Anti-vegfa fusion construct and methods of making and using same

Abstract

The present application provides an anti-VEGFA fusion construct formed by linking an anti-VEGFA antibody or antigen-binding fragment thereof with an anti-albumin antibody or antigen-binding fragment thereof, said fusion construct having strong VEGFA binding affinity and biological activity of inhibiting HUVEC proliferation stimulated by VEGFA, and the functions of the respective domains do not interfere with each other. The present application also provides a method for preparing the fusion construct, and use in the preparation of a product for treating and / or preventing a disease, said anti-VEGFA antibody or antigen-binding fragment thereof having activity of treating and / or diagnosing a disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an anti-VEGFA fusion construct and a preparation method and application thereof. BACKGROUND

[0002] Vascular endothelial growth factor A (VEGFA) is an important member of the VEGF family and is a major angiogenic factor that can promote the differentiation, proliferation, migration and invasion of vascular endothelial cells and regulate the formation and development of blood vessels. VEGFA promotes angiogenesis mainly by binding and activating the receptor VEGFR2 (VEGF Receptor 2) and its downstream pathway. The binding of VEGFA to the extracellular domain of VEGFR2 leads to receptor dimerization, protein kinase activation, tyrosine phosphorylation and activation of downstream signaling pathways, stimulating the proliferation of endothelial cells and increasing vascular permeability. Activation of the VEGF signaling pathway occurs in a variety of diseases, from cancer, autoimmune to retinopathy, etc. Inhibition of the VEGF signaling pathway can treat diseases related to abnormal vascular proliferation, such as wet age-related macular degeneration (wAMD). Currently, there are several anti-VEGF protein drugs on the market for the treatment of tumors and eye neovascular-related diseases.

[0003] The first anti-VEGFA protein drug is the monoclonal antibody bevacizumab (Avastin), and in addition to anti-VEGFA protein drugs are ranibizumab (Lucentis), aflibercept and Brolucizumab (Beovu) and so on. Among them, free aflibercept has a half-life of about 11 days in human aqueous humor, longer than ranibizumab (7 days) and bevacizumab (9 days), and the administration interval of aflibercept is every 2 months. Currently, the dose of aflibercept is increased from 2 mg to 8 mg, which has been proved in clinical trials to further extend the administration interval to every 3 or 4 months. Brolucizumab (Beovu) can be prepared into a protein solution of 120 mg / mL, and the protein amount of a single intravitreal injection can reach 6 mg, which is the largest dose of a single injection at present. In the administration scheme of brolucizumab, patients receive an injection every month for the first three months, and then every 3 months, and the visual improvement effect is not worse than the control group of aflibercept (injected every 2 months). The administration route of these anti-VEGF protein drugs is intravitreal injection, and the administration volume does not exceed 100 μL. The longest administration interval of brolucizumab is 3 months, and the administration interval of the rest of the drugs is 1-2 months once. Frequent intraocular injections increase the risk of intraocular pressure rise, intraocular inflammation, retinal detachment and other risks caused by injections, so there is an urgent need for long-acting anti-VEGFA therapeutic drugs to reduce the number of injections for patients.

[0004] The methods for reducing the frequency of administration in the prior art include increasing the concentration of protein drugs, so that the dose of a single injection of drugs is increased; other means also include enhancing the biological activity of anti-VEGFA antibodies to reduce the minimum effective concentration; or enhancing the half-life of the drug to achieve it. The development of long-acting anti-VEGFA therapeutic drugs has achieved certain results in clinical trials, but there are also some problems, such as high-concentration protein preparations often have increased viscosity, which is prone to form protein aggregates or poor stability technical problems, increasing the risk of intravitreal injection; in addition, there are problems such as insufficient biological effect or insignificant half-life enhancement, so the design and development of anti-VEGFA drugs still need further research and improvement.

[0005] Heavy chain antibodies are IgG2 and IgG3 antibodies that only contain heavy chains and do not contain light chains in the serum of camelids (camels, dromedaries and llamas, etc.). The variable domain of heavy chain (VHH) of the heavy chain antibody has independent antigen binding activity and is the smallest functional fragment in natural antibodies, which is called nanobody or single domain antibody. The molecular weight of the nanobody is about 13 kDa, it has high thermal stability and good water solubility, and has great potential to be developed into a high-concentration protein preparation. Albumin is the most abundant protein in plasma (concentration is about 40 mg / mL), which is essential for maintaining plasma osmotic pressure, and albumin is an important transport carrier for endogenous ligands (such as fatty acids, metal ions, hormones, etc.) and exogenous ligands (such as drugs).

[0006] The molecular weight of albumin is 66.5 kDa, and it can bind to the cell surface receptor FcRn (neonatal Fc receptor) under weakly acidic conditions, and the binding force is weak under neutral conditions. Therefore, after albumin binds to FcRn, it is endocytosed into endosomes, and as the endosome environment is acidified, the binding force between albumin and FcRn is enhanced, and FcRn is re-brought to the cell surface or transcellular transport is achieved. Therefore, albumin can realize a longer half-life by utilizing the recycling mechanism of FcRn. The half-life of human blood albumin in plasma is about 3 weeks, so small molecule compounds combined with albumin, or proteins expressed by fusion with albumin (such as the marketed drug and ) have significantly improved pharmacokinetic properties, longer half-life, and can potentially extend the dosing interval to provide a better drug regimen for patients.

[0007] Anti-albumin antibodies or antigen-binding fragments thereof can bind to different species of albumin with high affinity, and the anti-albumin antibodies can be linked to a biologically active effector molecule, such as an anti-VEGFA antibody or antigen-binding fragment, to form a fusion construct. The fusion construct can improve the pharmacokinetic properties by binding to albumin and obtain a longer half-life. However, the design of the structure of the fusion construct, the connection between the domains, and whether the activity of the biologically active effector molecule will be affected after binding to albumin still require a lot of creative work. The present application hopes to use nanobody technology for the development and design of anti-albumin antibodies or antigen-binding fragments and anti-VEGFA fusion constructs, and to provide a more optimal anti-VEGFA fusion construct. SUMMARY

[0008] In order to overcome the defects of the prior art, the present application takes a nanobody binding to VEGFA as a basic active unit, and designs monovalent and bivalent nanobodies to prepare an anti-VEGFA fusion protein with good stability and strong biological activity. In addition, the anti-VEGFA antibody or antigen binding fragment is connected to an anti-albumin antibody or antigen binding fragment through a linker to prepare a fusion construct of the anti-VEGFA antibody or antigen binding fragment and the anti-albumin antibody or antigen binding fragment. The fusion construct has strong VEGFA binding affinity and biological activity of inhibiting HUVEC proliferation stimulated by VEGFA, and the functions of the domains in the fusion construct do not interfere with each other. Specifically,

[0009] In a first aspect, the present application provides an anti-VEGFA fusion construct, which comprises an anti-albumin antibody or antigen binding fragment and an anti-VEGFA antibody or antigen binding fragment, wherein the anti-VEGFA antibody or antigen binding fragment is connected to the anti-albumin antibody or antigen binding fragment.

[0010] Preferably, the anti-albumin antibody or antigen binding fragment comprises CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region.

[0011] The amino acid sequence of CDR-H1 comprises SEQ ID NO: 1, or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0012] The amino acid sequence of CDR-H2 comprises SEQ ID NO: 41 (GISVX1X2SFLDYADAVKG) or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 41.

[0013] The amino acid sequence of CDR-H3 comprises SEQ ID NO: 3 or an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3.

[0014] In the SEQ ID NO: 41 (GISVX1X2SFLDYADAVKG), X can be any natural amino acid residue, such as alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), valine (V).

[0015] In one embodiment of the present application, X1X2in SEQ ID NO: 41 (GISVX1X2SFLDYADAVKG) represents DS, DA, EG or DG.

[0016] In one embodiment of the present application, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups (see Table 42 for details):

[0017] A) SEQ ID NO: 1, 2, 3;

[0018] B) SEQ ID NO: 1, 10, 3;

[0019] C) SEQ ID NO: 1, 11, 3;

[0020] D) SEQ ID NO: 1, 12, 3.

[0021] Table 42 CDR sequences of anti-albumin candidate antibodies

[0022]

[0023] Preferably, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are arranged in the order from N-terminal to C-terminal.

[0024] The anti-albumin antibody or antigen-binding fragment thereof comprises a heavy chain variable region. The division of the amino acid of the CDR region of the antibody in the present application adopts the Kabat numbering system.

[0025] Preferably, the anti-albumin antibody or antigen-binding fragment thereof comprises a humanized sequence, and the modification site of the humanized sequence is located in the non-CDR region, and further preferably, the modification site of the humanized sequence is located in the framework region of the variable region and / or the constant region.

[0026] Preferably, the structure of the anti-albumin antibody or antigen-binding fragment thereof comprises a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single chain antibody fragment (scFv) or a linear antibody.

[0027] The anti-albumin antibody or antigen-binding fragment thereof can be a single domain antibody or a nanobody.

[0028] In one embodiment of the present application, the anti-albumin antibody or antigen-binding fragment thereof is a nanobody. Compared with full-length IgG antibodies, Fabs and scFvs, nanobodies have higher molar concentration and can bind more antigen molecules at the same mass.

[0029] The anti-albumin antibody or antigen-binding fragment thereof can bind to mammalian albumin.

[0030] Preferably, the mammal includes a human or a non-human mammal, which can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, and the like. Preferably, the non-human mammal includes, but is not limited to, a pig, a cow, a sheep, a horse, an ass, a fox, a raccoon dog, a mink, a camel, a dog, a cat, a rabbit, a mouse (e.g., a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey, and the like.

[0031] Preferably, the amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences of SEQ ID NOs: 4, 6-9, 13-15, or an amino acid sequence having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 4, 6-9, 13-15.

[0032] In one specific embodiment of the present application, the amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof is as shown in any one of SEQ ID NOs: 4, 6-9, 13-15.

[0033] The anti-albumin antibody or antigen-binding fragment thereof can be constructed using any conventional method known in the art. For example, artificial synthesis or eukaryotic expression or prokaryotic expression.

[0034] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR-H3 of a heavy chain variable region.

[0035] wherein the amino acid sequence of CDR-H1 comprises SEQ ID NO: 16, or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 16;

[0036] the amino acid sequence of CDR-H2 comprises SEQ ID NO: 17, or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 17;

[0037] the amino acid sequence of CDR-H3 comprises SEQ ID NO: 18, or an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 18.

[0038] The anti-VEGFA antibody or antigen-binding fragment thereof comprises a heavy chain variable region.

[0039] The structure of the anti-VEGFA antibody or antigen-binding fragment thereof includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single chain antibody fragment (scFv), or a linear antibody.

[0040] The anti-VEGFA antibody or antigen-binding fragment thereof can be a single domain antibody or a nanobody.

[0041] The anti-VEGFA antibody or antigen-binding fragment thereof can be a humanized antibody or a fully human antibody.

[0042] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof includes a humanized sequence, and the modification site of the humanized sequence is located in a non-CDR region, and further preferably, the modification site of the humanized sequence is located in a framework region and / or a constant region of a variable region.

[0043] In one specific embodiment of the present application, the anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody, which has a higher molar concentration and can bind more antigen molecules compared to full-length IgG antibodies, Fabs, and scFvs at the same mass.

[0044] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof can bind to human or monkey VEGFA protein.

[0045] Preferably, the amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences of SEQ ID NOs: 19-28, or has at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 19-28.

[0046] Preferably, the fusion construct comprises one or more anti-VEGFA antibodies or antigen-binding fragments thereof.

[0047] Preferably, the fusion construct comprises one or more anti-albumin antibodies or antigen-binding fragments thereof.

[0048] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof is directly or indirectly connected to the anti-VEGFA antibody or antigen-binding fragment thereof.

[0049] Preferably, the anti-albumin antibody or antigen-binding fragment thereof is directly or indirectly connected to the anti-VEGFA antibody or antigen-binding fragment thereof.

[0050] Preferably, the anti-albumin antibody or antigen-binding fragment thereof is directly or indirectly linked to the anti-VEGFA antibody or antigen-binding fragment thereof.

[0051] Preferably, the indirect linkage is via a linker, a functional domain, and / or a linker for conjugation.

[0052] The linker is selected from the group consisting of 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.

[0053] The functional domain is a combination of one or more of an Fc fragment, a serum albumin, a cytokine, a transferrin, or a scaffold protein.

[0054] The linker for conjugation includes a functional group linker, preferably, the functional group linker includes a thiol, an amino, a hydroxyl, and / or a carboxyl reactive group, which allows covalent conjugation between the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof.

[0055] Preferably, the N-terminus and / or C-terminus of the anti-VEGFA antibody or antigen-binding fragment thereof is linked to the C-terminus and / or N-terminus of another anti-VEGFA antibody or antigen-binding fragment thereof via a connecting peptide.

[0056] In one embodiment of the present application, the fusion construct comprises, in order from N-terminus to C-terminus, a first anti-VEGFA antibody or antigen-binding fragment thereof, a connecting peptide, and a second anti-VEGFA antibody or antigen-binding fragment thereof.

[0057] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof is directly or indirectly linked to the N-terminus, C-terminus, and / or internal residues of the anti-albumin antibody or antigen-binding fragment thereof.

[0058] In one embodiment of the present application, the fusion construct comprises two anti-VEGFA antibodies or antigen-binding fragments thereof, which are directly or indirectly linked to the N-terminus, C-terminus, and / or internal residues of the anti-albumin antibody or antigen-binding fragment thereof.

[0059] For example, in the fusion construct, the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof are linked in the following order from N-terminus to C-terminus:

[0060] 1) anti-albumin antibody or antigen-binding fragment thereof, connecting peptide, anti-VEGFA antibody or antigen-binding fragment thereof;

[0061] 2) an anti-VEGFA antibody or antigen-binding fragment thereof, a linker peptide, an anti-albumin antibody or antigen-binding fragment thereof;

[0062] 3) an anti-albumin antibody or antigen-binding fragment thereof, a linker peptide, a first anti-VEGFA antibody or antigen-binding fragment thereof, a linker peptide, a second anti-VEGFA antibody or antigen-binding fragment thereof; or,

[0063] 4) a first anti-VEGFA antibody or antigen-binding fragment thereof, a linker peptide, an anti-albumin antibody or antigen-binding fragment thereof, a linker peptide, a second anti-VEGFA antibody or antigen-binding fragment thereof, and so on;

[0064] In some embodiments, the linker can not comprise a linker peptide.

[0065] In some embodiments, the two or more anti-VEGFA antibodies or antigen-binding fragments thereof are the same anti-VEGFA antibody or antigen-binding fragment thereof, partially same anti-VEGFA antibody or antigen-binding fragment thereof, or different anti-VEGFA antibody or antigen-binding fragment thereof.

[0066] In some embodiments, the fusion construct comprises any one of the amino acid sequences of SEQ ID NOs: 29-35, or has at least 80% identity to any one of the amino acid sequences of SEQ ID NOs: 29-35.

[0067] In some embodiments, the fusion construct comprises an Fc fragment.

[0068] In some embodiments, the Fc fragment comprises SEQ ID NO: 38, or has at least 80% identity to the amino acid sequence of SEQ ID NO: 38.

[0069] In some embodiments, the fusion construct further comprises a secretion peptide. The secretion peptide is linked to the N-terminus of the fusion construct. In some embodiments, the secretion peptide has the amino acid sequence of SEQ ID NO: 36.

[0070] In some embodiments, the fusion construct further comprises a tag.

[0071] In some embodiments, the tag is linked to the C-terminus of any one of the antibodies or antigen-binding fragments thereof, or the fusion construct.

[0072] In some embodiments, the fusion construct can inhibit or compete with the binding of other anti-VEGFA antibodies (preferably, antibodies that bind to the same or overlapping epitopes as the anti-VEGFA antibodies of the present application) to VEGFA.

[0073] Preferably, the fusion construct can also inhibit or compete with the binding of other anti-albumin antibodies (preferably antibodies that bind to the same or overlapping epitopes as the anti-albumin antibodies of the present application) to albumin.

[0074] In a second aspect of the present application, there is provided a nucleic acid encoding an antibody or antigen binding fragment as described herein, or a fusion construct as described above. For example, the nucleic acid comprises DNA and / or mRNA. For example, the nucleic acid is a therapeutic nucleic acid.

[0075] In some embodiments, the nucleic acid is DNA encoding an antibody or antigen binding fragment as described herein, or a fusion construct as described above.

[0076] Preferably, the nucleotide sequence encoding CDR-H1 of the anti-albumin antibody or antigen binding fragment thereof comprises any of the nucleotide sequences of SEQ ID NO: 42 or 51, or a degenerate sequence thereof, or has at least 80% identity to any of the nucleotide sequences of SEQ ID NO: 42 or 51.

[0077] Preferably, the nucleotide sequence encoding CDR-H2 of the anti-albumin antibody or antigen binding fragment thereof comprises any of the nucleotide sequences of SEQ ID NO: 43, 52-54, or a degenerate sequence thereof, or has at least 80% identity to any of the nucleotide sequences of SEQ ID NO: 43, 52-54.

[0078] Preferably, the nucleotide sequence encoding CDR-H3 of the anti-albumin antibody or antigen binding fragment thereof comprises any of the nucleotide sequences of SEQ ID NO: 44 or 55, or a degenerate sequence thereof, or has at least 80% identity to any of the nucleotide sequences of SEQ ID NO: 44 or 55.

[0079] Preferably, the nucleotide sequence encoding CDR-H1 of the anti-VEGFA antibody or antigen binding fragment thereof comprises any of the nucleotide sequences of SEQ ID NO: 59, or a degenerate sequence thereof, or has at least 80% identity to any of the nucleotide sequences of SEQ ID NO: 59.

[0080] Preferably, the nucleotide sequence encoding CDR-H2 of the anti-VEGFA antibody or antigen binding fragment thereof comprises any of the nucleotide sequences of SEQ ID NO: 60, or a degenerate sequence thereof, or has at least 80% identity to any of the nucleotide sequences of SEQ ID NO: 60.

[0081] Preferably, the nucleotide sequence encoding CDR-H3 of the anti-VEGFA antibody or antigen binding fragment thereof comprises any one of the nucleotide sequences in SEQ ID NO: 61 or a degenerate sequence thereof, or has at least 80% identity to any one of the nucleotide sequences in SEQ ID NO: 61.

[0082] More preferably, the nucleotide sequence encoding the anti-VEGFA antibody or antigen binding fragment thereof comprises any one of the nucleotide sequences in SEQ ID NO: 62-71 or a degenerate sequence thereof, or has at least 80% identity to any one of the nucleotide sequences in SEQ ID NO: 62-71, and has a nucleotide sequence encoding the anti-VEGFA antibody or antigen binding fragment thereof.

[0083] More preferably, the nucleotide sequence encoding the anti-albumin antibody or antigen binding fragment thereof comprises any one of the nucleotide sequences in SEQ ID NO: 45, 47-50, 56-58 or a degenerate sequence thereof, or has at least 80% identity to any one of the nucleotide sequences in SEQ ID NO: 45, 47-50, 56-58, and has a nucleotide sequence encoding the anti-albumin antibody or antigen binding fragment thereof.

[0084] More preferably, the nucleotide sequence encoding the fusion construct described above comprises any one of the nucleotide sequences in SEQ ID NO: 72-78 or a degenerate sequence thereof, or has at least 80% identity to any one of the nucleotide sequences in SEQ ID NO: 72-78, and has a nucleotide sequence encoding the fusion construct described above.

[0085] In some embodiments, the nucleic acid is mRNA. One or more modification techniques can be used to generate more stable mRNA. Known mRNA modification techniques can be broadly classified into three categories: synthesizing mRNA with artificially synthesized non-natural ribonucleic acids instead of natural ribonucleic acids; adding 5' caps, 3' poly(A) "tails", and UTR (untranslated region) sequences; and effectively protecting mRNA using special new formulation techniques. Among them, the preferred mRNA modification technique can be to synthesize mRNA with artificially synthesized non-natural ribonucleic acids instead of natural ribonucleic acids. Chemical modifications on eukaryotic mRNA can be broadly classified into three categories: methylation, pseudouridine (Ψ), and hypoxanthine. For example, the chemical modification can be selected from the group consisting of: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0086] In a third aspect of the present application, a vector is provided, wherein the vector comprises the nucleic acid as described above.

[0087] The vector can be expressed in vivo or in vitro or ex vivo. Preferably, the vector is a prokaryotic expression vector, a viral expression vector, or a eukaryotic expression vector, etc.

[0088] For example, a viral expression vector can be used. The viral expression vector can comprise viral-derived DNA or RNA sequences for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus AAV). The virus and viral expression vector can be used for in vitro, ex vivo, and / or in vivo delivery. For example, the recombinant vector can be comprised in a delivery vehicle. In certain embodiments, the delivery vehicle comprises the antibody or antigen-binding fragment described herein, the nucleic acid described herein, and / or the recombinant vector described herein, and optionally comprises a liposome and / or a lipid nanoparticle (LNP). For example, the delivery vehicle can be introduced into a cell by a physical delivery method. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery. For example, LNPs can encapsulate the nucleic acid described herein in cationic lipid particles (e.g., liposomes) and can be relatively easily delivered to a cell.

[0089] In a fourth aspect of the present application, a host cell is provided, wherein the host cell comprises the nucleic acid as described above or the vector as described above.

[0090] The host cell can be a eukaryotic cell or a prokaryotic cell.

[0091] The eukaryotic cell includes animal and plant cells, fungi, etc., such as T cells, yeast cells, HEK293 cells, CHO cells, etc.

[0092] The prokaryotic cell is, for example, E. coli, etc.

[0093] In a fifth aspect of the present application, a method for preparing a host cell comprising the above-mentioned nucleic acid or the above-mentioned vector is provided, and the method comprises introducing the above-mentioned nucleic acid or vector into the host cell.

[0094] In a sixth aspect of the present application, a method for preparing the above-mentioned fusion construct is provided, and the method comprises culturing the above-mentioned host cell to express the fusion construct.

[0095] In a seventh aspect of the present application, a product for treating and / or diagnosing a disease is provided, and the product comprises any one of the following:

[0096] A) the above-mentioned fusion construct;

[0097] B) the above-mentioned nucleic acid;

[0098] C) the above-mentioned vector; or,

[0099] D) the above-mentioned host cell.

[0100] Preferably, the anti-VEGFA antibody or antigen-binding fragment thereof has the activity of treating and / or diagnosing a disease.

[0101] Preferably, the product can be a diagnostic kit or a drug or a diagnostic chip, etc.

[0102] The disease can be a disease related to the VEGFA signaling pathway. Further preferably, the disease can be a tumor, abnormal vascular proliferation, an ophthalmic disease involving neovascularization (such as ocular fundus vascular disease), etc.

[0103] Preferably, the fusion construct blocks VEGFA-mediated vascular endothelial cell proliferation or inhibits angiogenesis.

[0104] In an eighth aspect of the present application, a method for detecting VEGFA is provided, and the method comprises contacting a sample to be detected with the above-mentioned fusion construct, and then detecting the content of the complex formed by VEGFA and the above-mentioned fusion construct.

[0105] The detection method is for detecting the presence or content of VEGFA. The presence indicates the presence or absence, and the content can be the expression amount or the protein concentration, etc.

[0106] Preferably, the VEGFA is from a mammal, more preferably the mammal is from a human, a mouse or a monkey.

[0107] In a ninth aspect of the present application, there is provided a method of blocking VEGFA-mediated proliferation of vascular endothelial cells or inhibiting angiogenesis, the method comprising contacting a vascular endothelial cell with the fusion construct, the nucleic acid, the vector, the host cell or the therapeutic and / or diagnostic product for disease described above.

[0108] Preferably, the method comprises diluting the fusion construct, the nucleic acid, the vector, the host cell or the therapeutic and / or diagnostic product for disease described above with a serum-free medium for vascular endothelial cells, and then incubating with an antigen (e.g. VEGFA165).

[0109] Preferably, the method comprises the step of discarding the complete medium for vascular endothelial cells in the culture plate after incubation.

[0110] Preferably, the antigen and the fusion construct, the nucleic acid, the vector, the host cell or the therapeutic and / or diagnostic product for disease described above are mixed with the vascular endothelial cells and added to a culture plate for vascular endothelial cells, and incubated on the culture plate.

[0111] Preferably, the method comprises detecting after incubation.

[0112] Preferably, the incubation time is 0.5-5h, for example 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5h.

[0113] 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.

[0114] 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. Preferably, the incubation is carried out in a 5% CO2incubator.

[0115] Preferably, the incubation time is 24-120h, preferably 48-96h, for example 24, 36, 48, 68, 72, 96, 120h, etc.

[0116] Preferably, the detection is detection of the number of viable vascular endothelial cells.

[0117] In a tenth aspect of the present application, a method for treating and / or preventing a disease is provided, the method comprising administering to an individual the fusion construct, the nucleic acid, the vector, the host cell, the product for treating and / or preventing a disease described above.

[0118] The disease includes a disease associated with VEGFA signaling pathway. Further preferably, the disease can be a tumor, abnormal vascular proliferation, an ophthalmic disease involving angiogenesis (e.g., ocular fundus vascular disease), etc.

[0119] Preferably, the target cell is selected from a cell expressing VEGFA, such as a myocardial cell, a proximal tubular cell, a hepatocyte, a vascular endothelial cell, a granulocyte, a specialized epithelial cell, a mesenchymal cell, a macrophage, a platelet, a dendritic cell, an activated T cell, a retinal pigment epithelial cell, a Muller cell in the retina, an astrocyte, an osteoblast, a bronchial and alveolar epithelial cell, a pericyte, a vascular smooth muscle cell, a myofibroblast, a keratinocyte, a juxtaglomerular cell, or a tumor cell, etc.

[0120] In an eleventh aspect of the present application, the fusion construct, the nucleic acid, the vector, or the host cell described above is used for preparing a product for treating and / or preventing a disease, the anti-VEGFA antibody or antigen-binding fragment thereof having an activity of treating and / or preventing a disease.

[0121] The disease includes a disease associated with VEGFA signaling pathway. Further preferably, the disease can be a tumor, abnormal vascular proliferation, an ophthalmic disease involving angiogenesis (e.g., ocular fundus vascular disease), etc.

[0122] Preferably, the product includes a product for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis.

[0123] The product described in the present application can be a kit, a drug, a chip, an antibody-drug conjugate, etc.

[0124] As used herein, "fusion construct" defines a fusion of an anti-albumin antibody or antigen-binding fragment thereof of the present application with another compound. The fusion construct can comprise one or more anti-albumin antibodies or antigen-binding fragments thereof, which can be the same or different. The fusion construct can comprise one or more additional compounds, which can also be the same or different. The compound can be a proteinaceous compound or a non-proteinaceous compound. In the case where the compound is a proteinaceous compound or the fusion construct comprises only anti-albumin antibodies or antigen-binding fragments thereof, the fusion construct can also be referred to as a fusion protein. In the case where the compound is fused to the anti-albumin antibody or antigen-binding fragment thereof in a conjugated form, the fusion construct can also be referred to as a conjugate.

[0125] The "medicament" of the present application can be used for treating a human or a non-human animal, such as a non-human mammal. The medicament can comprise a pharmaceutically acceptable carrier, adjuvant or salt commonly used in the art. The medicament can be administered by any suitable route, such as a gastrointestinal route (e.g., oral) or a non-gastrointestinal route (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.). The medicament can be in any suitable dosage form, such as a gastrointestinal dosage form or a non-gastrointestinal dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nose drops, sublingual tablets, suppositories, aerosols, effervescent tablets, dripping pills, gels, etc. The various dosage forms of the medicament can be prepared according to conventional methods in the pharmaceutical art.

[0126] The medicament can contain the fusion construct, the nucleic acid, the vector, the host cell, etc. of the present application in an amount of 0.01-99.5% by weight (e.g., 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 medicament can be prepared as a reagent having 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).

[0127] 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.

[0128] The "pharmaceutically acceptable" of the present application refers to neither significantly stimulating the organism nor inhibiting the biological activity and characteristics of the active substance of the administered product.

[0129] The "… method" of the present application can be for the diagnosis and treatment of diseases, or for the diagnosis and treatment of non-diseases.

[0130] The "antigen-binding fragment" of the present application is a part of the antibody that retains the specific binding activity of the antibody, i.e. any part of the antibody can specifically bind to the epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd and variants of these fragments. For example, the heavy chain and / or light chain of the antibody, the heavy chain variable region and / or the light chain variable region of the antibody, or a single or more than two CDRs from the heavy chain or light chain of the antibody. Among them,

[0131] Nanobody or single-domain antibody refers to the variable domain of heavy chain (VHH) of antibody, which has independent antigen binding activity.

[0132] Chimeric antibody refers to an antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, while the rest of the heavy chain and / or light chain is derived from a different source or species.

[0133] Single-chain antibody is an antibody formed by connecting the variable domain of heavy chain and the variable domain of light chain through a linker.

[0134] Fab, i.e. a monovalent fragment composed of VL, VH, CL and CH1 domains.

[0135] Fab', a Fab fragment with one or more cysteine residues at the C-terminus of the CH1 domain.

[0136] F(ab')2, a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region.

[0137] Fd, an Fd fragment composed of VH and CH1 domains.

[0138] Fv, a fragment of an antibody consisting of the VL and VH domains of a single arm of an antibody.

[0139] dAb fragment, an antibody fragment consisting of a VH domain.

[0140] The "linear antibody" described in the present application includes one or more pairs of tandem antibody fragments, which can be Fd segments (VH-CH1), single-chain antibodies (scFv), antibody fragments (Fab), or single-domain antibodies (VHH), which are linked together by a linker peptide to form a continuous antibody structure.

[0141] wherein VH represents a heavy chain variable region, VL represents a light chain variable region, CL represents a light chain constant region, and CH represents a heavy chain constant region.

[0142] The "Fc" region described in the present application contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are dimerized by two or more disulfide bonds of the hinge region and held together by hydrophobic interactions of the CH3 domains.

[0143] The "comprising" or "including" described in the present application is an open-ended writing, when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid can be composed of the sequence, or at one end or both ends of the protein or nucleic acid, there can be additional amino acids or nucleotides, but still have the same or similar activity as the original sequence.

[0144] The "homology" or "identity" as used herein refers to the degree of sequence relatedness between a test sequence and a reference sequence as determined by alignment of the test and reference sequences and comparison of a subset of sequence positions that includes, but is 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%, 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% of the test sequence is identical to the reference sequence.

[0145] The "humanized antibody" as used herein refers to an antibody in which the framework and / or constant region portions (e.g., CH regions) of the antibody or all or a portion of the antibody are encoded by human antibody genes. In one embodiment of the application, the CDR regions of the antibody are not humanized.

[0146] The "subject" as used herein can be a human or a non-human mammal, which can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, and the like. Preferably, the non-human mammal includes, but is not limited to, a pig, a cow, a sheep, a horse, a donkey, a fox, a raccoon dog, a mink, a camel, a dog, a cat, a rabbit, a mouse (e.g., a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey, and the like.

[0147] The "treatment" as used herein refers to slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0148] The "prevention" as used herein refers to a manner performed in order to prevent or delay the occurrence of a disease or a condition or a symptom in the body.

[0149] "Diagnosis" as used herein refers to the determination of whether a patient has had, has, or will have a disease or condition, or the determination of the progression or likely progression of a disease.

[0150] "Tumor" as used herein can be any undesirable cell proliferation (or any disease that manifests itself as undesirable cell proliferation), neoplasm, or increased predisposition or risk for undesirable cell proliferation, neoplasm, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). Neoplasm can be any abnormal growth or proliferation of cells, and can be located in any tissue. Examples of tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., kidney epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, 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, testicle, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, white blood cells. Further preferred, the tumor is selected from the group consisting of prostate cancer, breast cancer, liver cancer, glioma (e.g., neuroglioma), intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, squamous cell carcinoma of the tongue, nasopharyngeal carcinoma, ovarian cancer, placental villous carcinoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myelocytic leukemia, acute myelogenous leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic carcinoma, hematological cancer, head and neck cancer, or oropharyngeal cancer.

[0151] "Ocular vascular disease" as used herein refers to the general class of diseases that occur in the retinal arteries or veins, or the general class of diseases associated with choroidal neovascularization. It 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.

[0152] In summary, the beneficial technical effects of the present application include:

[0153] 1) The anti-VEGFA fusion construct obtained by the present application has strong VEGFA binding affinity (K D= 2.4-5.4 pM), which is much higher than the existing VEGFA antibody drugs, and can compete with VEGFR2 to bind VEGFA.

[0154] 2) The fusion construct can inhibit VEGFA-stimulated HUVEC proliferation, and the IC50 value is similar to that of the receptor drug aflibercept. In VEGFA-stimulated reporter cells, the fusion construct can inhibit luciferase expression and luciferin luminescence. In HUVEC cells, the fusion construct can inhibit VEGFA-activated VEGFR2 phosphorylation.

[0155] 3) The functions of the individual domains in the fusion construct do not interfere with each other. The fusion construct of the present application connects the anti-albumin antibody and the anti-VEGFA antibody, and still has strong human albumin binding affinity (K D = 35-69 nM) and VEGFA binding affinity (K D = 2.4-5.4 pM).

[0156] 4) In vivo, the fusion construct has the potential to inhibit VEGFA-stimulated vascular proliferation, and by binding to albumin, it is carried into the specific albumin-rich tissue microenvironment, potentially increasing the selectivity of the fusion construct to be delivered to specific tissues. Moreover, since the molecular weight of the fusion construct increases after binding to albumin, it can also participate in the binding of albumin to FcRn and the recycling mechanism to prolong the half-life, so the fusion construct of the anti-VEGFA antibody and the anti-albumin antibody has the potential to achieve long-acting effect in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0157] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings, in which:

[0158] Figure 1 : SDS-PAGE of antibody Alb Nb and antibody 3005 with His tag after Ni column purification, (A) is the SDS-PAGE of antibody Alb Nb, (B) is the SDS-PAGE of antibody 3005, the cell culture supernatant after transient transfection of antibody Alb Nb is labeled as Input, the flow-through liquid that cannot bind to the Ni column is labeled as FT, antibody Alb Nb that binds to the Ni column and is eluted is labeled as E, and Marker is labeled as M.

[0159] Figure 2 : ELISA test of antibody 3005-Fc and Alb Nb-Fc binding to albumin of different species, (A) is the HSA binding test, (B) is the MSA binding test, (C) is the rat albumin binding test.

[0160] Figure 3: SDS-PAGE gel of purified 3005 humanized antibody with His tag after Ni column purification.

[0161] Figure 4 : ELISA binding activity test of antibody 3005 and its humanized antibodies binding to MSA.

[0162] Figure 5 : ELISA binding activity test of antibody 3005 and its humanized antibodies 3005Hz6 and control antibody Alb Nb binding to HSA and rat albumin, (A) is HSA binding test, (B) is rat albumin binding test.

[0163] Figure 6 : Binding and dissociation curves of humanized antibody 3005Hz6 to HSA and MSA (SPR method), (A) is the binding and dissociation curves of antibody 3005Hz6 to HSA, (B) is the binding and dissociation curves of antibody 3005Hz6 to MSA.

[0164] Figure 7 : SDS-PAGE gel of purified humanized antibody 3005Hz6 mutant proteins 3005Hz6 (EG), 3005Hz6 (DS) and 3005Hz6 (DA), (A) is the SDS-PAGE gel of 3005Hz6 (EG), 3005Hz6 (DS), the cell culture supernatant after transient expression of 3005Hz6 (EG), 3005Hz6 (DS) is marked as Input, the flow-through that cannot bind to Ni column is marked as FT, and the eluted purified product is marked as E, (B) is the SDS-PAGE gel of 3005Hz6 (DA).

[0165] Figure 8 : ELISA activity test of humanized antibody 3005Hz6 and its mutant proteins 3005Hz6 (DA), 3005Hz6 (DS) and 3005Hz6 (EG) binding to HSA.

[0166] Figure 9 : SDS-PAGE gel of fusion constructs V1SA-3005Hz6, 3005Hz6-V1SA and V1DP-3005Hz6 after Ni column purification, (A) is the SDS-PAGE gel of V1SA-3005Hz6, 3005Hz6-V1SA, (B) is the SDS-PAGE gel of V1DP-3005Hz6, wherein the cell culture supernatant after transient expression of each protein is marked as Input, the flow-through that cannot bind to Ni column is marked as FT, and the eluted purified product is marked as E.

[0167] Figure 10Fusion construct of humanized antibody 3005Hz6 with monovalent anti-VEGFA nanobody, ELISA activity test of humanized antibody 3005Hz6 binding to HSA.

[0168] Figure 11 VEGFR2 competition ELISA activity test of fusion construct and monovalent nanobody V1-SA1.

[0169] Figure 12 SDS-PAGE of fusion construct of humanized antibody 3005Hz6 with bivalent anti-VEGFA nanobody after purification, (A) is the SDS-PAGE of V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA, where the flow-through is labeled as FT, the elution products of V1SA-3005Hz6-V1SA are labeled as A1, A2 components, and the elution products of 3005Hz6-2V1SA are labeled as A5, A6 components, (B) is the SDS-PAGE of unlabeled 3005Hz6-2V1SA protein product, (C) is the SDS-PAGE of V1DP-3005Hz6-V1DP, (D) is the SDS-PAGE of 3005Hz6-2V1DP.

[0170] Figure 13 ELISA test of fusion construct V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and 3005Hz6-2V1DP binding to HSA.

[0171] Figure 14 SPR method for testing the affinity of proteins to HSA, where (A) is the binding and dissociation curve of fusion construct V1SA-3005Hz6-V1SA to HSA, (B) is the binding and dissociation curve of fusion construct 3005Hz6-2V1SA to HSA, (C) is the binding and dissociation curve of antibody 3005Hz6 to HSA.

[0172] Figure 15 SPR method for testing the affinity of proteins to VEGFA, where (A) is the binding and dissociation curve of V1SA-3005Hz6-V1SA to VEGFA, (B) is the binding and dissociation curve of 3005Hz6-2V1SA to VEGFA, (C) is the binding and dissociation curve of positive control aflibercept to VEGFA.

[0173] Figure 16Figure 6: Fusion constructs inhibit VEGFA-induced HUVEC proliferation, where panel (A) shows the results for V1SA-3005Hz6-V1SA, a pre-mix of V1SA-3005Hz6-V1SA and HSA (V1SA-3005Hz6-V1SA + HSA), and panel (B) shows the results for 3005Hz6-2V1SA, a pre-mix of 3005Hz6-2V1SA and HSA (3005Hz6-2V1SA + HSA).

[0174] Figure 17 Figure 7: Fusion constructs inhibit VEGFA-induced reporter cell assay, where panel (A) shows the results for V1SA-3005Hz6-V1SA and V1SA-3005Hz6-V1SA + HSA, and panel (B) shows the results for 3005Hz6-2V1SA and 3005Hz6-2V1SA + HSA. DETAILED DESCRIPTION

[0175] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0176] Example 1: Immunization of Llama and Construction of Antibody Library

[0177] 1. Immunization of Llama and Titer Detection

[0178] The llama was immunized with emulsified human serum albumin HSA (purchased from Sigma-Aldrich, item number: A3782). 1 mg of human serum albumin was used for each immunization, and the llama was immunized twice a week for a total of three times. After the second immunization, the serum was collected one week later for titer detection.

[0179] The serum titer detection method is as follows:

[0180] 1) The HSA antigen was diluted to 1 μg / mL with CBS buffer, and 100 μL / well was added to the enzyme-labeled plate and incubated at 4°C overnight;

[0181] 2) The plate was washed with PBST (PBS containing 0.1% Tween 20, pH = 7.4) for 3 times. 200 μL of skimmed milk powder was added to each well, and incubated at room temperature for 1 h, and then washed with PBST for 3 times;

[0182] 3) Add sample: dilute the immune serum and negative serum 1000 times, 3000 times, 9000 times, 27000 times, 81000 times, 243000 times, respectively, take 100 μL and add to the blocked hole, room temperature for 1 h, PBST wash plate 3 times;

[0183] 4) Secondary antibody: add 100 μL of HRP labeled Goat Anti-Alpaca IgG to each well, room temperature for 1 h, PBST wash plate 3 times;

[0184] 5) TMB color development: add TMB color developing liquid 100 μL / well in the enzyme labeled plate, color development for 15 min; add stop solution 50 μL / well, put the enzyme labeled plate into the enzyme label instrument, read the light absorption value (OD450) at 450 nm.

[0185] The serum titer detection of the second and third alpaca immunization is shown in Table 1, and it can be seen that the signal value of the serum combined with human serum albumin is above 0.59 after being diluted 243000 times, and all reach the library construction standard. The peripheral blood of the alpaca after the third immunization is used for phage library construction.

[0186] Table 1 Serum titer detection of the second and third alpaca immunization after albumin

[0187]

[0188] 2, Phage library construction

[0189] Peripheral blood of alpaca is extracted, total RNA is extracted from the separated PBMC of alpaca using Trizol, and cDNA reverse transcription is carried out, and nanobody specific primer is used to amplify the variable region VH fragment (length about 700 bp), and the amplification is carried out twice. The above obtained fragment and pcomb3X vector are cut with SfiI enzyme, and after cutting, they are mixed in appropriate proportion and connected with T4 ligase. After connection, it is used for electric transformation of XL1-Blue competent cells. According to the colony growth of the competent cell diluent on the containing resistance plate, the transformation library capacity of alpaca is calculated as 6.24 x 10 8 .

[0190] Example 2 Antibody library screening to obtain antibodies cross-binding human, mouse and cynomolgus monkey albumin

[0191] 1, Phage library screening

[0192] The screening process is as follows:

[0193] 1) Coating antigen human serum albumin HSA and mouse serum albumin MSA (purchased from Sigma-Aldrich, item number: A3559), and setting negative control (coating enzyme-labeled plate with 3% skim milk powder), incubating at room temperature for 1 hour. Discarding blocking buffer and washing with PBST.

[0194] 2) Adding about 5 x 10 12 pfu library to antigen plate, incubating with antigen at 37°C for 2 hours, washing plate with 0.1% PBST.

[0195] 3) Eluting with low-pH glycine-HCl, and then neutralizing with Tris-HCl until pH is 7.4.

[0196] 4) Mixing eluent with E. coli XL1-Blue, incubating at 37°C and shaking.

[0197] 5) After adding bacteriophage, incubating overnight at 30°C, collecting supernatant.

[0198] 6) Repeating 1)-5) to obtain second-round library.

[0199] 7) For second-round amplified library obtained under coating MSA condition, performing antigen binding ELISA test. In ELISA experiment, coating HSA and MSA with CBS buffer respectively, and ELISA detection result is shown in Table 2. It can be seen that, under this enrichment condition, bacteriophage with stronger ability to bind HSA and MSA is also obtained.

[0200] Table 2 Detection result of second-round bacteriophage library obtained under coating MSA condition for binding HSA and MSA

[0201]

[0202] Summary: Antigen binding ELISA of second-round bacteriophage library obtained under coating MSA condition shows positive binding to HSA and MSA, indicating that bacteriophage binding to HSA and MSA is enriched, and single-molecule level binding screening of bacteriophage can be carried out.

[0203] 2, Single-molecule screening and sequencing of bacteriophage

[0204] Experimental procedure is as follows:

[0205] 1) From second-round bacteriophage library obtained under coating MSA condition, 96 single-molecule colonies are selected for bacteriophage expression, and HSA, MSA and monkey albumin are coated respectively for ELISA binding activity detection.

[0206] 2) Coating antigen HSA, MSA and monkey albumin, and protein concentration is 0.5 μg / mL when coating, coating overnight at 4°C.

[0207] 3) Blocking: 3% milk powder, 200 μL / well, room temperature for 1 hour.

[0208] 4) Adding 10-fold diluted phage expression supernatant, 100 μL / well, room temperature for 1 hour.

[0209] 5) Adding detection antibody Anti-M13 Antibody (HRP labeled) (concentration of 0.2 μg / mL), 100 μL / well, room temperature for 1 hour.

[0210] 6) Adding TMB color developing solution 200 μL / well, color developing for 20 minutes, adding stop solution 50 μL / well. Measuring OD450 by microplate reader.

[0211] From the 96 phage clones of the second round of phage library enriched by coating MSA, clone No. 3005 was selected, and the ELISA detection results are shown in Table 3.

[0212] Table 3 Activity detection of phage monoclonal 3005 binding to HSA, MSA and monkey albumin

[0213]

[0214] 7) After DNA sequencing and codon translation, the amino acid sequence of the candidate nanobody was obtained. Among them, the CDR region (CDR-H1, CDR-H2, CDR-H3) amino acid sequence of the preferred anti-albumin nanobody 3005 is shown in Table 4, the CDR region nucleic acid sequence is shown in Table 5, the antibody heavy chain variable region (VH) amino acid sequence is shown in Table 6, and the nucleic acid sequence is shown in Table 7.

[0215] Table 4 CDR region amino acid sequence of anti-albumin nanobody 3005

[0216]

[0217] Table 5 Nucleic acid sequence encoding CDR region of anti-albumin nanobody 3005

[0218]

[0219] Table 6 Amino acid sequence of anti-albumin nanobody 3005

[0220]

[0221] Table 7 Nucleic acid sequence of anti-albumin nanobody 3005

[0222]

[0223] Example 3 Activity detection of anti-albumin candidate antibody binding to different species of albumin

[0224] 1. Expression of anti-albumin candidate antibodies

[0225] Candidate antibodies were expressed in mammalian cells, and after protein purification, antigen binding activity tests were performed to verify the activity of the candidate antibodies in binding to albumin at the protein level.

[0226] The expression plasmids of His-tagged antibody 3005 and positive control antibody Alb Nb were constructed by gene synthesis, wherein the positive control Alb Nb antibody is a nanobody against human serum albumin, and the antibody amino acid sequence is from SEQ ID NO: 62 in patent US2007 / 0269422 A1.

[0227] A tPA secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36) was added to the N-terminus of the above antibody, a linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody, and the gene sequence was connected to the expression vector pCDNA3.1(+) through enzyme cutting sites NheI and XbaI. After plasmid sequencing was correct, endotoxin-free bulk extraction was performed, transient expression of suspension 293F cells was carried out, and affinity purification was performed using a Ni column.

[0228] After the His-tagged antibody Alb Nb and antibody 3005 were purified by the Ni column, the results of SDS-PAGE staining are shown in Figure 1 (A) and Figure 1 (B), and the molecular weight is consistent with the expected value (~ 15 kDa). In Figure 1 (A), the cell culture supernatant after transient transfection of antibody Alb Nb is marked as Input, the flow-through liquid that cannot bind to the Ni column is marked as FT, and the antibody Alb Nb that binds to the Ni column and is eluted is marked as E.

[0229] Meanwhile, the fusion protein of antibody 3005 and positive control antibody Alb Nb and Fc was constructed, and a tPA secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36) was added to the N-terminus of the antibody coding sequence by primer design. The amino acid sequence of human IgG1 Fc was added to the C-terminus of the antibody sequence by homologous recombination:

[0230] The amino acid sequence of IgG1 Fc is:

[0231] PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

[0232] NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

[0233] CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38)

[0234] The anti-albumin nanobody-Fc construct gene fragment was inserted into the mammalian cell expression vector pCDNA3.1(+). The amino acid sequence of the anti-albumin nanobody-Fc fusion protein is shown in Table 8, and the nucleic acid sequence is shown in Table 9.

[0235] Table 8 Amino acid sequence of anti-albumin nanobody-Fc fusion protein

[0236]

[0237] Table 9 Nucleic acid sequence encoding anti-albumin nanobody-Fc fusion protein

[0238]

[0239]

[0240] After the expression plasmid of the Fc fusion protein was sequenced correctly, endotoxin-free large-scale extraction was performed, and the target protein was obtained by transient expression of suspended 293F cells and Protein A affinity purification.

[0241] 2. Detection of the activity of anti-albumin candidate antibody binding to albumin

[0242] The affinity of monovalent nanobody binding to different species of albumin was tested by Bio-Layer Interferometry (BLI) method. Eight NTA sensors were used to bind to the same his tag monovalent nanobody (concentration of 1 μg / mL), and the antigens were human serum albumin HSA (Baxter AG), mouse albumin MSA (purchased from Equitech-bio, item number: MSA62-1000), and rat albumin (purchased from abcam, item number: ab198656). In the BLI experiment, the concentration gradient of the antigen was set to 200, 100, 50, 25, 12.5, 6.25, 3.125 nM, the antigen and antibody were combined for 180 seconds, and dissociated for 360 seconds.

[0243] Table 10 shows the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of his-tagged monovalent Nanobody® 3005 binding to different species of albumin after fitting to the "1 : 1" model. D

[0244] Table 10 shows the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of his-tagged monovalent Nanobody® 3005 binding to different species of albumin after fitting to the "1 : 1" model. D

[0245]

[0246] The data show that 3005 can bind to HSA and MSA with high affinity, and also to rat albumin.

[0247] The activity of 3005 binding to different species of albumin was again tested using an ELISA method, the experimental method being as follows:

[0248] The antigens were diluted in ELISA coating buffer, with a final concentration of 1 pg / mL for human serum albumin HSA (Baxter AG), 2 pg / mL for mouse albumin MSA (purchased from Equitech-bio, item number: MSA62-1000) and 2 pg / mL for rat albumin (purchased from abcam, item number: ab198656). The antigen dilutions were added to the enzyme-coated plates, 100 pL / well, and incubated overnight at 4°C. After blocking with 5% skimmed milk powder, the candidate antibody 3005-Fc and the positive control antibody Alb Nb-Fc were added in gradient dilutions (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM) in PBST (PBS containing 0.1% Tween 20, pH = 7.4), and incubated at 37°C for 1 hour. After washing, a goat anti-human IgG Fc antibody labelled with HRP (purchased from Abbkine, item number: A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 pL of TMB (purchased from Tiangen, item number: PA107-01) was added to each well, and the colour was developed at 37°C for 15 minutes before adding 50 pL of stop solution. The absorbance (OD450) was measured at 450 nm using a microplate reader.

[0249] The results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin are shown in Table 11. Figure 2

[0250] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of the antibodies 3005-Fc and Alb Nb-Fc to different species of albumin.

[0251] Table 11 shows the results of the ELISA test for the binding of Protein Binding HSA EC50 (nM) Binding MSA EC50 (nM) Binding rat albumin EC50 (nM) 3005-Fc 0.24 0.23 0.40 Alb Nb-Fc 0.19 0.27 Weak binding

[0252] The results show that antibody 3005-Fc can efficiently bind to HSA and MSA, and the binding activity is similar to that of the positive control Alb Nb-Fc. The positive control antibody Alb Nb-Fc has weak binding activity to rat albumin, but antibody 3005-Fc has good binding activity to rat serum albumin.

[0253] Example 4 Humanization of anti-albumin candidate antibody

[0254] 1. Humanization and expression and purification of candidate antibody

[0255] The humanization was performed by grafting the CDR regions of the antibody. The candidate nanobody 3005 (SEQ ID NO: 4) was used as the starting antibody for the humanization of the antibody. First, the homology of the sequence of the candidate antibody 3005 to human antibody sequences was compared by using the antibody database IGBLAST and IMGT, and then the framework region sequences with high homology to the candidate antibody 3005 were combined with the 3 CDR region sequences (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) of the candidate antibody 3005 to construct a humanized antibody.

[0256] A secretion signal peptide was added to the N-terminus of the antibody, and a linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody. The DNA sequence encoding the antibody was obtained by gene synthesis, and then the DNA sequence was connected to the expression vector pCDNA3.1(+) through the restriction enzyme sites NheI and XbaI. The plasmid was correctly sequenced and endotoxin-free extracted in large quantities, and then transiently expressed in suspension 293F cells and affinity purified by Ni column.

[0257] The amino acid sequence of the humanized antibody of nanobody 3005 is shown in Table 12, and the nucleic acid sequence is shown in Table 13.

[0258] Table 12 Amino acid sequence of humanized nanobody 3005

[0259]

[0260] Table 13 Nucleic acid sequence encoding humanized nanobody 3005

[0261]

[0262]

[0263] After the humanized protein with His tag was purified by Ni column, it was separated by SDS-PAGE electrophoresis, and the molecular weight was consistent with the expected value (~ 13 kDa). The staining results are shown in Figure 3 .

[0264] 2. Detection of binding activity of humanized antibodies

[0265] This example used ELISA method to detect the activity of the original candidate antibody 3005 and the humanized antibody binding to mouse albumin MSA.

[0266] In the experiment, mouse albumin MSA was coated in the enzyme-labeled plate (the concentration of the coated protein was 5 μg / mL), 100 μL / well, 4°C overnight. After blocking with 5% skim milk, the dilutions of the His-tagged proteins to be tested (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM) were prepared with PBST (0.1% Tween 20 in PBS, pH = 7.4). The proteins to be tested were the original antibody 3005 and the four humanized antibodies. After the protein dilutions were added to the enzyme-labeled plate, they were incubated at 37°C for 1 hour, then washed and added with HRP-labeled mouse anti-His tag monoclonal antibody (purchased from Proteintech, item number: HRP-66005), and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB (purchased from Tiangen Biochemical, item number: PA107-01) was added to each well, and color development was performed at 37°C for 15 minutes, followed by the addition of 50 μL of stop solution. The absorbance (OD450) at 450 nm was measured by an enzyme-labeled instrument.

[0267] The results of the ELISA test of the antibody 3005 and its humanized antibodies binding to MSA are shown in Table 13. Figure 4 The EC50 of the binding ELISA experiment is summarized in Table 14.

[0268] Table 14 Summary of ELISA experiment EC50 and maximum binding value of antibody 3005 and its humanized antibodies binding to MSA

[0269] Antibody Binding MSA EC50 (nM) Max binding value OD450 3005 2.3 1.38 3005Hz3 22.0 0.89 3005Hz4 49.8 0.79 3005Hz5 31.6 0.80 3005Hz6 2.9 1.34

[0270] The results show that the activity of the humanized antibody 3005Hz6 binding to MSA is similar to that of the original antibody 3005. The activities of the other humanized antibodies binding to MSA are poorer than that of 3005Hz6.

[0271] ELISA method was used to detect the activity of the original candidate antibody 3005 and the humanized 3005Hz6 binding to HSA and rat albumin.

[0272] In the experiment, HSA or rat albumin (the concentration of coated protein was 2 μg / mL) was coated in the enzyme-labeled plate, 100 μL / well, 4°C coated overnight. After blocking with 5% skim milk, the diluent of the His-tagged protein to be tested (0.0001, 0.001, 0.01, 0.1, 0.3, 1, 10, 100 nM) was prepared with PBST (0.1% Tween 20 in PBS, pH = 7.4), and the tested protein was the positive control antibody Alb Nb, and the original antibody 3005, the humanized antibody 3005Hz6. After the protein diluent was added to the enzyme-labeled plate, it was incubated at 37°C for 1 hour, and then the HRP-labeled mouse anti-His tag monoclonal antibody (purchased from Proteintech, item number: HRP-66005) was added after washing the plate, and it was incubated at room temperature for 45 minutes. After washing the plate, 100 μL TMB (purchased from Tiangeng Biochemical, item number: PA107-01) was added to each well, and after color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured by an enzyme-labeled instrument.

[0273] The ELISA results are shown in Figure 5 (A) and Figure 5 (B). Figure 5 The EC50 values of the binding ELISA curve fitting of the antibody 3005 and the humanized antibody 3005Hz6 are shown in Table 15.

[0274] Table 15 Summary of EC50 of the antibody 3005 and the humanized antibody 3005Hz6 binding to HSA and rat albumin

[0275] Antibody name Binding HSA EC50 (nM) Binding rat albumin EC50 (nM) 3005 0.31 2.74 3005Hz6 0.26 1.64 Alb Nb 0.66 Weak binding

[0276] From Figure 5 and Table 15, it can be seen that the activity of the humanized antibody 3005Hz6 binding to HSA and rat albumin is comparable to that of the original antibody 3005, and both are stronger than the positive control antibody Alb Nb.

[0277] The surface plasmon resonance (SPR) method was used to detect the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D). Biacore 8K instrument (Cytiva) was used in the experiment, Series S Sensor Chip NTA was used to capture humanized antibody 3005Hz6 with His tag, the analyte was gradient concentration of HSA or MSA, and the analyte was diluted with mobile phase buffer (composition: 10 mM HEPES, 150 mM NaCl, 0.05% v / v Tween-20, pH = 7.4). The binding time was 150 seconds, and the dissociation time was 900 seconds. The binding and dissociation curves of antibody 3005Hz6 with HSA are shown in Figure 6 (A), and the binding and dissociation curves of antibody 3005Hz6 with MSA are shown in Figure 6 (B). The "Two state reaction" model was used to fit the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of 3005Hz6 binding to HSA and MSA, and the results are shown in Table 16.

[0278] Table 16 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of humanized antibody 3005Hz6 with HSA and MSA

[0279] Ligand Analyte ka1 (1 / Ms) kd1 (1 / s) ka2 (1 / s) kd2 (1 / s) K D (M) 3005Hz6 HSA 9.04E+04 1.56E-03 3.67E-04 8.08E-04 1.19E-08 3005Hz6 MSA 7.57E+04 1.51E-02 5.01E-03 3.02E-04 1.14E-08

[0280] From Table 16, it can be seen that the affinities of humanized antibody 3005Hz6 binding to HSA and MSA are similar, and the K D determined by SPR method is about 11 nM.

[0281] In Table 10 of Example 3, the K D values of original antibody 3005 binding to HSA and MSA determined by BLI method are about 5 nM, and the numerical values are within 3 times of the K D values in Table 16, so it is considered that the affinities of humanized antibody 3005Hz6 binding to HSA and MSA are similar to those of original antibody 3005.

[0282] Example 5 Risk site modification of humanized antibody 3005Hz6 and activity test

[0283] 1. Mutation of risk site of humanized antibody 3005Hz6 and expression and purification

[0284] By analyzing the sequence of 3005Hz6 antibody, the CDR-H2 region (SEQ ID NO: 2) contains the amino acid motif DG which is easy to isomerize aspartic acid D, therefore the DG amino acid motif of CDR-H2 region of 3005Hz6 is modified to DA, DS or EG respectively to reduce the risk of protein structure and activity changes. The modified antibody name and its CDR region amino acid sequence are shown in Table 17, the nucleic acid sequence is shown in Table 18, the antibody heavy chain variable region (VH) amino acid sequence is shown in Table 19, and the nucleic acid sequence is shown in Table 20.

[0285] Table 17 CDR region amino acid sequence of humanized antibody 3005Hz6 mutant

[0286]

[0287]

[0288] Table 18 Nucleic acid sequence encoding CDR region of humanized antibody 3005Hz6 mutant

[0289]

[0290] Table 19 Amino acid sequence of humanized nanobody 3005Hz6 mutant

[0291]

[0292] Table 20 Nucleic acid sequence encoding humanized nanobody 3005Hz6 mutant

[0293]

[0294]

[0295] A secretion signal peptide is added to the N-terminus of the antibody 3005Hz6 mutant, and a linker GGGGS (SEQ ID NO: 37) and 6xHis are added to the C-terminus of the antibody. The DNA sequence encoding is obtained by gene synthesis (see Table 20), and then connected to the expression vector pCDNA3.1(+) through the restriction enzyme sites NheI and XhoI. The plasmid is sequenced correctly and endotoxin-free extracted in large quantities, and transiently expressed by suspension 293F cells. The cell culture supernatant is harvested and subjected to Ni column affinity purification. The SDS-PAGE result of the purified product is shown in Figure 7 (A), wherein the cell culture supernatant of 3005Hz6 (EG), 3005Hz6 (DS) transiently expressed is labeled as Input, the flow-through liquid that cannot be combined with the Ni column is labeled as FT, and the eluted purified product is labeled as E. The cell supernatant of 3005Hz6 (DA) protein transiently expressed is subjected to Ni column purification, and then subjected to cation exchange purification. The SDS-PAGE result of the purified protein is shown inFigure 7 (B). The molecular weight of the three mutant proteins is 13 kDa.

[0296] 2. Activity test of humanized antibody 3005Hz6 mutant

[0297] ELISA was performed to test the HSA binding activity of humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG). In the ELISA experiment, 2 μg / mL HSA was coated, and 3005Hz6 or its mutants were added in a concentration gradient. The level of antibody binding to HSA was detected by HRP-labeled mouse anti-His tag monoclonal antibody (purchased from Proteintech, product number: HRP-66005). The results of the ELISA experiment are shown in Figure 8 , and the EC50 of the ELISA experiment is shown in Table 21.

[0298] Table 21 Summary of EC50 of humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG) binding to HSA

[0299] Antibody name Binding HSA EC50 (nM) 3005Hz6 0.35 3005Hz6 (DA) 0.44 3005Hz6 (DS) 0.28 3005Hz6 (EG) 0.40

[0300] As can be seen from Table 21, the humanized antibody 3005Hz6 and its mutant proteins have similar HSA binding activity.

[0301] Example 6 Fusion construct of monovalent anti-VEGFA nanobody and humanized anti-albumin antibody

[0302] 1. Design of fusion construct of monovalent anti-VEGFA nanobody and humanized anti-albumin antibody 3005Hz6

[0303] A fusion construct of anti-VEGFA antibody and anti-albumin antibody 3005Hz6 was constructed using anti-VEGFA nanobody as the effector molecule.

[0304] The screening technology of anti-VEGFA nanobody in this embodiment can refer to the patent with application number PCT / CN2023 / 118600, which is briefly summarized as follows:

[0305] The anti-VEGFA nanobody sequence used in this example was screened from a human VEGFA immunized llama library. The construction and screening process of the phage library is as follows: human VEGFA165 (no label, purchased from: Yiqiao God, item number: HPLC-10008-HNAH, hereinafter referred to as VEGFA) was emulsified and immunized to a llama, and a phage display library was constructed. After 4 rounds of enrichment screening, 96-well monoclonal was picked, and the binding activity of phage to VEGFA was detected. After plasmid sequencing and codon translation, the amino acid sequence of the candidate nanobody was obtained. The CDR region amino acid sequence of the preferred anti-VEGFA nanobody V1 is shown in Table 22, and the nucleic acid sequence is shown in Table 23. The amino acid sequences of the nanobody V1 and its 9 humanized antibodies are shown in Table 24, and the nucleic acid sequences are shown in Table 25.

[0306] Table 22 CDR region amino acid sequence of anti-VEGFA nanobody V1

[0307]

[0308] Table 23 Nucleic acid sequence encoding the CDR region of anti-VEGFA nanobody V1

[0309]

[0310] Table 24 Amino acid sequence of anti-VEGFA nanobody V1 and its humanized antibodies

[0311]

[0312]

[0313] Table 25 Nucleic acid sequence encoding anti-VEGFA nanobody V1 and its humanized antibodies

[0314]

[0315]

[0316] Antibodies with high thermal stability are more conducive to the production and long-term storage of antibodies, so the thermal stability Tm values (melting temperature) of different anti-VEGFA nanobodies were compared. For anti-VEGFA nanobody V1 and its 9 humanized antibodies, the Tm value was determined using the UNCLE instrument (unchained labs), and the results are shown in Table 26.

[0317] Table 26 Thermal stability Tm values of anti-VEGFA nanobody V1 and its 9 humanized antibodies

[0318] Anti-VEGFA Nanobody name Tm (°C) V1-DP 72.4 V1-SA1 71.0 V1 67.0 V1-SA3 66.7 V1-3m 66.4 V1-2m 65.8 V1-com-78 64.5 V1-com-87 63.5 V1-com-74-78-97 60.6 V1-4m 59.4

[0319] From Table 26, it can be seen that the Tm values of V1-DP and V1-SA1 are higher, indicating that the thermal stability of these two antibodies is likely to be superior to other humanized antibodies.

[0320] The humanized anti-VEGFA nanobodies V1-SA1 (SEQ ID NO: 20) and V1-DP (SEQ ID NO: 21) with higher melting temperature Tm were selected for the design of fusion constructs with the humanized anti-albumin antibody 3005Hz6. According to different tandem sequences, a total of 3 fusion constructs were designed with GGS as the linker, and the amino acid sequences are shown in Table 27, and the nucleic acid sequences are shown in Table 28.

[0321] Table 27 Amino acid sequences of fusion constructs of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobodies

[0322]

[0323] Table 28 Nucleic acid sequences encoding fusion constructs of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobodies

[0324]

[0325]

[0326] A secretion signal peptide was added at the N-terminus of the fusion construct, and a linker GGGGS (SEQ ID NO: 37) and 6xHis were added at the C-terminus of the antibody. The DNA sequence encoding was obtained by gene synthesis (see Table 28), and then connected to the expression vector pCDNA3.1(+) through the restriction enzyme sites NheI and XhoI. The plasmid was sequenced correctly and endotoxin-free extracted, and transiently expressed using suspension 293F cells. The cell culture supernatant after transfection was harvested and subjected to Ni column affinity purification. The SDS-PAGE results of the purified product are shown in Figure 9 , where the cell culture supernatant after transfection of each protein is labeled as Input, the flow-through liquid that cannot be bound to the Ni column is labeled as FT, and the purified product eluted is labeled as E, and the molecular weight of the target protein is about 28 kDa.

[0327] 2. Activity detection of fusion constructs of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobodies

[0328] This example detects whether the two nanobody elements constituting the fusion construct maintain the original activity.

[0329] The activity of the anti-albumin nanobody 3005Hz6 in the fusion construct to bind HSA was detected. The detection method was the same as that in Example 4, "2. Activity detection of humanized antibodies". The ELISA results are shown in Figure 10The EC50values are shown in Table 29.

[0330] Table 29 Summary of EC50of fusion constructs or 3005Hz6 binding to HSA

[0331] Fusion construct name Binding HSA EC50 (nM) V1SA-3005Hz6 0.32 3005Hz6-V1SA 0.33 V1DP-3005Hz6 0.39 3005Hz6 0.25

[0332] From Table 29, it can be seen that the activity of fusion constructs binding to HSA is similar to that of 3005Hz6 alone, indicating that fusion of 3005Hz6 with other antibody fragments does not affect the binding activity of 3005Hz6 to HSA. Moreover, the anti-VEGFA antibody can be fused at the N- or C-terminus of the anti-albumin antibody, and the different positions do not affect the binding activity of the fusion protein to albumin.

[0333] The activity of the anti-VEGFA nanobody in the fusion construct was detected by VEGFR2 competitive ELISA method. The activity of the fusion construct and the VEGFA antibody alone was compared.

[0334] Specifically, the enzyme-labeled plate was coated with goat anti-human IgG Fc protein (purchased from Solarbio, product number: SPA105) at a concentration of 5 μg / mL at 4°C overnight. After washing the plate with PBST and blocking with milk powder, VEGFR2 extracellular region-human IgG1 Fc fusion protein (VEGFR2-Fc, prepared in-house) was added. The amino acid sequence of VEGFR2-Fc is:

[0335] ASVGLPSVSLDLPRLSIQKDILTIKANTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCFYRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCARYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYRIYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHWYWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEGKNKTVSTLVIQAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSLWCTADRSTFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIMELKNASLQDQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTITGNLENQTTSIGESIEVSCTASGNPPPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYTCQACSVLGCAKVEAFFIIEGAQEKTNLEPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39)

[0336] The fusion construct with concentration gradient was incubated with biotin-modified VEGFA165-Avi-His at a constant final concentration (0.4 nM) for 1 hour. VEGFA165-Avi-His was prepared internally by adding a linker and Avi tag and 6xHis tag at the C-terminus of human VEGFA165. The VEGFA165-Avi-His expression plasmid was co-transfected with BirA enzyme expression plasmid into 293F cells, and the protein was biotin-modified on the Avi tag during expression by BirA enzyme catalysis. The amino acid sequence of VEGFA165-Avi-His is:

[0337] APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGL ECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCK CSCKNTDSRCKARQLELNERTCRCDKPRRGSGSGLNDIFEAQKIEWHEGGGGSHHHHHH (SEQ ID NO: 40).

[0338] The mixture of the fusion construct and VEGFA165-Avi-His was added to the enzyme- labeled plate that had captured VEGFR2-Fc, and incubated at 37°C for 1 hour. The biotin signal of VEGFA165-Avi-His was detected by HRP-labeled streptavidin antibody Streptavidin-HRP (purchased from: RiboBio, item number: D111054-0001), incubated at room temperature for 45 minutes, and after washing the plate, 100 μL / well of TMB developing solution was added to the enzyme-labeled plate, developed for 15 min; 50 μL / well of stop solution was added, and the enzyme-labeled plate was placed into an enzyme-labeled instrument to read the light absorption value (OD450) at 450 nm.

[0339] The competition of VEGFR2 by the fusion construct and monovalent nanobody V1-SA1 is shown in Figure 11 The IC50 summary of the VEGFR2 competition experiment is shown in Table 30.

[0340] Table 30 IC50 summary of the competition of VEGFR2 by the fusion construct and monovalent nanobody V1-SA1

[0341] Fusion construct name VEGFR2 competition ELISA IC50 (nM) Ratio of IC50 of fusion construct to IC50 of V1-SA1 V1SA-3005Hz6 0.84 1.08 3005Hz6-V1SA 0.75 0.96 V1DP-3005Hz6 0.63 0.81 V1-SA1 0.78 1.00 3005Hz6 N / A N / A

[0342] From Table 30, the VEGFR2 competitive activity of the three fusion constructs of 3005Hz6 in series with V1-SA1 or V1-DP is similar to the activity of monovalent nanobody V1-SA1 (IC50 ratio of 0.81-1.08), indicating that the anti-VEGFA antibody does not affect the activity of anti-VEGFA after fusion with 3005Hz6, and the anti-VEGFA antibody can be fused to the N or C terminus of the anti-albumin antibody, without affecting the activity of the anti-VEGFA antibody in the fusion construct.

[0343] The data in Table 29 and Table 30 show that the fusion construct of monovalent anti-VEGFA nanobody (V1-SA1 or V1-DP) and anti-albumin nanobody (3005Hz6) has strong competitive activity for VEGFR2 and binding activity for HSA. This indicates that in the fusion construct, the antibody elements that bind to different antigens can independently function, and the mutual interference between the antibody elements is small. Therefore, the antibody that binds to other antigens can be fused with the humanized anti-albumin antibody 3005Hz6 for expression, to construct a multifunctional fusion construct.

[0344] Example 7 Fusion construct of bivalent anti-VEGFA nanobody and humanized anti-albumin antibody

[0345] 1. Design of fusion construct of bivalent anti-VEGFA nanobody and anti-albumin nanobody

[0346] Since VEGFA is a homodimer in vivo, to improve the efficiency of nanobody binding to VEGFA and enhance the efficiency of nanobody blocking the interaction of VEGFA / VEGFR, the anti-VEGFA nanobody is in a bivalent state, i.e., two nanobodies with the same sequence are connected in series. To increase the molecular weight of the bivalent anti-VEGFA nanobody in vivo and prolong the half-life of the bivalent antibody by binding to albumin, the bivalent anti-VEGFA nanobody is fused with the humanized anti-albumin antibody 3005Hz6 for expression. The amino acid sequences of the fusion construct of humanized antibody 3005Hz6 and bivalent anti-VEGFA nanobody V1-SA1 or V1-DP are shown in Table 31, and the nucleic acid sequences are shown in Table 32.

[0347] Table 31 Amino acid sequences of fusion construct of bivalent anti-VEGFA nanobody and humanized anti-albumin antibody 3005Hz6

[0348]

[0349]

[0350] Table 32 Nucleic acid sequences encoding fusion construct of bivalent anti-VEGFA nanobody and humanized anti-albumin antibody 3005Hz6

[0351]

[0352]

[0353]

[0354] The DNA sequence encoding the fusion construct was obtained by gene synthesis (see Table 32) with a secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36) added at the N-terminus, a linker GGGGS (SEQ ID NO: 37) and 6xHis added at the C-terminus of the antibody, and then cloned into the expression vector pCDNA3.1(+) through NheI and XhoI restriction sites. The plasmid was sequenced correctly and endotoxin-free extracted, and transiently expressed in suspension 293F cells. The cell culture supernatant was harvested and subjected to Ni column affinity purification. For the fusion construct 3005Hz6-2V1SA (SEQ ID NO: 33), a non-tagged protein was also constructed, which was cloned into the expression vector after adding a secretion signal peptide at the N-terminus of the protein and transiently expressed in 293F cells.

[0355] The V1SA-3005Hz6-V1SA (SEQ ID NO: 32) and 3005Hz6-2V1SA (SEQ ID NO: 33) purified by Ni column were subjected to cation exchange purification, and the purified products were subjected to SDS-PAGE, and the results are shown in Figure 12 (A), wherein the flow-through is labeled as FT, the eluted products of V1SA-3005Hz6-V1SA are labeled as A1 and A2 components, and the eluted products of 3005Hz6-2V1SA are labeled as A5 and A6 components. The 3005Hz6-2V1SA without any tag was subjected to cation exchange, and the SDS-PAGE of the purified product is shown in Figure 12 (B), and the V1DP-3005Hz6-V1DP and 3005Hz6-2V1DP were subjected to Ni column purification, and the SDS-PAGE of the purified products is shown in Figure 12 (C). The molecular weight of the fusion construct is about 41 kDa.

[0356] 2. ELISA test of the fusion construct and binding and dissociation rate determination (SPR)

[0357] The activity of binding HSA in fusion constructs V1SA-3005Hz6-V1SA (SEQ ID NO: 32), 3005Hz6-2V1SA (SEQ ID NO: 33) and 3005Hz6-2V1DP (SEQ ID NO: 35) was detected by ELISA method. In the experiment, human serum albumin HSA (Baxter AG) was diluted to 2 μg / mL and added to the enzyme-labeled plate, 100 μL / well, coated at 4°C overnight. After blocking with 5% skim milk powder and washing with PBST (0.1% Tween 20 in PBS, pH = 7.4), the His-tagged fusion construct gradient dilution was added to the enzyme-labeled plate, 100 μL / well, incubated at 37°C for 1 hour, and after washing the plate with PBST, HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, item number: HRP-66005) was added, and incubated at room temperature for 45 minutes. After washing the plate, 100 μL of TMB (purchased from: Tiangeng Biochemical, item number: PA107-01) was added to each well, and after color development at 37°C for 15 minutes, 50 μL of stop solution was added. The absorbance (OD450) at 450 nm was measured by an enzyme-labeled instrument (purchased from: Thermo Scientific, model: Multiskan SkyHigh, item number: A51119700C).

[0358] The ELISA results are shown in Table 33. Figure 13

[0359] Table 33 Summary of EC50 of fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and 3005Hz6-2V1DP binding to HSA

[0360] Fusion construct name Binding HSA EC50 (nM) V1SA-3005Hz6-V1SA 0.35 3005Hz6-2V1SA 0.26 3005Hz6-2V1DP 0.30

[0361] As can be seen from Table 33, the fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and 3005Hz6-2V1DP all have strong HSA binding activity, indicating that the antibody 3005Hz6 in the fusion construct can exert the complete biological activity.

[0362] The affinity of fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA to HSA was determined by surface plasmon resonance (SPR) method.

[0363] ​Biacore 8K instrument (Cytiva) was used in the experiment, and Series S Sensor Chip NTA was used to capture His-tagged anti-albumin antibody 3005Hz6 or fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA, respectively. The analyte was HSA at gradient concentrations, and the analyte was diluted with mobile phase buffer (composition: 10 mM HEPES, 150 mM NaCl, 0.05% v / v Tween-20, pH = 7.4). The binding time of the ligand and the analyte was 150 seconds, and the dissociation time was 900 seconds. The binding and dissociation curves of the ligand and the analyte are shown in Figure 14

[0364] The "1:1 binding" model was used to fit the binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of 3005Hz6, V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA and HSA, and the results are shown in Table 34.

[0365] Table 34 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of anti-albumin antibody 3005Hz6 and fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and HSA

[0366] Ligand Analyte ka (1 / Ms) kd (1 / s) K D (M) 3005Hz6 HSA 1.09E+05 1.38E-03 1.27E-08 V1SA-3005Hz6-V1SA HSA 2.48E+04 1.72E-03 6.94E-08 3005Hz6-2V1SA HSA 4.11E+04 1.47E-03 3.58E-08

[0367] From Table 34, it can be seen that the fusion construct 3005Hz6-2V1SA has a higher affinity for HSA, K D approximating the K D of antibody 3005Hz6 and HSA (K D is 12.7-35.8 nM).

[0368] 3. Binding and dissociation rate determination of fusion constructs and VEGFA (SPR)

[0369] ​The affinities of fusion constructs V1SA-3005Hz6-V1SA (His-tag), 3005Hz6-2V1SA (no tag) and positive control aflibercept to VEGFA were determined by SPR method. SPR experiments were performed using Biacore 8K (Cytiva) with Series S Sensor Chip CAP to capture biotin-modified VEGFA165-Avi-His (SEQ ID NO: 40) at a capture level of about 40 RU, and the flow phase buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20) was used to dilute each protein, and a 2-fold protein concentration gradient was set. During the kinetic analysis, the binding time of the ligand to the analyte was 150 seconds, the dissociation time was 900 seconds, and the flow rate was 30 μL / min.

[0370] The binding and dissociation curves of V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and positive control aflibercept to VEGFA are shown in Figure 15 (A), Figure 15 (B) and Figure 15 (C), respectively. The binding and dissociation curves were fitted using the “1:1 binding” model to obtain the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ), and the results are shown in Table 35.

[0371] Table 35 Association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and positive control aflibercept to VEGFA

[0372] Analyte Ligand ka (1 / Ms) kd (1 / s) K D (M)]]> V1SA-3005Hz6-V1SA VEGFA165-Avi-His 7.35E+06 1.79E-05 2.43E-12 3005Hz6-2V1SA VEGFA165-Avi-His 7.86E+06 4.24E-05 5.39E-12 Aflibercept VEGFA165-Avi-His 1.19E+06 1.31E-04 1.10E-10

[0373] As can be seen from Table 35, under the SPR experimental conditions of capturing biotin-modified VEGFA165-Avi-His, the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA have very high binding affinity to VEGFA (KD= 2.4-5.4 pM), and under the same experimental conditions, the affinity of the positive control drug aflibercept to VEGFA is one order of magnitude lower than that of V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA, with a KD of about 110 pM.

[0374] Example 8 Study on the activity of fusion constructs in inhibiting VEGFA-stimulated HUVEC proliferation

[0375] This example uses primary human umbilical vein endothelial cells (HUVEC) to test whether the fusion construct can block VEGFA-stimulated HUVEC proliferation at the cellular level.

[0376] Since the fusion construct will bind to HSA in the human body, in this experiment, the difference in the activity of the fusion construct in inhibiting HUVEC proliferation was compared when pre-incubated with or without HSA.

[0377] In the experiment, the endothelial cell culture medium ECM (purchased from: Zhijiao Xin Zhou, item number: ZQ-1304) containing 0.5% FBS was used to prepare a solution of human VEGFA165 (purchased from: Jin Sui, item number: Z03073) at a concentration of 210 ng / mL. The fusion construct 3005Hz6-2V1SA gradient dilution solution with or without the same molar concentration of HSA (labeled as: 3005Hz6-2V1SA+HSA and 3005Hz6-2V1SA, respectively), the fusion construct V1SA-3005Hz6-V1SA gradient dilution solution with or without the same molar concentration of HSA (labeled as: V1SA-3005Hz6-V1SA+HSA and V1SA-3005Hz6-V1SA, respectively), and the positive control of the same molar concentration gradient of the marketed drug aflibercept (Bayer) were prepared. In a 96-well plate, 50 μL of the test protein (3005Hz6-2V1SA+HSA, 3005Hz6-2V1SA, V1SA-3005Hz6-V1SA+HSA, V1SA-3005Hz6-V1SA, and aflibercept) at different concentrations was mixed with 50 μL of VEGFA165 solution. Controls without VEGFA165 (expected to proliferate the slowest) and with only VEGFA165 (expected to proliferate the fastest) were set up. The mixture of the test protein and VEGFA165 was placed in a 37°C, 5% CO2 incubator for 1.5-2 hours. The primary cells HUVEC (purchased from: National Stem Cell Transformation Resource Library, item number: DFSC-EC-01) were digested and centrifuged, and then resuspended in ECM containing 0.5% FBS. The cells were added to the 96-well plate pre-mixed with the test protein and VEGFA165 at 50 μL of cell suspension per well, and 1.2 x 10 4cells, and the final concentration of VEGFA165 was 70 ng / mL. Incubate at 37°C, 5% CO2 incubator for 68-72 h. After incubation, add 16 μL CCK-8 staining solution (purchased from: Solarbio, item number: CA-1210) to each well, and incubate in a 37°C incubator for 2.5-4 hours. Read the absorbance value (wavelength 450 nm) by a microplate reader to detect the proliferation activity of HUVEC cells. The IC50 value is calculated by four-parameter nonlinear regression analysis using GraphPad Prism 8 software.

[0378] Calculate the inhibition rate of the test protein on cell proliferation at different concentrations. The formula for calculating the inhibition rate is:

[0379]

[0380] The results of the fusion construct V1SA-3005Hz6-V1SA (pre-incubated or not pre-incubated with HSA), 3005Hz6-2V1SA (pre-incubated or not pre-incubated with HSA) inhibiting VEGFA-stimulated HUVEC proliferation experiments are shown in Figure 16 (A), Figure 16 (B), respectively.

[0381] The IC50 and maximum inhibition rate of the fusion construct V1SA-3005Hz6-V1SA inhibiting HUVEC proliferation are shown in Table 36, and the IC50 and maximum inhibition rate of the fusion construct 3005Hz6-2V1SA inhibiting HUVEC proliferation are shown in Table 37.

[0382] Table 36 IC50 and maximum inhibition rate of fusion construct V1SA-3005Hz6-V1SA in HUVEC proliferation inhibition experiment

[0383]

[0384] Table 37 IC50 and maximum inhibition rate of fusion construct 3005Hz6-2V1SA in HUVEC proliferation inhibition experiment

[0385]

[0386]

[0387] The results of Table 36 and Table 37 show that both fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA can efficiently block the downstream signaling pathway activated by human VEGFA165 at the cellular level, and inhibit the HUVEC proliferation stimulated by human VEGFA165, with an activity similar to that of the positive control aflibercept (receptor drug). Moreover, the results show that whether the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA are pre-incubated with HSA or not, it has no significant effect on their HUVEC proliferation inhibition activity.

[0388] Example 9 Study on the inhibition of VEGFA-induced VEGFR2 phosphorylation activity by fusion constructs

[0389] This example uses primary HUVEC cells to verify the inhibitory effect of fusion constructs on VEGFA-induced VGFR2 phosphorylation.

[0390] In the experiment, after the primary HUVEC cells were digested and centrifuged, they were resuspended with ECM medium (containing 5% FBS, 1% epidermal growth factor, 1% penicillin-streptomycin) (purchased from: Zhijiao Xin Zhou, item number: ZQ-1304) at a density of 3x10 5 The cells were cultured in a 37°C, 5% CO2 incubator overnight.

[0391] The VEGFA165 and fusion constructs were configured with ECM medium, and the working concentration was 50 ng / mL (molar concentration was 1.3 nM). The untagged human VEGFA165 (purchased from: Kingsriver, item number: Z03073) was mixed with different concentrations of fusion constructs V1SA-3005Hz6-V1SA (pre-incubated with the same molar concentration of HSA) and 3005Hz6-2V1SA (pre-incubated with the same molar concentration of HSA) respectively. At the same time, controls without VEGFA165 and with only VEGFA165 were set up. After the mixture was placed at 37°C for 30 minutes, HUVEC cells pre-plated overnight were added, and treated for 5 minutes. The cells were washed once with PBS, and 65 μL of strong RIPA lysis buffer containing PMSF (purchased from: Beyotime, item number: P0013B) was added to each well. The lysis was performed on ice for 20 minutes. The lysis solution was transferred to a 1.5 mL centrifuge tube, centrifuged at 13000 rpm for 15 minutes (4°C), and the supernatant was collected. 5xLoading buffer was added, heated at 95°C for 6 minutes, and the supernatant was subjected to western blot to detect VEGFR2 phosphorylation. In the western blot experiment, the anti-GAPDH antibody for detecting the internal reference GAPDH was purchased from Proteintech, item number 60004-1-Ig, and the antibody for detecting the phosphorylation of tyrosine at position 1175 of VGFR2 (p-VEGFR2) was purchased from Cell Signaling Technology, item number 2478S.

[0392] The signal intensity of the phosphorylated VEGFR2 (p-VEGFR2) band and the GAPDH band in the immunoblotting experiment was quantified, and the signal intensity ratio of the two bands (p-VEGFR2 / GADPH) was calculated to obtain the inhibition rate of the test protein on VEGFR2 phosphorylation.

[0393] The calculation formula of the inhibition rate of the test protein on VEGFR2 phosphorylation is:

[0394]

[0395] The inhibition rate of the fusion construct V1SA-3005Hz6-V1SA on VEGFR2 phosphorylation is shown in Table 38.

[0396] Table 38 Inhibition rate of fusion construct V1SA-3005Hz6-V1SA on VEGFR2 phosphorylation

[0397]

[0398] Table 38 shows that V1SA-3005Hz6-V1SA can effectively inhibit the VEGFR2 phosphorylation of HUVEC cells induced by human VEGFA. The inhibition rate of VEGFR2 phosphorylation of V1SA-3005Hz6-V1SA (under the condition of incubation with HSA) can reach more than 90% when the molar concentration is 5 times that of VEGFA.

[0399] The inhibition rate of fusion construct 3005Hz6-2V1SA on VEGFR2 phosphorylation is shown in Table 39.

[0400] Table 39 Fusion construct 3005Hz6-2V1SA on VEGFR2 phosphorylation inhibition rate

[0401]

[0402] Table 39 shows that 3005Hz6-2V1SA can effectively inhibit the VEGFR2 phosphorylation of HUVEC cells induced by human VEGFA. The inhibition rate of VEGFR2 phosphorylation of 3005Hz6-2V1SA (under the condition of incubation with HSA) can reach more than 90% when the molar concentration is 3 times that of VEGFA.

[0403] Example 10 Study on the activity of fusion constructs in VEGF reporter gene cells

[0404] This example uses VEGF reporter gene system to evaluate the activity of fusion constructs in blocking the downstream signal pathway of VEGF.

[0405] HEK-293 / VEGF / NFAT stable reporter gene cell strain (purchased from China Institute for Food and Drug Control) is transfected from HEK-293 cells to co-express VEGFR2 (KDR) and NFAT-RE-luc 2p genes. When VEGFA recognizes and activates KDR receptor, it starts the intracellular downstream signal pathway, activates transcription factor NFAT to start the expression of Luciferase reporter gene. At this time, after adding luciferase substrate, chemiluminescence can be produced.

[0406] After digesting and centrifuging HEK-293 / VEGF / NFAT reporter gene cells, resuspend the cells with DMEM containing 1% FBS, and the cell density is 5x10 4The cells were seeded at 1 x 104cells / mL, 80 μL per well on white-bottom 96-well plates (purchased from: Costar, Cat# 3917) and incubated overnight at 37 °C in a 5% CO2incubator. The next day, VEGFA165 (purchased from: Jinsheng, Cat# Z03073) was prepared to 80 ng / mL with DMEM containing 1% FBS and mixed with different concentrations of V1SA-3005Hz6-V1SA (pre-incubated or not with equimolar concentration of HSA) or 3005Hz6-2V1SA (pre-incubated or not with equimolar concentration of HSA) for 30 min at 37 °C. The negative control was without VEGFA165 and the positive control was with VEGFA165 only. 20 μL of the mixture (test protein and VEGFA165) was added to 80 μL of cells and incubated in a 37 °C incubator for 6 hours. 100 μL of luciferase reporter substrate (purchased from: Yeasen, Cat# 11404ES60) was added to each well and the relative light units (RLU) of luciferase were measured using a multifunctional microplate reader (purchased from: Agilent BioTek, Model: Synergy H1).

[0407] Under the above experimental conditions, the results of activity detection of fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA inhibiting the luminescence of luciferin substrate are shown in Figure 17 (A), Figure 17 (B), respectively. The fitted IC50values are shown in Table 40 and Table 41, respectively.

[0408] Table 40 Summary of IC50of fusion construct V1SA-3005Hz6-V1SA inhibiting the luminescence of luciferin in VEGF reporter system with or without pre-incubation of HSA

[0409] Protein IC50 (nM) in VEGF reporter assay V1SA-3005Hz6-V1SA 0.24 V1SA-3005Hz6-V1SA + HSA 0.22

[0410] Table 41 Summary of IC50of fusion construct 3005Hz6-2V1SA inhibiting the luminescence of luciferin in VEGF reporter system with or without pre-incubation of HSA

[0411] Protein IC50 (nM) in VEGF reporter assay 3005Hz6-2V1SA 0.24 3005 Hz 6-2 V1 SA + HSA 0.25

[0412] Figure 17The results of Table 40 and Table 41 show that both fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA can block the activation of downstream signaling pathway stimulated by VEGFA165 at cellular level, thus reducing the expression of Luciferase reporter gene activated by transcriptional promoter NFAT nuclear activation. The IC50 of both fusion constructs is 0.24 nM. In this embodiment, whether the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA are pre-bound to HSA has no substantial effect on the blocking activity of VEGFA downstream signal.

[0413] In summary, the activity detection experiments at protein level and cellular level show that both fusion constructs V1SA-3005Hz6-V1SA (SEQ ID NO: 32) and 3005Hz6-2V1SA (SEQ ID NO: 33) consisting of bivalent anti-VEGFA nanobody (V1-SA1, SEQ ID NO: 20) and humanized anti-albumin nanobody (3005Hz6, SEQ ID NO: 9) have strong HSA binding activity and affinity to VEGFA. The fusion constructs can efficiently inhibit HUVEC proliferation and VEGFR2 phosphorylation induced by VEGFA165 and inhibit the luciferin luminescence of VEGF reporter gene cell at cellular level. These data show that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA have strong VEGFA neutralization activity, can efficiently block VEGFA downstream signaling pathway and inhibit VEGFA-induced cell proliferation, and can be potentially used for treating VEGFA-induced abnormal vascular proliferation related diseases such as wet AMD, diabetic macular edema, tumors, etc.

[0414] Meanwhile, the data also show that the antibody elements binding to different antigens in the fusion constructs can independently function, the mutual interference between the antibody elements is small, and the binding to HSA does not affect the biological function of the effector molecule. Therefore, it can be expected that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA can normally exert the activity of blocking VEGFA signaling pathway and inhibiting endothelial cell proliferation after binding to albumin in vivo.

[0415] The above describes preferred embodiments of the present application in detail, but the present application is not limited to the specific details in the above-described embodiments. Within the technical concept scope of the present application, the technical solutions of the present application can be subjected to various simple modifications, and these simple modifications all belong to the protection scope of the present application.

[0416] It should be further noted that various specific technical features described in the above specific embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. An anti-VEGFA fusion construct, characterized in that, The fusion construct comprises an anti-albumin antibody or antigen-binding fragment thereof and an anti-VEGFA antibody or antigen-binding fragment thereof, the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof are nano-antibodies, and the anti-VEGFA antibody or antigen-binding fragment thereof is connected to the anti-albumin antibody or antigen-binding fragment thereof. The anti-albumin antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region, wherein, the amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 are any one of the following groups: A) SEQ ID NO: 1, 2, 3; B) SEQ ID NO: 1, 10, 3; C) SEQ ID NO: 1, 11, 3; D) SEQ ID NO: 1, 12, 3; 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 SEQ ID NO: 16; the amino acid sequence of CDR-H2 is SEQ ID NO: 17; the amino acid sequence of CDR-H3 is SEQ ID NO:

18.

2. The fusion construct of claim 1, wherein, The anti-albumin antibody or antigen-binding fragment thereof or 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 fusion construct 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 fusion construct of claim 1, wherein, The amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof is any one of the amino acid sequences shown in SEQ ID NO: 4, 6-9, 13-15.

5. The fusion construct of claim 1, wherein, The amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof is any one of the amino acid sequences shown in SEQ ID NO: 19-28.

6. The fusion construct of claim 1, wherein, The anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof are directly or indirectly connected.

7. The fusion construct of claim 6, wherein, The indirect connection is through a linker, a functional domain and / or a linker for coupling; The linker is selected from a linking peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, a nucleic acid, a polysaccharide, a fatty chain, biotin, streptavidin or avidin; The functional domain is a combination of one or more of an Fc fragment, serum albumin, a cytokine, transferrin or a scaffold protein; The linker for coupling comprises a functional group linker.

8. The fusion construct of claim 7, wherein, The functional group linker comprises a thiol, an amino, a hydroxyl and / or a carboxyl reactive group.

9. The fusion construct of claim 6, wherein, The fusion construct comprises one or more anti-VEGFA antibodies or antigen-binding fragments thereof, which are directly or indirectly connected to the N-terminal, C-terminal and / or internal residues of the anti-albumin antibody or antigen-binding fragment thereof.

10. The fusion construct of any one of claims 1-9, wherein, The fusion construct is any one of SEQ ID NO: 29-35.

11. A nucleic acid, characterized in that, The nucleic acid encodes the fusion construct of any one of claims 1-10.

12. The nucleic acid of claim 11, wherein The nucleotide sequence encoding the anti-VEGFA antibody or antigen-binding fragment thereof is any one of SEQ ID NOs: 62-71 or a degenerate sequence thereof.

13. The nucleic acid of claim 11, wherein The nucleotide sequence encoding the anti-albumin antibody or antigen-binding fragment thereof is any one of SEQ ID NOs: 45, 47-50, 56-58 or a degenerate sequence thereof.

14. The nucleic acid of any one of claims 11-13, wherein, The nucleotide sequence encoding the fusion construct is any one of SEQ ID NOs: 72-78 or a degenerate sequence thereof.

15. A vector, characterized in that, The vector comprises the nucleic acid of any one of claims 11-14.

16. A host cell, characterized in that, The host cell comprises the nucleic acid of any one of claims 11-14 or the vector of claim 15.

17. A method of producing a fusion construct according to any one of claims 1 to 10, characterized by, The preparation method comprises culturing the host cell of claim 16 to express the fusion construct.

18. Use of the fusion construct of any one of claims 1-10, the nucleic acid of any one of claims 11-14, the vector of claim 15 or the host cell of claim 16 in the preparation of a product for treating and / or diagnosing a disease, wherein the disease is a solid tumor, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy, central retinal vein occlusion, pathological myopia or neovascular glaucoma.

19. A product for treating and / or diagnosing a disease, characterized in that, The product for treating and / or diagnosing a disease comprises any one of: A) the fusion construct of any one of claims 1-10; B) the nucleic acid of any one of claims 11-14; C) the vector of claim 15; or, D) the host cell of claim 16; The disease is a solid tumor, wet age-related macular degeneration, diabetic macular edema, diabetic retinopathy, central retinal vein occlusion, pathological myopia or neovascular glaucoma.

20. A method of detecting VEGFA, comprising: The detection method comprises contacting a sample to be detected with the fusion construct of any one of claims 1-10, and then detecting the content of the complex formed by VEGFA and the fusion construct, wherein the detection method is not for the purpose of diagnosing a disease.

21. The detection method of claim 20, wherein, The VEGFA is from a mammal.

22. The detection method of claim 21, wherein, The mammal is from a human, a mouse or a monkey.

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