Anti-VEGFA antibodies or antigen-binding fragments thereof, and uses thereof

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, specifically to an anti-VEGFA antibody or antigen-binding fragment thereof, and uses thereof. [Background technology]

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

[0003] Pathological angiogenesis occurs in various solid tumors, inflammatory responses, and retinal vascular disease. The later stages of tumor cell proliferation depend on new blood vessels to provide sufficient nutrients to support tumor growth and metastasis. Therefore, inhibiting angiogenesis is an important approach to cancer treatment. The first anti-VEGFA monoclonal antibody, bevacizumab, was approved in 2004 for the treatment of metastatic colorectal cancer. Overexpression of VEGFA is also closely associated with retinal vascular proliferation, including exudative age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy (DR). Therefore, anti-VEGFA drugs have become the first-line treatment for retinal vascular proliferation.

[0004] Currently, four major anti-VEGFA protein drugs are commercially available for the treatment of wet AMD, including ranibizumab, aflibercept, conbercept, and brolucizumab. Additionally, bevacizumab is also used off-label for the treatment of wet AMD. These anti-VEGFA protein drug classes include traditional monoclonal antibodies, antibody fragments (Fab and scFv), and receptor-Fc fusion proteins. However, no single-domain antibodies (VHH: variable domain heavy-chain antibodies) or nanobodies (Nb) are currently available.

[0005] Single-domain antibodies, or nanobodies, are the variable domains of heavy-chain antibodies that naturally lack light chains in camelids. They are the smallest stable antibody unit capable of fully binding to an antigen. Single-domain antibodies have a molecular weight of approximately 13 kDa, are highly thermostable, and have excellent water solubility. As a novel antibody form, nanobodies are attracting increasing attention for their development.

[0006] Thus, the present invention provides novel anti-VEGF antibodies or antigen-binding fragments thereof that are used to inhibit the binding of VEGFR1 and VEGR2 to VEGF, thereby inhibiting the downstream signaling pathway of VEGF and inhibiting cell proliferation stimulated by VEGF. Summary of the Invention

[0007] A first aspect of the present invention provides an anti-VEGFA antibody or antigen-binding fragment thereof, the anti-VEGFA antibody or antigen-binding fragment thereof comprising CDR-H1, CDR-H2, and CDR-H3 of a heavy chain variable region; the amino acid sequence of CDR-H1 comprises SYTMG (SEQ ID NO: 1) or an amino acid sequence having at least 80% identity to SYTMG (SEQ ID NO: 1); the amino acid sequence of CDR-H2 comprises AISKGGYKYX1X2VSLEA (SEQ ID NO: 2) or an amino acid sequence having at least 80% identity to AISKGGYKYX1X2VSLEA (SEQ ID NO: 2); The amino acid sequence of CDR-H3 comprises TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3) or an amino acid sequence having at least 80% identity to TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3).

[0008] Wherein, X in SEQ ID NO:2 or SEQ ID NO:3 may be any naturally occurring 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, 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.

[0010] In a specific embodiment of the present invention, the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 comprise 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 CDR-H1, CDR-H2, and CDR-H3 of antibody candidates [Table 1]

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

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

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

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

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

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

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

[0019] 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, and more preferably, the humanized modification site is located in the framework region and / or constant region of the antibody.

[0020] In one particular embodiment of the invention, the anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody, which has a higher molar concentration and can bind more antigen molecules per mass compared to a full-length IgG antibody, Fab, or scFv.

[0021] The anti-VEGFA antibody or antigen-binding fragment thereof can bind human or monkey VEGFA protein, wherein the human VEGFA protein and monkey VEGFA protein are identical in sequence.

[0022] Preferably, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences of SEQ ID NOs: 13 to 32, 35 to 61, and 64 to 67, or has at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 13 to 32, 35 to 61, and 64 to 67.

[0023] In one specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof is set forth in any one of SEQ ID NOs: 13-32, 35-61, and 64-67.

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

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

[0026] In a second aspect of the invention, there is provided an anti-VEGFA antibody or antigen-binding fragment thereof.

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

[0028] The anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences of SEQ ID NOs: 13 to 32, 35 to 61, and 64 to 67, or has at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 13 to 32, 35 to 61, and 64 to 67.

[0029] In one specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof is set forth in any of SEQ ID NOs: 13-32, 35-61, 64-67.

[0030] In a third aspect of the present invention, there is provided a method for screening for an anti-VEGFA antibody or antigen-binding fragment thereof, comprising immunizing an alpaca with human or monkey VEGFA protein.

[0031] In a fourth aspect, the present invention provides a fusion protein comprising the anti-VEGFA antibody or antigen-binding fragment thereof described above.

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

[0033] Preferably, the functional moiety includes, but is not limited to, one or a combination of two or more of serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, lipid chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and markers thereof, PEGylated moieties, or Fc fragments.

[0034] Preferably, the other target is IgG, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Endoglin (CD105), TGF, Integrin, Integrin receptor, interleukin (e.g., IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptor (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 antigen-binding fragment thereof is a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, an Fd fragment, an Fd' fragment, an Fv fragment, a dAb fragment, an isolated CDR region, an F(ab')2 fragment, a single domain antibody, a single-chain antibody molecule, or a linear antibody.

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

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

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

[0039] Among these, the anti-VEGFA antibodies or antigen-binding fragments thereof are directly or indirectly linked, 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 antibodies or antigen-binding fragments thereof against other targets, or other functional moieties, between other anti-VEGFA antibodies and antibodies or antigen-binding fragments thereof against other targets, or other functional moieties, between antibodies or antigen-binding fragments thereof against other targets, or other functional moieties, and between other anti-VEGFA antibodies or antigen-binding fragments thereof.

[0040] The indirectly linked may be linked by a linker, functional domain and / or coupling linker, wherein the linker is selected from a linking peptide, oligopeptide, oligopeptide polymer, polypeptide, polypeptide polymer, PEG, nucleic acid, polysaccharide, lipid chain, biotin, streptavidin or avidin.

[0041] The functional domains are one or a combination of two or more of an Fc fragment, serum albumin, a cytokine, transferrin, a scaffolding protein, an antibody or antigen-binding fragment thereof against VEGFA or other targets.

[0042] The linker for coupling includes one or a combination of two or more of a toxin, a drug, a nucleic acid, PEG, a radionuclide and its marker.

[0043] Preferably, said directly or indirectly linked means directly or indirectly linked to the N-terminus, C-terminus and / or internal residue of said anti-VEGFA antibody or antigen-binding fragment thereof, other anti-VEGFA antibodies and / or antibodies against other targets.

[0044] The order of linkage of the anti-VEGFA antibody or antigen-binding fragment thereof, other anti-VEGFA antibody, or other antibody against another target contained in the fusion protein may be such that the N-terminus, C-terminus, and / or internal residue of one antibody is linked to the N-terminus, C-terminus, and / or internal residue of another antibody.

[0045] In one specific embodiment of the present invention, the order of linkage of the anti-VEGFA antibody or antigen-binding fragment thereof, other anti-VEGFA antibody, or antibody against another target 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 specific embodiment of the present invention, the order of linkage of the anti-VEGFA antibody or antigen-binding fragment thereof, other anti-VEGFA antibody, or antibody against another target 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 comprises the amino acid sequence of any one of SEQ ID NOs: 64 to 67 or has at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 64 to 67.

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

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

[0051] In a fifth aspect of the present invention, there is provided a chimeric antigen receptor, the extracellular domain of which comprises an anti-VEGFA antibody or an antigen-binding fragment thereof as described above.

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

[0053] In a sixth aspect of the present invention, there is provided 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.

[0054] In a seventh aspect of the present invention, there is provided a vector comprising the above nucleic acid.

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

[0056] In an eighth aspect of the present invention, there is provided a host cell comprising the above nucleic acid or the above vector.

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

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

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

[0060] In a ninth aspect of the present invention, there is provided a method for producing a host cell, which comprises introducing the above-described nucleic acid or vector into a host cell and then inducing its expression.

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

[0062] In an eleventh aspect, the present invention provides an immune cell expressing the above-mentioned anti-VEGFA antibody or antigen-binding fragment thereof, or the above-mentioned chimeric antigen receptor.

[0063] 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.

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

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

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

[0067] The products for treating and / or diagnosing the diseases target cells expressing VEGFA, and the cells may be cardiomyocytes, proximal tubule cells, hepatocytes, vascular endothelial cells, granulocytes, 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, a drug, a chip, an antibody-drug conjugate, or the like. The disease is a disease associated with the VEGFA signaling pathway. More preferably, the disease may be a tumor, an ocular disease accompanied by abnormal vascular proliferation or angiogenesis (e.g., retinal vascular disease), or the like.

[0069] In a fourteenth aspect of the invention, there is provided an antibody-drug conjugate (ADC) comprising the anti-VEGFA antibody, or antigen-binding fragment thereof, of the invention covalently linked to a drug.

[0070] In a fifteenth aspect of the present invention, there is provided a method for detecting VEGFA, which comprises 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 between VEGFA and the anti-VEGFA antibody or its antigen-binding fragment.

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

[0072] In a sixteenth aspect of the present invention, there is provided a method for diagnosing a disease, comprising: obtaining a sample; contacting the sample with the anti-VEGFA antibody or antigen-binding fragment thereof, 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 between VEGFA and the anti-VEGFA antibody or antigen-binding fragment thereof.

[0073] The disease is a disease associated with the VEGFA signaling pathway. More preferably, the disease may be a tumor, an ocular disease accompanied by abnormal vascular proliferation or angiogenesis (for example, ocular fundus vascular disease), or the like.

[0074] In a seventeenth aspect of the present invention, there is provided a method for treating and / or preventing a disease, the method comprising administering to an individual the above-described anti-VEGFA antibody or antigen-binding fragment thereof, the above-described chimeric antigen receptor, the above-described fusion protein, the above-described nucleic acid, the above-described vector, the above-described host cell, the above-described immune cell, or a product for treating and / or diagnosing the above-described disease.

[0075] The disease is a disease associated with the VEGFA signaling pathway, and more preferably, may be a tumor, an ocular disease accompanied by abnormal vascular proliferation or angiogenesis (for example, ocular fundus vascular disease), or the like.

[0076] In an eighteenth aspect of the present invention, there is provided a method for inhibiting VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, the method comprising contacting a vascular endothelial cell with the above-mentioned anti-VEGFA antibody or antigen-binding fragment thereof, 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 anti-VEGFA antibody or antigen-binding fragment thereof, 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 in serum-free medium for vascular endothelial cells, followed by incubation with an antigen (e.g., VEGFA165).

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

[0079] Preferably, the antigen, the anti-VEGFA antibody or antigen-binding fragment thereof, 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 is added to a vascular endothelial cell culture plate and cultured in the culture plate.

[0080] Preferably, the detection is carried out after the culture.

[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 between room temperature and 45°C, preferably between 30 and 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, or 45° C. Preferably, the culture is carried out in an incubator with 5% CO 2 .

[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 is to detect the number of viable vascular endothelial cells.

[0086] In a nineteenth aspect of the present invention, there is provided a method for treating and / or preventing a disease, the method comprising contacting a target cell with the anti-VEGFA antibody or antigen-binding fragment thereof, 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.

[0087] The disease is a disease associated with the VEGFA signaling pathway. More preferably, the disease may be a tumor, an ocular disease accompanied by abnormal vascular proliferation or angiogenesis (for example, ocular fundus vascular disease), or the like.

[0088] Preferably, the target cells are selected from cells that express VEGFA, such as cardiomyocytes, proximal tubule cells, hepatocytes, vascular endothelial cells, granulocytes, 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] In a twentieth aspect of the present invention, there is provided use of the above-mentioned anti-VEGFA antibody or antigen-binding fragment thereof, 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 inhibiting angiogenesis, or an antibody-drug conjugate, an antibody diagnostic kit, or a tracer.

[0090] The disease may be a tumor, an abnormal vascular proliferation, an ocular disease associated with neovascularization (for example, ocular fundus vascular disease), or the like.

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

[0092] The present invention provides a novel anti-VEGFA antibody or antigen-binding fragment thereof that has high thermostability, high water solubility, small molecular weight, simple expression and purification processes, and high affinity for VEGFA, effectively inhibiting the VEGFA signaling pathway and pathological processes such as vascular endothelial cell proliferation and angiogenesis caused by VEGFA, and is expected to be used in the treatment of VEGFA-related diseases. It may also provide greater clinical value, such as better tissue penetration, lower production costs, and more convenient administration.

[0093] The "drug" of the present invention can be used to treat non-human animals, such as humans or non-human mammals. The drug may contain a pharmaceutically acceptable carrier, excipient, or salt commonly found in the art. The drug may be administered by any appropriate route, such as gastrointestinal (e.g., oral) or parenteral (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, rectal, etc.) routes. The drug may be in any appropriate dosage form, such as a gastrointestinal dosage form or a parenteral dosage form, preferably including, but not limited to, tablets, pills, powders, granules, capsules, troches, 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, drop pills, 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%, or 99.5%). The drug may be prepared as a reagent having a protein concentration of 1 to 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 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] The term "pharmaceutically acceptable" as used herein means that it does not significantly stimulate an 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 aimed at diagnosing or treating a disease, or may be aimed at something other than diagnosing or treating a disease.

[0096] The "antigen-binding fragment" of the present invention refers to a portion of an antibody that retains its specific binding activity, i.e., any portion of an 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 chains 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. Fab is a monovalent fragment consisting of the 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 the VH and CH1 domains. Fv is an Fv fragment consisting of the VL and VH domains of a single antibody arm. Fab' is a Fab fragment containing one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH is Fab' having at least one free thiol on a cysteine ​​residue in the constant domain, where VH represents the heavy chain variable region, VL represents the light chain variable region, and CL represents the light chain.

[0097] The "CH2" or "CH3" of the present invention refers to the CH2 or CH3 of the heavy chain constant region. A complete heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Specifically, the CH2 domain refers to the portion of an antibody heavy chain polypeptide extending from approximately EU position 231 to EU position 340 (according to the Kabat 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 an 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 comprising the CH2 and CH3 domains of an antibody, which form a dimer via two or more disulfide bonds in the hinge region and are held together by hydrophobic interactions in the CH3 domain.

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

[0100] The terms "comprise" or "comprise" of the present invention are open expressions and when used to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist 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.

[0101] The "homology" of the present invention, in relation to the protein sequence or nucleotide sequence used, means that a person skilled in the art can determine that the sequence used has 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 1 This means that the sequence can be adjusted as needed for actual work to have a homology of at least (including, but not limited to) 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%, or 99.9%.

[0102] The "humanized antibody" of the present invention refers to an antibody in which the framework region and / or constant region portion (e.g., CH region) of the antibody, or the entire antibody, is encoded by a human antibody gene. In a specific embodiment of the present invention, the CDR region of the antibody is not modified.

[0103] The "individual" of the present invention may be a human or a non-human mammal, and the non-human mammal may be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, a pig, a cow, a sheep, a horse, a donkey, a fox, a raccoon dog, a mink, a camel, a dog, a cat, a rabbit, a rodent (e.g., a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey.

[0104] The term "treatment" as used herein means to slow, interrupt, prevent, control, arrest, reduce or reverse the progression or severity of a sign, symptom, disorder, condition or disease after the disease has begun to develop, but does not necessarily mean completely eliminating all signs, symptoms, conditions or disorders associated with the disease.

[0105] The term "prevention" as used herein means a method carried out to prevent or delay the occurrence of a disease, condition or symptom in the body.

[0106] The term "diagnosis" as used herein means determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or determining the progression or likelihood of future progression of a disease.

[0107] The tumor of the present invention can be any unwanted cell proliferation (or any disease manifesting as unwanted cell proliferation), neoplasm, or increased tendency or risk of unwanted cell proliferation, neoplasm, or tumor. It can be benign or malignant, primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be present in any tissue. Exemplary tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, mammary gland, cecum, central nervous system (including or excluding the brain), 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, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, retina, 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, 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, tongue squamous cell carcinoma, nasopharyngeal cancer, 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, neurilemmoma, 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, oropharyngeal cancer, etc.

[0108] The term "fundus vascular disease" as used herein refers collectively to diseases occurring in the retinal arteries or veins, or diseases associated with choroidal neovascularization, including, but 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 drawings]

[0109] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. [Figure 1] 1 is a Coomassie brilliant blue stained gel image of different batches of purified antibody candidates. [Figure 2] FIG. 1 shows the results of antibody concentration-dependent changes in VEGFA binding signals for various antibody candidates. [Figure 3] FIG. 1 shows the results of competitive ELISA of various antibody candidates. [Figure 4] Percent inhibition of HUVEC cell proliferation by various antibody candidates. [Figure 5] ELISA detection of antibody V1 binding activity against human VEGFA121. [Figure 6] ELISA detection of mouse VEGFA164 binding activity of antibody V1. [Figure 7] ELISA detection of rat VEGFA164 binding activity of antibody V1. [Figure 8] 1 is a Coomassie brilliant blue stained gel image after purification of humanized antibody. [Figure 9] ELISA test of VEGFA binding of humanized antibodies. [Figure 10] ELISA test of VEGFR2 competition of humanized antibodies. [Figure 11] ELISA test of VEGFR2 competition of humanized antibodies against V30 and V43. [Figure 12] Binding-dissociation curve (SPR) of V1-SA1 and VEGFA165. [Figure 13] Melting temperatures (Tm) of various humanized antibodies. [Figure 14] 1 shows an SDS-PAGE gel image of humanized antibody V1-SA1 and positive control antibody BI-VEGF ab after purification. [Figure 15] Figure 10: Coomassie brilliant blue stained gel image after purification of monovalent nanobody-Fc fusion proteins. [Figure 16] ELISA test of VEGFA binding of monovalent nanobody-Fc fusion proteins. [Figure 17]Binding-dissociation curve (SPR) of V1-SA1-Fc-m1 and VEGFA165. DETAILED DESCRIPTION OF THE INVENTION

[0110] The following describes the technical solutions in the embodiments of the present invention clearly and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some of the embodiments of the present invention, not all of the embodiments, and all other embodiments that can be obtained by those skilled in the art based on the embodiments of the present invention without any creative work fall within the scope of protection of the present invention.

[0111] Example 1: Alpaca immunization and preliminary antibody screening

[0112] 1. Alpaca immunity Human VEGFA165 (untagged, purchased from Yikyo Shinshu Co., Ltd., product number: HPLC-10008-HNAH, hereafter simply referred to as VEGFA) was emulsified and used to immunize alpacas. Two alpacas were selected, and each animal was immunized once every two weeks with 1 mg of VEGFA protein. Serum was collected from the second immunization and titers were measured. Of these, alpaca 1# was immunized four times with the antigen, and alpaca 2# was immunized three times with the antigen. Serum titers were measured, 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 (designated VEGFR2-biotin). VEGFA was diluted to 0.5 μg / mL in CBS buffer, coated onto an ELISA plate, and left overnight at 4°C. After blocking with 3% nonfat dry milk, 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 the 0.5 μg / mL VEGFR2-biotin solution was added to each blocked well and left at room temperature for 1 hour. The plate was then washed three times with PBST (PBS containing 0.1% Tween 20, pH 7.4). Streptavidin-HRP was added and left at room temperature for 1 hour. The plate was then washed three times with PBST. TMB was added to allow color development for 15 minutes. Stop solution was added, and the absorbance at 450 nm was read using a microplate reader. The results of the competitive activity detection of the sera from the two alpacas are shown in Tables 2 and 3.

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

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

[0116] The results show that antibodies that inhibit the binding of VEGFR2 to VEGFA were detected in both alpacas 1# and 2# and can be used for library construction.

[0117] 3. Construction of antibody library A 40 mL sample 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 lysis solution, the white blood cells were collected and cryopreserved with Trizol. Prior to library construction, total RNA was extracted from the isolated alpaca PBMCs using Trizol, cDNA reverse transcription was performed, and 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 ratios, and ligated with T4 ligase. After ligation, the resulting fragments were subjected to electrotransformation of XL1-Blue competent cells. Based on colony growth, the transformed library volumes for the two alpacas were calculated to be 6.52 × 10 for each. 8 and 4.12 × 10 8 A clone was randomly selected from the transformant colonies and subjected to sequencing verification, and the sequencing results showed the correct nanobody sequence.

[0118] 4. Antibody Library Screening Phage display techniques were used to screen for phages capable of binding to VEGFA.

[0119] For the 1# alpaca library, after four rounds of enrichment screening, a single clone was selected from one 96-well plate (the screening method was VEGF-4 th The binding activity of the phage to VEGFA was detected. The fourth round of phage was also subjected to competitive elution. Phages that bound to VEGF were competitively eluted using 10 μg / mL of the VEGFR2 extracellular domain VEGFR2-ECD-His (manufactured in-house). Antibodies that could compete with VEGFR2 for binding to VEGFA were expected to be obtained. Five 96-well plates were selected from the obtained phage, and their binding activity to VEGFA was detected (the screening method was the same as for VEGF-5). th(Recorded as VEGFR2 competition). Furthermore, the phages obtained by VEGFR2 competitive screening were subjected to competitive elution screening using the reference monoclonal antibody bevacizumab (Avastin). That is, phages that bind to VEGFR were competitively eluted using 10 μg / mL bevacizumab antibody. One 96-well plate was selected for the obtained phages, and the binding activity of the phages to VEGFR was detected (the screening method was VEGF-6). th The detection phage was an HRP-labeled antibody that recognizes the phage coat protein, labeled as anti-M13 (HRP). The results of detecting positive clones with VEGFA binding activity among the monoclones listed above are shown in Table 4.

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

[0121] Furthermore, biotin-labeled VEGFA was used to screen for phage binding to more epitopes. The method was as follows: first, the library was coated with avidin, then incubated with biotinylated VEGFA. The 1# and 2# alpaca libraries were merged, and VEGFA-binding phages were obtained through incubation and elution of the libraries with biotinylated VEGFA. This process was repeated. After multiple rounds of enrichment, single clone colonies were screened in 96-well plates. 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: Results of screening for positive clones with VEGFA binding activity after merging libraries. [Table 5]

[0123] 5. Preliminary Screening of Antibody Sequences The amino acid sequences of the nanobody candidates (CDR regions + framework regions) were obtained by plasmid sequencing and codon translation of the above phages with VEGFA-binding activity, and antibodies with the same sequences were merged. Antibody candidates with different CDR sequences are shown in Table 6.

[0124] Table 6: Amino acid sequences of antibody candidates [Table 6] TIFF2025534252000008.tif249170TIFF2025534252000009.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 gene for each nanobody was amplified from the original phage plasmid by PCR and constructed into the pVRC8400 expression vector. The secretory 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 a 6xHis tag at the C-terminus of the antibody. At the same time, the coding gene sequence for a positive control antibody, BI-VEGF ab, was synthesized (this antibody is an anti-VEGF A nanobody, and its amino acid sequence is derived from SEQ ID NO: 57 in U.S. Patent No. 9,527,925 B2). A 6xHis tag was added to the C-terminus of the control antibody, BI-VEGF ab. The sequence of each antibody expression plasmid was determined, and if it was found to be accurate, endotoxin-free large-scale extraction was performed. 293F suspension cells were transiently transfected using the transfection reagent polyethylenimine (PEI, linear, MW = 25 kDa, purchased from Polysciences, product number 23966-1). Four or five days after transfection, the cell supernatant was collected and subjected to Ni bead affinity purification. The purified protein was analyzed by SDS-PAGE and found to have the expected molecular weight (approximately 15 kDa). Figure 1 shows a Coomassie Brilliant Blue-stained gel image of the purified positive control antibody BI-VEGF ab and some of the antibody candidates. Example 2: Testing antibody activity

[0127] 1. Testing VEGFA binding activity of antibody candidates The VEGFA antigen was diluted in ELISA coating buffer (final concentration: 0.3 μg / mL) and added to an ELISA plate at 100 μL / well for overnight coating at 4°C. After blocking with 5% nonfat dry milk, the positive control antibody BI-VEGF ab (an anti-VEGFA nanobody whose amino acid sequence is derived from SEQ ID NO: 57 described in U.S. Patent No. 9,527,925 B2; its expression plasmid construction and protein purification methods were the same as in step 6, "Expression and Purification of Antibody Candidates," in Example 1) and gradient dilutions of the antibody candidates to be tested (0.001, 0.01, 0.1, 1, 3, 10, and 100 nM) were added and incubated at 37°C for 1 hour. After washing, a His-Tag monoclonal antibody dilution (purchased from Proteintech, product number 66005-1-Ig) was added and incubated at 37°C for 1 hour. After washing the plate, a dilution of HRP-conjugated goat anti-mouse IgG antibody (HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L), purchased from Proteintech, product number SA00001-1) was added and incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from Tiangen, product number PA107-01) was added to each well. Color development was allowed to occur 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.

[0128] The change in VEGFA binding signal for some antibodies with antibody concentration is shown in FIG.

[0129] The VEGFA binding data in the ELISA test was fitted to obtain the EC50 of antibody binding to VEGFA, which 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 antibody candidates [Table 7]

[0131] The amino acid sequences of the CDR regions of the above antibodies have partial differences in amino acid residues, and the activity of the antibodies varies within an acceptable range.

[0132] Furthermore, surface plasmon resonance (SPR) technology was used to test the affinity of nanobodies for VEGFA. Using a Biacore 8K instrument (Cytiva), the antigen VEGFA165 (untagged, purchased from Yiqiao Shenzhen Co., Ltd., product number: HPLC-10008-HNAH) was coupled to a CM5 chip via amino coupling reagents (EDC and NHS) at a coupling yield of approximately 400 RU. The mobile phase buffer used during the experiment 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. For kinetic analysis, the antigen-antibody binding time was 240 seconds, the dissociation time was 1500 seconds, and the flow rate was 30 μL / min. The chip was regenerated with 100 mM HCl using multi-cycle mode. The binding and dissociation curves of the antibody candidates and VEGFA165 were fitted with a "1:1 binding" model to obtain the binding rate (k), dissociation rate (kd), and equilibrium dissociation constant (KD), as shown in Table 8.

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

[0134] Table 8 shows that the antibody candidates have high affinity for VEGFA165, and the KD values ​​are within 3-fold of the positive control, indicating that the affinity of the antibody candidates is equivalent to that of the positive control protein.

[0135] 2. VEGFR2 competitive activity test To further validate the activity of the antibody candidates in inhibiting the VEGFA-binding receptor VEGFR2, a competitive ELISA was performed.

[0136] The VEGFA antigen was diluted in ELISA coating buffer (final concentration: 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% nonfat dry milk, the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from Yiqiao Shenzhou Co., Ltd., product number: 10012-H02H) was mixed with a gradient dilution of the test antibody (0.01, 0.1, 1, 3, 10, 100 nM) at a final concentration of 1 nM. The mixture was then added to the 96-well plate and incubated at 37°C for 1 hour. After washing, a dilution of HRP-conjugated goat anti-human IgG antibody (purchased from Abbkine, product number: A21050) was added and incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from Tiangen, product number PA107-01) was added to each well. Color development was allowed to occur at 37°C for 15 minutes, followed by the addition of 50 μL of stop solution. The optical density at 450 nm (OD450) was measured using a microplate reader. The results of the competitive ELISA are shown in Figure 3.

[0137] A comparison of the VEGFR2 competitive activity (IC50 value and maximum inhibition rate) of the antibodies is shown in Table 9. 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: VEGFR2 competitive activity of antibody candidates (IC50 value and maximum inhibition rate) [Table 9] 3. Inhibitory activity of antibody candidates on HUVEC cell proliferation

[0139] Antibody candidates V1, V13, V15, and V30 were validated to inhibit VEGFA-stimulated proliferation of human umbilical vein endothelial cells (HUVECs).

[0140] Primary HUVEC cells (obtained from China Typical Culture Collection Center (CCTCC)) were digested to 5 × 10 4 The cells were diluted to 100 cells / mL and seeded at 100 μL / well in a 96-well plate. The medium was complete (F-12K + 0.1 mg / mL heparin + ECGS + 10% FBS). Drugs were added after 5 hours of adherent culture.

[0141] A mixture of VEGFA and antibody candidate dilutions was prepared in 1:1 DMEM / F12 (HAM) medium (serum-free, supplemented with penicillin and streptomycin, and glutamine, purchased from Biological Industries, part number 01-172-1ACS) at a final VEGFA concentration of 35 ng / mL. The concentrations of antibody candidate V1 were: 1,000,000 pM, 33,333.33 pM, 111,111.11 pM, 37,037.04 pM, 12,345.68 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 1,000,000 pM, 33,333.33 pM, 111,111.11 pM, 37,037.04 pM, 12,345.68 pM, 4,115.23 pM, 1,371.74 pM, 457.25 pM, 152.42 pM, and 5.65 pM.

[0142] The negative control antibody was a nanobody that binds to an unrelated antigen at a concentration gradient (3-fold decreasing): 1,000,000 pM, 33,333.33 pM, 111,111.11 pM, 37,037.04 pM, 12,345.68 pM, 4115.23 pM, 1371.74 pM, 457.25 pM, 152.42 pM.

[0143] A control without VEGFA (minimal cell proliferation) and a control with VEGFA but no antibody (maximal cell proliferation) were also included. VEGFA was incubated with each antibody dilution mixture at 37°C for 1 hour. After aspirating the original medium from the 96-well cell plate, the VEGFA and antibody dilution mixture was added to the cell plate. Two cell wells were set up for each concentration. The plates were cultured in a cell culture incubator at 37°C for 72 hours. CCK-8 solution (purchased from Solarbio, product number CA1210) was added to the 96-well plate, and the plate was incubated in the incubator for 3 hours. The optical density at 450 nm (OD450) was then measured using a microplate reader.

[0144] The percentage of inhibition of cell proliferation by antibody candidates at various concentrations was calculated based on the difference in readings from control wells (wells without VEGFA and wells containing only VEGFA but no antibody). The formula for calculating the percentage of inhibition is as follows:

number

[0145] The inhibition rate curve, maximum inhibition rate and IC50 of each antibody against HUVEC cell proliferation are shown in FIG. 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 were able to inhibit the VEGFA signaling pathway at the cellular level, i.e., inhibit VEGFA-stimulated HUVEC cell proliferation. At the highest antibody concentration (1 μM), antibody candidate V1 effectively inhibited HUVEC proliferation (maximum inhibition rate was close to 100%). Antibody candidate V30 showed relatively strong activity in terms of IC50.

[0148] Through ELISA tests for VEGFA binding and VEGFR2 competition, and detection of HUVEC proliferation inhibitory activity at the cellular level, it was shown that the antibody candidate could efficiently bind VEGFA and inhibit VEGFR2 binding to VEGFA, 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 ELISA, the same method as in "1. Testing the VEGFA-binding activity of antibody candidates" was used to detect the binding activity of His-tagged antibody candidate V1 to human VEGFA121, mouse VEGFA164, and rat VEGFA164. 0.3 μg / mL human VEGFA121 (purchased from Yiqiu Shenzhou Co., Ltd., product code: 10008-HNAH), mouse VEGFA164 (purchased from Yiqiu Shenzhou Co., Ltd., product code: 50159-MNAB), or rat VEGFA164 (purchased from Yiqiu Shenzhou Co., Ltd., product code: 80006-RNAB) was coated onto the antibody.

[0150] ELISA was used to detect the human VEGFA121 binding activity of antibody V1, as shown in FIG.

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

[0152] The binding activity of antibody V1 to mouse VEGFA164 is shown in FIG.

[0153] The rat VEGFA164 binding activity of antibody V1 is shown in FIG.

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

[0155] 5. Determining the Tm value of antibody candidates Eight antibody candidates (V1, V15, V29, V30, V31, V36, V40, and 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 device (unchainedlabs). The results are shown in Table 12. The results indicate that these antibody candidates have a certain degree of thermal stability.

[0156] Table 12: Melting temperatures 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 sequence of antibody candidate V1 was aligned with human antibody sequences using the antibody databases IGBLAST and IMGT to determine homology. Next, a fully humanized antibody was constructed by combining highly homologous human antibody framework regions with the CDR regions of the antibody candidate. The methods for constructing the expression plasmid and purifying the protein were the same as those in "6. Expression and purification of antibody candidates" in Example 1.

[0158] The purified antibodies were tested for VEGFA binding activity using ELISA. After the initial humanization of V1, the VEGFA binding activity of the antibodies was significantly reduced. Some amino acid mutations in the framework region were restored to obtain nine optimized V1 humanized antibodies, the amino acid sequences of which are shown in Table 13.

[0159] Table 13: Amino acid sequences of humanized antibody candidates [Table 13] TIFF2025534252000019.tif249170TIFF2025534252000020.tif249170TIFF2025534252000021.tif62170

[0160] These humanized antibodies with restored mutations were transiently expressed and purified. The experimental method for the first stage of purification was the same as in Example 1, "6. Expression and Purification of Antibody Candidates." Among these, the positive control BI-VEGF antibody and the humanized antibodies V1-DP and V1-SA1 were subjected to Ni-bead affinity purification and then purified by cation exchange chromatography (S column). Figure 8(A) shows an SDS-PAGE Coomassie Brilliant Blue-stained gel image of a portion of the purified protein. The molecular weight of the target protein was as expected (approximately 15 kDa).

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

[0162] Example 4: Activity detection of humanized anti-VEGFA nanobodies The VEGFA-binding activity of the obtained humanized antibodies was tested by ELISA. The experimental method was the same as in "1. Test of VEGFA-binding activity of antibody candidate" in Example 2. The ELISA test results for VEGFA binding of some of the humanized antibodies are shown in Figure 9.

[0163] The VEGFA binding data from the ELISA test was fitted to obtain the EC50 values ​​for the binding of the humanized antibodies to VEGFA, which are shown in Table 14. The data show that the antibody candidates after humanization have similar VEGFA binding activity to those before humanization (V40).

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

[0165] To further verify the activity of the humanized antibodies in inhibiting the VEGFA-binding receptor VEGFR2, a competitive ELISA was performed. The experimental method was the same as in "2. VEGFR2 competitive activity test" in Example 2, with the following differences: For the V1, V13, V15, and V43 humanized antibodies, the final concentration of the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from Yiqiao Shenzhou Co., Ltd., product 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 can be seen from the results, the V1 and V15 humanized antibodies' competitive activity with VEGFR2 for binding to VEGFA returned to the pre-humanization level (i.e., V1 and V15), respectively, and the remaining humanized antibodies exhibited similar VEGFR2 competitive activity. The competitive ELISA results for some 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 for VEGFR2 inhibition were performed on the humanized antibody V30 and the remaining humanized antibodies V43. The experimental method was the same as in Example 2, "2. VEGFR2 competitive activity test," with the following differences: The final concentration of the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from Yiqiu Shenzhou Co., Ltd., product number 10012-H02H) was 1 nM. The humanized antibody concentration gradients were 0.03, 0.3, 3, 10, 30, and 300 nM.

[0169] The results of the ELISA test of VEGFR2 competition of the humanized antibodies V30 and V43 are shown in FIG.

[0170] The VEGFR2 competitive activity of the antibodies (IC50 value and maximum inhibition rate) is shown in Table 16. The humanized antibodies were found to be able to compete with VEGFR2 for binding to VEGFA, and their activity was similar to that of the unhumanized antibody (V30). The formula for calculating the maximum inhibition rate is as follows:

number

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

[0172] Surface plasmon resonance (SPR) technology was used to couple the antigen VEGFA165 and test the affinity of the nanobodies for VEGFA before and after humanization. The experimental and data fitting methods were the same as in Example 2, "1. Testing the VEGFA binding activity of antibody candidates." The resulting KDs are shown in Table 17.

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

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

[0175] The binding and dissociation curves of V1-SA1 and VEGFA165 are shown in FIG. 12, and the fitted KDs are shown in Table 18.

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

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

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

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

[0180] 2. Colloid stability study of 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(+). The secretory peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 68) was added to the N-terminus of the antibody, and the flexible linker GGS and tag 6xHis were added to the C-terminus of the antibody. The two antibodies were transiently expressed and purified using Ni bead affinity. The experimental method was the same as in step 6, "Expression and Purification of Antibody Candidates," in Example 1. After purification, the antibodies were separated by SDS-PAGE, and the gel staining results are shown in Figure 14. This figure indicates that the molecular weights of the two proteins were as expected (approximately 15 kDa). The protein expression levels and purity determined by Coomassie Brilliant Blue staining of 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 V1-SA1 and BI-VEGF ab protein solutions were changed to 1x PBS (pH = 7.4) and the protein concentration was adjusted to 10 mg / mL. HPLC-SEC analysis of the proteins was performed (chromatography column: Zenix-C SEC-300, Sepax). The percentages of protein monomers, aggregates, and fragments are shown in Table 20. The percentage of V1-SA1 aggregates was found to be much lower than that of BI-VEGF ab, 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 containing antibodies V1-SA1 and BI-VEGF ab (protein concentration: 10 mg / mL) were sterilized and filtered in PBS, and then left at 37°C for a set period (0 and 2 days). The properties of the protein solutions were observed, and the colloidal stability of both solutions was compared. The results are shown in Table 21.

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

[0186] Using a dynamic light scattering nanoparticle size and potential analyzer (NS-90Z, OMEC) to measure the protein particle size of the antibody V1-SA1 and BI-VEGF ab solutions (diluted 4-fold with PBS) after incubation at 37°C for 0 and 2 days, respectively, the protein average particle size (Z-Average) and polydispersity index (PDI) are shown in Table 21. For the 0-time point sample, the average hydrated dynamic diameter and PDI of V1-SA1 were all smaller than those of BI-VEGF ab. Given the similar molecular weights of the two, this suggests 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 and a significantly lower proportion of high-molecular-weight protein aggregates than BI-VEGF ab.

[0187] As can be seen from Table 21, in samples stored 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 hydrated dynamic 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 hydrated dynamic diameter of BI-VEGF ab is much larger than that of V1-SA1 and significantly larger than that of the BI-VEGF ab solution at time 0, indicating that BI-VEGF ab forms additional high molecular weight protein aggregates under high temperature conditions.

[0188] Example 6: Production and activity studies of monovalent Nanobody-Fc fusion proteins 1. Production of Monovalent Nanobody-Fc Fusion Proteins In this example, fusions of monovalent nanobodies with IgG1 Fc (SEQ ID NO:62) or PPCP-Fc (SEQ ID NO:75, i.e., the remaining amino acid sequence after removal of DKTHTC in SEQ ID NO:62) or the IgG1 Fc mutant Fc-m1 (SEQ ID NO:63) were expressed.

[0189] Here, the IgG1 Fc amino acid sequence 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) The amino acid sequences of the constructed Nanobody-Fc fusion proteins are shown in Table 22.

[0190] Table 22: Amino acid sequences of Nanobody-Fc fusion proteins [Table 22] TIFF2025534252000033.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 a six-histidine tag), and the fusion proteins V15-PPCP-Fc, V15-9GS-PPCP-Fc, and V15-Fc, a linker (SGGS (SEQ ID NO:70)) and a six-histidine tag (HHHHHH (SEQ ID NO:71)) were added to the C-terminus of the Fc segment of the fusion proteins. During the construction of the monovalent nanobody-Fc fusion protein plasmid, the nanobody and the Fc fragment containing the linker and histidine tag were amplified by PCR, respectively, where the PCR template for the Fc fragment was derived from a gene-synthesized Fc gene. The nanobody and Fc fragment were then integrated into the vector pCDNA3.1(+) by two-fragment homologous recombination. The enzyme cleavage sites used were NheI and XbaI. The secretory peptide of the fusion protein is METDTLLLWVLLLWVPGSTG (SEQ ID NO: 72). The protein sequence of interest was then fused to the fusion protein. After successful sequencing of these fusion protein particles, endotoxin-free extraction of the plasmid was performed. 293F suspension cells were transiently transfected using the transfection reagent polyethylenimine (PEI, linear, MW = 25 kDa, purchased from Polysciences, product number 23966-1). Cell supernatants were collected on days 4 or 5 posttransfection and purified using Ni beads. SDS-PAGE analysis of the purified protein revealed that the molecular weight of the protein monomer was approximately 39 kDa under reducing conditions, as expected. A Coomassie Brilliant Blue-stained gel image of a portion of the purified monovalent nanobody-Fc fusion protein is shown in Figure 15.

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

[0193] For the fusion protein of humanized antibody V1-SA1 and Fc (V1-SA1-Fc-m1), 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 without additional amino acids at the C-terminus of Fc. This means that the fusion protein V1-SA1-Fc-m1 contains an Fc tag but not a histidine tag. The V1-SA1-Fc-m1 expression plasmid was transiently transfected into 293F cells, followed by affinity purification using Protein A beads. During protein elution, the target protein was eluted with 0.1 M citric acid (pH 3.0) and quickly neutralized to obtain the target protein.

[0194] 2. Activity Detection of Monovalent Nanobody-Fc Fusion Proteins The purified monovalent Nanobody-Fc fusion proteins were tested for VEGFA binding activity. The ELISA experiment method was the same as in Example 2, "1. Testing the VEGFA binding activity of antibody candidates." The ELISA results are shown in Figure 16 and the EC50 values ​​are shown in Table 23.

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

[0196] The data in the table show that both the monovalent nanobody-Fc fusion proteins and the linear bivalent nanobodies tested have potent VEGFA binding activity.

[0197] The affinity of the monovalent nanobody-Fc fusion protein (V1-SA1-Fc-m1) for VEGFA was tested using surface plasmon resonance (SPR) technology. V1-SA1-Fc-m1 was captured onto a Protein A chip using a Biacore 8K instrument (Cytiva). The V1-SA1-Fc-m1 was diluted to a protein concentration of 2 μg / mL in HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20) buffer during immobilization. The coupling yield was approximately 590 RU. The analyte used in the experiment was the antigen VEGFA165 (untagged, purchased from GenScript, product 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. For kinetic analysis, the binding time between the fusion protein and the antigen VEGFA165 was 180 s, the dissociation time was 900 s, and the flow rate was 30 μL / min. After each "binding-dissociation" cycle, the chip was regenerated with 10 mM glycine hydrochloride (pH 1.5). The binding and dissociation curves for the fusion protein and the antigen VEGFA165 are shown in Figure 17. The binding rate (k), dissociation rate (kd), and equilibrium dissociation constant (KD) were obtained by fitting using a "1:1 binding" model, as shown in Table 24.

[0198] Table 24: Binding rates (ka), dissociation rates (kd), and equilibrium dissociation constants (KD) of monovalent Nanobody-Fc fusion proteins with VEGFA165 [Table 24]

[0199] SPR data showed that the monovalent nanobody-Fc fusion protein V1-SA1-Fc-m1 had high affinity for VEGFA165 (KD = 0.148 nM). Table 18 and Figure 12 in "6. Activity Detection of Humanized Antibodies" in Example 2 above show that the KD of the monovalent nanobody V1-SA1 and VEGFA165 is 4.38 nM. Thus, after fusion to Fc, the monovalent nanobody exhibited nearly 30-fold higher affinity for VEGFA165 than without Fc, indicating that the anti-VEGFA antibody-Fc fusion protein has stronger target affinity and potentially better efficacy in inhibiting the VEGFA signaling pathway.

[0200] Although the 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 solutions of the present invention within the scope of the technical concept of the present invention, and all of these simple modifications fall within the protection scope of the present invention.

[0201] It should be noted that the specific technical features described in the above specific embodiments can be combined in any suitable manner as long as there is no contradiction, and in order to avoid unnecessary duplication, the present invention will not re-describe various possible combination manners.

Claims

1. An anti-VEGFA antibody or antigen-binding fragment thereof, heavy chain variable region CDR-H1, CDR-H2, and CDR-H3; the amino acid sequence of CDR-H1 comprises SYTMG (SEQ ID NO: 1) or an amino acid sequence having at least 80% identity with SYTMG (SEQ ID NO: 1); The amino acid sequence of CDR-H2 is AISKGGYKYX １ X ２ VSLEA (SEQ ID NO: 2) or AISKGGYKYX １ X ２ comprising an amino acid sequence having at least 80% identity to VSLEA (SEQ ID NO: 2); The amino acid sequence of CDR-H3 is TRAYGSSRLX ３ LAX ４ TYEY (SEQ ID NO: 3) or TRAYGSSRLX ３ LAX ４ An anti-VEGFA antibody or antigen-binding fragment thereof, comprising an amino acid sequence having at least 80% identity with TYEY (SEQ ID NO: 3).

2. X in SEQ ID NO:2 １ X ２ represents DS, DA, NT, DT, NA or NS, and X in SEQ ID NO: 3 ３ represents R or K, and X ４ represents D, N, E or K. The anti-VEGFA antibody or antigen-binding fragment thereof according to claim 1 .

3. The anti-VEGFA antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequences of the CDR-H1, CDR-H2, and CDR-H3 comprise any one 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

4. 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, and the modification site of the humanized sequence is located in a non-CDR region, preferably, the modification site of the humanized sequence is located in a framework region and / or a constant region of the antibody.

5. The anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, an Fd fragment, an Fd' fragment, an Fv fragment, a dAb fragment, an isolated CDR region, an F(ab')2 fragment, a single domain antibody, a single-chain antibody molecule, or a linear antibody.

6. The anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences of SEQ ID NOs: 13 to 32, 35 to 61, and 64 to 67, or has at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 13 to 32, 35 to 61, and 64 to 67.

7. The anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the anti-VEGFA antibody or antigen-binding fragment thereof binds to a human or monkey VEGFA protein.

8. A fusion protein comprising the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. The fusion protein further comprises an anti-VEGFA antibody or antigen-binding fragment thereof other than the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, an antibody or antigen-binding fragment thereof against another target, or another functional component; Preferably, the other functional component includes, but is not limited to, one or a combination of two or more of serum albumin, cytokine, transferrin, scaffold protein, oligopeptide, oligopeptide polymer, polypeptide, polypeptide polymer, polysaccharide, lipid chain, avidin, biotin, streptavidin, toxin, drug, nucleic acid, radionuclide and its marker, PEGylated component or Fc fragment; Preferably, the other target is IgG, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, Edoglin (CD105), TGF, integrin, integrin receptor, interleukin (e.g., IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptor (e.g., IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL1 0R, 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, 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. A chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises an anti-VEGFA antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

11. An immune cell characterized by expressing 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.

12. A nucleic acid encoding the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or encoding the fusion protein according to any one of claims 8 to 9.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell characterized in that it contains a nucleic acid according to claim 12 or a vector according to claim 13.

15. 8. A method for producing the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising introducing a nucleic acid or vector encoding the anti-VEGFA antibody or antigen-binding fragment thereof into a host cell, and then inducing its expression.

16. A method for screening for an anti-VEGFA antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7, comprising immunizing an alpaca with a human or monkey VEGFA protein.

17. A product for treating and / or diagnosing a disease, comprising: A) an anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; B) a fusion protein according to any one of claims 8 to 9, C) The chimeric antigen receptor of claim 10. D) The immune cell according to claim 11; E) the nucleic acid according to claim 12; F) A vector according to claim 13, or G) A product comprising any one of the host cells of claim 14.

18. Use of 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, or the host cell according to claim 14 in the manufacture of a product for treating and / or preventing a disease associated with VEGFA, in the manufacture of a product for inhibiting VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, or in the manufacture of an antibody-drug conjugate, an antibody diagnostic kit, or a tracer.

19. A method for detecting VEGFA, comprising: A method for detecting VEGFA, comprising contacting a test sample 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 a complex formed between VEGFA and the anti-VEGFA antibody or antigen-binding fragment thereof.

20. 1. A method for treating and / or preventing a disease, comprising:

19. A method for treating and / or diagnosing a disease, comprising administering 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.

21. 1. A method for treating and / or preventing a disease, comprising:

19. A method comprising contacting a target cell with 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.

22. 22. The method of claim 21, wherein the target cells are selected from VEGFA-expressing cells, such as cardiomyocytes, proximal tubule cells, hepatocytes, vascular endothelial cells, granulocytes, 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.

23. The method according to any one of claims 20 to 22, wherein the disease is a disease associated with the VEGFA signaling pathway, and is preferably selected from tumors and ocular diseases accompanied by abnormal vascular proliferation or angiogenesis (e.g., retinal vascular diseases).

24. 1. A method for inhibiting VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, comprising:

19. A method comprising contacting a vascular endothelial cell with 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.

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