An anti-vegfa antibody or antigen-binding fragment thereof and uses thereof

Abstract

The application discloses an anti-VEGFA antibody or an antigen binding fragment thereof and application thereof. The anti-VEGFA antibody or the antigen binding fragment thereof has high thermal stability, high water solubility, small molecular weight, simple expression and purification process, high affinity to VEGFA, and can effectively block a VEGFA signal path, effectively block pathological processes such as vascular endothelial cell proliferation and angiogenesis caused by VEGFA, and can be potentially applied to treatment of diseases related to VEGFA.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-VEGFA antibody or its antigen-binding fragment and its applications. Background Technology

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

[0003] Pathological angiogenesis occurs in various solid tumors, inflammatory responses, and retinal vascular diseases. Tumor cell proliferation in its later stages relies on the supply of nutrients from newly formed blood vessels 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 related to retinal vascular proliferation, such as in wet 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, there are four main anti-VEGFA protein drugs on the market for the treatment of wet AMD: ranibizumab, aflibercept, conbercept, and brolucizumab. In addition, bevacizumab is also used off-label for the treatment of wet AMD. These anti-VEGFA protein drugs include traditional monoclonal antibodies, antibody fragments Fab and scFv, and receptor-Fc fusion proteins. There are currently no variable domain of heavy-chain antibody (VHH) or nanobody (Nb) drugs on the market.

[0005] Single-domain antibodies, or nanobodies, are the variable domains of naturally occurring heavy-chain antibodies (lacking the light chain) found in camels. They are stable, minimal antibody units with complete antigen-binding function. Single-domain antibodies have a molecular weight of approximately 13 kDa, high thermal stability, and good water solubility. As a novel antibody form, the development of nanobodies is receiving increasing attention.

[0006] Therefore, the present invention provides a novel anti-VEGFA antibody or its antigen-binding fragment for blocking the binding of VEGFA to VEGFR1 and VEGR2, thereby inhibiting the downstream signaling pathways of VEGFA and inhibiting the cell proliferation stimulated by VEGFA. Summary of the Invention

[0007] In a first aspect, the present invention provides an anti-VEGFA antibody or an antigen-binding fragment thereof, said anti-VEGFA antibody or antigen-binding fragment thereof comprising CDR-H1, CDR-H2 and / or CDR-H3 of the heavy chain variable region.

[0008] The amino acid sequence of CDR-H1 contains SYTMG (SEQ ID NO: 1) or has at least 80% identity with SYTMG (SEQ ID NO: 1);

[0009] The amino acid sequence of CDR-H2 contains AISKGGYKYX1X2VSLEA (SEQ ID NO: 2) or an amino acid sequence that has at least 80% identity with AISKGGYKYX1X2VSLEA (SEQ ID NO: 2);

[0010] The amino acid sequence of CDR-H3 contains TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3) or an amino acid sequence that has at least 80% identity with TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3).

[0011] Wherein, X in SEQ ID NO:2 or SEQ ID NO:3 can be any natural amino acid residue, such as alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), and valine (V).

[0012] In one 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.

[0013] In one specific embodiment of the present invention, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 comprise any one of the following groups (see Table 1 for details):

[0014] A) SEQ ID NO: 1, 4, 9;

[0015] B) SEQ ID NO: 1, 5, 9;

[0016] C) SEQ ID NO: 1, 6, 10;

[0017] D)SEQ ID NO: 1, 4, 11;

[0018] E)SEQ ID NO: 1, 7, 12;

[0019] F)SEQ ID NO: 1, 6, 11;

[0020] G)SEQ ID NO: 1, 4, 10;

[0021] H)SEQ ID NO: 1, 5, 12;

[0022] I)SEQ ID NO: 1, 4, 12;

[0023] J)SEQ ID NO: 1, 8, 12;

[0024] K)SEQ ID NO: 1, 33, 34.

[0025] Table 1. Amino acid sequences of candidate antibodies CDR-H1, CDR-H2, and CDR-H3

[0026]

[0027] 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. In this application, the amino acid division of the antibody CDR region adopts the Kabat numbering system.

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

[0029] The structure of the anti-VEGFA antibody or its antigen-binding fragment is 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.

[0030] The anti-VEGFA antibody or its antigen-binding fragment can be a dual-target specific antibody or a three- or more target specific antibody (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more, etc.).

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

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

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

[0034] Preferably, the anti-VEGFA antibody or its antigen-binding fragment includes a humanized sequence, wherein the modification site of the humanized sequence is located in a non-CDR region; more preferably, the humanized modification site is located in the frame region and / or constant region of the antibody.

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

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

[0037] Preferably, the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment comprises any one of the amino acid sequences in SEQ ID NO: 13-32, 35-61, 64-67, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NO: 13-32, 35-61, 64-67.

[0038] In one specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment is as shown in any one of SEQ ID NO: 13-32, 35-61, 64-67.

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

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

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

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

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

[0044] In one specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment is as shown in any one of SEQ ID NO: 13-32, 35-61, 64-67.

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

[0046] In a fourth aspect, the present invention provides a fusion protein comprising the above-described anti-VEGFA antibody or its antigen-binding fragment.

[0047] Preferably, the fusion protein further comprises, in addition to the aforementioned anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies or antibodies targeting other targets or their antigen-binding fragments or other functional components.

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

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

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

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

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

[0053] Preferably, the fusion protein contains at least one antibody or antigen-binding fragment of another target or other functional component.

[0054] Specifically, the anti-VEGFA antibodies or their antigen-binding fragments are directly or indirectly connected to each other, to other anti-VEGFA antibodies or their antigen-binding fragments, to other target antibodies or their antigen-binding fragments or other functional components, to other anti-VEGFA antibodies and other target antibodies or their antigen-binding fragments or other functional components, to other target antibodies or their antigen-binding fragments or other functional components, and to other anti-VEGFA antibodies or their antigen-binding fragments.

[0055] The indirect connection can be a connection via a linker, a functional domain, and / or a linker for coupling. The linker is selected from linking peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, fatty acid chains, biotin, streptavidin, or avidin.

[0056] The functional domain is one or more of the following: Fc fragment, serum albumin, cytokines, transferrin, scaffold protein, VEGFA, or antibodies or antigen-binding fragments targeting other targets.

[0057] The linkers used for coupling include one or more of toxins, drugs, nucleic acids, PEG, radionuclides and their markers.

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

[0059] The fusion protein contains an anti-VEGFA antibody or its antigen-binding fragment, other anti-VEGFA antibodies or other target antibodies, and the linking sequence can be 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.

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

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

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

[0063] Preferably, the fusion protein comprises any amino acid sequence in SEQ ID NO: 64-67, or has at least 80% identity with any amino acid sequence in SEQ ID NO: 64-67.

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

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

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

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

[0068] In a sixth aspect, the present invention provides a nucleic acid that encodes the aforementioned anti-VEGFA antibody or its antigen-binding fragment, the aforementioned fusion protein, or the aforementioned chimeric antigen receptor.

[0069] In a seventh aspect, the present invention provides a carrier comprising the above-described nucleic acid.

[0070] The vector can express the expression 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. Examples include E. coli vectors, bacteriophages, etc.

[0071] In an eighth aspect, the present invention provides a host cell comprising the above-described nucleic acid or the above-described vector.

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

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

[0074] Prokaryotic cells, such as Escherichia coli.

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

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

[0077] In an eleventh aspect, the present invention provides an immune cell that expresses the above-mentioned anti-VEGFA antibody or its antigen-binding fragment or the above-mentioned chimeric antigen receptor.

[0078] 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.

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

[0080] In a twelfth aspect of the present invention, a method for constructing immune cells is provided, the method comprising transfecting an immune cell with a nucleic acid sequence encoding the chimeric antigen receptor described herein into the immune cell for expression.

[0081] In a thirteenth aspect, the present invention provides a product for treating and / or diagnosing a disease, said product comprising any of the following:

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

[0083] B) The aforementioned fusion protein;

[0084] C) The chimeric antigen receptors mentioned above;

[0085] D) The above-mentioned immune cells

[0086] E) The aforementioned nucleic acids;

[0087] F) the aforementioned carrier; or,

[0088] G) The host cells mentioned above.

[0089] The product for treating and / or diagnosing diseases targets cells that express VEGFA, and these cells may be cardiomyocytes, proximal renal tubular cells, hepatocytes, vascular endothelial cells, granulocytes, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells, or tumor cells, etc.

[0090] Preferably, the product can be a reagent kit, drug, chip, or antibody-drug conjugate, etc. The disease is a disease related to the VEGFA signaling pathway. More preferably, it can be a tumor, abnormal angiogenesis, ophthalmic diseases involving angiogenesis (e.g., fundus vascular disease), etc.

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

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

[0093] The detection method described herein is to detect the presence or content of VEGFA. Here, "present" refers to the presence or absence of VEGFA, and "content" can refer to expression level or protein concentration, etc.

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

[0095] The diseases described are those related to the VEGFA signaling pathway. More preferably, these include tumors, abnormal angiogenesis, ophthalmic diseases involving angiogenesis (such as retinal vascular diseases), etc.

[0096] In a seventeenth aspect, the present invention provides a method for treating and / or preventing a disease, the method comprising administering to an individual the aforementioned anti-VEGFA antibody or its antigen-binding fragment, the aforementioned chimeric antigen receptor, the aforementioned fusion protein, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, the aforementioned immune cell, or the aforementioned product for treating and / or diagnosing a disease.

[0097] The diseases described are those related to the VEGFA signaling pathway. More preferably, these include tumors, abnormal angiogenesis, ophthalmic diseases involving angiogenesis (such as retinal vascular diseases), etc.

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

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

[0100] Preferably, the incubation process includes a step of discarding the complete culture medium for vascular endothelial cells in the culture plate.

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

[0102] Preferably, testing is performed after cultivation.

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

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

[0105] Preferably, the culture temperature is room temperature - 45°C, more preferably 30-40°C, such as 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc. Preferably, the culture is carried out in a 5% CO2 incubator.

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

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

[0108] Preferably, the method includes the treatment of a disease.

[0109] Preferably, the method described does not involve the treatment of the disease.

[0110] In a nineteenth aspect, the present invention provides a method for treating and / or preventing a disease, the method comprising contacting target cells with the above-described anti-VEGFA antibody or its antigen-binding fragment, the above-described chimeric antigen receptor, the above-described fusion protein, the above-described nucleic acid, the above-described carrier, the above-described host cell, the above-described immune cell, or the above-described product for treating and / or diagnosing a disease.

[0111] The diseases described are those related to the VEGFA signaling pathway. More preferably, these include tumors, abnormal angiogenesis, ophthalmic diseases involving angiogenesis (such as retinal vascular diseases), etc.

[0112] Preferably, the target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal renal tubular cells, hepatocytes, vascular endothelial cells, granule cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells, or tumor cells, etc.

[0113] In a twentieth aspect, the present invention provides the use of the above-described anti-VEGFA antibody or its antigen-binding fragment, the above-described chimeric antigen receptor, the above-described fusion protein, the above-described nucleic acid, the above-described carrier, the above-described host cell, or the above-described immune cell in the preparation of products for treating and / or preventing VEGFA-related diseases, or in the preparation of products for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, or in the preparation of antibody-drug conjugates or antibody diagnostic kits or tracers.

[0114] The diseases mentioned can include tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular diseases), etc.

[0115] The products described can be reagent kits, drugs, chips, antibody-drug conjugates, etc.

[0116] This invention provides a novel anti-VEGFA antibody or its antigen-binding fragment, which exhibits high thermal stability, high water solubility, small molecular weight, and simple expression and purification processes. It possesses high affinity for VEGFA and effectively blocks the VEGFA signaling pathway, thereby effectively inhibiting VEGFA-induced vascular endothelial cell proliferation and angiogenesis, and has potential applications in the treatment of VEGFA-related diseases. It may also bring greater clinical value, such as better tissue penetration, lower production costs, and more convenient administration methods.

[0117] The "medicine" described in this invention can be used to treat humans or non-human animals, such as non-human mammals. The medicine may contain pharmaceutically acceptable carriers, excipients, or salts common in the prior art. The medicine can be administered via any suitable route, such as gastrointestinal (e.g., oral) or non-gastrointestinal (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, intravenous infusion, intracerebral, intrathecal, transdermal, rectal, etc.). The medicine can be in any suitable dosage form, such as gastrointestinal or non-gastrointestinal dosage forms, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder inhalers, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pellets, gels, etc. Various dosage forms of the medicine can be prepared according to conventional pharmaceutical manufacturing methods. The drug may contain, by weight, 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the anti-VEGFA antibody or its antigen-binding fragment, the nucleic acid, the vector, the host cell, the immune cell, etc. The drug can be prepared as a reagent with a protein concentration of 1-300 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / mL). The single-dose dosage of the drug can be 0.1-1000 mg, for example 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg.

[0118] The term "pharmaceutically acceptable" as used in this invention refers to the biological activity and characteristics of the active substances in the applied product that neither significantly stimulate the organism nor inhibit it.

[0119] The “…method” described in this invention can be used for the diagnosis and treatment of diseases, or for the diagnosis and treatment of non-disease purposes.

[0120] The "antigen-binding fragment" described in this invention is a portion of an antibody that retains the specific binding activity of the antibody, meaning that any part of the antibody can specifically bind to an epitope on the antibody's target molecule. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd, and variants of these fragments. For example, the heavy and / or light chains of an antibody, the variable regions of the heavy and / or light chains of an antibody, or a single or more CDRs from the heavy or light chains of an antibody. Fab is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. F(ab')2 is a divalent fragment comprising two Fab fragments connected by disulfide bonds in a hinge region. Fd is an Fd fragment consisting of VH and CH1 domains. Fv is an Fv fragment consisting of the VL and VH domains of a single arm of the 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' fragment in which cysteine ​​residues in a constant domain have at least one free thiol group. Where VH represents the variable region of the heavy chain, VL represents the variable region of the light chain, and CL represents the light chain.

[0121] In this invention, "CH2" or "CH3" refers to CH2 or CH3 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 antibody heavy chain polypeptide portion extending from approximately EU position 231 to EU position 340 (according to Kabat's EU numbering system). The unique feature of the CH2 domain is that it does not pair tightly with another domain. The CH3 domain refers to the antibody heavy chain polypeptide portion extending from approximately EU position 341 to EU position 446.

[0122] The "Fc" region of this invention contains two heavy chain segments comprising the CH2 and CH3 domains of the antibody. The two heavy chain segments form a dimer by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.

[0123] The "linear antibody" described in this invention comprises a pair of tandem Fd segments (VH-CH1-VH-CH1).

[0124] The term "comprising" or "including" as used in this invention is an open-ended expression. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0125] The "homology" referred to in this invention means that, in the use of protein or nucleotide sequences, those skilled in the art can adjust the sequence according to actual work needs, so that the sequence used has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 3 Homology of 9%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, and 99.9%.

[0126] The "humanized antibody" described in this invention refers to an antibody whose framework region and / or constant region (e.g., CH region) are entirely encoded by a human antibody gene. In one specific embodiment of this invention, the CDR region of the antibody has not been modified.

[0127] The "individual" referred to in this invention can be a human or a non-human mammal. The non-human mammal can 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, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0128] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, alleviating, reducing, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0129] The term "prevention" as used in this invention refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in the body.

[0130] The term "diagnosis" as used in this invention refers to 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 of a disease or its possible future progression.

[0131] The tumor described in this invention can be any abnormal cell proliferation (or any disease that itself manifests as abnormal cell proliferation), vegetation, or an increased tendency or risk of abnormal cell proliferation, vegetation, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A vegetation can be any abnormal growth or proliferation of cells and can be located 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 (including or excluding the cerebrum), 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 nodes, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, 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., neuroglioma), colorectal 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 carcinoma, 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, epidermoid carcinoma, colon cancer, thymic carcinoma, hematologic malignancy, head and neck cancer, or oropharyngeal carcinoma.

[0132] The term "fundus vascular disease" as used in this invention refers to diseases occurring in the retinal arteries or veins, or a general term for diseases related to choroidal angiogenesis. This includes, but is not limited to, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc. Attached Figure Description

[0133] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0134] Figure 1 Cologne gel images of candidate antibodies purified from different batches.

[0135] Figure 2 Figure: Results of VEGFA binding signal changes with antibody concentration for different candidate antibodies.

[0136] Figure 3 : ELISA results of competition between different candidate antibodies.

[0137] Figure 4 Inhibition rate of different candidate antibodies on HUVEC cell proliferation.

[0138] Figure 5 ELISA was used to detect the activity of antibody V1 binding to human VEGFA121.

[0139] Figure 6 ELISA was used to detect the activity of antibody V1 binding to mouse VEGFA164.

[0140] Figure 7 ELISA was used to detect the activity of antibody V1 binding to rat VEGFA164.

[0141] Figure 8 Cologne gel image after purification of humanized antibody.

[0142] Figure 9 ELISA test for humanized antibody binding to VEGFA.

[0143] Figure 10 ELISA test for competing VEGFR2 with humanized antibodies.

[0144] Figure 11 Humanized antibodies of V30 and V43 compete for VEGFR2 in ELISA assays.

[0145] Figure 12 : Binding dissociation curves (SPR) of V1-SA1 and VEGFA165.

[0146] Figure 13 Melting temperature (Tm) of different humanized antibodies.

[0147] Figure 14 SDS-PAGE gel images of purified humanized antibody V1-SA1 and positive control antibody BI-VEGF ab.

[0148] Figure 15 Cologne gel image of purified monovalent nanobody-Fc fusion protein.

[0149] Figure 16 ELISA assay for binding VEGFA to monovalent nanobody-Fc fusion protein.

[0150] Figure 17 : Binding dissociation curve (SPR) of V1-SA1-Fc-m1 and VEGFA165. Detailed Implementation

[0151] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0152] Example 1: Alpaca Immunization and Antibody Screening

[0153] 1. Alpaca Immunization

[0154] Human VEGFA165 (unlabeled, purchased from: I-Pharmaceutical, catalog number: HPLC-10008-HNAH, hereinafter 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 2 weeks. Starting from the second immunization, serum titers were measured. Alpaca #1 was immunized with the antigen 4 times, and alpaca #2 was immunized with the antigen 3 times. Serum titers of both met the library construction standards.

[0155] 2. Detection of competitive activity of serum after immunization in blocking the binding of VEGFA and VEGFR2

[0156] The extracellular domain of the VEGF receptor, VEGFR2-ECD, was biotin-labeled (named VEGFR2-biotin). VEGFA was diluted to 0.5 μg / mL with CBS buffer and coated onto the microplate, then incubated overnight at 4°C. After blocking with 3% skim milk powder and washing, alpaca serum and negative serum (alpaca serum before antigen immunization) were diluted 5-, 15-, 45-, 135-, 405-, 1215-, and 3645-fold, respectively, to a final concentration of 0.5 μg / mL in VEGFR2-biotin solution. 100 μL of each solution was added to the sealed wells and incubated at room temperature for 1 h. The plates were then washed three times with PBST (PBS containing 0.1% Tween 20, pH 7.4), streptavidin-HRP was added, and the plates were incubated at room temperature for 1 h. After washing three times with PBST, TMB was added for color development for 15 minutes. Stop solution was added, and the absorbance was read at 450 nm using a microplate reader. The results of serum competitive activity assays in the two alpacas are shown in Table 2-3.

[0157] Table 2 Results of competitive activity assay in alpaca serum (No. 1)

[0158]

[0159] Table 3 Results of competitive activity assay in alpaca serum (No. 2)

[0160]

[0161]

[0162] The results show that antibodies blocking the binding of VEGFA and VEGFR2 were detected in both alpacas #1 and #2, which can be used for library construction.

[0163] 3. Antibody library construction

[0164] 40 mL of peripheral blood was drawn from alpaca using a lancet. Red blood cells were lysed according to the instructions for the red blood cell lysis buffer, and white blood cells were collected and cryopreserved in Trizol. Before library construction, total RNA and cDNA were extracted from isolated alpaca PBMCs using Trizol and reverse transcribed. The variable region VH fragment was amplified using nanobody-specific primers. The obtained fragment and the pcomb3X vector were digested with SfiI enzyme, and the digested fragments were ligated using T4 ligase at an appropriate ratio. After ligation, the ligation was performed on XL1-Blue competent cells via electrotransformation. Based on colony growth, the transformation library capacity for the two alpacas was calculated to be 6.52 × 10⁻⁶. 8 and 4.12×10 8 Clones were randomly selected from the transformant colonies and sent for sequencing verification. The sequencing results showed that the nanobody sequences were correct.

[0165] 4. Antibody library screening

[0166] Phage display method was used to screen phages that can bind VEGFA.

[0167] For the #1 alpaca library, after four rounds of enrichment screening, one plate of 96 wells of monoclonal antibodies was selected (the screening method is denoted as VEGF-4). th Enrichment was performed to detect the binding activity of phages to VEGFA. Simultaneously, competitive elution was used on the fourth round of phages: phages bound to VEGF were competitively eluted using 10 μg / mL VEGFR2 extracellular domain VEGFR2-ECD-His (prepared internally), with the aim of obtaining antibodies that could competitively bind VEGFA to VEGFR2. Five 96-well plates were picked from the obtained phages to detect the binding activity of phages to VEGFA (screening method denoted as VEGF-5). th (VEGFR2 competition). Furthermore, the phages obtained through VEGFR2 competitive screening were further screened using the control antibody Bevacizumab (Avastin). Specifically, phages bound to VEGFA were competitively eluted with 10 μg / mL Bevacizumab antibody. One 96-well plate was picked from each phage obtained, and the binding activity of the phage to VEGFA was detected (screening method denoted as VEGF-6). th(Bevacizumab competition) The detection of bacteriophages was performed using an HRP-labeled antibody that recognizes the phage coat protein, labeled as anti-M13(HRP). The detection results of positive clones with VEGFA binding activity among the above monoclonal samples are shown in Table 4.

[0168] Table 4. Detection results of positive clones with VEGFA activity after screening of Alpaca Library #1.

[0169]

[0170]

[0171] In addition, biotin-labeled VEGFA was used to screen phages that bound more epitopes. The method was as follows: Avidin was first coated onto the phage, followed by incubation with biotinylated VEGFA. After merging alpaca libraries #1 and #2, VEGFA-bound phages were obtained through incubation and elution of the libraries with biotinylated VEGFA. This process was repeated. After multiple rounds of enrichment, a single colony was selected from a 96-well plate. The VEGFA-binding activity of the phages in the supernatant was detected by ELISA. The results of positive clones with binding activity are shown in Table 5.

[0172] Table 5 shows the detection results of positive clones binding VEGFA activity after library merging.

[0173]

[0174]

[0175] 5. Initial screening antibody sequence

[0176] The above-mentioned phages with VEGFA binding activity were subjected to plasmid sequencing and codon translation to obtain the amino acid sequences of candidate nanobodies (CDR region + framework region). Antibodies with the same sequence were combined, and candidate antibodies with different CDR sequences are shown in Table 6.

[0177] Table 6. Amino acid sequences of candidate antibodies

[0178]

[0179]

[0180] 6. Expression and purification of candidate antibodies

[0181] Select some candidate antibodies from the table above, express them in mammalian cells, purify them, and perform ELISA tests to repeatedly verify the antibody's VEGFA binding activity.

[0182] The encoding genes of each nanobody were amplified from the original phage plasmids using PCR and constructed into the pVRC8400 expression vector. The N-terminus of each antibody was augmented with a secretory peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 68), and the C-terminus was augmented with a flexible linker GGGGS (SEQ ID NO: 69) and a 6×His tag. Simultaneously, the encoding gene sequence of the positive control antibody BI-VEGF ab was synthesized (this antibody is an anti-VEGFA nanobody; the antibody amino acid sequence is from SEQ ID NO: 57 in patent US 9527925 B2), and the C-terminus of the control antibody BI-VEGF ab was augmented with a 6×His tag. After confirming the correct sequencing of the expression plasmids for each antibody, endotoxin-free large-scale extraction was performed. 293F suspension cells were transiently transfected using polyethyleneimine (PEI, linear, MW = 25 kDa, purchased from Polysciences, catalog number: 23966-1). Cell supernatant was collected on day 4 or 5 post-transfection for Ni-bead affinity purification. The purified protein was analyzed by SDS-PAGE, and the molecular weight was as expected (~15 kDa). Cologne staining gel images of the positive control antibody BI-VEGF ab and some candidate antibodies after purification are shown below. Figure 1 As shown.

[0183] Example 2: Antibody Activity Test

[0184] 1. Activity assay of candidate antibody binding to VEGFA

[0185] Dilute the antigen VEGFA with ELISA coating buffer (final concentration 0.3 μg / mL), add 100 μL / well to the microplate, and coat 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, the antibody amino acid sequence of which comes from SEQ ID NO:57 in patent US 9527925 B2, and the construction method of its expression plasmid and the protein purification method are the same as the steps in "6. Expression and purification of candidate antibodies" in Example 1) and the gradient dilution buffer of the candidate antibody to be tested (0.001, 0.01, 0.1, 1, 3, 10, 100 nM) were added. After incubation at 37°C for 1 hour, the plate was washed and then His-Tag monoclonal antibody dilution buffer (purchased from Proteintech, catalog number: 66005-1-Ig) was added. After incubation at 37°C for 1 hour, the plate was washed and then HRP-conjugated goat anti-mouse IgG antibody dilution buffer (HRP-conjugated Affinipure Goat Anti-Mouse IgG) was added. Anti-Mouse IgG (H+L), purchased from Proteintech (catalog number: SA00001-1), was incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from Tiangen Biotech (catalog number: PA107-01)) was added to each well, and the plate was incubated at 37°C for 15 minutes. Then, 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured using a microplate reader.

[0186] The signal of partial antibody binding to VEGFA changes with antibody concentration, such as... Figure 2 As shown.

[0187] The VEGFA binding data in the ELISA test were fitted to obtain the EC50 of antibody binding to VEGFA, as shown in Table 7. It can be seen that all 10 candidate antibodies in the table have high VEGFA binding activity.

[0188] Table 7. VEGFA binding activity assay of candidate antibodies

[0189] Antibody name Combined with VEGFA-EC50 (nM) V1 0.53 V15 0.89 V29 0.77 V30 0.60 V31 0.67 V33 0.64 V36 0.36 V40 0.84 V41 0.57 V43 0.53

[0190] The amino acid sequences of the CDR regions of the above antibodies are partially different, and the antibody activity fluctuates within an acceptable range.

[0191] In addition, the affinity of nanobodies for VEGFA was tested using surface plasmon resonance (SPR) technology. A Biacore 8K instrument (Cytiva) was used, with antigen VEGFA165 (unlabeled, purchased from Sinocare, catalog number: HPLC-10008-HNAH) as the ligand, coupled to the CM5 chip via amino-coupled reagents (EDC and NHS), with a coupling amount of approximately 400 RU. The mobile phase buffer used in the experiment was HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v / v Tween 20). The analytes were the candidate antibodies and the positive control antibody BI-VEGF ab, diluted at concentrations of 80 nM, 40 nM, 20 nM, 10 nM, 5 nM, and 2.5 nM. For kinetic analysis, the binding time between the antigen and antibody was 240 seconds, the dissociation time was 1500 seconds, and the flow rate was 30 μL / min. A multi-cycle mode was used, with the chip regenerated using 100 mM hydrochloric acid. The binding and dissociation curves of the candidate antibody with 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.

[0192] Table 8 shows the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of candidate antibodies to VEGFA165.

[0193] ligands Analytes ka(1 / Ms) kd(1 / s) KD(M) VEGFA165 V1 4.09E+05 1.01E-04 2.47E-10 VEGFA165 V13 3.86E+05 8.65E-05 2.24E-10 VEGFA165 V15 3.47E+05 9.61E-05 2.77E-10 VEGFA165 V29 2.91E+05 1.27E-04 4.36E-10 VEGFA165 V30 6.35E+05 1.36E-04 2.14E-10 VEGFA165 V31 2.77E+05 1.72E-04 6.20E-10 VEGFA165 V33 3.23E+05 1.68E-04 5.22E-10 VEGFA165 V36 4.19E+05 1.20E-04 2.85E-10 VEGFA165 V40 3.53E+05 1.08E-04 3.05E-10 VEGFA165 V41 3.31E+05 1.60E-04 4.82E-10 VEGFA165 V43 4.18E+05 8.09E-05 1.93E-10 VEGFA165 BI-VEGF ab 4.00E+05 1.24E-04 3.10E-10

[0194] As shown in Table 8, the candidate antibody has a high affinity for VEGFA165, and the KD value is less than 3 times that of the positive control. Therefore, it is considered that the affinity of the candidate antibody is comparable to that of the positive control protein.

[0195] 2. Competitive VEGFR2 activity assay

[0196] To further verify the activity of the candidate antibody in blocking VEGFA binding to the receptor VEGFR2, a competitive ELISA was conducted.

[0197] Dilute the antigen VEGFA with ELISA coating buffer (final concentration 0.53 μg / mL) and add 100 μL / well to a 96-well microplate. Coat overnight at 4°C. Block with 5% skim milk powder. Mix the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from: E.T. Biotech, catalog number: 10012-H02H) at a final concentration of 1 nM with a gradient dilution of the antibody to be tested (0.01, 0.1, 1, 3, 10, 100 nM), add to a 96-well plate, and incubate at 37°C for 1 hour. After washing, add HRP-conjugated goat anti-human IgG antibody dilution (purchased from: Abbkine, catalog number: A21050) and incubate at room temperature for 45 minutes. After washing the plate, add 100 μL of TMB (purchased from Tiangen Biotech, catalog number: PA107-01) to each well, incubate at 37℃ for 15 minutes, then add 50 μL of stop solution. Measure the absorbance (OD450) at 450 nm using a microplate reader. Competitive ELISA results are shown below. Figure 3 .

[0198] Table 9 shows a comparison of the antibody activity (IC50 value and maximum inhibition rate) competing with VEGFR2. It can be seen that the 12 candidate antibodies in the table can competitively bind to VEGFA with VEGFR2. The formula for calculating the maximum inhibition rate is:

[0199]

[0200] Table 9. Candidate antibodies competing for VEGFR2 activity (IC50 value and maximum inhibition rate)

[0201]

[0202]

[0203] 3. The inhibitory activity of candidate antibodies on HUVEC cell proliferation

[0204] To validate the blocking effect of candidate antibodies V1, V13, V15 and V30 on the proliferation of VEGFA-stimulated human umbilical vein endothelial cells (HUVECs).

[0205] Primary HUVEC cells (from the China Center for Type Culture Collection (CCTCC)) were digested and diluted to 5×10^4 cells / mL, and seeded 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 h of adherent culture, the cells were treated with the drug.

[0206] A mixture of VEGFA and candidate antibody dilutions was prepared using DMEM / F12(HAM) 1:1 medium (serum-free, supplemented with penicillin and streptomycin, containing glutamine, purchased from Biological Industries, catalog number: 01-172-1ACS). The final concentration of VEGFA in each dilution was 35 ng / mL. The concentration gradients of candidate antibody V1 were 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, 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.

[0207] The negative control antibody is a nanobody that binds to an unrelated antigen, and its concentration gradient is as follows (decreasing by 3 times): 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, and 152.42 pM.

[0208] Simultaneously, controls were set up with VEGFA-free (least cell proliferation) and VEGFA-only (maximum cell proliferation) without any antibodies. The mixture of VEGFA and each antibody dilution was incubated at 37°C for 1 hour. After aspirating the original culture medium from the 96-well cell culture plate, the mixture of VEGFA and each antibody dilution was added to the cell culture plate. Two wells were set up for each concentration. Cells were incubated at 37°C for 72 hours. CCK-8 solution (purchased from Solarbio, catalog number: CA1210) was added to the 96-well plate, and the plate was incubated for 3 hours. The absorbance (OD450) at 450 nm was measured using a microplate reader.

[0209] Based on the difference in readings between control wells (without VEGFA and those containing only VEGFA without any antibody), the inhibition rate of the candidate antibody on cell proliferation at different concentrations was calculated. The formula for calculating the inhibition rate is:

[0210]

[0211] The inhibition rate curves of each antibody against HUVEC cell proliferation, as well as the maximum inhibition rate and IC50, are shown. Figure 4 As shown in Table 10.

[0212] Table 10. Maximum inhibitory rate and IC50 value of each antibody against HUVEC cell proliferation.

[0213] Candidate antibodies V1 V13 V15 V30 Negative control IC50(pM) 2693 3939 7119 1270 / Maximum inhibition rate (%) 103.4 94.7 84.2 87.4 0

[0214] Therefore, all four candidate antibodies 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), candidate antibody V1 effectively inhibited HUVEC proliferation (maximum inhibition rate close to 100%). However, based on IC50 data, candidate antibody V30 showed relatively stronger activity.

[0215] The results, obtained through ELISA assays of VEGFA binding and ELISA assays of competing VEGFR2, as well as cellular-level assays of HUVEC proliferation inhibition, indicate that the candidate antibody can efficiently bind to VEGFA and block VEGFA binding to VEGFR2, thereby inhibiting the VEGFA-VEGFR signaling pathway and suppressing vascular endothelial cell proliferation.

[0216] 4. Detection of the activity of candidate antibodies in binding to human VEGFA121 and mouse VEGFA164

[0217] The ELISA assay was performed using the same method as in "1. Activity test of candidate antibody binding to VEGFA" in this embodiment, namely, coated with 0.3 μg / mL human VEGFA121 (purchased from: Sinocare, catalog number: 10008-HNAH) or mouse VEGFA164 (purchased from: Sinocare, catalog number: 50159-MNAB) or rat VEGFA164 (purchased from: Sinocare, catalog number: 80006-RNAB), and the activity of His-tagged candidate antibody V1 binding to human VEGFA121, mouse VEGFA164 and rat VEGFA164 was detected.

[0218] The activity of antibody V1 binding to human VEGFA121, such as... Figure 5 As shown in Table 11.

[0219] Table 11 Activity of antibody V1 binding to human VEGFA121

[0220]

[0221]

[0222] Antibody V1 binding activity against mouse VEGFA164 is as follows Figure 6 As shown.

[0223] Antibody V1 binding activity against rat VEGFA164 is as follows Figure 7 As shown.

[0224] In summary, candidate antibody V1 exhibits high activity in binding to human VEGFA121.

[0225] 5. Detection of Tm values ​​for candidate antibodies

[0226] Eight candidate antibodies (V1, V15, V29, V30, V31, V36, V40, and V43) were selected, and the buffer solution for the antibodies was changed to: 10 mM His, pH 6.5, 40 mM NaCl, 5% sucrose, and 0.01% Tween-20. The melting temperature (Tm) of the antibodies was determined using an Unchained Labs instrument, and the results are shown in Table 12. The results indicate that these candidate antibodies all possess a certain degree of thermal stability.

[0227] Table 12 Melting temperatures (Tm) of candidate antibodies

[0228] Antibody name Tm (°C) V1 72.6 V15 68.7 V29 65.3 V30 72.8 V31 65.6 V36 64.6 V40 74.3 V43 62.4

[0229] Example 3: Humanization of anti-VEGFA nanobodies

[0230] Candidate antibody V1 was used as the starting antibody for humanization. First, the sequence of candidate antibody V1 was compared with the human antibody sequence using the antibody databases IGBLAST and IMGT for homology. Then, the highly homologous human antibody framework region was combined with the CDR region of the candidate antibody to construct a complete humanized antibody. The method for constructing the expression plasmid and purifying the protein were the same as steps "6. Expression and Purification of Candidate Antibody" in Example 1.

[0231] The purified antibodies were tested for VEGFA binding activity using ELISA. After the initial humanization of V1, the VEGFA binding activity of the antibodies decreased significantly. The mutations of a few amino acids in the framework region were restored to obtain nine optimized humanized V1 antibodies, and their amino acid sequences are shown in Table 13.

[0232] Table 13 Amino acid sequences of humanized candidate antibodies

[0233]

[0234]

[0235]

[0236]

[0237] These humanized antibodies that restored the mutations underwent transient transduction expression and purification. The first step of purification was the same as step "6. Expression and Purification of Candidate Antibodies" in Example 1. The positive control BI-VEGF ab and the humanized antibodies V1-DP and V1-SA1 were purified using Ni bead affinity chromatography followed by cation exchange chromatography (S column). A portion of the purified proteins were obtained via SDS-PAGE staining. Figure 8 As shown in (A), the molecular weight of the target protein is as expected (~15kDa).

[0238] The CDR regions of candidate antibodies V13, V15, V30, V40, and V43 were transplanted into the framework region after the humanization recovery mutation in V1. These candidate antibodies underwent humanization modification, expression, and purification. Some of the purified humanized antibodies are shown below. Figure 8 As shown in (B)(C)(D), the molecular weight of the target protein is as expected (~15kDa).

[0239] Example 4: Activity Detection of Humanized Anti-VEGFA Nanobodies

[0240] The obtained humanized antibodies were tested for VEGFA binding activity using an ELISA method, the same as described in "1. VEGFA Binding Activity Test of Candidate Antibodies" in Example 2. ELISA results for VEGFA binding of some humanized antibodies are shown below. Figure 9 As shown.

[0241] The VEGFA binding data from the ELISA test were fitted to obtain the EC50 of the humanized antibody binding to VEGFA, as shown in Table 14. The data show that the VEGFA binding activity of the candidate antibody after humanization is similar to that before humanization (V40).

[0242] Table 14 EC50 values ​​of humanized antibodies binding to VEGFA

[0243] Antibody name Combined with VEGFA-EC50 (nM) V15-2m 2.04 V15-4m 1.42 V30-3H-2m 1.38 V40-3H-2m 1.64 V40-3H-4m 2.05 V40-4H-2m 2.82 V40-4H-4m 2.34 V40 2.03

[0244] To further verify the activity of humanized antibodies in blocking VEGFA binding to the receptor VEGFR2, a competitive ELISA was conducted. The experimental method was similar to "2. Competitive VEGFR2 Activity Assay" in Example 2, with the following differences: For the humanized antibodies V1, V13, V15, and V43, the final concentration of the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from: Sinocare, catalog number: 10012-H02H) was 2 nM. The concentration gradient of the V1 humanized antibody was: 0.01, 0.1, 1, 10, 30, 100, 1000 nM. The concentration gradient of the V13, V15, and V43 humanized antibodies was: 0.03, 0.3, 3, 10, 30, 300 nM.

[0245] The results showed that the humanized antibodies V1 and V15 competed with VEGFR2 for VEGFA binding activity, reverting to their pre-humanization state (i.e., V1 and V15), respectively. The remaining humanized antibodies exhibited similar competitive VEGFR2 activity. The competitive ELISA results for some humanized antibodies are shown below. Figure 10 The IC50 and maximum inhibition rate are shown in Table 15. The formula for calculating the maximum inhibition rate is:

[0246]

[0247] Table 15 Competitive ELISA results and maximum inhibition rate of humanized antibodies

[0248]

[0249]

[0250] In addition, ELISA tests were performed on the humanized antibody V30 and the remaining humanized antibodies V43 to block VEGFR2. The experimental method was similar to "2. Competitive VEGFR2 Activity Assay" in Example 2, except that the final concentration of the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from: Sinocare, catalog number: 10012-H02H) was 1 nM. The concentration gradient of the humanized antibody was: 0.03, 0.3, 3, 10, 30, 300 nM.

[0251] The ELISA test results of the competition between humanized antibodies V30 and V43 for VEGFR2 are as follows: Figure 11 As shown.

[0252] Table 16 shows the antibody's competitive activity against VEGFR2 (IC50 value and maximum inhibition rate). It can be seen that the humanized antibody can competitively bind to VEGFA with VEGFR2, and its activity is similar to that of the unhumanized antibody (V30). The formula for calculating the maximum inhibition rate is:

[0253]

[0254] Table 16. Antibody competition for VEGFR2 activity (IC50 value and maximum inhibition rate)

[0255] Antibody name Competing with VEGFR2-IC50 (nM) Maximum inhibition rate (%) V30 5.72 94.8 V30-2H-78 4.97 95.5 V30-2H-87 5.81 94.2 V30-2H-2m 5.64 94.7 V43-1H-78 5.62 95.5 V43-1H-87 5.27 95.4 V43-1H-2m 5.99 94.5 V43-3H-78 8.63 93.4 V43-3H-87 7.15 91.6

[0256] Surface plasmon resonance (SPR) technology was used to conjugate the antigen VEGFA165, and the affinity of the nanobody for VEGFA was tested before and after humanization. The experimental methods and data fitting methods were the same as those in "1. Activity test of candidate antibody binding to VEGFA" in Example 2. The obtained KD values ​​are shown in Table 17.

[0257] Table 17. Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of humanized antibodies to VEGFA.

[0258] ligands Analytes ka(1 / Ms) kd(1 / s) KD(M) VEGFA165 V1 4.09E+05 1.01E-04 2.47E-10 VEGFA165 V1-2m 3.39E+05 8.24E-05 2.43E-10 VEGFA165 V1-com-78 3.47E+05 8.91E-05 2.57E-10 VEGFA165 V1-com-87 3.60E+05 9.08E-05 2.52E-10

[0259] Furthermore, the affinity of the humanized antibody V1-SA1 for VEGFA was tested using surface plasmon resonance (SPR) technology via antibody conjugation. A Biacore 8K instrument (Cytiva) was used, with antibody V1-SA1 as the ligand, conjugated to a CM5 chip via amino-conjugating reagents (EDC and NHS): the conjugation conditions were 10 mM sodium acetate (pH 4.0), and the V1-SA1 protein concentration was 20 μg / mL. The conjugation amount of V1-SA1 ligand was 371.8 RU. The mobile phase buffer used in 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 antigen VEGFA165 (unlabeled, purchased from GenScript, catalog number: Z03073), with dilution concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM. For kinetic analysis, the antigen-antibody binding time was 180 seconds, the dissociation time was 200 seconds, and the flow rate was 30 μL / min. A multi-cycle mode was used, with the chip regenerated using 10 mM glycine hydrochloride (pH = 1.5). The antigen-antibody binding curves were fitted using a "Two-state reaction" model to obtain the binding rate, dissociation rate, and KD.

[0260] The binding dissociation curves of V1-SA1 and VEGFA165 are shown below. Figure 12 The fitted KD values ​​are shown in Table 18.

[0261] Table 18. Binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of humanized antibody V1-SA1 to VEGFA.

[0262] ligands Analytes ka1(1 / Ms) kd1(1 / s) ka2(1 / s) kd2(1 / s) KD(M) V1-SA1 VEGFA165 1.06E+06 5.98E-02 9.41E-03 7.95E-04 4.38E-09

[0263] As shown in Tables 17 and 18, antibody V1 maintained a high affinity for VEGFA165 after humanization.

[0264] Example 5: Stability Study of Humanized Anti-VEGFA Nanobodies

[0265] 1. Compare the Tm values ​​of V1 humanized antibodies.

[0266] Antibodies with high thermal stability are more conducive to antibody production and long-term storage, so the melting temperatures (Tm) of different humanized antibodies were compared.

[0267] Nine humanized antibodies against V1 were used, with PBS (pH = 7.4) replaced in the buffer solution. Tm was measured using a UNCLE instrument (unchainedlabs). The Tm ranking is shown below. Figure 13 The Tm values ​​of humanized antibodies fluctuated within an acceptable range, with V1-DP and V1-SA1 having higher Tm values, indicating that the thermal stability of these two antibodies is likely superior to that of other humanized antibodies.

[0268] 2. Colloidal stability study of humanized antibody V1-SA1

[0269] The encoding genes of the humanized antibody V1-SA1 and the positive control antibody BI-VEGF ab were constructed into the expression vector pCDNA3.1(+). The N-terminus of the antibody was augmented with the secretory peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 68), and the C-terminus was augmented with the flexible linker GGS and the tag 6×His. Transient transfection and Ni-bead affinity purification of these two antibodies were performed using the same methods as described in "6. Expression and Purification of Candidate Antibodies" in Example 1. After purification, the antibodies were separated by SDS-PAGE, and the gel staining results are shown below. Figure 14 As can be seen from the figure, the molecular weights of the two proteins are as expected (~15kDa). The protein expression levels and SDS-PAGE staining purity are shown in Table 19. It can be seen that the expression level of V1-SA is much higher than that of BI-VEGF ab.

[0270] Table 19 Transient expression levels of humanized antibody V1-SA1 and positive control antibody BI-VEGF ab, and protein purity on SDS-PAGE gel.

[0271] protein name Expression level / L Protein purity in SDS-PAGE staining gel V1-SA1 37.3mg 100％ BI-VEGF ab 13.3mg 99.7％

[0272] Both V1-SA1 and BI-VEGF ab proteins were replaced with 1xPBS (pH=7.4), and the protein concentration was adjusted to 10 mg / mL. HPLC-SEC analysis was performed on the proteins (Zenix-C SEC-300, Sepax column). The percentages of protein monomers, aggregates, and fragments are shown in Table 20. It is evident that the proportion of aggregates in V1-SA1 is significantly lower than that in BI-VEGF ab, indicating that V1-SA1 is less prone to aggregation.

[0273] Table 20. Proportions of each component in SEC-HPLC analysis of antibody V1-SA1 and BI-VEGF ab

[0274] protein name Percentage of polymer aggregates (%) Monomer percentage (%) Protein fragment percentage (%) V1-SA1 0.20 98.55 1.25 BI-VEGF ab 3.25 95.49 1.27

[0275] Antibodies V1-SA1 and BI-VEGF ab (protein concentration of 10 mg / mL) were sterile filtered and placed at 37°C for a period of time (0 days and 2 days) after being replaced with PBS. The properties of the protein solution were observed and the colloidal stability of the two was compared. The results are shown in Table 21.

[0276] Table 21 Comparison of the properties, average particle size, and PDI of antibodies V1-SA1 and BI-VEGF ab after 0 and 2 days of storage at 37℃.

[0277]

[0278]

[0279] Based on the principle of dynamic light scattering, the protein particle size of antibody V1-SA1 and BI-VEGF ab solutions (diluted 4-fold with PBS) was measured using a nanoparticle size and potential analyzer (NS-90Z, Omec). The results are shown in Table 21. For the sample at time 0, the average hydration kinetic diameter and PDI of V1-SA1 were smaller than those of BI-VEGF ab. Considering their similar molecular weights, this indicates that BI-VEGF ab contains a certain proportion of high molecular weight protein aggregates in this buffer solution. Under the same conditions, V1-SA1 has a more uniform particle size distribution than BI-VEGF ab, and the proportion of high molecular weight protein aggregates is significantly lower.

[0280] As shown in Table 21, for samples placed at 37℃ for 2 days, V1-SA1 remained clear, while BI-VEGFab protein solution showed precipitation, indicating that V1-SA1 had better colloidal stability than BI-VEGF ab. At this time, the average hydration kinetic diameter of V1-SA1 was much smaller than that of BI-VEGF ab, and the change was small compared to time 0; while the average hydration kinetic diameter of BI-VEGF ab was much larger than that of V1-SA1, and it increased significantly compared to the BI-VEGF ab solution at time 0, indicating that BI-VEGF ab further formed high molecular weight protein aggregates under high temperature conditions.

[0281] Example 6: Preparation and Activity Study of Monovalent Nanobody-Fc Fusion Protein

[0282] 1. Preparation of monovalent nanobody-Fc fusion protein

[0283] This embodiment demonstrates the fusion expression of a monovalent nanobody with IgG1 Fc (SEQ ID NO: 62) or PPCP-Fc (SEQ ID NO: 75, which is the remaining amino acid sequence after removing DKTHTC from SEQ ID NO: 62) or the IgG1 Fc mutant Fc-m1 (SEQ ID NO: 63).

[0284] The amino acid sequence of IgG1 Fc is as follows:

[0285] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 62)

[0286] The amino acid sequence of PPCP-Fc is as follows:

[0287] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:75)

[0288] The amino acid sequence of Fc-m1, a mutant of IgG1 Fc, is as follows:

[0289] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 63)

[0290] The amino acid sequence of the constructed nanobody-Fc fusion protein is shown in Table 22.

[0291] Table 22 Amino acid sequence of nanobody-Fc fusion protein

[0292]

[0293]

[0294] To facilitate comparison of the VEGFA binding activity of the linearly tandem bivalent nanobody 2V15-9GS (SEQ ID NO: 74) (containing a 6-histidine tag on top of 2V15-9GS), and the fusion proteins V15-PPCP-Fc, V15-9GS-PPCP-Fc, and V15-Fc, a linker (SGGS (SEQ ID NO: 70)) and a 6-histidine tag (HHHHHH (SEQ ID NO: 71)) were added to the C-terminus of the Fc fragment of the fusion protein. During the plasmid construction of the monovalent nanobody-Fc fusion protein, the nanobody and the Fc fragment containing the linker and histidine tag were amplified separately using PCR. The PCR template for the Fc fragment was derived from the synthesized Fc gene. The nanobody and Fc were integrated into the vector pCDNA3.1(+) using homologous recombination of two fragments, with the restriction enzymes NheI and XbaI used. The secretory peptide of the fusion protein is METDTLLLWVLLLWVPGSTG (SEQ ID NO: 72), followed by the sequence of the target protein. After successful sequencing of these fusion protein plasmids, endotoxin-free plasmid extraction was performed. 293F suspension cells were transiently transfected using polyethyleneimine (PEI, linear, MW = 25 kDa, purchased from Polysciences, catalog number: 23966-1). Cell supernatant was collected on day 4 or 5 post-transfection for Ni-bead purification. SDS-PAGE analysis of the purified protein showed that the molecular weight of the protein monomer under reducing conditions was approximately 39 kDa, consistent with expectations. Cosmetic gel images of some purified monovalent nanobody-Fc fusion proteins are shown below. Figure 15 As shown.

[0295] 2V15-9GS (SEQ ID NO: 74)

[0296] QLQLVESGGGSVQPGGSLRLSCEVSGRTFASYTMGWFRQAPGKEREFVVAISKGGYKYDSVSLEARFTISKDNTKNTVYLQMNSLKPEDTAVYYCAGTRAYGSSRLRLAETYEYWGQGTQVTVSSGGGG SGGGSQLQLVESGGGSVQPGGSLRLSCEVSGRTFASYTMGWFRQAPGKEREFVVAISKGGYKYDSVSLEARFTISKDNTKNTVYLQMNSLKPEDTAVYYCAGTRAYGSSRLRLAETYEYWGQGTQVTVSS

[0297] For the humanized antibody V1-SA1 fusion protein (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 above method, but the C-terminus of Fc did not contain any additional amino acids; that is, the fusion protein V1-SA1-Fc-m1 contains an Fc tag but no histidine tag. After transient transfection of the V1-SA1-Fc-m1 expression plasmid into 293F cells, it was purified using Protein A bead affinity chromatography. During protein elution, the target protein was eluted with 0.1M citric acid (pH 3.0) and rapidly neutralized to obtain the target protein.

[0298] 2. Activity detection of monovalent nanobody-Fc fusion protein

[0299] The purified monovalent nanobody-Fc fusion protein was subjected to a VEGFA binding activity assay using the same ELISA method as described in Example 2, "1. VEGFA Binding Activity Assay of Candidate Antibody". The ELISA results are as follows: Figure 16 As shown in Table 23, EC50 is as follows.

[0300] Table 23 VEGFA binding activity assay of monovalent nanobody-Fc fusion protein

[0301] Monovalent nanobody-Fc fusion protein or tandem bivalent nanobody Combined with VEGFA's EC50 (nM) V15-PPCP-Fc 0.64 V15-9GS-PPCP-Fc 0.35 V15-Fc 0.43 2V15-9GS 0.25

[0302] The data in the table show that both the tested monovalent nanobody-Fc fusion protein and the linearly tandem divalent nanobody exhibit strong VEGFA binding activity.

[0303] The affinity of the monovalent nanobody-Fc fusion protein (V1-SA1-Fc-m1) for VEGFA was tested using surface plasmon resonance (SPR) technology. A Biacore 8K instrument (Cytiva) was used to capture V1-SA1-Fc-m1 onto a ProteinA chip using V1-SA1-Fc-m1 as the ligand. During immobilization, V1-SA1-Fc-m1 was diluted to a protein concentration of 2 μg / mL with HBS-EP mobile phase buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20), resulting in a conjugation capacity of approximately 590 RU. During the experiment, the analyte was antigen VEGFA165 (unlabeled, purchased from GenScript, catalog number: Z03073), with dilution concentrations 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 of the fusion protein to antigen VEGFA165 was 180 seconds, the dissociation time was 900 seconds, and the flow rate was 30 μL / min. After each binding-dissociation cycle, the chip was regenerated under 10 mM glycine hydrochloride (pH 1.5). The binding-dissociation curves of the fusion protein and antigen VEGFA165 are shown below. Figure 17 As shown in Table 24, the binding rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) of the monovalent nanobody-Fc fusion protein with VEGFA165 were obtained using a 1:1 binding model.

[0304] ligands Analytes ka(1 / Ms) kd(1 / s) KD(M) V1-SA1-Fc-m1 VEGFA165 8.77E+05 1.30E-04 1.48E-10

[0305] SPR data show that the monovalent nanobody-Fc fusion protein V1-SA1-Fc-m1 has a high affinity for VEGFA165 (KD = 0.148 nM). This is consistent with Table 18 of "6. Activity Detection of Humanized Antibodies" in Example 2 above. Figure 12 As can be seen from the data, the KD of the monovalent nanobody V1-SA1 with VEGFA165 is 4.38 nM. Therefore, the affinity of the monovalent nanobody for VEGFA165 after fusion with Fc is nearly 30 times higher than that of the unfused Fc, indicating that the anti-VEGFA antibody-Fc fusion protein has stronger target affinity and potentially better efficacy in blocking the VEGFA signaling pathway.

[0306] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0307] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An anti-VEGFA antibody or antigen-binding fragment thereof, characterized in that, The anti-VEGFA antibody or antigen-binding fragment thereof is a single-domain antibody, and the anti-VEGFA antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region, wherein, the amino acid sequence of CDR-H1 is SYTMG (SEQ ID NO: 1); the amino acid sequence of CDR-H2 is AISKGGYKYX1X2VSLEA (SEQ ID NO: 2); the amino acid sequence of CDR-H3 is TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3); 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, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are 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.

2. The anti-VEGFA antibody or antigen-binding fragment thereof of claim 1, wherein, 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.

3. The anti-VEGFA antibody or antigen-binding fragment thereof of claim 2, wherein, The modification site of the humanized sequence is located in a framework region and / or a constant region of the antibody.

4. The anti-VEGFA antibody or antigen-binding fragment thereof of claim 1, wherein, The anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody.

5. The anti-VEGFA antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences in SEQ ID NO: 13, 16-19, 22, 25-32, 35-61, 64-67.

6. The anti-VEGFA antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, The anti-VEGFA antibody or antigen-binding fragment thereof binds to human or monkey VEGFA protein.

7. A fusion protein, characterized in that, The fusion protein comprises the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-6.

8. The fusion protein of claim 7, wherein, The fusion protein further comprises an anti-VEGFA antibody or antigen-binding fragment thereof or an antibody or antigen-binding fragment thereof of another target or another functional component in addition to the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-6, and the other functional component comprises one or more of serum albumin, a cytokine, transferrin, a scaffold protein, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, a polysaccharide, a fatty chain, avidin, biotin, a toxin, a drug, a nucleic acid, a radionuclide, a label thereof or a combination of Fc fragments.

9. The fusion protein of claim 8, wherein, The avidin comprises streptavidin.

10. The fusion protein of claim 9, wherein, The other target is selected from the group consisting of 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, 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, DLL 3 or 5T4.

11. The fusion protein of claim 10, wherein, The interleukin includes IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, and the interleukin receptor includes IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12R, IL15Rα, IL17R, IL23R.

12. A chimeric antigen receptor, characterized in that, The extracellular domain of the chimeric antigen receptor comprises the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-6.

13. An immune cell, characterized in that, The immune cell expresses the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-6 or the chimeric antigen receptor according to claim 12.

14. A nucleic acid, characterized in that, The nucleic acid encodes the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-6 or the fusion protein according to any one of claims 7-11.

15. A vector, characterized in that, The vector comprises the nucleic acid according to claim 14.

16. A host cell, characterized in that, The host cell comprises the nucleic acid according to claim 14 or the vector according to claim 15.

17. A method of producing an anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, The preparation method comprises introducing the nucleic acid or vector encoding the anti-VEGFA antibody or antigen-binding fragment thereof into a host cell, and then inducing its expression.

18. A product for treating and / or diagnosing a disease, characterized in that, The product for treating and / or diagnosing diseases comprises any one of the following: A) the anti-VEGFA antibody or antigen-binding fragment thereof according to any one of claims 1-6; B) the fusion protein according to any one of claims 7-11; C) the chimeric antigen receptor of claim 12; D) the immune cell of claim 13; E) the nucleic acid of claim 14; F) the vector of claim 15; or, G) the host cell of claim 16; said disease is non-Hodgkin lymphoma, breast cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy, central retinal vein occlusion, pathological myopia or neovascular glaucoma.

19. Use of the anti-VEGFA antibody or antigen-binding fragment thereof of any one of claims 1-6, the fusion protein of any one of claims 7-11, the chimeric antigen receptor of claim 12, the immune cell of claim 13, the nucleic acid of claim 14, the vector of claim 15, or the host cell of claim 16 in the manufacture of a medicament for treating and / or preventing a disease associated with VEGFA, wherein the disease is non-Hodgkin lymphoma, breast cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy, central retinal vein occlusion, pathological myopia or neovascular glaucoma.

20. Use of the anti-VEGFA antibody or antigen-binding fragment thereof of any one of claims 1-6, the fusion protein of any one of claims 7-11, the chimeric antigen receptor of claim 12, the immune cell of claim 13, the nucleic acid of claim 14, the vector of claim 15, or the host cell of claim 16 in the manufacture of a medicament for blocking VEGFA-mediated proliferation of vascular endothelial cells or inhibiting a disease associated with angiogenesis, wherein the disease is non-Hodgkin lymphoma, breast cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy, central retinal vein occlusion, pathological myopia or neovascular glaucoma.

21. Use of the anti-VEGFA antibody or antigen-binding fragment thereof of any one of claims 1-6, the fusion protein of any one of claims 7-11, the chimeric antigen receptor of claim 12, the immune cell of claim 13, the nucleic acid of claim 14, the vector of claim 15, or the host cell of claim 16 in the manufacture of an antibody diagnostic kit for a disease associated with VEGFA, wherein the disease is non-Hodgkin lymphoma, breast cancer, colorectal cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy, central retinal vein occlusion, pathological myopia or neovascular glaucoma.

22. Use of the anti-VEGFA antibody or antigen-binding fragment thereof of any one of claims 1-6, the fusion protein of any one of claims 7-11, the chimeric antigen receptor of claim 12, the immune cell of claim 13, the nucleic acid of claim 14, the vector of claim 15, or the host cell of claim 16 in the manufacture of a VEGFA tracer.

23. A method of detecting VEGFA, comprising contacting a sample with an antibody of any one of claims 1-22 and detecting binding of the antibody to the sample. The detection method comprises contacting a sample to be detected with the anti-VEGFA antibody or antigen-binding fragment thereof of any one of claims 1-6, and then detecting the content of the complex formed by VEGFA and the anti-VEGFA antibody or antigen-binding fragment thereof, wherein the detection method is not for the purpose of diagnosing a disease.

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