VEGFA (vascular endothelial growth factor A)-resistant fusion protein as well as preparation method and application thereof

Abstract

The invention provides an anti-VEGFA fusion protein and a preparation method and application thereof, the anti-VEGFA fusion protein has high affinity with VEGFA, can effectively block combination of VEGFA and a receptor VEGFA, block a VEGFA signal channel and effectively block vascular endothelial cell proliferation, angiogenesis and other pathological processes caused by VEGFA, and can be potentially applied to treatment of VEGFA-related diseases.

Description

An anti-VEGFA fusion protein and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an anti-VEGFA fusion protein and a preparation method and application thereof. Background Art

[0002] Abnormal vascular proliferation is closely associated with the development and progression of a variety of diseases, such as various solid tumors and retinal vascular diseases. Studies have shown that vascular endothelial growth factor A (VEGFA) is an important factor in promoting endothelial cell proliferation and angiogenesis. Therefore, inhibiting the VEGFA signaling pathway can be used to treat diseases related to abnormal vascular proliferation. For example, wet age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is caused by excessive expression of VEGFA in the retina, leading to choroidal angiogenesis and disrupting normal retinal function.

[0003] Currently, there are four main anti-VEGFA 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-VEGF drugs are administered via local injection, such as intravitreal injection, with a volume not exceeding 100 μL. The longest dosing interval for brolucizumab is 3 months, while the dosing interval for the other drugs is once every 1-2 months. Frequent intraocular injections increase the risk of elevated intraocular pressure, intraocular inflammation, and retinal detachment. Therefore, there is an urgent clinical need for long-acting anti-VEGFA therapeutics to reduce the number of injections required.

[0004] Increasing the affinity between a drug and its target, i.e., reducing the KD, can lower the drug's minimum effective concentration. Among drugs used to treat wet AMD, ranibizumab has a strong affinity for VEGFA (KD = 46 pM), while aflibercept and conbercept have a 100-fold stronger affinity for VEGFA than ranibizumab (KD = 0.5 pM).

[0005] The diffusion rate of drugs in the vitreous humor directly influences the dosing interval and is related to properties such as charge, molecular weight, and molecular shape. Drugs with larger molecular weights diffuse more slowly and have longer half-lives. In a pharmacokinetic study conducted in the vitreous humor of Dutch belted rabbits, the half-life of ranibizumab (molecular weight: 48 kDa) was 2.81 days, that of bevacizumab (molecular weight: 149 kDa) was 4.22 days, and that of aflibercept (molecular weight: 115 kDa) was 4.58 days.

[0006] Therefore, the development of high-affinity, long-half-life anti-VEGFA fusion proteins is of great significance for improving the compliance of wet AMD patients.

[0007] Single-domain antibodies (SDOs), or nanobodies, are the variable domains of heavy-chain antibodies naturally found in camelids, lacking their light chains. They represent the smallest stable antibody unit with complete antigen-binding function. With a molecular weight of approximately 13 kDa, SDOs exhibit high thermal stability and good water solubility. Their inherent properties offer great potential for development into high-concentration protein formulations.

[0008] Therefore, the present invention uses nanobodies as the basic active unit for binding to VEGFA, designs the bivalent nature of the nanobodies, and fuses them with Fc fragments to prepare an anti-VEGFA fusion protein with high affinity.

[0009] Summary of the Invention

[0010] In a first aspect of the present invention, an anti-VEGFA fusion protein is provided, wherein the anti-VEGFA fusion protein comprises two or more anti-VEGFA antibodies or antigen-binding fragments thereof, and an Fc fragment, wherein the anti-VEGFA antibodies or antigen-binding fragments thereof comprise CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region; wherein,

[0011] the amino acid sequence of CDR-H1 comprises SYTMG (SEQ ID NO: 1) or an amino acid sequence at least 80% identical to SYTMG (SEQ ID NO: 1);

[0012] The amino acid sequence of CDR-H2 comprises AISKGGYKYX1X2VSLEA (SEQ ID NO: 2) or an amino acid sequence that is at least 80% identical to AISKGGYKYX1X2VSLEA (SEQ ID NO: 2);

[0013] The amino acid sequence of CDR-H3 comprises TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3) or an amino acid sequence that is at least 80% identical to TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3).

[0014] Preferably, the anti-VEGFA fusion protein comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) anti-VEGFA antibodies or antigen-binding fragments thereof.

[0015] In a specific embodiment of the present invention, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NOs: 1, 2 and 3, respectively.

[0016] Preferably, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in order from N-terminus to C-terminus. The amino acid division of the CDR region of the antibody in this application adopts the Kabat numbering system.

[0017] Preferably, the anti-VEGFA antibodies or antigen-binding fragments thereof are directly linked to each other, or the anti-VEGFA antibodies or antigen-binding fragments thereof are directly linked to the Fc fragment.

[0018] Preferably, the other anti-VEGFA antibodies or antigen-binding fragments thereof, the other anti-VEGFA antibodies or antigen-binding fragments thereof and the anti-VEGFA antibody or antigen-binding fragment thereof, the other anti-VEGFA antibody or antigen-binding fragment thereof and the Fc fragment, and the Fc fragment and the anti-VEGFA antibody or antigen-binding fragment thereof are directly or indirectly connected.

[0019] Preferably, the direct or indirect connection includes direct or indirect connection to the N-terminus, C-terminus and / or internal residues of the anti-VEGFA antibody or antigen-binding fragment thereof, other anti-VEGFA antibodies or antigen-binding fragments thereof and / or Fc fragment.

[0020] Preferably, the N-terminus, C-terminus and / or internal residues of the anti-VEGFA antibody or antigen-binding fragment thereof in the anti-VEGFA fusion protein are linked to the N-terminus, C-terminus and / or internal residues of other anti-VEGFA antibodies or antigen-binding fragments thereof or Fc fragments.

[0021] In a specific embodiment of the present invention, the N-terminus of the anti-VEGFA antibody or antigen-binding fragment thereof in the anti-VEGFA fusion protein is linked to the N-terminus or C-terminus of another anti-VEGFA antibody or antigen-binding fragment thereof.

[0022] In a specific embodiment of the present invention, the C-terminus of the anti-VEGFA antibody or antigen-binding fragment thereof in the anti-VEGFA fusion protein is linked to the N-terminus or C-terminus of another anti-VEGFA antibody or antigen-binding fragment thereof.

[0023] In a specific embodiment of the present invention, the C-terminus of the anti-VEGFA antibody or antigen-binding fragment thereof or the C-terminus of other anti-VEGFA antibodies or antigen-binding fragments thereof in the anti-VEGFA fusion protein is linked to the N-terminus of the Fc fragment.

[0024] Preferably, the indirect connection is through a joint connection.

[0025] Preferably, the linker is selected from a connecting peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, a nucleic acid, a polysaccharide, a fatty chain, biotin, streptavidin, or avidin. Preferably, the connecting peptide can be a rigid linker, a flexible linker, or nonsense amino acids. More preferably, the amino acid sequence of the connecting peptide comprises one or a combination of two or more of SEQ ID NOs: 77-91, 152-153, and 156.

[0026] Preferably, the two or more anti-VEGFA antibodies or antigen-binding fragments thereof are anti-VEGFA antibodies or antigen-binding fragments thereof with completely identical sequences, anti-VEGFA antibodies or antigen-binding fragments thereof with partially identical sequences, or anti-VEGFA antibodies or antigen-binding fragments thereof with completely different sequences.

[0027] Preferably, the anti-VEGFA fusion protein further comprises a secretory peptide. The secretory peptide is linked to the N-terminus of the anti-VEGFA fusion protein. Further preferably, the amino acid sequence of the secretory peptide may be SEQ ID NO: 151 or 155.

[0028] In a specific embodiment of the present invention, the Fc fragment comprises SEQ ID NO: 134, 135 or 158.

[0029] In a specific embodiment of the present invention, the anti-VEGFA fusion protein comprises, from N-terminus to C-terminus, a first anti-VEGFA antibody or an antigen-binding fragment thereof, a connecting peptide, and a second anti-VEGFA antibody or an antigen-binding fragment thereof.

[0030] In a specific embodiment of the present invention, the anti-VEGFA fusion protein comprises, from N-terminus to C-terminus, a secretory peptide, a first anti-VEGFA antibody or an antigen-binding fragment thereof, a connecting peptide, and a second anti-VEGFA antibody or an antigen-binding fragment thereof.

[0031] In a specific embodiment of the present invention, the anti-VEGFA fusion protein comprises, from N-terminus to C-terminus, a secretory peptide, a first anti-VEGFA antibody or an antigen-binding fragment thereof, a connecting peptide, a second anti-VEGFA antibody or an antigen-binding fragment thereof, and an Fc fragment.

[0032] In a specific embodiment of the present invention, the anti-VEGFA fusion protein comprises, from N-terminus to C-terminus, a secretory peptide, a first anti-VEGFA antibody or an antigen-binding fragment thereof, a connecting peptide, a second anti-VEGFA antibody or an antigen-binding fragment thereof, and a tag (e.g., 6×His).

[0033] In a specific embodiment of the present invention, X1X2 in SEQ ID NO: 2 represent DS, DA, NT, DT, NA or NS; X3 in SEQ ID NO: 3 represents R or K, and X4 represents D, N, E or K.

[0034] Preferably, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups (see Table 1):

[0035] A) SEQ ID NO: 1, 4, 9;

[0036] B) SEQ ID NO: 1, 5, 9;

[0037] C) SEQ ID NO: 1, 6, 10;

[0038] D) SEQ ID NO: 1, 4, 11;

[0039] E) SEQ ID NO: 1, 7, 12;

[0040] F) SEQ ID NO: 1, 6, 11;

[0041] G) SEQ ID NO: 1, 4, 10;

[0042] H) SEQ ID NO: 1, 5, 12;

[0043] I) SEQ ID NO: 1, 4, 12;

[0044] J) SEQ ID NO: 1, 8, 12;

[0045] K) SEQ ID NO: 1, 33, 34.

[0046] Table 1 Amino acid sequences of CDR-H1, CDR-H2 and CDR-H3

[0047] Preferably, the antibody or antigen-binding fragment thereof is a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a F(ab')2 fragment, a single domain antibody, a single chain antibody molecule or a linear antibody.

[0048] Preferably, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 13-32, 35-61, or has at least 80% identity with any one of SEQ ID NOs: 13-32, 35-61. Specific sequence numbers are shown in Table 2.

[0049] Table 2 Antibody amino acid sequences

[0050] Preferably, the anti-VEGFA fusion protein binds to human or monkey VEGFA protein, wherein the human VEGFA protein and the monkey VEGFA protein have the same sequence.

[0051] The anti-VEGFA antibody or antigen-binding fragment thereof can be a humanized antibody or a fully human antibody.

[0052] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof comprises a humanized sequence, and the modification site of the humanized sequence is located in a non-CDR region. Further preferably, the humanized modification site is located in the framework region and / or constant region of the antibody.

[0053] In a specific embodiment of the present invention, the amino acid sequence of the anti-VEGFA fusion protein comprises any one of SEQ ID NOs: 62-76, 92-133, 136-150 and 159, or has at least 80% identity with any one of SEQ ID NOs: 62-76, 92-133, 136-150 and 159.

[0054] The second aspect of the present invention provides a chimeric antigen receptor, wherein the extracellular domain of the chimeric antigen receptor comprises the above-mentioned anti-VEGFA fusion protein.

[0055] Preferably, the chimeric antigen receptor further comprises any transmembrane region and / or intracellular signaling region conventional in the prior art.

[0056] The third aspect of the present invention provides a nucleic acid encoding the above-mentioned anti-VEGFA fusion protein or the above-mentioned chimeric antigen receptor.

[0057] The fourth aspect of the present invention provides a vector comprising the above nucleic acid.

[0058] The vector can be expressed in vivo, in vitro, or in vitro. Preferably, the vector is a prokaryotic expression vector, a viral expression vector, or a eukaryotic expression vector, such as an E. coli vector, a bacteriophage, or the like.

[0059] The fifth aspect of the present invention provides a cell comprising the above-mentioned vector or the above-mentioned nucleic acid.

[0060] The cells can be eukaryotic cells or prokaryotic cells.

[0061] Eukaryotic cells include animal and plant cells, such as T cells, yeast cells, HEK293 cells, or CHO cells.

[0062] Prokaryotic cells such as Escherichia coli.

[0063] In a sixth aspect, the present invention provides a method for preparing an anti-VEGFA fusion protein, which comprises introducing the above-mentioned nucleic acid into a host cell and inducing its expression.

[0064] In a seventh aspect, the present invention provides an immune cell, wherein the immune cell expresses the above-mentioned anti-VEGFA fusion protein or the above-mentioned chimeric antigen receptor.

[0065] Preferably, the immune cells include but are not limited to lymphocytes (such as T cells, B cells, NK cells), dendritic cells, monocytes / macrophages, granulocytes, and mast cells.

[0066] Preferably, the immune cells are CAR-immune cells.

[0067] In an eighth aspect, the present invention provides a method for constructing an immune cell, the method comprising transfecting a nucleic acid sequence encoding the chimeric antigen receptor of the present invention into an immune cell for expression.

[0068] In a ninth aspect, the present invention provides a product for treating and / or diagnosing a disease, wherein the product for treating and / or diagnosing a disease comprises any one of the following:

[0069] A) the anti-VEGFA fusion protein described above;

[0070] B) the chimeric antigen receptor described above;

[0071] C) the nucleic acid described above;

[0072] D) the above-mentioned vector;

[0073] E) the cell described above; or,

[0074] F) The immune cells described above.

[0075] The product for treating and / or diagnosing the disease targets cells expressing VEGFA, and the cells may be cardiomyocytes, proximal renal tubular cells, hepatocytes, vascular endothelial cells, granular cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

[0076] Preferably, the product can be a kit, a drug, a chip, an antibody-drug conjugate, etc.

[0077] The diseases are diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0078] In a tenth aspect, the present invention provides an antibody-drug conjugate (ADC), which comprises the anti-VEGFA fusion protein of the present invention covalently bound to a drug.

[0079] In an eleventh aspect of the present invention, a method for detecting VEGFA is provided, comprising contacting a sample to be tested with the anti-VEGFA fusion protein, and then detecting the content of a complex formed between VEGFA and the anti-VEGFA fusion protein.

[0080] The detection method is to detect the presence or content of VEGFA, wherein the presence refers to the presence or absence, and the content can be the expression level or protein concentration.

[0081] In a twelfth aspect of the present invention, a method for diagnosing a disease is provided, which comprises taking a sample, contacting the sample with the anti-VEGFA fusion protein, the chimeric antigen receptor, the nucleic acid, the vector, the cell, the immune cell or the product for treating and / or diagnosing the disease, and detecting the content of the complex formed by VEGFA and the anti-VEGFA fusion protein.

[0082] The diseases are diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0083] The thirteenth aspect of the present invention provides an anti-VEGFA fusion protein, the chimeric antigen receptor, the nucleic acid, the vector, the cell, or the immune cell for use in preparing a product for treating and / or preventing a disease associated with VEGFA, or for use in preparing a product for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, or for use in preparing an antibody-drug conjugate or an antibody diagnostic kit or a tracer.

[0084] In the fourteenth aspect of the present invention, a method for treating and / or preventing a disease is provided, which comprises administering the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease to an individual.

[0085] The diseases are diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular diseases), and the like.

[0086] In a fifteenth aspect, the present invention provides a method for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, the method comprising contacting vascular endothelial cells with the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cells, the above-mentioned immune cells or the above-mentioned product for treating and / or diagnosing the disease.

[0087] Preferably, the method comprises diluting the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease with a culture medium containing vascular endothelial cells, and then incubating with the antigen (e.g., VEGFA165).

[0088] Preferably, after incubation, the method further comprises the step of resuspending the vascular endothelial cells in a culture medium.

[0089] Preferably, the resuspended vascular endothelial cells, antigen, and the aforementioned anti-VEGFA fusion protein, the aforementioned chimeric antigen receptor, the aforementioned nucleic acid, the aforementioned vector, the aforementioned cells, the aforementioned immune cells, or the aforementioned product for treating and / or diagnosing a disease are cultured on a culture plate.

[0090] Preferably, the detection is performed after culturing.

[0091] Preferably, the incubation time is 0.5-5 h, preferably 1-3 h, such as 0.5, 1, 2, 3, 4, 5 h.

[0092] Preferably, the incubation temperature is room temperature-45°C, preferably 30-40°C, for example 25, 30, 35, 36, 37, 38, 39, 40, 45°C, etc.

[0093] Preferably, the culture temperature is room temperature-45° C., preferably 30-40° C., such as 25, 30, 35, 36, 37, 38, 39, 40, 45° C., etc. Preferably, the culture is carried out in an incubator with 5% CO 2 .

[0094] Preferably, the culture time is 1-5 days, preferably 2-4 days, for example 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, 5 days, etc.

[0095] Preferably, the detection is the detection of the number of living vascular endothelial cells.

[0096] In the sixteenth aspect of the present invention, a method for treating and / or preventing a disease is provided, which comprises contacting the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing the disease with a target cell.

[0097] The diseases are diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular diseases), and the like.

[0098] Preferably, the target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

[0099] The seventeenth aspect of the present invention provides a use of the above-mentioned anti-VEGFA fusion protein, the above-mentioned chimeric antigen receptor, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell, the above-mentioned immune cell or the above-mentioned product for treating and / or diagnosing a disease in blocking the VEGFA signaling pathway.

[0100] Preferably, the application includes:

[0101] A) Use in the preparation of drugs for treating and / or preventing tumors;

[0102] B) Application in the treatment and / or prevention of tumors;

[0103] C) use in the preparation of a medicament for treating and / or preventing ophthalmic diseases involving angiogenesis (e.g., fundus vascular diseases), preferably age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc.;

[0104] D) treatment and / or prevention of ophthalmic diseases involving angiogenesis (e.g., fundus vascular diseases), preferably age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc.; and / or,

[0105] E) Use in inhibiting abnormal vascular proliferation, preferably blocking the proliferation of vascular endothelial cells.

[0106] The anti-VEGFA fusion protein of the present invention has a high affinity for VEGFA, can effectively block the binding of VEGFA to the receptor VEGFR, block the VEGFA signaling pathway, and effectively block pathological processes such as vascular endothelial cell proliferation and angiogenesis caused by VEGFA, and can be potentially used in the treatment of VEGFA-related diseases.

[0107] The "medicine" of the present invention can be used to treat humans or non-human animals, such as non-human mammals. The medicine can include pharmaceutically acceptable carriers, excipients or salts common in the prior art. The medicine can be administered by any suitable route, such as enteral administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.). The medicine can be in any suitable dosage form, such as enteral administration or parenteral administration, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc. The various dosage forms of the medicine can be prepared according to the conventional production methods of pharmaceutical field. The drug may contain the anti-VEGFA fusion protein, the nucleic acid, the vector, the cells, the immune cells, etc. in a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%). The drug can be prepared as a reagent with a protein concentration of 1-300 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / mL). The single dose of the drug can be 0.1-1000 mg, for example, 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg.

[0108] The term "pharmaceutically acceptable" as used herein means that the pharmaceutical composition neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0109] The "method" described in the present invention can be used for the diagnosis and treatment of diseases or for non-disease diagnosis and treatment purposes.

[0110] The "antigen-binding fragment" described in the present invention is a portion of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody that is capable of specifically binding to an epitope on the antibody's target molecule. It includes, for example, Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, and variants of these fragments. For example, the heavy and / or light chains of an antibody, the heavy and / or light chain variable regions of an antibody, or a single or two or more CDRs from the heavy or light chains of an antibody. Fab is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. F(ab')2 is a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge 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 arm of an antibody. Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH is a Fab' in which the cysteine ​​residue in the constant domain carries at least one free thiol group. Among them, VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain, and CH1 is CH1 that constitutes the heavy chain constant region.

[0111] The "Fc" region of the present invention contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments 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.

[0112] The "humanized antibody" of the present invention refers to an antibody whose framework region and / or constant region portion (e.g., CH region) or all of the antibody are encoded by human antibody genes. In one embodiment of the present invention, the CDR region of the antibody has not been modified.

[0113] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0114] The term "prevention" used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.

[0115] The "individual" described in the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, a commercial animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0116] The terms "comprising" or "including" as used in the present invention are open-ended. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0117] As used herein, a "tumor" can be any undesirable cell proliferation (or any disease manifesting as undesirable cell proliferation), neoplasm, or a predisposition or increased risk of undesirable cell proliferation, neoplasm, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsils, trachea, uterus, vulva, leukocytes. Further preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., glioma), intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal 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, neurilemoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0118] The "homology" mentioned in the present invention refers to the fact that when using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the sequences used have (but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 112%, %, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0120] Figure 1: Electrophoresis gel image of purified bivalent nanobody (linker is 9GS).

[0121] Figure 2: ELISA test results of bivalent nanobody (linker is 9GS) binding to VEGFA.

[0122] Figure 3: ELISA test results of bivalent nanobody (linker is 9GS) competing for VEGFR2.

[0123] Figure 4: Comparison of VEGFA binding activity between bivalent and monovalent nanobodies.

[0124] Figure 5: Comparison of the competitive activity of bivalent and monovalent nanobodies against VEGFR2.

[0125] Figure 6: Cochlear-stained gel image of purified heterologous bivalent nanobodies.

[0126] Figure 7: ELISA test of heterologous bivalent nanobodies binding to VEGFA.

[0127] Figure 8: ELISA test of heterologous bivalent nanobody competing for VEGFR2.

[0128] Figure 9: Cochlear-stained gel after purification of bivalent nanobodies with different linkers.

[0129] Figure 10: Activity test of bivalent nanobodies binding to VEGFA with different linkers in series.

[0130] Figure 11: ELISA test of bivalent nanobody competing for VEGFR2.

[0131] Figure 12: Electrophoresis gel images of bivalent V1 with different linkers stored at 4°C for 30 days.

[0132] Figure 13: Absorbance of bivalent V1 with different linkers after storage at 4°C for 30 days.

[0133] Figure 14: Electrophoresis gel images of bivalent V1 with different linkers after storage at 40°C for 7 days.

[0134] Figure 15: Absorbance of bivalent V1 with different linkers after being stored at 40°C for 7 days and then at room temperature for 23 days.

[0135] Figure 16: Test-stained gel image of the purified bivalent humanized nanobody.

[0136] Figure 17: ELISA test of bivalent humanized Nanobodies binding to VEGFA.

[0137] Figure 18: Cootisol-stained gel image of purified monovalent nanobody-Fc fusion protein.

[0138] Figure 19: ELISA test of VEGFA binding by monovalent Nanobody-Fc fusion proteins or tandem bivalent Nanobodies.

[0139] Figure 20: Binding and dissociation curves (SPR) of monovalent Nanobody-Fc fusion protein (V1-SA1-Fc-m1) with VEGFA165.

[0140] Figure 21: Cochlear-stained gel image of the purified bivalent nanobody-Fc fusion protein.

[0141] Figure 22: ELISA test of bivalent Nanobody-Fc fusion protein binding to VEGFA.

[0142] Figure 23: Binding and dissociation curves (SPR) of bivalent nanobody-Fc fusion protein with VEGFA165.

[0143] Figure 24: Binding and dissociation curves (SPR) of bivalent Nanobody-Fc fusion protein and Aflibercept to VEGFA165.

[0144] Figure 25: Activity test of the bivalent nanobody-Fc fusion protein 2V1-SA1-3GS-Fc binding to VEGFA from different species.

[0145] FIG. 26 : 2V1-SA1-3GS-Fc and Aflibercept inhibit VEGFA-stimulated HUVEC proliferation. DETAILED DESCRIPTION

[0146] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0147] Example 1: Preparation and activity study of bivalent nanobodies

[0148] 1. Bivalent design of nanobodies

[0149] In order to improve the efficiency of nanoantibodies binding to VEGFA and enhance the efficiency of nanoantibodies in blocking VEGFA / VEGFR interactions, two nanoantibodies with the same sequence were fused in series to construct a homologous bivalent nanoantibody, and biological activity studies were carried out.

[0150] First, a linker 9GS (amino acid sequence: GGGGSGGGS (SEQ ID NO: 78)) rich in glycine and serine (GS) with a flexible and variable spatial structure was selected to connect two nanobodies with the same sequence in series. The coding genes of each nanobody were amplified by PCR from the expression plasmid of the monovalent nanobody and constructed into the pVRC8400 expression vector, wherein the nucleotide sequence of the secretory peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 151) was added to the N-terminus of the first nanobody, and the nucleotide sequence encoding the linker 9GS was added to its C-terminus. The nucleotide sequence encoding the second nanobody was then connected, and the nucleotide sequence encoding the linker GGGGS (SEQ ID NO: 152) and the tag 6×His were added to the C-terminus of the second nanobody in sequence. The two nanobody nucleotide fragments were homologously recombined with the vector plasmid using a seamless cloning kit (purchased from: Biyuntian, product number: D7010M) to form the correct expression plasmid. At the same time, the coding gene sequence for the positive control antibody BI-VEGF ab was synthesized (this antibody is an anti-VEGFA Nanobody, the antibody amino acid sequence is derived from SEQ ID NO: 57 in patent US 9527925B2). The C-terminus of the control antibody BI-VEGF ab was tagged with 6×His. The names and numbers of the exemplary bivalent Nanobody sequences constructed are shown in Table 3.

[0151] Table 3 Names and numbers of amino acid sequences of bivalent nanobodies

[0152] After the expression plasmid of the bivalent nanobody was sequenced correctly, endotoxin-free extraction was performed. 293F suspension cells were transiently transfected using the transfection reagent polyethylenimine (PEI, linear, MW = 25 kDa, purchased from Polysciences, Cat. No. 23966-1). Cell supernatants were collected on the 4th or 5th day after transfection and purified using Ni beads. The purified protein was analyzed by SDS-PAGE, and the molecular weight was consistent with the expected value (~30 kDa). A cross-stained gel image of some of the purified bivalent nanobodies is shown in Figure 1.

[0153] 2. Activity detection of bivalent nanoantibodies binding to VEGFA

[0154] The ELISA method was used to detect the activity of the purified bivalent nanobody binding to VEGFA.

[0155] The antigen human VEGFA165 (untagged, purchased from Sino Biological, Cat. No. HPLC-10008-HNAH, hereinafter referred to as VEGFA) was diluted in ELISA coating buffer to a final concentration of 0.3 μg / mL and added to the ELISA plate at 100 μL / well for overnight coating at 4°C. After blocking with 5% skim milk powder, a serial dilution of each bivalent nanobody was added (0.001, 0.01, 0.1, 1, 3, 10, 100 nM). The antibody dilution solution used was PBS (pH 7.4) containing 0.1% Tween-20, also known as PBST. After incubation at 37°C for 1 hour, the plate was washed three times with PBST and diluted with a His-Tag mouse monoclonal antibody (Proteintech, Catalog No. 66005-1-Ig) in buffer. The plate was then incubated at 37°C for 1 hour. After washing, HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (Proteintech, Catalog No. SA00001-1) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader. The signal changes of some bivalent nanobodies binding to VEGFA with antibody concentration are shown in Figure 2.

[0156] The EC50 of the bivalent nanobody binding to VEGFA is shown in Table 4. It can be seen from the table that all 9 candidate antibodies have high VEGFA binding activity.

[0157] Table 4 EC50 values ​​of bivalent nanobodies binding to VEGFA

[0158] 3. Activity detection of bivalent nanoantibodies competing for VEGFR2

[0159] Competitive ELISA was used to further verify the activity of the bivalent nanobody in blocking VEGFA binding to the receptor VEGFR2.

[0160] The antigen VEGFA was diluted with ELISA coating buffer (final concentration of 0.53 μg / mL) and added to a 96-well ELISA plate at 100 μL / well for overnight coating at 4°C. After blocking with 5% skim milk powder, the VEGFR2 extracellular domain VEGFR2-ECD-Fc (purchased from Sino Biological, Cat. No. 10012-H02H) with a final concentration of 1 nM was mixed with a gradient dilution of the divalent nanoantibody to be tested (0.01, 0.1, 1, 3, 10, 100 nM), added to the 96-well plate, and incubated at 37°C for 1 hour. After washing the plate three times with PBST, HRP-conjugated goat anti-human IgG antibody dilution (HRP-conjugated Goat Anti-Human IgG, purchased from Proteintech, Cat. No. A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, catalog number PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader. The competitive ELISA results are shown in Figure 3.

[0161] The comparison of the activity (IC50 value) of the bivalent nanobody in competing for VEGFR2 is shown in Table 5. It can be seen that the bivalent nanobody has a strong competitive activity for VEGFR2, and the competitive activity is stronger than that of the positive control monovalent nanobody BI-VEGF ab.

[0162] Table 5 Bivalent Nanobody Competitive Activity of VEGFR2 (IC50 Values)

[0163] Example 2: Comparison of VEGFA binding and blocking activity of bivalent and monovalent nanobodies

[0164] 1. Comparison of VEGFA binding activity between bivalent and monovalent nanobodies

[0165] The activity of bivalent Nanobodies (2V1-9GS, 2V15-9GS, 2V31-9GS, 2V43-9GS) and the corresponding monovalent Nanobodies in binding to VEGFA were compared using ELISA. The experimental method was the same as that in Example 1, "2. Detection of the activity of bivalent Nanobodies binding to VEGFA".

[0166] The changes in the signals of monovalent and bivalent nanoantibodies binding to VEGFA with antibody concentration are shown in Figure 4. The EC50 values ​​of monovalent and bivalent nanoantibodies binding to VEGFA are shown in Table 6. It can be seen from the table that the activity of bivalent nanoantibodies binding to VEGFA is stronger than that of monovalent nanoantibodies.

[0167] Table 6 EC50 values ​​of monovalent and bivalent Nanobodies binding to VEGFA

[0168] In addition, surface plasmon resonance (SPR) technology was used to exemplify the binding affinity of monovalent and bivalent nanobodies to VEGFA. Using a Biacore 8K instrument (Cytiva), the antigen VEGFA165 (unlabeled, purchased from Sino Biological, Cat. No. HPLC-10008-HNAH) was coupled to a CM5 chip via amino coupling reagents (EDC and NHS) at a coupling capacity of approximately 400 RU. The mobile phase buffer 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 monovalent and bivalent nanobodies, and their dilution concentrations were: 20 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.3125 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. Multi-cycle mode was used, and the chip was regenerated with 100 mM hydrochloric acid. The binding and dissociation curves of the antibody with VEGFA165 were fitted using a "1:1 binding" model. The obtained association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) are shown in Table 7.

[0169] Table 7 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of antibodies to VEGFA165

[0170] As shown in Table 7, the bivalent nanobody has a high affinity for VEGFA (KD = 22 pM), while the monovalent nanobody has an even higher affinity for VEGFA (KD = 116-719 pM), with a difference of 5.3-32.7 times. This indicates that the monovalent antibody can significantly increase its affinity for binding to the target after divalent modification.

[0171] 2. Comparison of bivalent and monovalent nanobodies in the competition for VEGFR2 activity

[0172] Competition ELISA was used to compare the activities of monovalent and bivalent nanobodies in blocking VEGFA / VEGFR2 interaction.

[0173] The experimental method is the same as that in "3. Detection of the activity of bivalent nanobody competing for VEGFR2" in Example 1. The ELISA results of bivalent nanobody 2V15-9GS and monovalent nanobody V15 competing for VEGFR2 are shown in Figure 5.

[0174] The activity of monovalent and bivalent nanobodies in competing for VEGFR2 (IC50 and maximum inhibition rate) is shown in Table 8.

[0175] The formula for calculating the maximum inhibition rate is:

[0176] The results showed that the bivalent nanobody had stronger competitive activity against VEGFR2 than the monovalent nanobody.

[0177] Table 8 Monovalent and bivalent nanobody competitive activity of VEGFR2 (IC50 and maximum inhibition rate)

[0178] Example 3: Design and activity evaluation of heterologous bivalent nanobodies

[0179] 1. Design and preparation of heterologous bivalent nanobodies

[0180] (1) Monovalent antibody Tm value detection

[0181] Eight monovalent antibodies (V1, V15, V29, V30, V31, V36, V40, and V43) were selected and their buffer was replaced with 10 mM His, pH 6.5, 40 mM NaCl, 5% sucrose, and 0.01% Tween-20. The melting temperatures (Tm) of the antibodies were measured using a UNCLE instrument (Unchained Labs). The results are shown in Table 9. These candidate antibodies all exhibited a certain degree of thermal stability.

[0182] Table 9 Melting temperature Tm of monovalent antibodies

[0183] (2) Design and preparation of heterologous bivalent nanobodies

[0184] Monovalent Nanobodies with different amino acid sequences were concatenated via the linker 9GS to construct heterologous bivalent Nanobodies. Nanobodies V1, V15, V30, and V40 with higher melting temperatures (Tm) were selected to construct heterologous bivalent Nanobodies. A total of six heterologous bivalent Nanobodies were expressed. Their names, sequence names, and numbers are shown in Table 10.

[0185] Table 10 Amino acid sequence names and numbers of heterologous bivalent Nanobodies

[0186] For the six heterologous bivalent nanobodies, the plasmid construction, expression, and purification methods were the same as in "1. Design of Bivalent Nanobodies" in Example 1. A cross-stained gel image of the antibodies after Ni-bead affinity purification is shown in Figure 6. The molecular weight of the target protein is approximately 30 kDa.

[0187] 2. Study on the activity of heterologous bivalent nanobodies

[0188] The purified heterologous bivalent Nanobody was evaluated for VEGFA binding activity using the same experimental method as in "2. Detection of bivalent Nanobody binding to VEGFA" in Example 1. The ELISA results of heterologous bivalent Nanobody binding to VEGFA are shown in FIG7 .

[0189] The EC50 of heterologous bivalent Nanobodies binding to VEGFA is shown in Table 11. The results showed that the six heterologous bivalent Nanobodies had similar activities in binding to VEGFA (EC50 of 0.3-0.4 nM), which was similar to the activity of homologous bivalent Nanobodies (two Nanobodies with the same sequence in series) (EC50 of approximately 0.24 nM, see Table 4 in Example 1).

[0190] The ELISA results of heterologous bivalent nanobody competing for VEGFR2 are shown in Figure 8. The IC50 and inhibition rate of the competition ELISA are shown in Table 11. The results show that the heterologous bivalent nanobody has a strong activity of competing with VEGFR2 for binding to VEGFA.

[0191] Table 11 ELISA results of heterologous bivalent nanobodies competing for VEGFR2

[0192] Example 4: Effects of different linkers on the activity of bivalent nanobodies

[0193] In the above examples, the linkers connecting the two Nanobodies were mostly flexible 9GS, and the spatial orientations of the two Nanobodies were relatively independent and did not interfere with each other. However, different linker properties (such as different lengths, hydrophilicity, secondary structure, etc.) may affect the spatial conformation of the antibodies and the antigen binding efficiency. Therefore, different linkers were prepared and evaluated to determine the effects of different linkers on the activity and stability of bivalent Nanobodies.

[0194] 1. Design linkers between nanobodies and prepare bivalent nanobodies with different linkers

[0195] To understand the impact of linker properties on the bioactivity and physicochemical stability of bivalent nanobodies, 16 linkers were designed, varying in linker length, potential secondary structure, and hydrophilicity. The amino acid sequences of the linkers are shown in Table 12.

[0196] Table 12 Linker amino acid sequence *WL stands for Whitlow Linker.

[0197] Using Nanobodies V1 and V15 as model antibodies, bivalent Nanobodies with different linkers were constructed. Their expression vector construction, transient transfection, and purification methods were the same as those in "1. Bivalent Nanobody Design" in Example 1. The sequence names and numbers of bivalent Nanobodies with different linkers are shown in Table 13.

[0198] Table 13 Bivalent Nanobody Amino Acid Sequence Names and Numbers

[0199] The stained gel images of some antibodies after Ni-bead affinity purification are shown in Figure 9. The molecular weight of the target protein is approximately 30 kDa.

[0200] 2. Study on the activity of bivalent nanobodies with different linkers

[0201] The purified bivalent Nanobodies were evaluated for VEGFA binding activity using the same experimental method as described in "2. Detection of VEGFA binding activity of bivalent Nanobodies" in Example 1. The ELISA results for VEGFA binding of some bivalent Nanobodies are shown in Figure 10 , and the EC50 values ​​for VEGFA binding are shown in Table 14 .

[0202] For the bivalent nanobody V1, different linkers had little effect on the EC50 of bivalent V1 (EC50 ranged from 0.2 to 0.3 nM). Furthermore, the activity of bivalent V1 without a linker (2V1-NL) was comparable to that of bivalent V1 with a linker. The activity of bivalent V15, constructed using different linkers in series, was also similar.

[0203] Table 14 EC50 values ​​of bivalent Nanobodies binding to VEGFA

[0204] The ELISA results of bivalent nanobody competition for VEGFR2 are shown in Figure 11. The IC50 and inhibition rate of the competition ELISA are shown in Table 15. The results showed that for V15, the competitive activity of bivalent V15 (2V15-20S) formed by connecting the linker 20S in series was slightly worse than that of bivalent V15 formed by connecting other linkers in series.

[0205] Table 15 IC50 and inhibition rate of ELISA of bivalent nanobodies competing for VEGFR2

[0206] Example 5: Effect of different linkers on the stability of bivalent nanobodies

[0207] 1. Effects of different linkers on the stability of bivalent nanobodies at 4°C

[0208] The buffer for the bivalent V1 antibodies: 2V1-3GS, 2V1-(WL)2, and 2V1-EAAAK-Turn was replaced with 10 mM His, pH 6.5, 40 mM NaCl, 5% sucrose, and 0.01% Tween-20. The protein concentration was uniformly adjusted to 0.5 mg / mL. At time 0, the protein solution was clarified and incubated at 4°C for 30 days before SDS-PAGE and absorbance analysis. The protein staining gel is shown in Figure 12. The results show that the bivalent antibody 2V1-EAAAK-Turn exhibited degradation bands with a molecular weight lower than the main band. However, no significant degradation fragments were observed in the 2V1-3GS and 2V1-(WL)2 lanes, indicating good chemical stability.

[0209] In addition, the absorbance of the samples at a wavelength of 600 nm (OD600) was measured, and a comparison chart is shown in Figure 13. As can be seen, 2V1-3GS exhibited the lowest absorbance and the clearest solution, suggesting that the 2V1-3GS protein exhibited relatively low aggregation compared to the other proteins. Therefore, among the three linkers, 3GS was more effective in maintaining the physical and chemical stability of the bivalent V1.

[0210] 2. Effects of different linkers on the stability of bivalent nanobodies at high temperatures

[0211] The buffer for the bivalent V1 antibodies: 2V1-9GS, 2V1-30GS, 2V1-39GS, 2V1-(WL)2, 2V1-(AP)5, 2V1-(AP)10, and 2V1-EAAAK-Rigid was replaced with 10 mM His, pH 6.5, 40 mM NaCl, 5% sucrose, and 0.01% Tween-20. The protein concentration was uniformly adjusted to 0.5 mg / mL. At time 0, the protein solution was clarified and incubated at 40°C for 7 days before being subjected to SDS-PAGE. The stained gel is shown in Figure 14. The results show that the bivalent antibody 2V1-EAAAK-Turn exhibits a degradation band with a molecular weight lower than the main band. However, no obvious degradation fragments were observed in the 2V1-3GS and 2V1-(WL)2 lanes, indicating good chemical stability.

[0212] The results showed that under accelerated conditions of 40°C, among the seven bivalent V1s, only 2V1-9GS and 2V1-(AP)10 could maintain most proteins from degradation.

[0213] After 7 days at 40°C, the samples were placed at room temperature for another 23 days, and their absorbance at 600 nm (OD600) was measured. A comparison chart is shown in Figure 15. As can be seen, the 2V1-9GS solution exhibited the lowest absorbance and the clearest solution, suggesting that the 2V1-9GS protein exhibited relatively low levels of aggregation. Therefore, among the seven linkers, the bivalent V1 containing 9GS exhibited superior physical and chemical stability.

[0214] By comparing the chemical and physical stabilities of bivalent nanobodies containing different linkers at 4°C and 40°C, bivalent nanobodies containing linkers 3GS and 9GS had better stability performance.

[0215] Example 6: Activity study of bivalent humanized nanobodies

[0216] 1. Bivalent design of humanized nanobodies

[0217] In the previous embodiment, the original unhumanized antibody was bivalent and the linker was compared. Next, bivalent humanized nanobodies were constructed with linkers 3GS and 9GS or without linker (NL). Antibody humanization is to first compare the homology of the candidate antibody sequence with the human antibody sequence (through the databases IGBLAST and IMGT), and then graft the CDR region of the candidate antibody into the human antibody framework region with high homology to form a complete humanized antibody. In order to maintain the activity of the antibody, the individual amino acids in the human antibody framework region need to be restored and mutated to the sequence of the corresponding position of the candidate antibody. The humanized antibodies of V1, V13, V15, V30, V40, and V43 are used as elements to construct bivalent humanized nanobodies with different linkers. The amino acid sequence names and numbers of the constructed bivalent humanized nanobodies are shown in Table 16.

[0218] Table 16 Bivalent humanized Nanobody amino acid sequence names and numbers

[0219] The plasmid construction, expression, and purification methods for the bivalent Nanobody were the same as those described in "1. Bivalent Nanobody Design" in Example 1. A partial protein-stained gel after purification is shown in Figure 16. The molecular weight of the target protein is approximately 30 kDa.

[0220] 2. Activity test of bivalent humanized nanobodies

[0221] The purified bivalent humanized Nanobodies were evaluated for VEGFA binding activity using the same experimental method as in Example 1, "2. Detection of Bivalent Nanobody Binding to VEGFA." The ELISA results for VEGFA binding of bivalent humanized Nanobodies are shown in Figure 17. The ELISA EC50 values ​​are shown in Table 17. The results show that the vast majority of bivalent humanized Nanobodies can efficiently bind to VEGFA.

[0222] Table 17 EC50 of bivalent humanized Nanobodies binding to VEGFA ELISA

[0223] Example 7: Preparation and activity study of monovalent nanobody-Fc fusion protein

[0224] 1. Preparation of monovalent nanobody-Fc fusion protein

[0225] To achieve the bivalency of nanobodies, in addition to linearly concatenating two nanobodies, dimerization domains or fragments, such as the Fc of IgG1 antibody (SEQ ID NO: 134), PPCP-Fc (SEQ ID NO: 158, the remaining amino acid sequence after removing DKTHTC in SEQ ID NO: 134), or the IgG1 Fc mutant Fc-m1 (SEQ ID NO: 135), can also be used to connect two nanobodies in the same molecule.

[0226] Among them, the IgG1 Fc amino acid sequence is:

[0227] The amino acid sequence of PPCP-Fc is:

[0228] The amino acid sequence of the IgG1 Fc mutant Fc-m1 is:

[0229] Therefore, the expression of monovalent Nanobody-Fc fusion proteins was carried out. The amino acid sequence names and numbers of the constructed Nanobody-Fc fusion proteins are shown in Table 18.

[0230] Table 18 Nanobody-Fc fusion protein amino acid sequence names and numbers

[0231] To facilitate comparison of the VEGFA-binding activity of the linearly tandem bivalent Nanobody 2V15-9GS (containing a 6-histidine tag) with the fusion proteins V15-PPCP-Fc, V15-9GS-PPCP-Fc, and V15-Fc, a linker (SGGS (SEQ ID NO: 153)) and a 6-histidine tag (HHHHHH (SEQ ID NO: 154)) were added to the C-terminus of the Fc segment of the fusion protein. During plasmid construction of the monovalent Nanobody-Fc fusion protein, the Nanobody and the Fc fragment containing the linker and histidine tag were amplified separately by PCR, with the PCR template for the Fc fragment being derived from a gene-synthesized Fc gene. The Nanobody and Fc fragments were integrated into the vector pCDNA3.1(+) using homologous recombination between the two fragments, using the restriction enzyme sites NheI and XbaI. The secretory peptide of the fusion protein is: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 155), which is then fused to the target protein sequence. After successful sequencing of these fusion protein plasmids, endotoxin-free plasmid extraction was performed, and 293F suspension cells were transiently transfected using the transfection reagent polyethyleneimine (Polyethylenimine, abbreviated as PEI, linear, MW = 25kDa, purchased from: Polysciences, Cat. No.: 23966-1). Cell supernatant was collected on the 4th or 5th day after transfection and Ni bead purification was performed. The purified protein was analyzed by SDS-PAGE. The molecular weight of the protein monomer under reducing conditions was approximately 39kDa, which was in line with the expected molecular weight. The stained gel image of some monovalent nanobody-Fc fusion proteins after purification is shown in Figure 18.

[0232] For the humanized antibody V1-SA1 and Fc fusion protein (V1-SA1-Fc-m1), the linker amino acid sequence between V1-SA1 and Fc-m1 is EPKSA (SEQ ID NO: 156). An expression plasmid was constructed using the above method, except that the C-terminus of the Fc tag did not contain additional amino acids. This means that the fusion protein V1-SA1-Fc-m1 contained an Fc tag but not a histidine tag. After transient transfection of the V1-SA1-Fc-m1 expression plasmid into 293F cells, affinity purification using Protein A beads was performed. Elution of the target protein was performed using 0.1 M citric acid (pH 3.0) and rapid neutralization to obtain the target protein.

[0233] 2. Activity detection of monovalent nanobody-Fc fusion protein

[0234] The purified monovalent Nanobody-Fc fusion protein was tested for VEGFA binding activity using the same ELISA method as described in "2. Detection of bivalent Nanobody binding activity to VEGFA" in Example 1. The ELISA results are shown in Figure 19 and the EC50 values ​​are shown in Table 19.

[0235] Table 19 Monovalent Nanobody-Fc fusion protein or tandem bivalent Nanobody VEGFA binding activity test

[0236] As can be seen from the data in the table, the tested monovalent Nanobody-Fc fusion protein and the linear tandem bivalent Nanobody both have strong binding activity to VEGFA.

[0237] Surface plasmon resonance (SPR) was used to test the affinity of a monovalent nanobody-Fc fusion protein (V1-SA1-Fc-m1) for VEGFA. Using a Biacore 8K instrument (Cytiva), V1-SA1-Fc-m1 was used as the ligand and captured on a Protein A chip. During immobilization, V1-SA1-Fc-m1 was diluted to a protein concentration of 2 μg / mL in the mobile phase buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20), resulting in a conjugated capacity of approximately 590 RU. During the experiment, the analyte was the antigen VEGFA165 (untagged, purchased from GenScript, Cat. No. Z03073). The dilution concentrations were 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0.390625 nM. Kinetic analysis was performed with a binding time of 180 seconds and a dissociation time of 900 seconds for the fusion protein and the antigen VEGFA165. The flow rate was 30 μL / min. After each binding-dissociation cycle, the chip was regenerated using 10 mM glycine hydrochloride (pH 1.5). The binding and dissociation curves of the fusion protein V1-SA1-Fc-m1 and the antigen VEGFA165 ( FIG. 20 ) were fitted using the “1:1 binding” model to obtain the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD), as shown in Table 20 .

[0238] Table 20 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of fusion protein V1-SA1-Fc-m1 and antigen VEGFA165

[0239] From the SPR data, it can be seen that the monovalent nanobody-Fc fusion protein V1-SA1-Fc-m1 has a very high affinity for VEGFA165 (KD = 0.148 nM).

[0240] Example 8: Preparation and activity study of bivalent nanobody-Fc fusion protein

[0241] 1. Expression and purification of bivalent nanobody-Fc fusion protein

[0242] The monovalent Nanobody-Fc fusion protein in Example 7 has a high affinity for VEGFA. The results of Example 2 show that the activity of the bivalent Nanobody in binding to VEGFA is higher than that of the monovalent Nanobody. Therefore, a bivalent Nanobody-Fc fusion protein was constructed, that is, the bivalent Nanobody in Table 16 was fused with IgG1 Fc (SEQ ID NO: 134) for expression. The amino acid sequence names and numbers of some bivalent Nanobody-Fc fusion proteins are shown in Table 21.

[0243] Table 21 Bivalent Nanobody Fc Fusion Protein Amino Acid Sequence Names and Numbers

[0244] An expression plasmid was constructed for the above-mentioned bivalent nanobody-Fc fusion protein, and the expression plasmid was transiently transfected and purified.

[0245] The specific method is to construct expression plasmids using gene synthesis or PCR based on the amino acid sequence of each fusion protein in the table. For PCR, two identical Nanobody genes and Fc were integrated into the vector pCDNA3.1(+) using the following restriction sites: NheI and XbaI. The secretory peptide of the fusion protein is: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 155), which is then fused to the target protein sequence. After sequencing confirmation and endotoxin-free plasmid extraction, 293F suspension cells were transiently transfected with the transfection reagent polyethylenimine (PEI, linear, MW = 25 kDa, purchased from Polysciences, Cat. No. 23966-1). Cell supernatants were collected on the 4th or 5th day after transfection and affinity purified with Protein A beads. The target protein was eluted with 0.1 M citric acid (pH 3.0) for protein elution. The protein elution product was quickly neutralized by adding 0.5 times the volume of 1 M sodium citrate (pH = 8.5). The protein was concentrated and the solution was replaced before use in subsequent testing.

[0246] An exemplary purified bivalent nanobody-Fc fusion protein is shown in a cross-stained gel in FIG21 . Under reducing SDS-PAGE conditions, the protein has a molecular weight of approximately 53 kDa.

[0247] 2. Study on the activity of bivalent nanobody-Fc fusion protein

[0248] The activity of the purified bivalent nanobody-Fc fusion protein in binding to VEGFA was detected by ELISA. The ELISA method is as follows: human VEGFA165 (unlabeled, purchased from Sino Biological, catalog number: HPLC-10008-HNAH) was diluted with ELISA coating buffer to a final concentration of 0.3 μg / mL, coated overnight at 4°C, blocked with 5% skim milk powder (prepared with PBST), and then different concentrations of fusion protein were added (the highest concentration was 100 nM, the lowest concentration was 0.0001 nM, 10-fold concentration gradient, prepared with PBST). The plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, the detection antibody recognizing the Fc tag: HRP-labeled Goat Anti-Human IgG antibody (purchased from Abbkine, catalog number: A21050) was added. The plate was incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, catalog number: PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.

[0249] ELISA results for some fusion proteins are shown in Figure 22, where the negative control is an IgG1 Fc fusion protein that binds to an unrelated target. EC50 values ​​are shown in Table 22.

[0250] Table 22 EC50 of fusion protein binding to VEGFA

[0251] Surface plasmon resonance (SPR) was used to determine the affinity of bivalent nanobody-Fc fusion proteins (2V1-SA1-3GS-Fc, 2V1-SA1-NL-Fc, and 2V1-DP-3GS-Fc) for VEGFA165. Using a Biacore 8K instrument (Cytiva), VEGFA165 (untagged, purchased from Sino Biological, Cat. No. HPLC-10008-HNAH) was coupled to a CM5 chip in "low immobilization" mode. (Because these proteins have a high affinity for VEGFA165 and dissociate very slowly, the amount of VEGFA165 coupled was reduced.) Each fusion protein was diluted in HBS-EP mobile phase buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v / v Tween 20) to the following concentrations: 0.0244nM, 0.0977nM, 0.3906nM, 1.5625nM, and 6.25nM. Kinetic analysis was performed with an association time of 120 seconds and a dissociation time of 780 seconds at a flow rate of 40μL / min. Multi-cycle mode was used, and the chip was regenerated with 100mM hydrochloric acid. Binding and dissociation curves of the fusion proteins with VEGFA165 were fitted using a "1:1 binding" model to obtain association rates (ka), dissociation rates (kd), and equilibrium dissociation constants (KD).

[0252] The binding and dissociation curves of the bivalent nanobody-Fc fusion protein binding to VEGFA165 are shown in Figure 23, and the fitted KD is shown in Table 23.

[0253] Table 23 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of bivalent nanobody-Fc fusion protein to VEGFA165

[0254] Table 23 shows that the three bivalent Nanobody-Fc fusion proteins have a high affinity for VEGFA165 (KD is 3.84-6.83 pM).

[0255] In addition, the antigen human VEGFA165 from different reagent suppliers was replaced, and the KD of the fusion protein and VEGFA165 was repeatedly tested. Using a similar SPR experimental method to the above, the KD values ​​of the fusion protein (2V1-SA1-3GS-Fc) and the positive control drug Aflibercept with VEGFA165 were determined. Specifically, using a Biacore 8K instrument (Cytiva), in "low fixed amount" mode, VEGFA165 (unlabeled, purchased from: GenScript, Cat. No.: Z03073) was coupled to a CM5 chip, and the protein was diluted with the mobile phase buffer HBS-EP. The concentrations of the 2V1-SA1-3GS-Fc dilutions were: 0.091nM, 0.1828nM, 0.3656nM, 0.73125nM, 1.4625nM, 2.925nM, and 5.85nM. The concentrations of aflibercept dilutions were 0.3125 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM. For kinetic analysis, the antigen-antibody binding time was 150 seconds, the dissociation time was 1000 seconds, and the flow rate was 30 μL / min. Multi-cycle mode was used, and the chip was regenerated with 10 mM glycine hydrochloride (pH = 1.5). The binding and dissociation curves of the test protein with VEGFA165 were fitted using a "1:1 binding" model to obtain the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD), as shown in Table 24. The binding and dissociation curves (SPR) of the bivalent nanobody-Fc fusion protein (2V1-SA1-3GS-Fc) and aflibercept with VEGFA165 are shown in Figure 24.

[0256] Table 24 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (KD) of 2V1-SA1-3GS-Fc and Aflibercept to VEGFA165

[0257] Comparing the KD values ​​in Table 23 and Table 24, it can be seen that the fusion protein 2V1-SA1-3GS-Fc has extremely high affinity for human VEGFA165 from different reagent suppliers, and the KD values ​​are all at the pM level. The KD values ​​are significantly smaller than Aflibercept (approximately 1 / 12 of Aflibercept), which indicates that the affinity of the fusion protein 2V1-SA1-3GS-Fc to VEGFA165 is higher than that of Aflibercept.

[0258] The above SPR test was carried out using a similar method (coupled antigen VEGFA165) to compare the KD values ​​of monovalent nanoantibodies and bivalent nanoantibodies with VEGFA (Table 7) and the KD values ​​of the above bivalent nanoantibody-Fc fusion protein with VEGFA (Table 23, Table 24). The KD values ​​of antibodies with different constructs with VEGFA are summarized in Table 25.

[0259] Table 25 Summary of KD values ​​of antibodies with different constructs and VEGFA

[0260] As can be seen from Table 25, the affinity of bivalent Nanobodies and bivalent Nanobody-Fc fusion proteins for antigens is increased by 22.5 times and 105.5 times, respectively, compared to monovalent Nanobodies. Therefore, as the number of antibodies or antigen-binding fragments in a protein molecule increases, the affinity of the protein molecule for the antigen will increase significantly.

[0261] ELISA was used to test the cross-binding activity of 2V1-SA1-3GS-Fc with VEGFA proteins from different species. Mouse VEGFA164 was purchased from Sino Biological under the catalog number 50159-MNAB, and rat VEGFA164 was purchased from Sino Biological under the catalog number 80006-RNAB. Rabbit VEGFA121 was transiently expressed in 293F cells and purified using Ni beads. The nucleotide sequence of rabbit VEGFA121 was synthesized and cloned into the pCDNA3.1(+) vector. The amino acid sequence of rabbit VEGFA121 is:

[0262] The protein's own secretory peptide was used at the N-terminus, and a flexible linker (GGGGS (SEQ ID NO: 152)) and a 6-histidine tag (HHHHHH (SEQ ID NO: 154)) were sequentially added to the protein's C-terminus.

[0263] The ELISA experimental method used was similar to that of "2. Activity detection of bivalent nanoantibodies binding to VEGFA" in Example 1. Different concentrations of 2V1-SA1-3GS-Fc were added to ELISA plates coated with different antigens. After incubation for 1 hour, the detection antibody recognizing the Fc tag was added: HRP-labeled Goat Anti-Human IgG antibody (purchased from Abbkine, product number: A21050). The plate was incubated at room temperature for 45 minutes. After washing, TMB color development and termination of the reaction, the OD450 was read using a microplate reader.

[0264] The ELISA data for VEGFA binding are shown in FIG25 , and the EC50 values ​​are shown in Table 26 .

[0265] Table 26 EC50 or maximum binding value of 2V1-SA1-3GS-Fc binding to VEGFA from different species

[0266] From the above, it can be seen that 2V1-SA1-3GS-Fc only has high binding activity to human VEGFA165, can weakly bind to rabbit VEGFA121, and cannot bind to mouse and rat VEGFA164.

[0267] The cellular efficacy of the fusion protein 2V1-SA1-3GS-Fc, specifically its ability to block the VEGFA signaling pathway, was verified using a primary HUVEC (HUVEC) proliferation inhibition assay. The positive control was the marketed drug Aflibercept, and the negative control was a human IgG1 antibody that binds to HIV (hIgG1 control). The number of viable HUVEC cells was determined using the CellTiter-Glo luminescent cell viability assay. HUVECs (purchased from ScienCell, Cat. No. 8000) were routinely passaged in complete culture medium: ECM + 5% FBS + 1% ECGS (purchased from ScienCell, Cat. Nos. 1001, 0010, and 5352, respectively). At the beginning of the experiment, the protein to be tested was diluted to the required concentration with low serum medium (ECM + 1% FBS + 0.05% ECGS): 1000nM to 0.0128nM, 5-fold concentration gradient (the final antibody concentration when HUVEC cells were incubated was 250nM to 0.0032nM). The antibody dilution was mixed with an equal volume of 200ng / mL VEGFA165 solution and incubated in a cell culture incubator at 37°C for 2 hours. The mixture was added to a 96-well plate (50μL / well), and the HUVEC cells were then resuspended in low serum medium and transferred to a 96-well plate at a volume of 50μL / well (ensuring that 6×10 cells were seeded per well). 3 HUVEC cells). The culture plate with cells, VEGF, and protein dilution added was recorded as Cell+VEGF+Protein. At the same time, control groups were set up: only cells without VEGF and protein (Cell only, expected cell proliferation is the slowest), and cells and VEGF without protein (Cell and VEGF only, expected cell proliferation is the fastest). The 96-well plate was placed in an incubator at 37°C and 5% CO2 for 3 days. After the incubation period, CellTiter-Glo reagent (purchased from Promega, catalog number: G7573) was added to each well and mixed thoroughly. The luminescence intensity was measured by a microplate reader (Envision model, PerkinElmer). The IC50 value was calculated by four-parameter nonlinear regression analysis using GraphPad Prism 9 software.

[0268] The experimental results are shown in Figure 26. The fitted IC50 and maximum inhibition rate are shown in Table 27. The inhibition rate calculation formula is: Inhibition (%) = {1-[(Cell+VEGF+Protein)-(Cell only) / (Cell and VEGF only)-(Cell only)]}*100

[0269] Table 27 IC50 and maximum inhibition rate of HUVEC proliferation inhibition

[0270] It can be seen from this that the IC50 of the fusion protein 2V1-SA1-3GS-Fc in inhibiting HUVEC proliferation is approximately half of that of the positive control Aflibercept, indicating that the fusion protein 2V1-SA1-3GS-Fc can effectively block the downstream signaling pathway activated by VEGFA at the cellular level, inhibiting VEGFA-stimulated HUVEC proliferation, and the activity of the fusion protein 2V1-SA1-3GS-Fc at the cellular level is stronger than that of the positive control Aflibercept.

[0271] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0272] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An anti-VEGFA fusion protein, characterized in that The anti-VEGFA fusion protein comprises two or more anti-VEGFA antibodies or antigen-binding fragments thereof, and an Fc fragment, wherein the anti-VEGFA antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein, the amino acid sequence of CDR-H1 comprises SYTMG (SEQ ID NO: 1) or an amino acid sequence at least 80% identical to SYTMG (SEQ ID NO: 1); The amino acid sequence of CDR-H2 comprises AISKGGYKYX1X2VSLEA (SEQ ID NO: 2) or an amino acid sequence that is at least 80% identical to AISKGGYKYX1X2VSLEA (SEQ ID NO: 2); The amino acid sequence of CDR-H3 comprises TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3) or an amino acid sequence that is at least 80% identical to TRAYGSSRLX3LAX4TYEY (SEQ ID NO: 3).

2. The anti-VEGFA fusion protein according to claim 1, characterized in that The anti-VEGFA antibodies or antigen-binding fragments thereof are directly or indirectly linked to each other, or to the Fc fragment.

3. The anti-VEGFA fusion protein according to claim 2, characterized in that The N-terminus, C-terminus and / or internal residues of the anti-VEGFA antibody or antigen-binding fragment thereof in the anti-VEGFA fusion protein are linked to the N-terminus, C-terminus and / or internal residues of other anti-VEGFA antibodies or antigen-binding fragments thereof or Fc fragments.

4. The anti-VEGFA fusion protein according to any one of claims 1 to 3, characterized in that The two or more anti-VEGFA antibodies or antigen-binding fragments thereof are anti-VEGFA antibodies or antigen-binding fragments thereof with completely identical sequences, anti-VEGFA antibodies or antigen-binding fragments thereof with partially identical sequences, or anti-VEGFA antibodies or antigen-binding fragments thereof with completely different sequences.

5. The anti-VEGFA fusion protein according to any one of claims 1 to 4, characterized in that X1 and X2 in SEQ ID NO: 2 represent DS, DA, NT, DT, NA or NS; X3 in SEQ ID NO: 3 represents R or K, and X4 represents D, N, E or K.

6. The anti-VEGFA fusion protein according to any one of claims 1 to 5, characterized in that The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 include any of the following groups: A) SEQ ID NO: 1, 4, 9; B) SEQ ID NO: 1, 5, 9; C) SEQ ID NO: 1, 6, 10; D) SEQ ID NO: 1, 4, 11; E) SEQ ID NO: 1, 7, 12; F) SEQ ID NO: 1, 6, 11; G) SEQ ID NO: 1, 4, 10; H) SEQ ID NO: 1, 5, 12; I) SEQ ID NO: 1, 4, 12; J) SEQ ID NO: 1, 8, 12; K) SEQ ID NO: 1, 33, 34.

7. The anti-VEGFA fusion protein according to any one of claims 1 to 6, characterized in that The anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a F(ab')2 fragment, a single domain antibody, a single chain antibody molecule or a linear antibody.

8. The anti-VEGFA fusion protein according to any one of claims 1 to 7, characterized in that The amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 13-32, 35-61, or has at least 80% identity with any one of SEQ ID NOs: 13-32, 35-61.

9. The anti-VEGFA fusion protein according to any one of claims 1 to 8, characterized in that The amino acid sequence of the anti-VEGFA fusion protein comprises any one of SEQ ID NOs: 62-76, 92-133, 136-150 and 159, or has at least 80% identity with any one of SEQ ID NOs: 62-76, 92-133, 136-150 and 159.

10. A chimeric antigen receptor, characterized in that The extracellular domain of the chimeric antigen receptor comprises the anti-VEGFA fusion protein according to any one of claims 1 to 9.

11. A nucleic acid encoding the anti-VEGFA fusion protein according to any one of claims 1 to 9 or the chimeric antigen receptor according to claim 10.

12. A vector comprising the nucleic acid of claim 11.

13. A cell comprising the nucleic acid of claim 11 or the vector of claim 12.

14. An immune cell, characterized in that The immune cell expresses the anti-VEGFA fusion protein according to any one of claims 1 to 9 or the chimeric antigen receptor according to claim 10.

15. A method for preparing an anti-VEGFA fusion protein, characterized in that: The preparation method comprises introducing the nucleic acid according to claim 11 into a host cell and inducing its expression.

16. A product for treating and / or diagnosing a disease, characterized in that The product for treating and / or diagnosing a disease comprises any of the following: A) the anti-VEGFA fusion protein according to any one of claims 1 to 9; B) the chimeric antigen receptor of claim 10; C) the nucleic acid of claim 11; D) the vector according to claim 12; E) the cell of claim 13; or F) The immune cell according to claim 14.

17. Use of the anti-VEGFA fusion protein according to any one of claims 1 to 9, the chimeric antigen receptor according to claim 10, the nucleic acid according to claim 11, the vector according to claim 12, the cell according to claim 13, the immune cell according to claim 14, or the product for treating and / or diagnosing a disease according to claim 16 in blocking the VEGFA signaling pathway; Preferably, the application includes: A) Use in the preparation of drugs for treating and / or preventing tumors; B) use in the preparation of a medicament for treating and / or preventing ophthalmic diseases involving angiogenesis (e.g., fundus vascular diseases), preferably age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, neovascular glaucoma, etc.; and / or, C) Use in inhibiting abnormal vascular proliferation, preferably blocking the proliferation of vascular endothelial cells.

18. A method for treating and / or preventing a disease, characterized in that: The method comprises administering to an individual the anti-VEGFA fusion protein of any one of claims 1 to 9, the chimeric antigen receptor of claim 10, the nucleic acid of claim 11, the vector of claim 12, the cell of claim 13, the immune cell of claim 14, or the product for treating and / or diagnosing a disease of claim 16.

19. A method for treating and / or preventing a disease, comprising contacting the anti-VEGFA fusion protein of any one of claims 1 to 9, the chimeric antigen receptor of claim 10, the nucleic acid of claim 11, the vector of claim 12, the cell of claim 13, the immune cell of claim 14, or the product for treating and / or diagnosing a disease of claim 16 with a target cell.

20. The method according to claim 19, characterized in that The target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

21. The method according to any one of claims 18 to 20, characterized in that: The disease is a disease related to the VEGFA signaling pathway, preferably a tumor, abnormal vascular proliferation, or angiogenesis-related ophthalmic disease.

22. A method for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, characterized in that: The method comprises contacting vascular endothelial cells with the anti-VEGFA fusion protein of any one of claims 1 to 9, the chimeric antigen receptor of claim 10, the nucleic acid of claim 11, the vector of claim 12, the cell of claim 13, the immune cell of claim 14, or the product for treating and / or diagnosing a disease of claim 16.

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