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

Abstract

The invention provides an anti-VEGFA fusion construct, the fusion construct is formed by connecting an anti-VEGFA antibody or an antigen binding fragment thereof and an anti-albumin antibody or an antigen binding fragment thereof, the fusion construct has very strong VEGFA binding affinity and biological activity for inhibiting VEGFA stimulated HUVEC proliferation, and functions of all structural domains do not influence each other. The invention also provides a method for preparing the fusion construct and application of the fusion construct in preparation of products for treating and / or preventing diseases, and the anti-VEGFA antibody or the antigen binding fragment thereof has activity for treating and / or diagnosing diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an anti-VEGFA fusion construct, a preparation method thereof, and an application thereof. Background Art

[0002] Vascular endothelial growth factor A (VEGFA) is an important member of the VEGF family and a major angiogenic factor, which 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 to and activating the receptor VEGFR2 (VEGF Receptor 2) and its downstream pathways. 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, ranging from cancer, autoimmunity to retinopathy, etc. Inhibiting the VEGF signaling pathway can treat diseases associated with abnormal blood vessel hyperplasia, such as wet age-related macular degeneration (wAMD). Currently, multiple anti-VEGF protein drugs have been marketed for the treatment of tumors and diseases related to ocular neovascularization.

[0003] The first anti-VEGFA protein drug is the monoclonal antibody bevacizumab (bevacizumab / Avastin). Other anti-VEGFA protein drugs include ranibizumab (ranibizumab / Lucentis), aflibercept and brolucizumab (Beovu), etc. Among them, the half-life of free aflibercept in human aqueous humor is about 11 days, which is longer than ranibizumab (7 days) and bevacizumab (9 days). The dosing interval of aflibercept is once every 2 months. At present, the dosage of aflibercept is increased from 2mg to 8mg, and clinical trials have shown that the dosing interval can be further extended to once every 3 or 4 months. Brolucizumab (Beovu) can be prepared into a 120mg / mL protein solution, and the amount of protein injected into the vitreous cavity can reach 6mg, which is the maximum dose for a single injection. In the dosing regimen of brolucizumab, patients received an injection once a month for the first three months, and then once every three months. The visual improvement effect was no worse than that of the control group, aflibercept (injected once every two months). These anti-VEGF protein drugs are administered by intravitreal injection, and the administration volume does not exceed 100 μL. The longest dosing interval for brolucizumab is 3 months, and the dosing interval for other drugs is once every 1 to 2 months. Frequent intraocular injections increase the risk of increased intraocular pressure, intraocular inflammation, retinal detachment, etc., so there is an urgent need for long-acting anti-VEGFA therapeutic drugs in clinical practice to reduce the number of injections for patients.

[0004] The existing methods for reducing the frequency of dosing include increasing the concentration of protein drugs to increase the dose of a single injection of the drug; other means include enhancing the biological activity of anti-VEGFA antibodies, reducing the minimum effective concentration; or enhancing the half-life of the drug. At present, the development of long-acting anti-VEGFA therapeutic drugs has achieved certain results in clinical practice, but there are also some problems, such as high-concentration protein preparations often have increased viscosity, easy to form protein aggregates or poor stability, which increases the risk of vitreous injection; in addition, there are problems such as insufficient biological effects or insignificant half-life enhancement. Therefore, the design and development of anti-VEGFA drugs needs further research and improvement.

[0005] Heavy chain antibodies are IgG2 and IgG3 antibodies that only contain heavy chains but no light chains and originate from the serum of camelids (camels, dromedaries and llamas, etc.). The variable domain of heavy chain (VHH) of heavy chain antibodies has independent antigen binding activity and is the smallest functional fragment of natural antibodies at present. It is called nano antibody or single domain antibody. The molecular weight of nano antibody is about 13kDa, with high thermal stability and good water solubility. It has great potential to be developed into protein preparations with higher concentrations. Albumin is the most abundant protein in plasma (concentration is about 40mg / mL). It is essential for maintaining plasma osmotic pressure. At the same time, 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 66.5kDa, and it can bind to the cell surface receptor FcRn (neonatal Fc receptor) under weakly acidic conditions, but the binding force is weaker under neutral conditions. Therefore, after albumin binds to FcRn, it is internalized into the endosome. As the endosomal environment becomes acidic, the binding force between albumin and FcRn increases, and it is brought back to the cell surface by FcRn, or transported across cells. Therefore, albumin can achieve a longer half-life with the help of the recycling mechanism of FcRn. The half-life of human albumin in plasma is about 3 weeks. Therefore, small molecule compounds that bind to albumin, or proteins expressed by fusion with albumin (such as marketed drugs) and ), has significantly improved pharmacokinetic properties, obtains a longer half-life, can potentially extend the dosing interval, and provide patients with a better medication regimen.

[0007] Anti-albumin antibodies or antigen-binding fragments thereof can bind to albumins of different species with high affinity. At the same time, the anti-albumin antibodies can be connected to biologically active effector molecules, such as anti-VEGFA antibodies or antigen-binding fragments, to form fusion constructs. The fusion constructs 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 mode between the various domains, and whether the activity of the biologically active effector molecules will be affected after binding to albumin still require a lot of creative work. The present application hopes to use nano-antibody technology for the development of anti-albumin antibodies or antigen-binding fragments, and the development and design of anti-VEGFA fusion constructs, to provide a better anti-VEGFA fusion construct. Summary of the invention

[0008] In order to overcome the defects of the prior art, the present application uses nano antibodies that bind to VEGFA as the basic active unit, and designs the monovalent and divalent nano antibodies 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 antibody or antigen-binding fragment of another antigen through a linker, such as an anti-albumin antibody, 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 a strong VEGFA binding affinity and a biological activity of inhibiting the proliferation of HUVEC stimulated by VEGFA. In addition, the functions of the various domains in the fusion construct do not affect each other. Specifically,

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

[0010] Preferably, the anti-albumin antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the 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 to the amino acid sequence shown in SEQ ID NO: 1;

[0012] The amino acid sequence of CDR-H2 comprises SEQ ID NO: 41 (GISVX 1 X 2 SFLDYADAVKG) or an amino acid sequence that is at least 80% identical to the amino acid sequence of 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 to the amino acid sequence shown in SEQ ID NO: 3.

[0014] Wherein, the SEQ ID NO: 41 (GISVX 1 X 2X in SFLDYADAVKG) 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 a specific embodiment of the present invention, SEQ ID NO: 41 (GISVX 1 X 2 X in SFLDYADAVKG) 1 X 2 Stands for DS, DA, EG, or DG.

[0016] In a specific embodiment of the present invention, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 include any 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 Anti-albumin candidate antibody CDR sequences

[0022]

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

[0024] The anti-albumin antibody or antigen-binding fragment thereof comprises a heavy chain variable region. The amino acids in the antibody CDR region are divided using the Kabat numbering system in this application.

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

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

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

[0028] In a specific embodiment of the present invention, the anti-albumin antibody or antigen-binding fragment thereof is a nanobody. Compared with full-length IgG antibody, Fab, and scFv, at the same mass, the nanobody has a higher molar concentration and can bind to more antigen molecules.

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

[0030] Preferably, the mammals include humans or non-human mammals, and the non-human mammals may be wild animals, zoo animals, economic animals, pets, experimental animals, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

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

[0032] In a specific embodiment of the present invention, 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 by any conventional method in the prior art, such as 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 the 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 that is at least 80% identical 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 shown in 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 its antigen-binding fragment includes nanobody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fv fragment, dAb fragment, F(ab')2 fragment, single chain antibody (single chain antibody fragment, scFv) or linear antibody.

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

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

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

[0043] In a specific embodiment of the present invention, the anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody. Compared with full-length IgG antibody, Fab, and scFv, at the same mass, the nanobody has a higher molar concentration and can bind to more antigen molecules.

[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 SEQ ID NOs: 19-28, or has at least 80% identity with any one 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 linked 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 connected to the anti-albumin antibody or antigen-binding fragment thereof.

[0051] Preferably, the indirect connection is through a linker, a functional domain and / or a linker for coupling.

[0052] Wherein, the linker is selected from connecting peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, fatty chains, biotin, streptavidin or avidin.

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

[0054] The linker used for coupling includes a functional group linker. Preferably, the functional group linker includes a thiol, amino, hydroxyl and / or carboxyl reactive group, which can covalently couple the anti-albumin antibody or its antigen-binding fragment with the anti-VEGFA antibody or its antigen-binding fragment.

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

[0056] In a specific embodiment of the present invention, the sequence from N-terminus to C-terminus includes a first anti-VEGFA antibody or an antigen-binding fragment thereof, a connecting peptide, and a second anti-VEGFA antibody or an antigen-binding fragment thereof.

[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 a specific embodiment of the present invention, the fusion construct comprises two anti-VEGFA antibodies or antigen-binding fragments thereof, and the two anti-VEGFA antibodies or antigen-binding fragments thereof are directly or indirectly connected 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, from N-terminus to C-terminus, the connection order of the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof is as follows:

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

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

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

[0063] 4) a first anti-VEGFA antibody or antigen-binding fragment thereof, a connecting peptide, an anti-albumin antibody or antigen-binding fragment thereof, a connecting peptide, a second anti-VEGFA antibody or antigen-binding fragment thereof, and the like;

[0064] Wherein, the connection may not include a connecting peptide.

[0065] Preferably, the two or more anti-VEGFA antibodies or antigen-binding fragments thereof are anti-VEGFA antibodies or antigen-binding fragments thereof with completely identical sequences, anti-VEGFA antibodies or antigen-binding fragments thereof with partially identical sequences, or anti-VEGFA antibodies or antigen-binding fragments thereof with completely different sequences.

[0066] In a specific embodiment, the fusion construct comprises any one of the amino acid sequences in SEQ ID NOs: 29-35, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NOs: 29-35.

[0067] Preferably, the fusion construct comprises an Fc fragment.

[0068] Further preferably, the Fc fragment comprises SEQ ID NO: 38, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 38.

[0069] Preferably, the fusion construct further comprises a secretory peptide. The secretory peptide is connected to the N-terminus of the fusion construct. Further preferably, the amino acid sequence of the secretory peptide may be SEQ ID NO: 36.

[0070] Preferably, the fusion construct further comprises a tag.

[0071] Preferably, the tag is connected to the C-terminus of any antibody or antigen-binding fragment thereof, or fusion construct.

[0072] Preferably, the fusion construct can inhibit or compete for 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 invention) to VEGFA.

[0073] Preferably, the fusion construct can also inhibit or compete for 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 invention) to albumin.

[0074] In a second aspect of the present invention, a nucleic acid is provided, which encodes the antibody or antigen-binding fragment described in the present application, or the above-mentioned fusion construct. 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, which encodes the antibody or antigen-binding fragment described herein, or the fusion construct described above.

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

[0077] Preferably, the nucleotide sequence encoding CDR-H2 in the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 43, 52-54 or a degenerate sequence thereof, or has at least 80% identity with any one of SEQ ID NOs: 43, 52-54.

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

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

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

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

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

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

[0084] More preferably, the nucleotide sequence encoding the above-mentioned fusion construct comprises any one of the nucleotide sequences in SEQ ID NOs: 72-78 or a degenerate sequence thereof, or has at least 80% identity with any one of the nucleotide sequences in SEQ ID NOs: 72-78, and has a nucleotide sequence encoding the function of the above-mentioned fusion construct.

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

[0086] The third aspect of the present invention provides a vector, wherein the vector comprises the above-mentioned nucleic acid.

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

[0088] For example, a viral expression vector can be used. The viral expression vector may include a virally derived DNA or RNA sequence for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an 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 may be included in a delivery vector. In certain embodiments, the delivery vector includes an antibody or Fab as described herein, a nucleic acid as described herein, and / or a recombinant vector as described herein, and optionally includes liposomes and / or lipid nanoparticles (LNPs). For example, a delivery vector may be introduced into a cell by a physical delivery method. The example of a physical method includes microinjection, electroporation, and hydrodynamic delivery. For example, nucleic acid as described herein may be wrapped in a cationic lipid particle (e.g., liposome) by LNPs, and may be delivered to a cell relatively easily.

[0089] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the above-mentioned nucleic acid or the above-mentioned vector.

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

[0091] Eukaryotic cells include animal and plant cells, fungi, etc., such as T cells, yeast cells, HEK293 cells or CHO cells, etc.

[0092] Prokaryotic cells such as Escherichia coli.

[0093] The fifth aspect of the present invention provides a method for preparing a host cell comprising the above-mentioned nucleic acid or the above-mentioned vector, and the preparation method comprises introducing the above-mentioned nucleic acid or vector into the host cell.

[0094] The sixth aspect of the present invention provides a method for preparing the above-mentioned fusion construct, which comprises culturing the above-mentioned host cell to express the fusion construct.

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

[0096] A) the fusion construct described above;

[0097] B) the nucleic acid described above;

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

[0099] D) the host cell described above.

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

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

[0102] The disease is a disease related to the VEGFA signaling pathway, and more preferably may be a tumor, abnormal vascular proliferation, angiogenesis-related ophthalmic disease (eg, fundus vascular disease), and the like.

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

[0104] The eighth aspect of the present invention provides a method for detecting VEGFA, which comprises contacting a sample to be tested 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 to detect the presence or content of VEGFA, wherein the presence refers to the presence or absence, and the content can be the expression level or protein concentration, etc.

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

[0107] The ninth aspect of the present invention provides a method for blocking VEGFA-mediated vascular endothelial cell proliferation or inhibiting angiogenesis, the method comprising contacting vascular endothelial cells with the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned product for treating and / or diagnosing the disease.

[0108] Preferably, the method comprises diluting the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned product for treating and / or diagnosing a disease with a serum-free culture medium of vascular endothelial cells, and then incubating with an antigen (eg, VEGFA165).

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

[0110] Preferably, the antigen and the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell or the above-mentioned product for treating and / or diagnosing a disease are mixed with vascular endothelial cells, added to a vascular endothelial cell culture plate, and cultured on the culture plate.

[0111] Preferably, the detection is performed after culturing.

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

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

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

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

[0117] The tenth aspect of the present invention provides a method for treating and / or preventing a disease, the method comprising applying the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, the above-mentioned product for treating and / or diagnosing the disease to an individual.

[0118] The diseases include diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0119] Preferably, the target cells are selected from cells expressing VEGFA, such as cardiomyocytes, proximal tubular cells, hepatocytes, vascular endothelial cells, granular cells, specialized epithelial cells, mesenchymal cells, macrophages, platelets, dendritic cells, activated T cells, retinal pigment epithelial cells, Muller cells in the retina, astrocytes, osteoblasts, bronchial and alveolar epithelial cells, pericytes, vascular smooth muscle cells, myofibroblasts, keratinocytes, renal mesangial cells or tumor cells, etc.

[0120] In the eleventh aspect of the present invention, the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector or the above-mentioned host cell is used in the preparation of a product for treating and / or preventing a disease, and the anti-VEGFA antibody or its antigen-binding fragment has the activity of treating and / or diagnosing a disease.

[0121] The diseases include diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.

[0122] Preferably, the product includes a product that blocks VEGFA-mediated endothelial cell proliferation or inhibits angiogenesis.

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

[0124] As used herein, "fusion construct" defines the fusion of an anti-albumin antibody or antigen-binding fragment thereof of the present invention with another compound. The fusion construct may contain one or more anti-albumin antibodies or antigen-binding fragments thereof, and the multiple anti-albumin antibodies or antigen-binding fragments thereof may be the same or different. The fusion construct may contain one or more additional compounds, and the multiple additional compounds may also be the same or different. The compound may be a protein compound or a non-protein compound. In the case where the compound is a protein compound or the fusion construct only contains multiple anti-albumin antibodies or antigen-binding fragments thereof, the fusion construct may 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 coupled form, the fusion construct may also be referred to as a conjugate.

[0125] The "drug" of the present invention can be used to treat humans or non-human animals, such as non-human mammals. The drug can contain pharmaceutically acceptable carriers, excipients or salts common in the prior art. The drug can be administered by any suitable route, such as gastrointestinal administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, rectal, etc.). The drug can be in any suitable dosage form, such as a gastrointestinal dosage form or a parenteral 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, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, drop pills, gels, etc. The various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field.

[0126] The drug may contain the fusion construct, the nucleic acid, the vector, the host cell, etc. in a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%). The drug can be prepared as a reagent with a protein concentration of 1-300 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / mL).

[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 term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0129] The "method" described in the present invention can be used for the purpose of diagnosis and treatment of diseases, or for the purpose of diagnosis and treatment of non-diseases.

[0130] The "antigen-binding fragment" of the present invention is a part of an antibody that retains the specific binding activity of the antibody, that is, 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 an antibody, the heavy chain variable region and / or light chain variable region of an antibody, or a single or more than two CDRs from the heavy chain or light chain of an antibody. Among them,

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

[0132] Chimeric antibodies are antibodies in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0133] Single-chain antibody is an antibody composed of the heavy chain variable region and the light chain variable region connected by a linker.

[0134] Fab, a monovalent fragment consisting of the VL, VH, CL and CH1 domains.

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

[0136] F(ab')2 is a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region.

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

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

[0139] The dAb fragment is an antibody fragment consisting of the VH domain.

[0140] The "linear antibody" described in the present invention includes one or more pairs of antibody fragments connected in series, wherein the antibody fragment can be an Fd segment (VH-CH1), a single-chain antibody (scFv), an antibody fragment (Fab), or a single-domain antibody (VHH), and these fragments are connected in series through connecting peptides to form a continuous antibody structure.

[0141] Among them, VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain constant region, and CH represents the heavy chain constant region.

[0142] The "Fc" region of the present invention contains two heavy chain fragments including the CH2 and CH3 domains of an antibody. The two heavy chain fragments form dimers by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.

[0143] The words “comprising” or “including” described in the present invention are open-ended expressions. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0144] The "homology" or "identity" mentioned in the present invention refers to that in terms of using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 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% homology / identity.

[0145] The "humanized antibody" of the present invention refers to the framework region and / or constant region portion (such as CH region) of the antibody or all or part of the antibody encoded by human antibody genes. In a specific embodiment of the present invention, the CDR region of the antibody has not been humanized.

[0146] The "individual" described in the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0147] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0148] The term "prevention" as used herein refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in an organism.

[0149] The term "diagnosis" as used herein refers to finding out whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression of a disease or its possible future progression.

[0150] The "tumor" of the present invention can be any undesirable cell proliferation (or any disease that manifests itself as undesirable cell proliferation), neoplasm, or increased tendency or risk of 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 cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblastic cells, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, leukocytes. Further preferably, the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma (e.g., glioma), intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, rhabdomyosarcoma, tongue squamous cell carcinoma, nasopharyngeal carcinoma, ovarian cancer, placental choriocarcinoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T-cell lymphoma), leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, neurilemoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic myeloid leukemia, acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid carcinoma, colon cancer, thymic cancer, blood cancer, head and neck cancer, or oropharyngeal cancer.

[0151] The "retinal vascular disease" mentioned in the present invention refers to the general term for diseases occurring in the retinal arteries or veins, or diseases related to choroidal angiogenesis, including but 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 invention include:

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

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

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

[0156] 4) In vivo, the fusion construct has the potential to inhibit VEGFA-stimulated angiogenesis, and by binding to albumin, it is carried by albumin into a specific tissue microenvironment enriched with albumin, potentially improving 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 and recycling mechanism of albumin and FcRn to extend the half-life. Therefore, the fusion construct of anti-VEGFA antibody and anti-albumin antibody has the potential to achieve long-term effects in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0158] Figure 1 : Cootipol stained gel images of His-tagged antibodies Alb Nb and 3005 after purification by Ni column, (A) is the Cootipol stained gel image of antibody Alb Nb, (B) is the Cootipol stained gel image of antibody 3005, the supernatant of cell culture medium after transient transfection of antibody Alb Nb is marked as Input, the flow-through that cannot bind to the Ni column is marked as FT, the antibody Alb Nb that binds to the Ni column and is eluted is marked as E, and the marker is marked as M.

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

[0160] Figure 3: A stained gel image of 3005 humanized antibody with His tag after purification by Ni column.

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

[0162] Figure 5 : ELISA binding activity test of antibody 3005 and its humanized antibody 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 with HSA and MSA (SPR method), (A) is the binding and dissociation curves of antibody 3005Hz6 with HSA, (B) is the binding and dissociation curves of antibody 3005Hz6 with MSA.

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

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

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

[0167] Fig.10: Fusion construct of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody, and ELISA activity detection of humanized antibody 3005Hz6 binding to HSA.

[0168] Fig.11 : VEGFR2 competition ELISA activity test of fusion constructs and monovalent nanobody V1-SA1.

[0169] Fig.12 : SDS-PAGE image of the fusion construct of humanized antibody 3005Hz6 and divalent anti-VEGFA nanobody after purification, (A) is the gel stained image of V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA, wherein the flow-through is marked as FT, the elution products of V1SA-3005Hz6-V1SA are marked as A1 and A2 components, and the elution products of 3005Hz6-2V1SA are marked as A5 and A6 components, (B) is the gel stained image of the unlabeled 3005Hz6-2V1SA protein product, (C) is the gel stained image of V1DP-3005Hz6-V1DP, and (D) is the gel stained image of 3005Hz6-2V1DP.

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

[0171] Fig.14 :The affinity of the protein to HSA was tested using the SPR method, wherein Figure (A) shows the binding and dissociation curves of the fusion construct V1SA-3005Hz6-V1SA and HSA, Figure (B) shows the binding and dissociation curves of the fusion construct 3005Hz6-2V1SA and HSA, and Figure (C) shows the binding and dissociation curves of the antibody 3005Hz6 and HSA.

[0172] Fig.15 : The affinity of the protein to VEGFA was tested using the SPR method, wherein Figure (A) shows the binding and dissociation curves of V1SA-3005Hz6-V1SA and VEGFA, Figure (B) shows the binding and dissociation curves of 3005Hz6-2V1SA and VEGFA, and Figure (C) shows the binding and dissociation curves of the positive control aflibercept and VEGFA.

[0173] Fig.16: Experiment on inhibition of VEGFA-induced HUVEC proliferation by fusion construct, wherein Figure (A) shows the test results of V1SA-3005Hz6-V1SA, and the premix of V1SA-3005Hz6-V1SA and HSA (V1SA-3005Hz6-V1SA+HSA), and Figure (B) shows the test results of 3005Hz6-2V1SA, and the premix of 3005Hz6-2V1SA and HSA (3005Hz6-2V1SA+HSA).

[0174] Fig.17 : The fusion construct inhibits VEGFA-induced reporter gene cell experiment, wherein Figure (A) shows the test results of V1SA-3005Hz6-V1SA and V1SA-3005Hz6-V1SA+HSA, and Figure (B) shows the test results of 3005Hz6-2V1SA and 3005Hz6-2V1SA+HSA. DETAILED DESCRIPTION

[0175] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0176] Example 1 Alpaca immunization and antibody library construction

[0177] 1. Alpaca immunization and titer testing

[0178] Human serum albumin HSA (purchased from Sigma-Aldrich, catalog number: A3782) was emulsified and used to immunize alpacas. 1 mg of human serum albumin was used for each immunization, and the immunization was performed once every 2 weeks for a total of three times. From the second immunization, serum was collected one week after the immunization to test the titer.

[0179] The serum titer test method is:

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

[0181] 2) Wash the plate 3 times with PBST (PBS containing 0.1% Tween20, pH=7.4). Add 200 μL skim milk powder to each well, leave at room temperature for 1 hour, and wash the plate 3 times with PBST;

[0182] 3) Sample addition: Dilute the immunized serum and negative serum by 1000-fold, 3000-fold, 9000-fold, 27000-fold, 81000-fold, and 243000-fold, respectively, and add 100 μL of each to the sealed wells, place at room temperature for 1 h, and wash the plate 3 times with PBST;

[0183] 4) Secondary antibody: Add 100 μL of HRP-labeled Goat Anti-Alpaca IgG to each well, leave at room temperature for 1 hour, and wash the plate three times with PBST;

[0184] 5) TMB color development: Add 100 μL / well of TMB color development solution to the ELISA plate and color development for 15 min; add 50 μL / well of stop solution, place the ELISA plate into the ELISA reader, and read the light absorption value at 450 nm (OD450).

[0185] The serum titer detection after the second and third alpaca immunization is shown in Table 1. It can be seen that after the serum was diluted 243,000 times, the signal value of binding to human serum albumin was above 0.59, which met the library construction standard. The peripheral blood of the alpaca after the third immunization was used for phage library construction.

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

[0187]

[0188] 2. Phage library construction

[0189] Peripheral blood was drawn from alpacas, and total RNA was extracted from the isolated alpaca PBMC using Trizol, and reverse transcription of cDNA was performed. The variable region VH fragment (about 700 bp in length) was amplified twice using nanoantibody-specific primers. The above-obtained fragments and the pcomb3X vector were digested with SfiI enzyme, and then mixed in appropriate proportions and connected with T4 ligase. After connection, it was used for electroporation of XL1-Blue competent cells. According to the colony growth of the dilution of competent cells on the resistance-containing plate, the transformation library capacity of the alpaca was calculated to be 6.24×10 8 .

[0190] Example 2 Screening of antibody library to obtain antibodies that cross-bind to human, mouse, and cynomolgus monkey albumin

[0191] 1. Phage library screening

[0192] The screening process is:

[0193] 1) Coating antigens human serum albumin HSA and mouse albumin MSA (purchased from Sigma-Aldrich, catalog number: A3559), and setting up a negative control (coating the ELISA plate with 3% skim milk powder), incubate at room temperature for 1 hour. Discard the blocking buffer and wash with PBST.

[0194] 2) Add about 5×10 12 pfu library was incubated with antigen at 37°C for 2 hours, and the plate was washed with 0.1% PBST.

[0195] 3) Elution was performed with low pH glycine-HCl, and then neutralization was performed with Tris-HCl until the pH was 7.4.

[0196] 4) The eluate was mixed with E. coli XL1-Blue and incubated at 37°C with shaking.

[0197] 5) After adding phage, culture at 30°C overnight and collect the supernatant.

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

[0199] 7) For the second round amplification library obtained under the condition of coating with MSA, an antigen binding ELISA test was performed. In the ELISA experiment, CBS buffer was used to coat HSA and MSA respectively, and the ELISA test results are shown in Table 2. It can be seen that phages that can strongly bind to HSA and MSA were also obtained under this enrichment condition.

[0200] Table 2 Detection results of the second round of phage library obtained by enrichment under MSA coating conditions combined with HSA and MSA

[0201]

[0202] Summary: The antigen binding ELISA of the second-round phage library obtained by MSA enrichment showed positive binding to HSA and MSA, indicating that phages binding to HSA and MSA were enriched, and binding screening at the phage monoclonal level can be carried out.

[0203] 2. Phage monoclonal screening and sequencing

[0204] The experimental process is:

[0205] 1) 96 monoclonal colonies were selected from the second round phage library enriched by coating with MSA for phage expression and coated with HSA, MSA and monkey albumin for ELISA binding activity detection.

[0206] 2) The coating antigens were HSA, MSA, and monkey albumin. The protein concentration during coating was 0.5 μg / mL and the coating was carried out at 4°C overnight.

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

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

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

[0210] 6) Add 200 μL / well of TMB colorimetric solution, color for 20 minutes, add 50 μL / well of stop solution, and measure OD450 with an ELISA reader.

[0211] From the 96 phage clones of the second round phage library enriched by MSA, clone number 3005 was selected, and the ELISA test 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 is obtained. Among them, the amino acid sequence of the CDR region (CDR-H1, CDR-H2, CDR-H3) of the preferred anti-albumin nanobody 3005 is shown in Table 4, the nucleic acid sequence of the CDR region is shown in Table 5, the amino acid sequence of the antibody heavy chain variable region (VH) is shown in Table 6, and the nucleic acid sequence is shown in Table 7.

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

[0216]

[0217] Table 5 Nucleic acid sequences encoding the CDR regions of anti-albumin nanobody 3005

[0218]

[0219] Table 6 Anti-albumin Nanobody 3005 amino acid sequence

[0220]

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

[0222]

[0223] Example 3 Detection of the activity of anti-albumin candidate antibodies binding to albumins from different species

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

[0225] The candidate antibody is expressed in mammalian cells, and the antigen binding activity test is performed after protein purification to verify the activity of the candidate antibody in binding to albumin at the protein level.

[0226] By gene synthesis, expression plasmids of His-tagged antibody 3005 and positive control antibody Alb Nb were constructed, 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] The tPA secretion signal peptide was added to the N-terminus of the above antibody: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36), the 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 the restriction sites NheI and XbaI. After the plasmid sequencing was correct, it was extracted in large quantities without endotoxin, transient expression in suspended 293F cells was carried out, and affinity purification was performed using a Ni column.

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

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

[0230] Amino acid sequence of IgG1 Fc:

[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 sequences encoding anti-albumin nanobody-Fc fusion protein

[0238]

[0239]

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

[0241] 2. Detection of albumin binding activity of anti-albumin candidate antibodies

[0242] The bio-layer interferometry (BLI) method was used to test the affinity of monovalent nanoantibodies binding to albumins of different species. Eight NTA sensors were used to bind to the same his tag monovalent nanoantibody (concentration of 1 μg / mL), and the antigens were human serum albumin HSA (Baxter AG), mouse albumin MSA (purchased from: Equitech-bio, catalog number: MSA62-1000) and rat albumin Rat albumin (purchased from: abcam, catalog number: ab198656). The concentration gradient of the antigen in the BLI experiment was set to 200, 100, 50, 25, 12.5, 6.25, 3.125 nM, and the antigen and antibody bound for 180 seconds and dissociated for 360 seconds.

[0243] Table 10 shows the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of monovalent nanobody 3005 with his tag binding to albumin from different species after "1:1" model fitting. D ).

[0244] Table 10 Binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K) of nanobody 3005 binding to albumin from different species D )

[0245]

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

[0247] The ELISA method was used to test the activity of 3005 binding to albumin of different species. The experimental method was:

[0248] The antigen was diluted with ELISA coating buffer, the final concentration of human serum albumin HSA (Baxter AG) was 1 μg / mL, the final concentration of mouse albumin MSA (purchased from: Equitech-bio, catalog number: MSA62-1000) was 2 μg / mL, and the final concentration of rat albumin (purchased from: abcam, catalog number: ab198656) was 2 μg / mL. The antigen dilution was added to the ELISA plate, 100 μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder, PBST (PBS containing 0.1% Tween20, pH = 7.4) was added to the candidate antibody 3005-Fc and the gradient dilution of the positive control antibody Alb Nb-Fc (0.0001, 0.001, 0.01, 0.1, 1, 10, 100 nM), incubated at 37°C for 1 hour, and HRP-labeled goat anti-human IgG Fc antibody (purchased from: Abbkine, item number: A21050) was added after washing the plate, and incubated at room temperature for 45 minutes. After washing the plate, 100 μL TMB (purchased from: Tiangen 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 with an enzyme marker.

[0249] The ELISA test results of antibody 3005-Fc and Alb Nb-Fc binding to albumin from different species are as follows Figure 2 The EC50 of the binding ELISA experiments are summarized in Table 11.

[0250] Table 11 Summary of EC50 of ELISA experiments of antibody 3005-Fc and positive control Alb Nb-Fc binding to albumin from different species

[0251] protein HSA binding EC50 (nM) Binding to MSAEC50 (nM) Binding to rat albumin EC50 (nM) 3005-Fc 0.24 0.23 0.40 Alb Nb-Fc 0.19 0.27 Weak Binding

[0252] The results showed that antibody 3005-Fc could efficiently bind to HSA and MSA, and the binding activity was similar to that of the positive control Alb Nb-Fc. The positive control antibody Alb Nb-Fc had a very weak binding activity to rat albumin, but antibody 3005-Fc had a good binding activity to rat serum albumin.

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

[0254] 1. Humanization transformation, expression and purification of candidate antibodies

[0255] Humanization was performed by transplanting the CDR region of the antibody. The candidate nanobody 3005 (SEQ ID NO: 4) was used as the starting antibody to perform humanization of the antibody. The homology between the candidate antibody 3005 sequence and the human antibody sequence was first compared through the antibody databases IGBLAST and IMGT, and then the human antibody framework region sequence with high homology was combined with the three CDR region sequences of the candidate antibody 3005 (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) 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 coding DNA sequence was obtained by gene synthesis and then linked to the expression vector pCDNA3.1(+) through the restriction sites NheI and XbaI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin, transiently expressed in suspended 293F cells and affinity purified using a Ni column.

[0257] The amino acid sequence of the humanized modified 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 3005 humanized nanobody

[0259]

[0260] Table 13 Nucleic acid sequences encoding 3005 humanized modified nanobodies

[0261]

[0262]

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

[0264] 2. Binding activity detection of humanized antibodies

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

[0266] In the experiment, mouse albumin MSA (coated protein concentration is 5μg / mL) was coated in the ELISA plate, 100μL / well, coated overnight at 4℃. After blocking with 5% skim milk powder, PBST (PBS containing 0.1% Tween20, pH=7.4) was used to prepare the dilutions of the His-tagged protein to be tested (0.0001, 0.001, 0.01, 0.1, 1, 10, 100nM). The proteins to be tested were the original antibody 3005 and four humanized antibodies. After the protein dilutions were added to the ELISA plate, incubated at 37℃ for 1 hour, and after washing the plate, HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL TMB (purchased from Tiangen Biochemical, catalog 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 at 450 nm (OD450) was measured using an enzyme reader.

[0267] The ELISA test results of antibody 3005 and its humanized antibody binding to MSA are as follows Figure 4 The EC50 of the binding ELISA experiments is summarized in Table 14.

[0268] Table 14 Summary of EC50 and maximum binding values ​​of ELISA experiments of antibody 3005 and its humanized antibodies binding to MSA

[0269] Antibody Binding to MSA EC50 (nM) Maximum 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 showed that the MSA binding activity of humanized antibody 3005Hz6 was similar to that of original antibody 3005. The MSA binding activity of other humanized antibodies was worse than that of 3005Hz6.

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

[0272] In the experiment, HSA or rat albumin (coated protein concentration is 2 μg / mL) was coated in the ELISA plate, 100 μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder, PBST (PBS containing 0.1% Tween20, pH = 7.4) was used to prepare the dilution of the His-tagged protein to be tested (0.0001, 0.001, 0.01, 0.1, 0.3, 1, 10, 100 nM). The proteins to be tested were positive control antibody Alb Nb, as well as original antibody 3005 and humanized antibody 3005Hz6. After the protein dilution was added to the ELISA plate, it was incubated at 37°C for 1 hour. After washing the plate, HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL TMB (purchased from Tiangen Biochemical, catalog 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 at 450 nm (OD450) was measured using an enzyme reader.

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

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

[0275] Antibody Name EC50 of HSA binding (nM) EC50 for binding to rat albumin (nM) 3005 0.31 2.74 3005Hz6 0.26 1.64 Alb 0.66 Weak Binding

[0276] from Figure 5 As shown in Table 15, the activity of humanized antibody 3005Hz6 in binding to HSA and rat albumin is comparable to that of original antibody 3005, and both are stronger than positive control antibody Alb Nb.

[0277] The surface plasmon resonance (SPR) method was used to detect the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (Kd) of the humanized antibody 3005Hz6 binding to HSA and MSA. D). In the experiment, a Biacore 8K instrument (Cytiva) was used to capture the His-tagged humanized antibody 3005Hz6 using a Series S Sensor Chip NTA. The analytes were HSA or MSA at gradient concentrations, and the analytes were diluted with a mobile phase buffer (components: 10mM HEPES, 150mM 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 the antibody 3005Hz6 and HSA are shown in Figure 6 As shown in (A), the binding and dissociation curves of antibody 3005Hz6 and 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) of 3005Hz6 binding to HSA and MSA. D ), the results are shown in Table 16.

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

[0279] Ligand Analytes ka1(1 / Ms) kd1(1 / s) ka2(1 / s) kd2(1 / s) <![CDATA[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] As shown in Table 16, the affinity of humanized antibody 3005Hz6 for binding to HSA and MSA is similar, and the K D About 11nM.

[0281] In combination with Table 10 of Example 3, the K values ​​of the original antibody 3005 and HSA and MSA were determined by BLI method. D The value is about 5 nM, which is consistent with the K in Table 16 D The difference is within 3 times, so it is believed that the affinity of humanized antibody 3005Hz6 for binding to HSA and MSA is similar to that of original antibody 3005.

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

[0283] 1. Risk site mutation of humanized antibody 3005Hz6 and its expression and purification

[0284] Through the analysis of the sequence of 3005Hz6 antibody, its CDR-H2 region (SEQ ID NO: 2) contains an amino acid motif DG that is easy to isomerize aspartic acid D, so the DG amino acid motif in the CDR-H2 region of 3005Hz6 is modified and mutated to DA, DS or EG respectively to reduce the risk of structural and activity changes in the protein. The name of the modified antibody 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 sequences of humanized antibody 3005Hz6 mutants

[0286]

[0287]

[0288] Table 18 Nucleic acid sequences encoding the CDR regions of humanized antibody 3005Hz6 mutants

[0289]

[0290] Table 19 Amino acid sequences of humanized Nanobody 3005Hz6 mutants

[0291]

[0292] Table 20 Nucleic acid sequences encoding humanized Nanobody 3005Hz6 mutants

[0293]

[0294]

[0295] A secretion signal peptide was added to the N-terminus of the antibody 3005Hz6 mutant, and a linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence (see Table 20) was obtained by gene synthesis, and then linked to the expression vector pCDNA3.1 (+) through the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. Transient expression was performed using suspended 293F cells, and the cell culture supernatant was harvested for Ni column affinity purification. The SDS-PAGE results of the purified product are shown in Figure 7 (A), where the supernatant of the culture medium of cells transiently expressing 3005Hz6 (EG) and 3005Hz6 (DS) is marked as Input, the flow-through that cannot bind to the Ni column is marked as FT, and the eluted purified product is marked as E. The supernatant of cells transiently expressing 3005Hz6 (DA) protein was purified by Ni column and then purified by cation exchange. The SDS-PAGE results of the purified protein are shown in Figure 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 the humanized antibody 3005Hz6 mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG). In the ELISA experiment, 2μg / mL HSA was coated, and 3005Hz6 or its mutants were added in a concentration gradient. The antibody level binding to HSA was detected by HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005). The results of the ELISA experiment are shown in Figure 8 , the EC50 of 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 EC50 of HSA binding (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 activities.

[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] Using anti-VEGFA nanobody as the effector molecule, a fusion construct of anti-VEGFA antibody and anti-albumin antibody 3005Hz6 was constructed.

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

[0305] The anti-VEGFA nanobody sequence used in this example was screened from the human VEGFA immune alpaca library, and the construction and screening process of the phage library was as follows: human VEGFA165 (unlabeled, purchased from Yiqiao Shenzhou, catalog number: HPLC-10008-HNAH, hereinafter referred to as VEGFA) was emulsified and immunized with alpacas to construct a phage display library. After 4 rounds of enrichment screening, a single clone of 1 plate with 96 wells was picked to detect the binding activity of phage to VEGFA. After plasmid sequencing and codon translation, the amino acid sequence of the candidate nanobody was obtained for the phage with VEGFA binding activity, among which the amino acid sequence of the CDR region 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 sequence of nanobody V1 and its 9 humanized antibodies is shown in Table 24, and the nucleic acid sequence is shown in Table 25.

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

[0307]

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

[0309]

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

[0311]

[0312]

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

[0314]

[0315]

[0316] Antibodies with high thermal stability are more conducive to antibody production and long-term storage, so the thermal stability Tm values ​​(melting temperature) of different anti-VEGFA nanobodies were compared. The Tm values ​​of anti-VEGFA nanobody V1 and its 9 humanized antibodies were measured using the UNCLE instrument (unchainedlabs), 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(℃) 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] It can be seen from Table 26 that the Tm values ​​of V1-DP and V1-SA1 are relatively high, indicating that the thermal stability of these two antibodies is likely to be better than that of other humanized antibodies.

[0320] Humanized anti-VEGFA nanobodies with higher melting temperatures Tm values: V1-SA1 (SEQ ID NO: 20) and V1-DP (SEQID NO: 21) were selected for the design of fusion constructs with humanized anti-albumin antibody 3005Hz6. According to different tandem sequences, a total of three fusion constructs with GGS as the linker were designed. 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 nanobody

[0322]

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

[0324]

[0325]

[0326] A secretion signal peptide was added to the N-terminus of the fusion construct, and a linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence (see Table 28) was obtained by gene synthesis, and then linked to the expression vector pCDNA3.1 (+) through the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin. Transient expression was performed using suspended 293F cells, and the cell culture supernatant was harvested for Ni column affinity purification. The SDS-PAGE results of the purified product are shown in Fig. 9 The supernatant of the cell culture medium after transient transfection of each protein is marked as Input, the flow-through that cannot bind to the Ni column is marked as FT, and the eluted purified product is marked as E. The molecular weight of the target protein is approximately 28 kDa.

[0327] 2. Activity detection of the fusion construct of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody

[0328] This example tests whether the two Nanobody elements constituting the fusion construct maintain their original activity.

[0329] The activity of anti-albumin nanobody 3005Hz6 binding to HSA in the fusion construct was detected. The detection method was the same as "2. Activity detection of humanized antibodies" in Example 4. The ELISA results are as follows: Fig.10EC50 values ​​are shown in Table 29.

[0330] Table 29 Summary of EC50 of fusion constructs or 3005Hz6 binding to HSA

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

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

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

[0334] Specifically, the ELISA plate was coated with goat anti-human IgG Fc protein (purchased from Solarbio, catalog number: SPA105) at a coating 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 concentration gradient of the fusion construct was incubated with a constant final concentration (0.4 nM) of biotin-modified VEGFA165-Avi-His for 1 hour. VEGFA165-Avi-His was prepared in-house by adding a linker, an Avi tag, and a 6xHis tag to the C-terminus of human VEGFA165. The VEGFA165-Avi-His expression plasmid and the BirA enzyme expression plasmid were co-transfected into 293F cells. During the expression process, the protein was biotin-modified on the Avi tag by BirA enzyme catalysis. The amino acid sequence of VEGFA165-Avi-His is:

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

[0338] The mixture of fusion construct and VEGFA165-Avi-His was added to the ELISA plate with captured VEGFR2-Fc and incubated at 37°C for 1 hour. The biotin signal of VEGFA165-Avi-His was detected with HRP-labeled streptavidin antibody Streptavidin-HRP (purchased from: Sangon Biotechnology, catalog number: D111054-0001), incubated at room temperature for 45 minutes, and after washing the plate, 100 μL / well of TMB color development solution was added to the ELISA plate, and color development was performed for 15 minutes; 50 μL / well of stop solution was added, and the ELISA plate was placed in an ELISA reader to read the light absorption value (OD450) at 450 nm.

[0339] The competitive activity of fusion construct and monovalent nanobody V1-SA1 against VEGFR2 is shown in Fig.11 The IC50 of VEGFR2 competition experiment is summarized in Table 30.

[0340] Table 30 Summary of IC50 of fusion constructs and monovalent nanobody V1-SA1 competing for VEGFR2

[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] As can be seen from Table 30, the VEGFR2 competitive activity of the three fusion constructs formed by 3005Hz6 and V1-SA1 or V1-DP in series is similar to that of the monovalent nanoantibody V1-SA1 (IC50 ratio is 0.81-1.08), indicating that the fusion of anti-VEGFA antibody with 3005Hz6 does not affect the activity of anti-VEGFA. Moreover, 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 of 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 competition for VEGFR2 and binding activity to HSA. This shows that in the fusion construct, the antibody elements binding to different antigens can function independently, and the mutual interference between the antibody elements is small. Therefore, antibodies binding to other antigens can be fused with the anti-albumin humanized antibody 3005Hz6 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 a fusion construct of a bivalent anti-VEGFA nanobody and an anti-albumin nanobody

[0346] Since VEGFA is a homodimer in vivo, in order to improve the efficiency of nanoantibodies binding to VEGFA and to enhance the efficiency of nanoantibodies in blocking VEGFA / VEGFR interactions, anti-VEGFA nanoantibodies are in a divalent form, that is, two nanoantibodies with the same sequence are connected in series. In order to increase the molecular weight of the divalent anti-VEGFA nanoantibody in vivo and extend the half-life of the divalent antibody by binding to albumin, the divalent anti-VEGFA nanoantibody is fused with the humanized anti-albumin antibody 3005Hz6 for expression. The amino acid sequence of the humanized antibody 3005Hz6 and the divalent anti-VEGFA nanoantibody V1-SA1 or V1-DP fusion construct is shown in Table 31, and the nucleic acid sequence is shown in Table 32.

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

[0348]

[0349]

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

[0351]

[0352]

[0353]

[0354] The secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36) was added to the N-terminus of the fusion construct, and the linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody. The coding DNA sequence (see Table 32) was obtained by gene synthesis, and then connected to the expression vector pCDNA3.1 (+) through the restriction sites NheI and XhoI. The plasmid was sequenced correctly and extracted in large quantities without endotoxin, and transiently expressed using suspended 293F cells, and the cell culture supernatant was harvested and purified by Ni column affinity. For the fusion construct 3005Hz6-2V1SA (SEQ ID NO: 33), a tag-free protein was constructed at the same time, and after adding the secretion signal peptide to the N-terminus of the protein, it was connected to the expression vector and transiently expressed in 293F cells.

[0355] V1SA-3005Hz6-V1SA (SEQ ID NO: 32) and 3005Hz6-2V1SA (SEQ ID NO: 33) purified by Ni column were purified by cation exchange, and each component of the purified product was subjected to SDS-PAGE. The results are shown in Fig.12 (A), where the flow-through is labeled FT, the eluted products of V1SA-3005Hz6-V1SA are labeled A1 and A2, and the eluted products of 3005Hz6-2V1SA are labeled A5 and A6. Cation exchange was performed on 3005Hz6-2V1SA without any label, and the purified product staining is shown in Fig.12 (B) Ni column purification was performed on V1DP-3005Hz6-V1DP and 3005Hz6-2V1DP. The purified products were shown in the staining diagram. Fig.12 (C) The molecular weight of the fusion construct is approximately 41 kDa.

[0356] 2. ELISA testing of fusion constructs and association and dissociation rate assays (SPR)

[0357] The ELISA method was used to detect the activity of binding to HSA in the fusion constructs V1SA-3005Hz6-V1SA (SEQ ID NO: 32), 3005Hz6-2V1SA (SEQ ID NO: 33) and 3005Hz6-2V1DP (SEQ ID NO: 35). In the experiment, human serum albumin HSA (Baxter AG) was diluted to 2μg / mL and added to the ELISA plate, 100μL / well, and coated overnight at 4°C. After blocking with 5% skim milk powder and washing with PBST (PBS containing 0.1% Tween20, pH=7.4). The gradient dilutions of the fusion construct containing the His tag were added to the ELISA plate, 100μL / well, incubated at 37°C for 1 hour, and after washing with PBST, HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, catalog number: HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 μL TMB (purchased from Tiangen Biochemical, catalog 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 using an ELISA reader (purchased from Thermo Scientific, model: Multiskan SkyHigh, catalog number: A51119700C).

[0358] ELISA results Fig.13 EC50 values ​​are shown in Table 33.

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

[0360] Fusion construct name EC50 of HSA binding (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 complete biological activity.

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

[0363] In the experiment, a Biacore 8K instrument (Cytiva) was used, and the Series S Sensor Chip NTA was used to capture the His-tagged anti-albumin antibody 3005Hz6 or the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA. The analyte was HSA in gradient concentrations, and the analyte was diluted with a mobile phase buffer (components: 10mM HEPES, 150mM 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 Fig.14 shown.

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

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

[0366] Ligand Analytes ka(1 / Ms) kd(1 / s) <![CDATA[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] As shown in Table 34, the fusion construct 3005Hz6-2V1SA has a high affinity with HSA, K D Similar to the K of antibody 3005Hz6 and HSA D (K D is 12.7-35.8nM).

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

[0369] The affinity of the fusion constructs V1SA-3005Hz6-V1SA (His tag), 3005Hz6-2V1SA (untagged) and the positive control aflibercept to VEGFA was determined by the SPR method. The SPR experiment was carried out using Biacore 8K (Cytiva). The biotin-modified VEGFA165-Avi-His (SEQ ID NO: 40) was captured using the Series S Sensor Chip CAP. The capture level was about 40RU. Each protein was diluted with the mobile phase buffer HBS-EP (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% v / v Tween 20) and a 2-fold protein concentration gradient was set. For 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 30μL / min.

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

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

[0372] Analytes Ligand ka(1 / Ms) kd(1 / s) <![CDATA[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] It can be seen from Table 35 that under the SPR experimental conditions of capturing biotin-modified VEGFA165-Avi-His, the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA have extremely high binding affinity to VEGFA (KD=2.4-5.4pM). 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 approximately 110pM.

[0374] Example 8 Study on the inhibition of HUVEC proliferation activity stimulated by VEGFA by fusion construct

[0375] In this example, primary human umbilical vein endothelial cells (HUVEC) were used to test whether the fusion construct could 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 the proliferation of HUVECs when it was pre-incubated with or without HSA was compared.

[0377] In the experiment, endothelial cell culture medium ECM (purchased from Zhongqiao Xinzhou, catalog number: ZQ-1304) containing 0.5% FBS was used to prepare a human VEGFA165 (purchased from GenScript, catalog number: Z03073) solution with a concentration of 210 ng / mL, and gradient dilutions of the fusion construct 3005Hz6-2V1SA with or without the addition of the same molar concentration of HSA (labeled as: 3005Hz6-2V1SA+HSA and 3005Hz6-2V1SA), and gradient dilutions of the fusion construct V1SA-3005Hz6-V1SA with or without the addition of the same molar concentration of HSA (labeled as: V1SA-3005Hz6-V1SA+HSA and V1SA-3005Hz6-V1SA), and the positive control was the marketed drug aflibercept (Bayer) with the same molar concentration gradient. 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) with a concentration gradient was mixed with 50 μL of VEGFA165 solution. A control without VEGFA165 (expected to have the slowest proliferation) and a control with only VEGFA165 (expected to have the fastest proliferation) 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. Primary HUVEC cells (purchased from the National Stem Cell Transformation Resource Center, catalog number: DFSC-EC-01) were digested and centrifuged, and then resuspended in ECM containing 0.5% FBS. The cells were added to a 96-well plate premixed with the test protein and VEGFA165 at a rate of 50 μL per well. 1.2×10 4 cells, the final concentration of VEGFA165 was 70 ng / mL. 2Culture in an incubator for 68-72 hours. After the culture, add 16 μL CCK-8 staining solution (purchased from: Solarbio, catalog number: CA-1210) to each well and incubate in a 37°C incubator for 2.5-4 hours. Read the absorbance value (wavelength of 450nm) with an enzyme reader to detect the proliferation activity of HUVEC cells. The IC50 value was calculated by four-parameter nonlinear regression analysis using GraphPad Prism 8 software.

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

[0379]

[0380] The results of the experiments on inhibition of HUVEC proliferation stimulated by VEGFA by the fusion constructs V1SA-3005Hz6-V1SA (with or without pre-incubation of HSA) and 3005Hz6-2V1SA (with or without pre-incubation of HSA) are shown as follows: Fig.16 (A) Fig.16 (B) shown.

[0381] The IC50 and maximum inhibition rate of the fusion construct V1SA-3005Hz6-V1SA in inhibiting HUVEC proliferation are shown in Table 36, and the IC50 and maximum inhibition rate of the fusion construct 3005Hz6-2V1SA in 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 the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA can effectively block the downstream signaling pathway activated by human VEGFA165 at the cellular level, inhibiting the proliferation of HUVEC stimulated by human VEGFA165, and their activity is 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 has no significant effect on their HUVEC proliferation inhibitory activity.

[0388] Example 9 Study on the inhibitory activity of fusion construct on VEGFA-induced VEGFR2 phosphorylation

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

[0390] In the experiment, primary HUVEC cells were digested and centrifuged, and then resuspended in ECM medium (containing 5% FBS, 1% epidermal growth factor, and 1% penicillin-streptomycin) (purchased from Zhongqiao Xinzhou, catalog number: ZQ-1304) at 3×10 5 The cells were plated in a 96-well plate at a density of 100 μL / mL, and each well was filled with 100 μL of cell suspension. The cells were cultured overnight in a 37°C, 5% CO2 incubator.

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

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

[0393] The calculation formula for the inhibition rate of VEGFR2 phosphorylation by the tested protein is:

[0394]

[0395] The inhibition rate of VEGFR2 phosphorylation by the fusion construct V1SA-3005Hz6-V1SA 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 phosphorylation of VEGFR2 in HUVEC cells induced by human VEGFA. When the molar concentration of V1SA-3005Hz6-V1SA (incubated with HSA) is 5 times that of VEGFA, the inhibition rate of VEGFR2 phosphorylation can reach more than 90%.

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

[0400] Table 39 Inhibition rate of fusion construct 3005Hz6-2V1SA on VEGFR2 phosphorylation

[0401]

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

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

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

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

[0406] HEK-293 / VEGF / NFAT reporter gene cells were digested and centrifuged, and then resuspended in DMEM containing 1% FBS at a cell density of 5×10 4 80 μL / mL was plated on a 96-well plate with a white bottom (purchased from Costar, catalog number: 3917), and incubated at 37°C and 5% CO 2Incubate overnight in an incubator. The next day, prepare VEGFA165 (purchased from: GenScript, catalog number: Z03073) to 80 ng / mL in DMEM containing 1% FBS, and mix with different concentrations of V1SA-3005Hz6-V1SA (pre-incubated or not pre-incubated with equimolar concentrations of HSA) or 3005Hz6-2V1SA (pre-incubated or not pre-incubated with equimolar concentrations of HSA), and incubate at 37°C for 30 minutes. The negative control is without VEGFA165, and the positive control is only with VEGFA165. Add 20 μL of the mixture (test protein and VEGFA165) to 80 μL of cells and culture in a 37°C incubator for 6 hours. 100 μL of luciferase reporter gene substrate (purchased from Yeasen, catalog number: 11404ES60) was added to each well, and the relative light unit (RLU) of luciferin was measured using a multifunctional microplate reader (purchased from Agilent BioTek, model: Synergy H1).

[0407] Under the above experimental conditions, the results of the activity detection of the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA inhibiting the luminescence of the luciferin substrate are as follows: Fig.17 (A) Fig.17 (B) and the fitted IC50s are shown in Tables 40 and 41, respectively.

[0408] Table 40 Summary of IC50 of fusion construct V1SA-3005Hz6-V1SA in VEGF reporter gene system for inhibition of luciferin luminescence with or without pre-incubation of HSA

[0409] protein IC50 (nM) of VEGF reporter gene assay V1SA-3005Hz6-V1SA 0.24 V1SA-3005Hz6-V1SA+HSA 0.22

[0410] Table 41 Summary of IC50 of fusion construct 3005Hz6-2V1SA in VEGF reporter gene system for inhibition of luciferin luminescence with or without pre-incubation of HSA

[0411] protein IC50 (nM) of VEGF reporter gene assay 3005Hz6-2V1SA 0.24 3005Hz6-2V1SA+HSA 0.25

[0412] Fig.17 The results of Table 40 and Table 41 show that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA can block the activation of the downstream signaling pathway stimulated by VEGFA165 at the cellular level, thereby reducing the expression of the transcription promoter NFAT into the nucleus to activate the Luciferase reporter gene, and their IC50s are all 0.24nM. In this embodiment, whether the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA are pre-bound to HSA has little effect on the blocking activity of the VEGFA downstream signal.

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

[0414] At the same time, the data also show that the antibody elements that bind to different antigens in the fusion construct can function independently, the mutual interference between the antibody elements is small, and the binding to HSA does not affect the biological function of the effector molecules. Therefore, it can be expected that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA can normally block the VEGFA signaling pathway and inhibit endothelial cell proliferation after binding to albumin in vivo.

[0415] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0416] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An anti-VEGFA fusion construct, characterized in that The fusion construct comprises an anti-albumin antibody or an antigen-binding fragment thereof and an anti-VEGFA antibody or an antigen-binding fragment thereof, wherein the anti-VEGFA antibody or an antigen-binding fragment thereof is connected to the anti-albumin antibody or an antigen-binding fragment thereof.

2. The fusion construct according to claim 1, characterized in that The anti-albumin antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein, The amino acid sequence of CDR-H1 comprises SEQ ID NO: 1, or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO: 1; the amino acid sequence of CDR-H2 comprises SEQ ID NO:41 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:41; The amino acid sequence of CDR-H3 comprises SEQ ID NO:3 or an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID NO:

3.

3. The fusion construct according to claim 1 or 2, characterized in that X1X2 in the SEQ ID NO: 41 (GISVX1X2SFLDYADAVKG) represents DS, DA, EG or DG.

4. The fusion construct according to any one of claims 1 to 3, characterized in that: The amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 include any 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.

5. The fusion construct according to claim 1, characterized in that The anti-VEGFA antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; wherein, The amino acid sequence of CDR-H1 comprises SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 16; The amino acid sequence of CDR-H2 comprises SEQ ID NO: 17 or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 17; 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 shown in SEQ ID NO:

18.

6. The fusion construct according to any one of claims 1 to 5, characterized in that: The anti-albumin antibody or antigen-binding fragment thereof or anti-VEGFA antibody or antigen-binding fragment thereof comprises a humanized sequence, wherein the modification site of the humanized sequence is located in a non-CDR region, and preferably, the modification site of the humanized sequence is located in the framework region and / or constant region of the antibody.

7. The fusion construct according to any one of claims 1 to 6, characterized in that: The structures of the anti-albumin antibody or its antigen-binding fragment or anti-VEGFA antibody or its antigen-binding fragment include nanobodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, F(ab')2 fragments, single-chain antibodies or linear antibodies.

8. The fusion construct according to any one of claims 1 to 7, characterized in that: The amino acid sequence of the anti-albumin antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 4, 6-9, 13-15, or has at least 80% identity with any one of SEQ ID NOs: 4, 6-9, 13-15.

9. The fusion construct according to any one of claims 1 to 7, characterized in that: The amino acid sequence of the anti-VEGFA antibody or antigen-binding fragment thereof comprises any one of SEQ ID NOs: 19-28, or has at least 80% identity with any one of SEQ ID NOs: 19-28.

10. The fusion construct according to any one of claims 1 to 9, characterized in that: The anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof are directly or indirectly connected, and preferably, the indirect connection can be through a linker, a functional domain and / or a linker for coupling; Wherein, the linker is selected from a connecting peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, a nucleic acid, a polysaccharide, a fatty chain, biotin, streptavidin or avidin; The functional domain is a combination of one or more of an Fc fragment, serum albumin, cytokine, transferrin or a scaffold protein; the linker for coupling includes a functional group linker, preferably, the functional group linker includes a thiol, an amino, a hydroxyl and / or a carboxyl reactive group, More preferably, the fusion construct may include one or more anti-VEGFA antibodies or antigen-binding fragments thereof, wherein the anti-VEGFA antibodies or antigen-binding fragments thereof 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, Further preferably, the fusion construct comprises any one of SEQ ID NOs: 29-35, or has at least 80% identity with the amino acid sequence shown in SEQ ID NOs: 29-35.

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

12. The nucleic acid according to claim 11, characterized in that The nucleic acid comprises any nucleotide sequence in SEQ ID NO: 62-71 or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity with any nucleotide sequence in SEQ ID NO: 62-71 and having the function of encoding an anti-VEGFA antibody or an antigen-binding fragment thereof.

13. The nucleic acid according to claim 11 or 12, characterized in that The nucleic acid also comprises any nucleotide sequence of SEQ ID NO: 45, 47-50, 56-58 or a degenerate sequence thereof, or a nucleotide sequence that has at least 80% identity with any nucleotide sequence of SEQ ID NO: 45, 47-50, 56-58 and has the function of encoding an anti-albumin antibody or an antigen-binding fragment thereof.

14. The nucleic acid according to any one of claims 11 to 13, characterized in that The nucleic acid comprises any nucleotide sequence in SEQ ID NO: 72-78 or a degenerate sequence thereof, or has at least 80% identity with any nucleotide sequence in SEQ ID NO: 72-78, and has a nucleotide sequence encoding the function of the fusion construct.

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

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

17. A method for preparing the fusion construct according to any one of claims 1 to 10, characterized in that: The preparation method comprises culturing the host cell according to claim 16 to express the fusion construct.

18. Use of the fusion construct according to any one of claims 1 to 10, the nucleic acid according to any one of claims 11 to 14, the vector according to claim 15 or the host cell according to claim 16 in the preparation of a product for treating and / or diagnosing a disease.

19. A product for treating and / or diagnosing a disease, characterized in that The product for treating and / or diagnosing a disease comprises any of the following: A) the fusion construct of any one of claims 1 to 10; B) the nucleic acid according to any one of claims 11 to 14; C) the vector according to claim 15; or D) The host cell according to claim 16.

20. A method for detecting VEGFA, characterized in that: The detection method comprises contacting the sample to be tested with the fusion construct described in any one of claims 1-10, and then detecting the content of the complex formed by VEGFA and the fusion construct. Preferably, the VEGFA is from a mammal, and more preferably, the mammal is from a human, a mouse or a monkey.

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