endothelial cells (HUVECs) stimulated by VEGFA.

[0140] Primary HUVEC cells (obtained from the China Center for Typical Cell Cultures (CCTCC)) were digested, resulting in 5 × 10 4 The drug was diluted to cells / mL and inoculated into 96-well plates at 100 μL / well. The culture medium was complete medium (F-12K + 0.1 mg / mL heparin + ECGS + 10% FBS). After 5 hours of adhesion culture, the drug was added.

[0141] Mixtures of VEGFA and diluted antibody candidates were prepared using DMEM / F12(HAM) 1:1 medium (serum-free, penicillin and streptomycin added, containing glutamine, purchased from Biological Industries, catalog number: 01-172-1ACS). The final concentration of VEGFA in each dilution was 35 ng / mL, and the concentration gradients for antibody candidate V1 were 1,000,000 pM, 33,333.33 pM, 11,1111.11 pM, 37,037.04 pM, 12,345.68 pM, 41,15.23 pM, 13,71.74 pM, 457.25 pM, 152.42 pM, 50.81 pM, 16.94 pM, and 5.65 pM. The concentration gradients for V13, V15, and V30 are 1,000,000 pM, 33,333.33 pM, 111,111.11 pM, 37,037.04 pM, 12,345.68 pM, 41,15.23 pM, 1371.74 pM, 457.25 pM, 152.42 pM, and 5.65 pM.

[0142] The negative control antibody is a nanobody that binds to an unrelated antigen, with a concentration gradient (3-fold decrease): 1,000,000 pM, 33,333.33 pM, 11,111.11 pM, 37,037.04 pM, 12,345.68 pM, 41,15.23 pM, 13,71.74 pM, 457.25 pM, and 152.42 pM.

[0143] In addition, controls without VEGFA (minimal cell proliferation) and controls containing only VEGFA but no antibodies (maximum cell proliferation) were established. VEGFA was incubated with a mixture of each antibody diluent at 37°C for 1 hour. After aspirating and removing the original medium from the 96-well cell plate, the mixture of VEGFA and each antibody diluent was added to the cell plate. Two cell wells were prepared for each concentration. The cells were cultured in a 37°C cell culture incubator for 72 hours. CCK-8 solution (purchased from Solarbio, catalog number: CA1210) was added to the 96-well plate, and the culture plate was incubated in the incubator for 3 hours. The absorbance at 450 nm (OD450) was then measured using a microplate reader.

[0144] The inhibition rates of various antibody candidates against cell proliferation were calculated based on the differences in readings from control wells (wells without VEGFA and wells containing only VEGFA but no antibody). The formula for calculating the inhibition rate is as follows:

number

[0145] The inhibition rate curves, maximum inhibition rate, and IC50 for each antibody against HUVEC cell proliferation are shown in Figure 4 and Table 10.

[0146] Table 10: Maximum inhibition rate and IC50 value of each antibody against HUVEC cell proliferation [Table 10]

[0147] Therefore, all four antibody candidates can inhibit the VEGFA signaling pathway at the cellular level, meaning they can inhibit VEGFA-stimulated HUVEC cell proliferation. At the highest antibody concentration (1 μM), antibody candidate V1 can effectively inhibit HUVEC proliferation (maximum inhibition rate close to 100%). In terms of IC50, antibody candidate V30 was found to have relatively strong activity.

[0148] Through ELISA studies of VEGFA binding, VEGFR2 competition, and detection of HUVEC proliferation inhibitory activity at the cellular level, it was demonstrated that the antibody candidate efficiently binds VEGFA, inhibits VEGFA binding by VEGFR2, thereby inhibiting VEGFA-VEGFR signaling and inhibiting vascular endothelial cell proliferation.

[0149] 4. Detection of human VEGFA121 and mouse VEGFA164 binding activity of antibody candidates Using an ELISA experiment, the same method as in Example "1. Testing the VEGFA-binding activity of the antibody candidate" was employed. Specifically, 0.3 μg / mL of human VEGFA121 (purchased from Yiqiao Shenzhou Co., Ltd., catalog number: 10008-HNAH), mouse VEGFA164 (purchased from Yiqiao Shenzhou Co., Ltd., catalog number: 50159-MNAB), or rat VEGFA164 (purchased from Yiqiao Shenzhou Co., Ltd., catalog number: 80006-RNAB) was coated, and the binding activity of the His-tagged antibody candidate V1 to human VEGFA121, mouse VEGFA164, and rat VEGFA164 was detected.

[0150] The human VEGFA121 binding activity of antibody V1 was detected using ELISA, as shown in Figure 5 and Table 11.

[0151] Table 11: Human VEGFA121 binding activity of antibody V1 [Table 11]

[0152] Figure 6 shows the mouse VEGFA164 binding activity of antibody V1.

[0153] Figure 7 shows the rat VEGFA164 binding activity of antibody V1.

[0154] In summary, antibody candidate V1 shows high binding activity to human VEGFA121.

[0155] 5. Detection of Tm values ​​of antibody candidates Eight antibody candidates (V1, V15, V29, V30, V31, V36, V40, V43) were selected, and the buffer containing the antibodies was replaced with the following (10 mM His, pH=6.5, 40 mM NaCl, 5% sucrose, 0.01% Tween-20). The melting temperatures (Tm) of the antibodies were measured using an UNCLE instrument (unchainedlabs), and the results are shown in Table 12. The results indicate that these antibody candidates possess a certain degree of thermal stability.

[0156] Table 12: Melting temperature Tm of antibody candidates [Table 12] Example 3: Humanization of anti-VEGFA nanobodies

[0157] Antibody candidate V1 was used as the starting antibody for humanization. First, the antibody candidate V1 sequence and the human antibody sequence were aligned via the antibody databases IGBLAST and IMGT to determine homology. Then, the human antibody framework region with high homology and the CDR region of the antibody candidate were combined to construct a complete humanized antibody. The method for constructing the humanized antibody expression plasmid and purifying the protein was the same as in "6. Expression and Purification of Antibody Candidate" in Example 1.

[0158] The purified antibodies were tested for VEGFA binding activity using the ELISA method. After the initial humanization of V1, the VEGFA binding activity of the antibodies decreased significantly. By restoring several amino acid mutations in the framework region, nine optimized V1 humanized antibodies were obtained, and their amino acid sequences are shown in Table 13.

[0159] Table 13: Amino acid sequences of candidate humanized antibodies [Table 13] TIFF0007897657000018.tif249170TIFF0007897657000019.tif249170TIFF0007897657000020.tif62170

[0160] These humanized antibodies, with the mutations restored, were transiently expressed and purified. The first step of the purification process was the same as in "6. Expression and Purification of Antibody Candidates" in Example 1. Of these, the positive control BI-VEGF ab and the humanized antibodies V1-DP and V1-SA1 were subjected to Ni bead affinity purification and purified by cation exchange chromatography (S column). Figure 8(A) shows the SDS-PAGE Coomassie brilliant blue stained gel image after the purification of some proteins. The molecular weight of the target protein was as expected (approximately 15 kDa).

[0161] The CDR regions of antibody candidates V13, V15, V30, V40, and V43 were transplanted into the humanized framework region of V1 after mutation restoration. These antibody candidates were then humanized, expressed, and purified. Some of the purified humanized antibodies are shown in Figures 8(B), (C), and (D). The molecular weight of the target protein was as expected (approximately 15 kDa).

[0162] Example 4: Detection of activity of humanized anti-VEGFA nanobodies The VEGFA-binding activity of the obtained humanized antibodies was tested using the ELISA method. The experimental method was the same as in "1. Testing the VEGFA-binding activity of antibody candidates" in Example 2. Figure 9 shows the ELISA test results for some of the humanized antibodies' VEGFA binding.

[0163] The EC50 of the humanized antibody's binding to VEGFA was obtained by fitting VEGFA binding data from an ELISA test, and is shown in Table 14. The data indicates that the humanized antibody candidate has similar VEGFA binding activity to the pre-humanized antibody (V40).

[0164] Table 14: EC50 values ​​of humanized antibody binding to VEGFA [Table 14]

[0165] To further verify the activity of humanized antibodies that inhibit the VEGFA-binding receptor VEGFR2, competitive ELISA was performed. The experimental method was the same as in "2. VEGFR2 Competitive Activity Test" in Example 2, but the differences are as follows: For humanized antibodies V1, V13, V15, and V43, the final concentration of the VEGFR2-ECD-Fc domain in the extracellular region of VEGFR2 (purchased from Yiqiao Shenzhou Co., Ltd., catalog number: 10012-H02H) was 2 nM. The concentration gradients for the V1 humanized antibody were 0.01, 0.1, 1, 10, 30, 100, and 1000 nM. The concentration gradients for the V13, V15, and V43 humanized antibodies were 0.03, 0.3, 3, 10, 30, and 300 nM.

[0166] As the results show, the humanized antibodies V1 and V15 reverted to their pre-humanization levels (i.e., V1 and V15) in terms of competitive activity against VEGFR2 for binding to VEGFA, while the remaining humanized antibodies showed similar VEGFR2 competitive activity. The competitive ELISA results for several humanized antibodies are shown in Figure 10, and the IC50 and maximum inhibition rates are shown in Table 15. The formula for calculating the maximum inhibition rate is as follows:

number

[0167] Table 15: Results of competitive ELISA and maximum inhibition rate of humanized antibodies [Table 15]

[0168] Furthermore, ELISA tests were performed to inhibit VEGFR2 using the humanized antibody of V30 and the remaining humanized antibodies of V43. The experimental method was the same as in "2. Test of competitive activity of VEGFR2" in Example 2, but with the following differences: The final concentration of the VEGFR2-ECD-Fc domain of the extracellular region of VEGFR2 (purchased from Yiqiao Shenzhou Co., Ltd., catalog number: 10012-H02H) was 1 nM. The concentration gradients of the humanized antibodies were 0.03, 0.3, 3, 10, 30, and 300 nM.

[0169] Figure 11 shows the results of ELISA tests for VEGFR2 competition between humanized antibodies V30 and V43.

[0170] Table 16 shows the competitive activity of the antibodies against VEGFR2 (IC50 value and maximum inhibition rate). It was found that the humanized antibody could compete with VEGFR2 for binding to VEGFA, and its activity was similar to that of the pre-humanized antibody (V30). The formula for calculating the maximum inhibition rate is as follows:

number

[0171] Table 16: Competitive activity of antibodies against VEGFR2 (IC50 value and maximum inhibition rate) [Table 16]

[0172] Using surface plasmon resonance (SPR) technology, the antigen VEGFA165 was coupled, and the affinity of the nanobodies to VEGFA before and after humanization was tested. The experimental method and data fitting method were the same as in "1. Testing the VEGFA-binding activity of antibody candidates" in Example 2. The obtained KDs are shown in Table 17.

[0173] Table 17: Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of humanized antibodies and VEGFA. [Table 17]

[0174] Furthermore, the affinity of the humanized antibody V1-SA1 to VEGFA was tested by coupling the antibody using surface plasmon resonance (SPR) technology. Using a Biacore 8K instrument (Cytiva), antibody V1-SA1 was coupled to a CM5 chip via amino coupling reagents (EDC and NHS) with the ligand. The coupling conditions were 10 mM sodium acetate (pH 4.0) and a V1-SA1 protein concentration of 20 μg / mL. The coupling amount of ligand V1-SA1 was 371.8 RU. During the experiment, the mobile phase buffer was HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20). The analytes were the antigen VEGFA165 (untagged, purchased from GenScript, catalog number Z03073), at dilutions of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. Kinetic analysis showed an antigen-antibody binding time of 180 seconds, a dissociation time of 200 seconds, and a flow rate of 30 μL / min. Using multicycle mode, the tips were regenerated with 10 mM glycine hydrochloride (pH=1.5). Antigen-antibody binding curves were fitted using a "two-state reaction" model to obtain binding rates, dissociation rates, and KD.

[0175] The binding and dissociation curves of V1-SA1 and VEGFA165 are shown in Figure 12, and the fitted KD values ​​are shown in Table 18.

[0176] Table 18: Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of humanized antibody V1-SA1 and VEGFA. [Table 18]

[0177] Tables 17 and 18 show that antibody V1 maintains a high affinity for VEGFA165 even after humanization.

[0178] Example 5: Study on the stability of humanized anti-VEGFA nanobodies 1. Comparison of Tm values ​​of V1 humanized antibodies Antibodies with high thermal stability are suitable for antibody production and long-term storage; therefore, the melting temperatures (Tm) of various humanized antibodies were compared.

[0179] For the nine humanized antibodies against V1, the buffer was replaced with PBS (pH=7.4) and the Tm was measured using an UNCLE instrument (unchainedlabs). The ranking of Tm is shown in Figure 13. The Tm values ​​of the humanized antibodies varied within an acceptable range. Among them, V1-DP and V1-SA1 had high Tm values, suggesting that these two antibodies likely have better thermal stability than the other humanized antibodies.

[0180] 2. Study on the colloidal stability of the humanized antibody V1-SA1 The coding genes for the humanized antibody V1-SA1 and the positive control antibody BI-VEGF ab were constructed in the expression vector pCDNA3.1(+). Here, the secreted peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 68) was added to the N-terminus of the antibody, and the flexible linker GGS and tag 6×His were added to the C-terminus. The two antibodies were transiently expressed and purified using Ni bead affinity. The experimental method was the same as in the "6. Expression and Purification of Antibody Candidates" step of Example 1. After purification, the proteins were separated by SDS-PAGE, and the gel staining results are shown in Figure 14. From this figure, it was found that the molecular weights of the two proteins were as expected (approximately 15 kDa). The protein expression levels and the purity of the Coomassie brilliant blue stain on SDS-PAGE are shown in Table 19. The expression level of V1-SA was found to be much higher than that of BI-VEGF ab.

[0181] Table 19: Transient expression of humanized antibody V1-SA1 and positive control antibody BI-VEGF ab, and protein purity on SDS-PAGE gel. [Table 19]

[0182] The liquids of V1-SA1 and BI-VEGF ab proteins were changed to 1x PBS (pH=7.4), the protein concentration was adjusted to 10 mg / mL, and HPLC-SEC analysis of the proteins was performed (chromatographic column: Zenix-C SEC-300, Sepax). The proportions of protein monomers, aggregates, and fragments are shown in Table 20. It was found that the proportion of V1-SA aggregates was much lower than that of BI-VEGF ab aggregates, indicating that V1-SA1 is less prone to aggregation.

[0183] Table 20: Percentage of each component of antibody V1-SA1 and BI-VEGF ab in SEC-HPLC analysis [Table 20]

[0184] The solutions were converted to PBS, and the antibodies V1-SA1 and BI-VEGF ab (protein concentration 10 mg / mL) were added. After sterile filtration, the solutions were left at 37°C for a set period (0 days and 2 days). The properties of the protein solutions were observed, and the colloidal stability of both was compared. The results are shown in Table 21.

[0185] Table 21: Comparison of properties, average particle size, and PDI of antibodies V1-SA1 and BI-VEGFab after being left at 37°C for 0 and 2 days, respectively. [Table 21]

[0186] Following the principle of dynamic light scattering, the particle size of the proteins in the above antibody V1-SA1 and BI-VEGF ab solutions (after being diluted four-fold with PBS) was measured using a nanoparticle size and potential analyzer (NS-90Z, OMEC) after being left at 37°C for a fixed period (0 days and 2 days). The average particle size (Z-Average) and polydispersity index (PDI) results are shown in Table 21. At time 0, the average hydration dynamics diameter and PDI of V1-SA1 were all smaller than those of BI-VEGF ab. Considering that the molecular weights of the two are similar, this indicates that BI-VEGF ab contains a certain proportion of high molecular weight protein aggregates in this buffer. Under the same conditions, V1-SA1 had a more uniform particle size distribution than BI-VEGF ab, and the proportion of high molecular weight protein aggregates was significantly lower.

[0187] As can be seen from Table 21, in samples left at 37°C for 2 days, V1-SA1 remained clear, while precipitation was observed in the BI-VEGF ab protein solution. This indicates that the colloidal stability of V1-SA1 is superior to that of BI-VEGF ab. In this case, the mean hydration dynamics diameter of V1-SA1 is much smaller than that of BI-VEGF ab, and its change is smaller than that at time 0. The mean hydration dynamics diameter of BI-VEGF ab is much larger than that of V1-SA1, and significantly larger than the mean hydration dynamics diameter of the BI-VEGF ab solution at time 0, indicating that BI-VEGF ab forms even higher molecular weight protein aggregates under high-temperature conditions.

[0188] Example 6: Research on the production and activity of monovalent nanobody-Fc fusion proteins 1. Production of monovalent nanobody-Fc fusion proteins In this example, fusion expression was performed between monovalent nanobodies and IgG1 Fc (SEQ ID NO: 62), PPCP-Fc (SEQ ID NO: 75, i.e., the remaining amino acid sequence after removing DKTHTC in SEQ ID NO: 62), or the IgG1 Fc mutant Fc-m1 (SEQ ID NO: 63).

[0189] The amino acid sequence of IgG1 Fc is as follows: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:62) The amino acid sequence of PPCP-Fc is as follows: PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:75) The amino acid sequence of the IgG1 Fc mutant Fc-m1 is as follows: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:63) Table 22 shows the amino acid sequence of the constructed nanobody-Fc fusion protein.

[0190] Table 22: Amino acid sequences of nanobody-Fc fusion proteins [Table 22] TIFF0007897657000032.tif52170

[0191] To facilitate comparison of the VEGFA-binding activity of the linear tandem bivalent nanobody 2V15-9GS (SEQ ID NO:74) (based on 2V15-9GS and containing six histidine tags), the fusion proteins V15-PPCP-Fc, V15-9GS-PPCP-Fc, and V15-Fc, a linker (SGGS (SEQ ID NO:70)) and six histidine tags (HHHHHH (SEQ ID NO:71)) were added to the C-terminus of the Fc segment of the fusion protein. During plasmid construction of the monovalent nanobody-Fc fusion protein, the nanobody and Fc containing the linker and histidine tags were amplified by PCR, where the PCR template for the Fc fragment was derived from the synthesized Fc gene. The nanobody and Fc were incorporated into the vector pCDNA3.1(+) by two-fragment homologous recombination. The enzymatic cleavage sites used were NheI and XbaI. The secreted peptide of the fusion protein is METDTLLLWVLLLWVPGSTG (SEQ ID NO: 72). Subsequently, the target protein sequence was fused. After successful sequencing of these fusion protein particles, endotoxin-free extraction of the plasmid was performed, and 293F suspension cells were transiently transfected using the transfection reagent polyethyleneimine (PEI, linear, MW=25kDa, purchased from Polysciences, catalog number: 23966-1). Cell supernatant was collected on day 4 or 5 post-transfection and purified by Ni beads. Analysis of the purified protein by SDS-PAGE showed that the molecular weight of the protein monomer under reducing conditions was approximately 39kDa, which was as expected. Figure 15 shows a Coomassie brilliant blue stained gel image of a portion of the purified monovalent nanobody-Fc fusion protein.

[0192] 2V15-9GS (SEQ ID NO: 74) QLQLVESGGGSVQPGGSLRLSCEVSGRTFASYTMGWFRQAPGKEREFVVAISKGGYKYDSVSLEARFTISKDNTKNTVYLQMNSLKPEDTAVYYCAGTRAYGSSRLRLAETYEYWGQGTQVTVSSGGGG SGGGSQLQLVESGGGSVQPGGSLRLSCEVSGRTFASYTMGWFRQAPGKEREFVVAISKGGYKYDSVSLEARFTISKDNTKNTVYLQMNSLKPEDTAVYYCAGTRAYGSSRLRLAETYEYWGQGTQVTVSS

[0193] In the case of the fusion protein (V1-SA1-Fc-m1) of the humanized antibody V1-SA1 and Fc, the linker amino acid sequence between V1-SA1 and Fc-m1 is EPKSA (SEQ ID NO: 73). The expression plasmid was constructed using the method described above, but it did not contain an additional amino acid at the C-terminus of Fc. That is, the fusion protein V1-SA1-Fc-m1 contains an Fc tag but not a histidine tag. After transiently transfecting 293F cells with the V1-SA1-Fc-m1 expression plasmid, affinity purification was performed using protein A beads. During protein elution, the target protein was eluted using 0.1M citrate (pH 3.0), rapidly neutralized, and the target protein was obtained.

[0194] 2. Detection of activity of monovalent nanobody-Fc fusion proteins The VEGFA-binding activity of the purified monovalent nanobody-Fc fusion protein was tested. The ELISA method was the same as in "1. Testing of VEGFA-binding activity of antibody candidates" in Example 2. The ELISA results are shown in Figure 16, and the EC50 is shown in Table 23.

[0195] Table 23: Testing of VEGFA binding activity of monovalent nanobody-Fc fusion proteins [Table 23]

[0196] The data in the table shows that both the monovalent nanobody-Fc fusion protein and the linear divalent nanobody tested possessed potent VEGFA-binding activity.

[0197] The affinity of the monovalent nanobody-Fc fusion protein (V1-SA1-Fc-m1) to VEGFA was tested using surface plasmon resonance (SPR) technology. Using a Biacore 8K instrument (Cytiva), V1-SA1-Fc-m1 was captured on a Protein A chip as a ligand. During immobilization, V1-SA1-Fc-m1 was diluted to a protein concentration of 2 μg / mL using mobile phase buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20), and the coupling amount was approximately 590 RU. During the experiment, the analyte was the antigen VEGFA165 (untagged, purchased from GenScript, catalog number: Z03073), at dilutions of 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0.390625 nM. Kinetic analysis showed a binding time of 180 seconds, a dissociation time of 900 seconds, and a flow rate of 30 μL / min for the fusion protein and VEGFA165 antigen. After each "binding-dissociation" cycle, the tip was regenerated with 10 mM glycine hydrochloride (pH 1.5). The binding and dissociation curves of the fusion protein and VEGFA165 antigen are shown in Figure 17. As shown in Table 24, the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) were obtained by fitting using a "1:1 binding" model.

[0198] Table 24: Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of monovalent nanobody-Fc fusion protein and VEGFA165. [Table 24]

[0199] SPR data revealed that the monovalent nanobody-Fc fusion protein V1-SA1-Fc-m1 has a high affinity for VEGFA165 (KD = 0.148 nM). From Table 18 and Figure 12 of "6. Activity Detection of Humanized Antibodies" in Example 2 above, it was found that the KD of monovalent nanobody V1-SA1 and VEGFA165 was 4.38 nM. Therefore, after fusion with Fc, the affinity of the monovalent nanobody to VEGFA165 was nearly 30 times higher than the affinity when not fused with Fc. This indicates that the anti-VEGFA antibody-Fc fusion protein has a stronger target affinity and potentially superior efficacy in inhibiting the VEGFA signaling pathway.

[0200] Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the specific details of the above embodiments, and many simple modifications can be made to the technical solution means of the present invention within the scope of the technical concept of the present invention, and all of these simple modifications fall within the scope of protection of the present invention.

[0201] Furthermore, the specific technical features described in the above-mentioned embodiments can be combined in any suitable manner, as long as they do not contradict each other, and in order to avoid unnecessary redundancy, the present invention will not describe various possible combinations again.

Claims

1. An anti-VEGFA antibody or its antigen-binding fragment, The anti-VEGFA antibody or its antigen-binding fragment is a single-domain antibody and comprises heavy chain variable regions CDR-H1, CDR-H2, and CDR-H3. An anti-VEGFA antibody or its antigen-binding fragment, characterized in that the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are SEQ ID NO: 1, 4, and 9, respectively.

2. The anti-VEGFA antibody or antigen-binding fragment according to claim 1, characterized in that the anti-VEGFA antibody or antigen-binding fragment comprises a humanized sequence, and the modification site of the humanized sequence is located in a non-CDR region.

3. The anti-VEGFA antibody or its antigen-binding fragment according to claim 1, characterized in that the anti-VEGFA antibody or its antigen-binding fragment is a nanobody.

4. The anti-VEGFA antibody or antigen-binding fragment according to claim 1, characterized in that the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment includes any of the amino acid sequences among SEQ ID NO: 13, 35-43, and 67.

5. The anti-VEGFA antibody or its antigen-binding fragment according to claim 1, characterized in that the anti-VEGFA antibody or its antigen-binding fragment binds to human or monkey VEGFA protein.

6. A fusion protein characterized by comprising the anti-VEGFA antibody or its antigen-binding fragment as described in claim 1.

7. The fusion protein according to claim 6, further comprising an anti-VEGFA antibody or its antigen-binding fragment other than the anti-VEGFA antibody or its antigen-binding fragment described in claim 1, an antibody or its antigen-binding fragment against another target, or another functional component.

8. The fusion protein according to claim 7, characterized in that the other functional components include one or more combinations of serum albumin, cytokines, transferrin, scaffolding proteins, polysaccharides, fatty acid chains, avidin, biotin, streptavidin, radionuclides, PEG-modified components, or Fc fragments.

9. Other targets include IgG, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Endoglin (CD105), TGF, Integrin, Integrin receptor, Interleukin, Interleukin receptor, 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, and Claudin. The fusion protein according to claim 7, characterized by being selected from 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, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, or 5T4.

10. The fusion protein according to claim 9, characterized in that the interleukin comprises IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, and IL-23, and the interleukin receptor comprises IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12R, IL15Rα, IL17R, and IL23R.

11. A chimeric antigen receptor, The chimeric antigen receptor is characterized in that the extracellular domain of the chimeric antigen receptor comprises the anti-VEGFA antibody or its antigen-binding fragment as described in claim 1.

12. An immune cell characterized by expressing the anti-VEGFA antibody or its antigen-binding fragment as described in claim 1.

13. An immune cell characterized by expressing the chimeric antigen receptor described in claim 11.

14. A nucleic acid characterized by encoding an anti-VEGFA antibody or its antigen-binding fragment as described in claim 1.

15. A nucleic acid characterized by encoding the fusion protein described in claim 6.

16. A vector characterized by comprising the nucleic acid described in claim 14.

17. A host cell characterized by containing the nucleic acid described in claim 14.

18. A method for producing an anti-VEGFA antibody or its antigen-binding fragment according to any one of claims 1 to 5, characterized by comprising introducing a nucleic acid or vector encoding an anti-VEGFA antibody or its antigen-binding fragment into a host cell and subsequently inducing its expression.

19. A method for obtaining an anti-VEGFA antibody or its antigen-binding fragment according to any one of claims 1 to 5, comprising immunizing an alpaca with human or monkey VEGFA protein.

20. The use of an anti-VEGFA antibody or its antigen-binding fragment according to any one of claims 1 to 5, a fusion protein according to any one of claims 6 to 10, a chimeric antigen receptor according to claim 11, an immune cell according to claim 12 or 13, a nucleic acid according to claim 14 or 15, a vector according to claim 16, or a host cell according to claim 17 in the manufacture of a product for treating and / or preventing a disease related to VEGFA, the manufacture of a product for inhibiting VEGFA-mediated vascular endothelial cell proliferation or angiogenesis, or in the manufacture of an antibody-drug conjugate, an antibody diagnostic kit or a VEGFA racer.

21. A method for detecting VEGFA, The method involves contacting a test sample with an anti-VEGFA antibody or its antigen-binding fragment according to any one of claims 1 to 5, and then detecting the content of a complex formed by VEGFA and the anti-VEGFA antibody or its antigen-binding fragment. The detection method is characterized by being performed in vitro on a non-human organism.

22. A method for inhibiting vascular endothelial cell proliferation or angiogenesis mediated by VEGFA, The method involves contacting vascular endothelial cells with an anti-VEGFA antibody or its antigen-binding fragment according to any one of claims 1 to 5, a fusion protein according to any one of claims 6 to 10, a chimeric antigen receptor according to claim 11, an immune cell according to claim 12 or 13, a nucleic acid according to claim 14 or 15, a vector according to claim 16, or a host cell according to claim 17. The method described above is characterized by being performed in vitro on a non-human organism.

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