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WO2025189977A1 - Construction de fusion anti-vegfa, son procédé de préparation et son utilisation - Google Patents

Construction de fusion anti-vegfa, son procédé de préparation et son utilisation

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Publication number
WO2025189977A1
WO2025189977A1 PCT/CN2025/073963 CN2025073963W WO2025189977A1 WO 2025189977 A1 WO2025189977 A1 WO 2025189977A1 CN 2025073963 W CN2025073963 W CN 2025073963W WO 2025189977 A1 WO2025189977 A1 WO 2025189977A1
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WIPO (PCT)
Prior art keywords
antibody
seq
vegfa
antigen
fusion construct
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Pending
Application number
PCT/CN2025/073963
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English (en)
Chinese (zh)
Inventor
黄金亮
刘慧琴
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Quaerite Biopharm Research Beijing Co Ltd
Original Assignee
Quaerite Biopharm Research Beijing Co Ltd
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Publication of WO2025189977A1 publication Critical patent/WO2025189977A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors

Definitions

  • the present invention relates to the field of biomedicine technology, and in particular to an anti-VEGFA fusion construct and a preparation method and application thereof.
  • Vascular endothelial growth factor A is an important member of the VEGF family and a major angiogenic factor that promotes the differentiation, proliferation, migration and invasion of vascular endothelial cells, and regulates 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.
  • VEGF Receptor 2 VEGF Receptor 2
  • 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.
  • VEGF signaling pathway Activation of the VEGF signaling pathway occurs in a variety of diseases, ranging from cancer, autoimmunity to retinal diseases. Inhibiting the VEGF signaling pathway can treat diseases related to abnormal vascular proliferation, such as wet age-related macular degeneration (wAMD).
  • wAMD wet age-related macular degeneration
  • anti-VEGF protein drugs are on the market for the treatment of tumors and diseases related to ocular neovascularization.
  • the first anti-VEGFA protein drug is the monoclonal antibody bevacizumab (Avastin).
  • Other anti-VEGFA protein drugs include ranibizumab (Lucentis), aflibercept, and brolucizumab (Beovu).
  • ranibizumab (Lucentis)
  • aflibercept aflibercept
  • brolucizumab (Beovu)
  • the dosing interval of aflibercept is once every 2 months.
  • the dose of aflibercept has been increased from 2 mg to 8 mg, 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 as a 120 mg/mL protein solution, and the amount of protein injected into the vitreous cavity can reach 6 mg, which is the maximum dose currently available for a single injection.
  • patients received an injection once a month for the first three months, and then once every three months.
  • the visual acuity 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, with a volume of no more than 100 ⁇ L.
  • the longest dosing interval for brolucizumab is three months, and the dosing interval for the 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.
  • Heavy-chain antibodies are IgG2 and IgG3 antibodies that originate from the serum of camelids (camels, dromedaries, and llamas, etc.) and contain only heavy chains but no light chains.
  • 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. It is called a nanobody or single-domain antibody.
  • Nanobodies have a molecular weight of approximately 13kDa, high thermal stability and good water solubility, and have great potential for development into higher-concentration protein preparations.
  • Albumin is the most abundant protein in plasma (at a concentration of approximately 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).
  • Albumin has a molecular weight of 66.5kDa and can bind to the cell surface receptor FcRn (neonatal Fc receptor) under weakly acidic conditions, but has a weaker binding force 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.
  • 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.
  • Anti-albumin antibodies or antigen-binding fragments thereof can bind to albumins of different species with high affinity.
  • the anti-albumin antibodies can be linked to bioactive effector molecules, such as anti-VEGFA antibodies or antigen-binding fragments, to form fusion constructs. Fusion constructs can improve pharmacokinetic properties by binding to albumin, resulting in a longer half-life.
  • the design of the fusion construct's structure, the connection between the various domains, and whether binding to albumin will affect the activity of the bioactive effector molecule still require a great deal of creative work.
  • This application hopes to utilize nanobody technology for the development of anti-albumin antibodies or antigen-binding fragments, as well as the development and design of anti-VEGFA fusion constructs, to provide a superior anti-VEGFA fusion construct.
  • the present application uses nanobodies that bind to VEGFA as the basic active unit, and designs monovalent and bivalent nanobodies to prepare anti-VEGFA fusion proteins with good stability and strong biological activity.
  • the anti-VEGFA antibody or antigen-binding fragment is connected to an antibody or antigen-binding fragment of another antigen, such as an anti-albumin antibody, through a linker to prepare a fusion construct of the anti-VEGFA antibody or antigen-binding fragment and the anti-albumin antibody or antigen-binding fragment.
  • This fusion construct has strong VEGFA binding affinity and biological activity that inhibits VEGFA-stimulated HUVEC proliferation.
  • the functions of the various domains in the fusion construct do not affect each other. Specifically,
  • an anti-VEGFA fusion construct comprising 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 linked to the anti-albumin antibody or an antigen-binding fragment thereof.
  • the anti-albumin antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region.
  • the amino acid sequence of CDR-H1 comprises SEQ ID NO: 1, or an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1;
  • the amino acid sequence of CDR-H2 comprises SEQ ID NO:41 (GISVX 1 X 2 SFLDYADAVKG) or an amino acid sequence 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 that has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 3.
  • X in SEQ ID NO: 41 can be any natural amino acid residue, such as alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), and valine (V).
  • A alanine
  • R arginine
  • N asparagine
  • D aspartic acid
  • C cysteine
  • E glutamic acid
  • G histidine
  • isoleucine (I) leucine
  • L lysine
  • K methionine
  • M phenylalanine
  • T proline
  • P serine
  • S tryptophan
  • X 1 X 2 in SEQ ID NO: 41 represents DS, DA, EG or DG.
  • amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups (see Table 42 for details):
  • amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are arranged in order from N-terminus to C-terminus.
  • the anti-albumin antibody or antigen-binding fragment thereof comprises a heavy chain variable region.
  • the division of the antibody CDR region amino acids adopts the Kabat numbering system.
  • the anti-albumin antibody or antigen-binding fragment thereof comprises a humanized sequence
  • the modification site of the humanized sequence is located in a non-CDR region.
  • the modification site of the humanized sequence is located in the framework region of the variable region and/or the constant region.
  • the structure of the anti-albumin antibody or its antigen-binding fragment includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single-chain antibody (scFv) or a linear antibody.
  • a nanobody a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single-chain antibody (scFv) or a linear antibody.
  • the anti-albumin antibody or antigen-binding fragment thereof can be a single domain antibody or a nanobody.
  • the anti-albumin antibody or antigen-binding fragment thereof is a nanobody.
  • nanobodies Compared with full-length IgG antibodies, Fab, and scFv, nanobodies have a higher molar concentration at the same mass and can bind to more antigen molecules.
  • the anti-albumin antibody or antigen-binding fragment thereof can bind to mammalian albumin.
  • the mammals include humans or non-human mammals, and the non-human mammals may be wild animals, zoo animals, commercial animals, pets, experimental animals, etc.
  • 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.
  • the amino acid sequence of the anti-albumin antibody or its antigen-binding fragment comprises any one of the amino acid sequences in SEQ ID NO: 4, 6-9, 13-15, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NO: 4, 6-9, 13-15.
  • amino acid sequence of the anti-albumin antibody or its antigen-binding fragment is as shown in any one of SEQ ID NOs: 4, 6-9, 13-15.
  • the anti-albumin antibody or antigen-binding fragment thereof can be constructed using any conventional method in the prior art, such as artificial synthesis or eukaryotic or prokaryotic expression.
  • the anti-VEGFA antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region;
  • 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 that has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 18.
  • the anti-VEGFA antibody or antigen-binding fragment thereof comprises a heavy chain variable region.
  • the structure of the anti-VEGFA antibody or its antigen-binding fragment includes a nanobody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single-chain antibody (scFv) or a linear antibody.
  • the anti-VEGFA antibody or antigen-binding fragment thereof can be a single domain antibody or a nanobody.
  • the anti-VEGFA antibody or antigen-binding fragment thereof can be a humanized antibody or a fully human antibody.
  • the anti-VEGFA antibody or antigen-binding fragment thereof comprises a humanized sequence
  • the modification site of the humanized sequence is located in a non-CDR region.
  • the modification site of the humanized sequence is located in the framework region of the variable region and/or the constant region.
  • the anti-VEGFA antibody or antigen-binding fragment thereof is a nanobody.
  • nanobodies Compared with full-length IgG antibodies, Fab, and scFv, nanobodies have a higher molar concentration at the same mass and can bind to more antigen molecules.
  • the anti-VEGFA antibody or antigen-binding fragment thereof can bind to human or monkey VEGFA protein.
  • the amino acid sequence of the anti-VEGFA antibody or its antigen-binding fragment comprises any one of the amino acid sequences in SEQ ID NO: 19-28, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NO: 19-28.
  • the fusion construct comprises one or more anti-VEGFA antibodies or antigen-binding fragments thereof.
  • the fusion construct comprises one or more anti-albumin antibodies or antigen-binding fragments thereof.
  • the anti-VEGFA antibody or antigen-binding fragment thereof is directly or indirectly linked to the anti-VEGFA antibody or antigen-binding fragment thereof.
  • the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof are directly or indirectly linked.
  • the anti-albumin antibody or antigen-binding fragment thereof is directly or indirectly connected to the anti-albumin antibody or antigen-binding fragment thereof.
  • the indirect connection is through a linker, a functional domain and/or a linker for coupling.
  • the linker is selected from connecting peptides, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, PEG, nucleic acids, polysaccharides, fatty chains, biotin, streptavidin or avidin.
  • the functional domain is a combination of one or more of an Fc fragment, serum albumin, cytokine, transferrin or scaffold protein.
  • the linker used for coupling includes a functional group linker.
  • 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.
  • 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 another anti-VEGFA antibody or antigen-binding fragment thereof via a connecting peptide.
  • 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.
  • 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.
  • 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 linked to the N-terminus, C-terminus and/or internal residues of the anti-albumin antibody or antigen-binding fragment thereof.
  • the order of connection of the anti-albumin antibody or antigen-binding fragment thereof and the anti-VEGFA antibody or antigen-binding fragment thereof from N-terminus to C-terminus is as follows:
  • anti-VEGFA antibodies or antigen-binding fragments thereof connecting peptides, anti-albumin antibodies or antigen-binding fragments thereof;
  • 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;
  • connection may not include a connecting peptide.
  • 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.
  • the fusion construct comprises any one of the amino acid sequences in SEQ ID NO: 29-35, or has at least 80% identity with any one of the amino acid sequences in SEQ ID NO: 29-35.
  • the fusion construct comprises an Fc fragment.
  • 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.
  • the fusion construct further comprises a secretory peptide.
  • the secretory peptide is attached to the N-terminus of the fusion construct.
  • the amino acid sequence of the secretory peptide may be SEQ ID NO: 36.
  • the fusion construct further comprises a tag.
  • the tag is connected to the C-terminus of any antibody or antigen-binding fragment thereof, or fusion construct.
  • the fusion construct can inhibit or compete with the binding of other anti-VEGFA antibodies (preferably antibodies that bind to the same or overlapping epitopes as the anti-VEGFA antibodies of the present invention) to VEGFA.
  • anti-VEGFA antibodies preferably antibodies that bind to the same or overlapping epitopes as the anti-VEGFA antibodies of the present invention
  • 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.
  • other anti-albumin antibodies preferably antibodies that bind to the same or overlapping epitopes as the anti-albumin antibodies of the present invention
  • a second aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment described herein, or the fusion construct described above.
  • the nucleic acid comprises DNA and/or mRNA.
  • the nucleic acid is a therapeutic nucleic acid.
  • the nucleic acid is DNA, which encodes the antibody or antigen-binding fragment described herein, or the fusion construct described above.
  • the nucleotide sequence encoding CDR-H1 in the anti-albumin antibody or its antigen-binding fragment comprises any nucleotide sequence in SEQ ID NO: 42 or 51 or a degenerate sequence thereof, or has at least 80% identity with any nucleotide sequence in SEQ ID NO: 42 or 51.
  • the nucleotide sequence encoding CDR-H2 in the anti-albumin antibody or its antigen-binding fragment comprises any one of the nucleotide sequences in SEQ ID NO: 43, 52-54 or its degenerate sequence, or has at least 80% identity with any one of the nucleotide sequences in SEQ ID NO: 43, 52-54.
  • the nucleotide sequence encoding CDR-H3 in the anti-albumin antibody or its antigen-binding fragment 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.
  • the nucleotide sequence encoding CDR-H1 in the anti-VEGFA antibody or its antigen-binding fragment 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.
  • the nucleotide sequence encoding CDR-H2 in the anti-VEGFA antibody or its antigen-binding fragment 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.
  • the nucleotide sequence encoding CDR-H3 in the anti-VEGFA antibody or its antigen-binding fragment 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.
  • the nucleotide sequence encoding the anti-VEGFA antibody or its antigen-binding fragment comprises any one of the 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 the nucleotide sequences in SEQ ID NO: 62-71 and has the function of encoding the anti-VEGFA antibody or its antigen-binding fragment.
  • 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.
  • the nucleotide sequence encoding the above-mentioned fusion construct comprises any one of the nucleotide sequences in SEQ ID NO: 72-78 or its degenerate sequence, or has at least 80% identity with any one of the nucleotide sequences in SEQ ID NO: 72-78, and has a nucleotide sequence encoding the function of the above-mentioned fusion construct.
  • the nucleic acid is mRNA.
  • One or more modification techniques can be used to produce more stable mRNA.
  • Known mRNA modification technologies 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 technologies to effectively protect mRNA.
  • 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 ( ⁇ ) and hypoxanthine.
  • the chemical modification can be selected from the group consisting of 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, dihydrops
  • the third aspect of the present invention provides a vector comprising the above-mentioned nucleic acid.
  • the vector can be expressed in vivo, in vitro or ex vivo.
  • the vector is a recombinant vector such as a prokaryotic expression vector, a viral expression vector or a eukaryotic expression vector.
  • a viral expression vector can be used.
  • the viral expression vector can include viral-derived DNA or RNA sequences for packaging into viruses (such as retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV).
  • viruses such as retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses AAV.
  • the virus and viral expression vector can be used for in vitro, ex vivo, and/or in vivo delivery.
  • the recombinant vector can be included in a delivery vector.
  • the delivery vector includes antibody or Fab as described herein, nucleic acid as described herein, and/or recombinant vector as described herein, and optionally includes liposomes and/or lipid nanoparticles (LNPs).
  • delivery vectors can be introduced into cells by physical delivery methods.
  • the example of physical method includes microinjection, electroporation, and hydrodynamic delivery.
  • nucleic acid as described herein can be wrapped in cationic lipid particles (such as liposomes) by LNPs, and can be delivered to cells relatively easily.
  • 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.
  • the host cell can be a eukaryotic cell or a prokaryotic cell.
  • Eukaryotic cells include animal and plant cells, fungi, etc., such as T cells, yeast cells, HEK293 cells or CHO cells, etc.
  • Prokaryotic cells such as Escherichia coli.
  • 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, wherein the preparation method comprises introducing the above-mentioned nucleic acid or vector into the host cell.
  • 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.
  • 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:
  • the anti-VEGFA antibody or antigen-binding fragment thereof has activity in treating and/or diagnosing diseases.
  • the product can be a diagnostic kit, a drug, or a diagnostic chip, etc.
  • the diseases are diseases related to the VEGFA signaling pathway, and more preferably, they may be tumors, abnormal vascular proliferation, ophthalmic diseases involving angiogenesis (such as fundus vascular disease), and the like.
  • the fusion construct blocks VEGFA-mediated vascular endothelial cell proliferation or inhibits angiogenesis.
  • the present invention provides a method for detecting VEGFA, comprising contacting a sample to be tested with the above-mentioned fusion construct, and then detecting the content of a complex formed between VEGFA and the above-mentioned fusion construct.
  • 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.
  • the VEGFA is from a mammal, more preferably, the mammal is from a human, mouse or monkey.
  • 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 a disease.
  • 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).
  • an antigen eg, VEGFA165
  • the method comprises the step of discarding the complete culture medium of the vascular endothelial cells in the culture plate.
  • 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.
  • the detection is performed after culturing.
  • 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.
  • 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.
  • 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.
  • the culture is carried out in an incubator with 5% CO2 .
  • the culture time is 24-120 hours, preferably 48-96 hours, for example 24, 36, 48, 68, 72, 96, 120 hours, etc.
  • the detection is the detection of the number of living vascular endothelial cells.
  • the tenth aspect of the present invention provides a method for treating and/or preventing a disease, which comprises administering the above-mentioned fusion construct, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned host cell, and the above-mentioned product for treating and/or diagnosing the disease to an individual.
  • 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.
  • 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.
  • 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, osteoblast
  • 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.
  • 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.
  • the product includes a product that blocks VEGFA-mediated vascular endothelial cell proliferation or inhibits angiogenesis.
  • the product described in this application may be a kit, a drug, a chip, an antibody-drug conjugate, etc.
  • the “medicine” of the present invention can be used to treat humans or non-human animals, such as non-human mammals.
  • the medicine can include pharmaceutically acceptable carriers, excipients or salts common in the prior art.
  • the medicine can be administered by any suitable route, such as enteral administration (e.g., oral) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).
  • enteral administration e.g., oral
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous, intradermal, intra-organ, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.
  • the medicine can be in any suitable dosage form, such as enteral administration or parenteral administration, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc.
  • enteral administration or parenteral administration preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills,
  • the drug may contain 0.01-99.5% (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the fusion construct, the nucleic acid, the vector, the host cell, and the like by weight.
  • 0.01-99.5% e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%
  • the 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).
  • a protein concentration of 1-300 mg/mL e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg/mL).
  • the single dose of the drug can be 0.1-1000 mg, for example, 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg.
  • pharmaceutically acceptable means that the pharmaceutical composition neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product.
  • the "method" described in the present invention can be used for the diagnosis and treatment of diseases or for non-disease diagnosis and treatment purposes.
  • 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 that 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.
  • Nanobodies or single-domain antibodies refer to the variable domain of the heavy chain (VHH) of an antibody, which has independent antigen-binding activity.
  • VHH variable domain of the heavy chain
  • 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.
  • Single-chain antibodies are antibodies composed of the variable regions of the heavy chain and the light chain connected by a linker peptide.
  • Fab a monovalent fragment composed of the VL, VH, CL and CH1 domains.
  • Fab' is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain.
  • F(ab')2 is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • Fd Fd fragment consisting of VH and CH1 domains.
  • Fv an Fv fragment consisting of the VL and VH domains of a single antibody arm.
  • dAb fragments are antibody fragments consisting of the VH domain.
  • the “linear antibody” described in the present invention comprises 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.
  • VH-CH1 Fd segment
  • scFv single-chain antibody
  • Fab antibody fragment
  • VHH single-domain antibody
  • VH represents the heavy chain variable region
  • VL represents the light chain variable region
  • CL represents the light chain constant region
  • CH represents the heavy chain constant region
  • the "Fc" region of the present invention contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody.
  • the two heavy chain fragments form a dimer by two or more disulfide bonds in the hinge region and are held together by the hydrophobic interaction of the CH3 domain.
  • the terms “comprising” or “including” as used in the present invention are open-ended.
  • 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.
  • the "homology” or “identity” mentioned in the present invention refers to the fact that when using protein sequences or nucleotide sequences, those skilled in the art can adjust the sequences according to actual work needs so that the 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%,
  • the "humanized antibody” of the present invention refers to an antibody whose framework region and/or constant region portion (e.g., CH region) or all or part of an antibody is encoded by a human antibody gene. In one embodiment of the present invention, the CDR region of the antibody has not been humanized.
  • the "individual" described in the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, a commercial animal, a pet, an experimental animal, etc.
  • the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.
  • treating means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.
  • prevention used in the present invention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in the body.
  • diagnosis refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression or possible future progression of a disease.
  • a "tumor” can be any undesirable cell proliferation (or any disease manifesting as undesirable cell proliferation), neoplasm, or a predisposition or increased risk of undesirable cell proliferation, neoplasm, or tumor. It can be benign or malignant, and can be primary or secondary (metastatic). A neoplasm can be any abnormal growth or proliferation of cells and can be located in any tissue.
  • tissues include adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the cerebrum), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelial cells), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsils, trachea,
  • retina vascular disease refers to a general term for diseases that occur in the retinal arteries or veins, or diseases associated with choroidal neovascularization. These diseases include, but are not limited to, age-related macular degeneration (AMD), diabetic macular edema (DME), diabetic retinopathy, central retinal vein occlusion, pathological myopia, and neovascular glaucoma.
  • AMD age-related macular degeneration
  • DME diabetic macular edema
  • retinopathy central retinal vein occlusion
  • pathological myopia and neovascular glaucoma.
  • the fusion construct inhibited VEGFA-stimulated HUVEC proliferation with an IC50 value similar to that of the receptor-based drug aflibercept.
  • VEGFA-stimulated reporter cells the fusion construct inhibited luciferase expression and luciferin luminescence.
  • HUVEC cells the fusion construct also inhibited VEGFA-activated VEGFR2 phosphorylation.
  • the fusion construct has the potential to inhibit VEGFA-stimulated angiogenesis.
  • VEGFA-stimulated angiogenesis By binding to albumin, it is carried by albumin into specific albumin-rich tissue microenvironments, potentially enhancing the selectivity of the fusion construct for delivery to specific tissues.
  • the fusion construct increases in molecular weight upon albumin binding, it can participate in the binding and recycling mechanisms of albumin to FcRn, thereby extending its half-life. Therefore, the fusion construct of an anti-VEGFA antibody and an anti-albumin antibody has the potential to achieve long-lasting effects in vivo.
  • FIG. 1 Cootisol-stained gel images of His-tagged antibodies Alb Nb and 3005 after purification via Ni column.
  • (A) is the Cootisol-stained gel image of antibody Alb Nb
  • (B) is the Cootisol-stained gel image of antibody 3005.
  • the supernatant of the 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
  • the marker is marked as M.
  • Figure 2 ELISA tests of antibody 3005-Fc and Alb Nb-Fc binding to albumins from different species, (A) is HSA binding test, (B) is MSA binding test, and (C) is rat albumin binding test.
  • Figure 3 A stained gel image of the His-tagged 3005 humanized antibody after Ni column purification.
  • Figure 4 ELISA binding activity test of antibody 3005 and its humanized antibody binding to MSA.
  • Figure 5 ELISA binding activity test of antibody 3005, 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.
  • FIG. 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.
  • FIG. 7 SDS-PAGE gel images of the purified humanized antibody 3005Hz6 mutant proteins 3005Hz6(EG), 3005Hz6(DS) and 3005Hz6(DA).
  • A is an 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, and the eluted purified product is marked as E.
  • (B) is an SDS-PAGE gel image of 3005Hz6(DA).
  • Figure 8 ELISA activity detection of humanized antibody 3005Hz6 and its mutant proteins 3005Hz6(DA), 3005Hz6(DS) and 3005Hz6(EG) binding to HSA.
  • FIG. 9 SDS-PAGE gel images of the fusion constructs V1SA-3005Hz6, 3005Hz6-V1SA and V1DP-3005Hz6 after Ni column purification
  • (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 purification product is marked as E.
  • Figure 10 Fusion construct of humanized antibody 3005Hz6 and monovalent anti-VEGFA nanobody, and ELISA activity detection of humanized antibody 3005Hz6 binding to HSA.
  • FIG. 11 VEGFR2 competition ELISA activity test of fusion constructs and monovalent Nanobody V1-SA1.
  • Figure 12 SDS-PAGE image of the fusion construct of humanized antibody 3005Hz6 and bivalent anti-VEGFA nanobody after purification
  • (A) is a 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 a gel stained image of the unlabeled 3005Hz6-2V1SA protein product
  • (C) is a gel stained image of V1DP-3005Hz6-V1DP
  • (D) is a gel stained image of 3005Hz6-2V1DP.
  • Figure 13 ELISA test of the fusion constructs V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA and 3005Hz6-2V1DP binding to HSA.
  • Figure 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.
  • 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
  • Figure (C) shows the binding and dissociation curves of the antibody 3005Hz6 and HSA.
  • Figure 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.
  • Figure 16 Fusion construct inhibits VEGFA-induced HUVEC proliferation experiment, wherein Figure (A) shows the test results of V1SA-3005Hz6-V1SA, and the premixture of V1SA-3005Hz6-V1SA and HSA (V1SA-3005Hz6-V1SA+HSA), and Figure (B) shows the test results of 3005Hz6-2V1SA, and the premixture of 3005Hz6-2V1SA and HSA (3005Hz6-2V1SA+HSA).
  • Figure 17 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.
  • HSA human serum albumin
  • Serum titer detection method is:
  • TMB color development Add 100 ⁇ L/well of TMB color development solution to the ELISA plate and develop for 15 min. Add 50 ⁇ L/well of stop solution, place the plate in a microplate reader, and read the absorbance at 450 nm (OD450).
  • the serum titer test 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.
  • the screening process is:
  • HSA human serum albumin
  • MSA mouse albumin
  • Antigen binding ELISA of the second-round phage library obtained by MSA enrichment showed positive binding to both 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.
  • Coating antigens HSA, MSA, and monkey albumin.
  • the protein concentration during coating is 0.5 ⁇ g/mL. Coating is carried out at 4°C overnight.
  • Blocking 3% milk powder, 200 ⁇ L/well, room temperature for 1 hour.
  • the amino acid sequence of the candidate Nanobody is obtained.
  • 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
  • the nucleic acid sequence is shown in Table 7.
  • the candidate antibody is expressed in mammalian cells, and after protein purification, the antigen binding activity test is performed to verify the activity of the candidate antibody in binding to albumin at the protein level.
  • the positive control Alb Nb antibody is a nanobody against human serum albumin, and the antibody amino acid sequence comes from SEQ ID NO: 62 in patent US2007/0269422 A1.
  • the tPA secretion signal peptide (MDAMKRGLCCVLLLCGAVFVSPS) (SEQ ID NO: 36) was added to the N-terminus of the above-mentioned antibody.
  • the linker GGGGS (SEQ ID NO: 37) and 6xHis were added to the C-terminus of the antibody.
  • the gene sequence was ligated into the expression vector pCDNA3.1(+) via the restriction enzyme sites NheI and XbaI. After correct plasmid sequencing, the antibody was extracted in an endotoxin-free manner, transiently expressed in suspension 293F cells, and affinity purified using a Ni column.
  • Figures 1(A) and 1(B) show SDS-PAGE results of His-tagged antibodies Alb Nb and 3005 after Ni column purification, demonstrating molecular weights consistent with expectations ( ⁇ 15 kDa).
  • Figure 1(A) the supernatant from the cell culture medium after transient transfection of antibody Alb Nb is labeled Input, the flow-through that failed to bind to the Ni column is labeled FT, and the antibody Alb Nb that bound to the Ni column and was eluted is labeled E.
  • a fusion protein of antibody 3005 and the positive control antibody Alb Nb with Fc was constructed.
  • the tPA secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 36) was added to the N-terminus of the antibody coding sequence through primer design.
  • the amino acid sequence of human IgG1 Fc was added to the C-terminus of the antibody sequence through homologous recombination:
  • 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.
  • Bio-layer interferometry was used to test the affinity of monovalent nanobodies for binding to albumins from different species.
  • Eight NTA sensors were used to bind to the same his-tag monovalent nanobody (at a concentration of 1 ⁇ g/mL).
  • the antigens were human serum albumin (HSA) (Baxter AG), mouse albumin (MSA) (purchased from Equitech-bio, Catalog No. MSA62-1000), and rat albumin (purchased from abcam, Catalog No. ab198656).
  • HSA human serum albumin
  • MSA mouse albumin
  • rat albumin purchased from abcam, Catalog No. ab198656
  • the BLI assay used a gradient of antigen concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM.
  • the binding time was 180 seconds, and the dissociation time was 360 seconds.
  • Table 10 shows the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of the monovalent Nanobody 3005 with a His tag binding to albumins of different species after "1:1" model fitting.
  • the ELISA method was used to test the activity of 3005 binding to albumin from different species.
  • the experimental method was as follows:
  • HSA human serum albumin
  • MSA mouse albumin
  • rat albumin abcam, Catalog No. ab198656
  • the candidate antibody 3005-Fc and a serial dilution of the positive control antibody Alb Nb-Fc were added to the plate in PBST (PBS containing 0.1% Tween 20, pH 7.4) and incubated at 37°C for 1 hour. After washing, HRP-conjugated goat anti-human IgG Fc antibody (Abbkine, Catalog No. A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 ⁇ L of TMB (Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 ⁇ L of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader.
  • PBST PBS containing 0.1% Tween 20, pH 7.4
  • HRP-conjugated goat anti-human IgG Fc antibody (Abbkine, Catalog No. A21050) was added and incubated at room temperature for 45 minutes. After washing, 100 ⁇ L of T
  • Humanization was performed by transplanting the CDR region of the antibody. Using candidate Nanobody 3005 (SEQ ID NO: 4) as the starting antibody, humanization was performed. The sequence of candidate Antibody 3005 was first compared with human antibody sequences using the antibody databases IGBLAST and IMGT. The highly homologous human antibody framework sequences were then combined with the three CDR region sequences of candidate Antibody 3005 (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) to construct a humanized antibody.
  • a secretion signal peptide was added to the antibody's N-terminus, and the linkers GGGGS (SEQ ID NO: 37) and 6xHis were added to the antibody's C-terminus.
  • the coding DNA sequence was synthesized and ligated into the expression vector pCDNA3.1(+) via the restriction enzyme sites NheI and XbaI.
  • the plasmid was sequenced correctly and extracted in an endotoxin-free bulk format. Transient expression was performed in suspension 293F cells and affinity purified using a Ni column.
  • 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.
  • the His-tagged humanized protein was purified by Ni column and separated by SDS-PAGE electrophoresis. The molecular weight was consistent with the expected value ( ⁇ 13 kDa). The staining results are shown in Figure 3.
  • the ELISA method was used to detect the binding activity of the original candidate antibody 3005 and the humanized antibody to mouse albumin MSA.
  • mouse albumin MSA coated protein concentration of 5 ⁇ g/mL
  • the test proteins were the original antibody 3005 and four humanized antibodies. After adding the protein dilutions to the ELISA plate, incubate at 37°C for 1 hour. After washing, HRP-conjugated mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat.
  • 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.
  • HSA or rat albumin coated protein concentration of 2 ⁇ g/mL was coated in an ELISA plate at 100 ⁇ L/well and coated overnight at 4°C.
  • the tested proteins included the positive control antibody Alb Nb, the original antibody 3005, and the humanized antibody 3005Hz6.
  • the protein dilutions were added to the ELISA plate and incubated at 37°C for 1 hour.
  • HRP-conjugated mouse anti-His tag monoclonal antibody purchased from Proteintech, Cat. No. HRP-66005
  • TMB purchased from Tiangen Biochemical, catalog number PA107-01
  • SPR Surface plasmon resonance
  • association and dissociation curves of the antibody 3005Hz6 with HSA are shown in Figure 6(A), and the association and dissociation curves of the antibody 3005Hz6 with MSA are shown in Figure 6(B).
  • the "Two state reaction” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of 3005Hz6 binding to HSA and MSA. The results are shown in Table 16.
  • the humanized antibody 3005Hz6 binds to HSA and MSA with similar affinities, with the KD determined by SPR being approximately 11 nM.
  • the K value of the original antibody 3005 for HSA and MSA, measured by the BLI method, was approximately 5 nM. This value is within 3 times of the K value in Table 16. Therefore, it is believed that the affinity of the humanized antibody 3005Hz6 for binding to HSA and MSA is similar to that of the original antibody 3005.
  • a secretory signal peptide was added to the N-terminus of the antibody 3005Hz6 mutant, and the linkers 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 (see Table 20) and then ligated into the expression vector pCDNA3.1(+) via the restriction enzyme cleavage sites NheI and XhoI.
  • the plasmid was sequenced correctly and extracted in a large amount without endotoxin.
  • Transient expression was performed in suspended 293F cells. The cell culture supernatant was harvested and purified by Ni column affinity purification. The SDS-PAGE results of the purified products are shown in Figure 7(A).
  • the supernatant of the culture medium of cells transiently expressing 3005Hz6(EG) and 3005Hz6(DS) is labeled as Input
  • the flow-through that cannot bind to the Ni column is labeled as FT
  • the eluted purified product is labeled as E.
  • the supernatant of cells transiently expressing 3005Hz6(DA) protein was purified by Ni column and then 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 was 13 kDa.
  • ELISA assays were performed to assess HSA binding activity of the humanized 3005Hz6 antibody mutants, 3005Hz6(DA), 3005Hz6(DS), and 3005Hz6(EG).
  • 2 ⁇ g/mL HSA was coated and a concentration gradient of 3005Hz6 or its mutants was added.
  • HSA binding was detected using an HRP-conjugated mouse anti-His tag monoclonal antibody (purchased from Proteintech, Cat. No. HRP-66005).
  • the ELISA results are shown in Figure 8, and the EC50 values are shown in Table 21.
  • the humanized antibody 3005Hz6 and its mutant proteins have similar HSA binding activities.
  • anti-VEGFA nanobody As the effector molecule, a fusion construct of anti-VEGFA antibody and anti-albumin antibody 3005Hz6 was constructed.
  • the anti-VEGFA nanobody sequences used in this example were screened from a human VEGFA-immunized alpaca library.
  • the phage library construction and screening process was as follows: Human VEGFA165 (untagged, purchased from Sino Biological, Cat. No. HPLC-10008-HNAH, hereinafter referred to as VEGFA) was emulsified and immunized against alpacas to construct a phage display library. After four rounds of enrichment screening, single clones were selected from a 96-well plate and tested for phage binding activity to VEGFA. Phage with VEGFA binding activity were sequenced and codon translated to obtain the amino acid sequences of candidate nanobodies.
  • 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 sequences of Nanobody V1 and its nine humanized antibodies are shown in Table 24, and the nucleic acid sequences are shown in Table 25.
  • V1-SA1 SEQ ID NO: 20
  • V1-DP SEQ ID NO: 21
  • the amino acid sequences are shown in Table 27, and the nucleic acid sequences are shown in Table 28.
  • a secretory signal peptide was added to the N-terminus of the fusion construct, and linkers 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 (see Table 28), 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 products are shown in Figure 9, where 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.
  • the HSA binding activity of the anti-albumin nanobody 3005Hz6 in the fusion construct was tested using the same method as in "2. Activity testing of humanized antibodies" in Example 4.
  • the ELISA results are shown in Figure 10, and the EC50 values are shown in Table 29.
  • the HSA binding activity of the fusion construct is similar to that of 3005Hz6 alone, indicating that the fusion of 3005Hz6 with other antibody fragments does not affect the HSA binding activity of 3005Hz6.
  • the anti-VEGFA antibody can be fused to the N- or C-terminus of the anti-albumin antibody; the different positions do not affect the albumin binding activity of the fusion protein.
  • the activity of the anti-VEGFA nanobody in the fusion construct was detected using a VEGFR2 competition ELISA method, and the activity of the fusion construct was compared with that of the VEGFA antibody alone.
  • VEGFR2 extracellular domain-human IgG1 Fc fusion protein (VEGFR2-Fc, prepared in-house) was added.
  • the amino acid sequence of VEGFR2-Fc is:
  • 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 was co-transfected with a BirA enzyme expression plasmid into 293F cells. During protein expression, the Avi tag was biotinylated by BirA enzyme.
  • the amino acid sequence of VEGFA165-Avi-His is:
  • the mixture of the 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 using HRP-labeled streptavidin-HRP (purchased from Sangon Biotechnology, catalog number: D111054-0001).
  • the plate was incubated at room temperature for 45 minutes. After washing, 100 ⁇ L/well of TMB colorimetric solution was added to the ELISA plate and color was developed for 15 minutes. 50 ⁇ L/well of stop solution was added, and the plate was placed in a microplate reader to read the absorbance at 450 nm (OD450).
  • the VEGFR2 competitive activity of the three fusion constructs composed of 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 anti-VEGFA activity.
  • the anti-VEGFA antibody can be fused to the N-terminus or C-terminus of the anti-albumin antibody without affecting the activity of the anti-VEGFA antibody in the fusion construct.
  • anti-VEGFA nanoantibodies are in a bivalent form, i.e., two nanoantibodies with the same sequence are linked in series.
  • the bivalent anti-VEGFA nanoantibody was fused with the humanized anti-albumin antibody 3005Hz6 for expression.
  • the amino acid sequence of the humanized antibody 3005Hz6 and the bivalent 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.
  • 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 was obtained by gene synthesis (see Table 32) and then ligated into 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 and affinity purified using a Ni column.
  • V1SA-3005Hz6-V1SA (SEQ ID NO: 32) and 3005Hz6-2V1SA (SEQ ID NO: 33) purified by Ni column were subjected to cation exchange purification, and the components of the purified products were subjected to SDS-PAGE.
  • the results are shown in Figure 12 (A), where the flowthrough is labeled FT, the eluted products of V1SA-3005Hz6-V1SA are labeled A1 and A2 components, and the eluted products of 3005Hz6-2V1SA are labeled A5 and A6 components.
  • Cation exchange was performed on 3005Hz6-2V1SA without any tag, and the purified product staining is shown in Figure 12 (B).
  • V1DP-3005Hz6-V1DP and 3005Hz6-2V1DP were purified by Ni column, and the purified product staining is shown in Figure 12 (C).
  • the molecular weight of the fusion construct is approximately 41 kDa.
  • HSA binding activity of the fusion constructs V1SA-3005Hz6-V1SA (SEQ ID NO: 32), 3005Hz6-2V1SA (SEQ ID NO: 33), and 3005Hz6-2V1DP (SEQ ID NO: 35) was assessed by ELISA.
  • Human serum albumin (HSA) (Baxter AG) was diluted to 2 ⁇ g/mL and added to an ELISA plate at 100 ⁇ L/well. The plates were coated overnight at 4°C. The plates were blocked with 5% skim milk powder and washed with PBST (PBS containing 0.1% Tween 20, pH 7.4).
  • a gradient dilution of the His-tagged fusion construct was added to an ELISA plate at 100 ⁇ L/well and incubated at 37°C for 1 hour. The plate was washed with PBST and then HRP-conjugated mouse anti-His tag monoclonal antibody (Proteintech, Catalog No. HRP-66005) was added and incubated at room temperature for 45 minutes. After washing, 100 ⁇ L of TMB (Tiangen Biochemical, Catalog No. PA107-01) was added to each well. After color development at 37°C for 15 minutes, 50 ⁇ L of stop solution was added. The absorbance at 450 nm (OD450) was measured using a microplate reader (Thermo Scientific, Multiskan SkyHigh, Catalog No. A51119700C).
  • 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.
  • the affinity of the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA to HSA was determined by surface plasmon resonance (SPR) method.
  • the experiments were performed using a Biacore 8K instrument (Cytiva) and a Series S Sensor Chip NTA to capture the His-tagged anti-albumin antibody 3005Hz6 or the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA, respectively.
  • the analyte was a gradient of HSA concentrations diluted in a mobile phase buffer (10 mM HEPES, 150 mM NaCl, 0.05% v/v Tween-20, pH 7.4).
  • the binding time between the ligand and the analyte was 150 seconds, and the dissociation time was 900 seconds.
  • the binding and dissociation curves for the ligand and analyte are shown in Figure 14.
  • the “1:1 binding” model was used to fit the association rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of 3005Hz6, V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA with HSA. The results are shown in Table 34.
  • the fusion construct 3005Hz6-2V1SA has a high affinity for HSA, with a K D similar to that of the antibody 3005Hz6 for HSA (K D is 12.7-35.8 nM).
  • V1SA-3005Hz6-V1SA His-tagged
  • 3005Hz6-2V1SA untagged
  • the positive control aflibercept for VEGFA was determined using SPR.
  • SPR experiments were performed using a Biacore 8K (Cytiva).
  • Biotinylated VEGFA165-Avi-His (SEQ ID NO: 40) was captured using a Series S Sensor Chip CAP at a capture level of approximately 40 RU.
  • Each protein was diluted in the mobile phase buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Tween 20) using a 2-fold protein concentration gradient.
  • Kinetic analysis was performed with a 150-second association time for the ligand and analyte, a 900-second dissociation time, and a flow rate of 30 ⁇ L/min.
  • the binding and dissociation curves of V1SA-3005Hz6-V1SA, 3005Hz6-2V1SA, and the positive control aflibercept with VEGFA are shown in Figures 15(A), 15(B), and 15(C), respectively.
  • the binding and dissociation curves were fitted using a "1:1 binding" model to obtain the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant ( KD ). The results are shown in Table 35.
  • 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.
  • HUVEC primary human umbilical vein endothelial cells
  • a human VEGFA165 solution (purchased from GenScript, catalog number: Z03073) at a concentration of 210 ng/mL was prepared using endothelial cell culture medium (ECM) containing 0.5% FBS (purchased from Zhongqiao Xinzhou, catalog number: ZQ-1304).
  • ECM endothelial cell culture medium
  • a gradient dilution of the fusion construct 3005Hz6-2V1SA with or without the addition of HSA at the same molar concentration (labeled as: 3005Hz6-2V1SA+HSA and 3005Hz6-2V1SA, respectively) and a gradient dilution of the fusion construct V1SA-3005Hz6-V1SA with or without the addition of HSA at the same molar concentration (labeled as: V1SA-3005Hz6-V1SA+HSA and V1SA-3005Hz6-V1SA, respectively) were prepared.
  • the positive control was the marketed drug aflibercept (Bayer) at the same molar concentration gradient.
  • test protein concentrations (3005Hz6-2V1SA+HSA, 3005Hz6-2V1SA, V1SA-3005Hz6-V1SA+HSA, V1SA-3005Hz6-V1SA, and aflibercept) were mixed with 50 ⁇ L of VEGFA165 solution.
  • a control without VEGFA165 (expected to result in the slowest proliferation) and a control with VEGFA165 alone (expected to result in the fastest proliferation) were set up.
  • the test protein and VEGFA165 mixture was incubated 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 No.
  • DFSC-EC-01 were digested and centrifuged, then resuspended in ECM containing 0.5% FBS. 50 ⁇ L of cell suspension was added per well to a 96-well plate premixed with the test protein and VEGFA165, with 1.2 ⁇ 10 cells added per well for a final VEGFA165 concentration of 70 ng/mL. The plates were incubated at 37°C, 5% CO2 for 68–72 hours. Following incubation, 16 ⁇ L of CCK-8 staining solution (purchased from Solarbio, Catalog No. CA-1210) was added to each well and incubated at 37°C for 2.5–4 hours. HUVEC cell proliferation activity was measured by absorbance readings (wavelength 450 nm) using a microplate reader. IC50 values were calculated using four-parameter nonlinear regression analysis using GraphPad Prism 8 software.
  • 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.
  • Tables 36 and 37 show that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA were both able to effectively block the downstream signaling pathways activated by human VEGFA165 at the cellular level, inhibiting the proliferation of HUVECs stimulated by human VEGFA165, with activity similar to that of the positive control, aflibercept (a receptor drug). Furthermore, the results showed that whether or not the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA were pre-incubated with HSA had no significant effect on their HUVEC proliferation inhibitory activity.
  • This example uses primary HUVEC cells to verify the inhibitory effect of the fusion construct on VEGFA-induced VGFR2 phosphorylation.
  • HUVEC cells were digested and centrifuged, then resuspended in ECM medium (containing 5% FBS, 1% epidermal growth factor, and 1% penicillin-streptomycin) (purchased from Zhongqiao Xinzhou, Cat. No. ZQ-1304).
  • ECM medium containing 5% FBS, 1% epidermal growth factor, and 1% penicillin-streptomycin
  • the cells were plated at a density of 3 ⁇ 10 5 cells/mL in a 96-well plate, with 100 ⁇ L of the cell suspension per well.
  • the cells were cultured overnight in a 37°C, 5% CO 2 incubator.
  • VEGFA165 and fusion constructs were prepared in ECM medium.
  • Untagged human VEGFA165 purchased from GenScript, Cat. No. Z03073 at a working concentration of 50 ng/mL (1.3 nM) was premixed with varying concentrations of the 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).
  • VEGFA165-free and VEGFA165-only controls were also prepared. The mixture was incubated at 37°C for 30 minutes before being added to HUVEC cells plated overnight and incubated for 5 minutes.
  • the cells were washed once with PBS, and 65 ⁇ L of strong RIPA lysis buffer (purchased from Beyotime, Cat. No. P0013B) containing PMSF was added to each well and lysed on ice for 20 minutes. Transfer the lysate to a 1.5 mL centrifuge tube, centrifuge at 13,000 rpm for 15 minutes (4°C), and collect the supernatant. Add 5 ⁇ Loading buffer, heat at 95°C for 6 minutes, and take the supernatant for western blot to detect VEGFR2 phosphorylation.
  • RIPA lysis buffer purchased from Beyotime, Cat. No. P0013B
  • the anti-GAPDH antibody for detecting the internal reference GAPDH was purchased from Proteintech, catalog number 60004-1-Ig, and the antibody for detecting phosphorylation of tyrosine 1175 of VGFR2 (p-VEGFR2) was purchased from Cell Signaling Technology, catalog number 2478S.
  • the signal intensities of the phosphorylated VEGFR2 (p-VEGFR2) band and the GAPDH band in the immunoblotting experiment 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.
  • V1SA-3005Hz6-V1SA can effectively inhibit human VEGFA-induced VEGFR2 phosphorylation in HUVEC cells.
  • 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%.
  • the inhibition rate of the fusion construct 3005Hz6-2V1SA on VEGFR2 phosphorylation is shown in Table 39.
  • Table 39 shows that 3005Hz6-2V1SA can effectively inhibit human VEGFA-induced VEGFR2 phosphorylation in HUVEC cells.
  • 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%.
  • This example uses a VEGF reporter gene system to evaluate the activity of the fusion construct in blocking the VEGF downstream signaling pathway.
  • 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.
  • VEGFA recognizes and activates the KDR receptor, it initiates the intracellular downstream signaling pathway and activates the transcription factor NFAT to initiate the expression of the luciferase reporter gene. At this time, chemiluminescence can be generated after adding luciferase substrate.
  • HEK-293/VEGF/NFAT reporter cells were digested and centrifuged, then resuspended in DMEM containing 1% FBS at a density of 5 ⁇ 10 cells/mL. The cells were plated in a white-bottomed 96-well plate (Costar, Catalog No. 3917) with 80 ⁇ L per well and incubated overnight at 37°C in a 5% CO2 incubator. The next day, VEGFA165 (GeneScript, Catalog No.
  • Z03073 was prepared at 80 ng/mL in DMEM containing 1% FBS and mixed with varying concentrations of V1SA-3005Hz6-V1SA (with or without preincubation with an equimolar concentration of HSA) or 3005Hz6-2V1SA (with or without preincubation with an equimolar concentration of HSA) and incubated at 37°C for 30 minutes. Negative controls included the absence of VEGFA165, while positive controls included the addition of VEGFA165 alone. 20 ⁇ L of the mixture (test protein and VEGFA165) was added to 80 ⁇ L of cells and incubated at 37°C for 6 hours. 100 ⁇ L of luciferase reporter gene substrate (purchased from Yeasen, Catalog No. 11404ES60) was added to each well, and the relative light units (RLU) of luciferase were measured using a multi-function microplate reader (purchased from Agilent BioTek, Model: Synergy H1).
  • RLU relative light units
  • the fusion constructs can effectively inhibit VEGFA165-induced HUVEC proliferation and VEGFR2 phosphorylation at the cellular level, and inhibit the fluorescein luminescence of VEGF reporter gene cells.
  • 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.
  • the data demonstrate that the antibody components within the fusion constructs, which bind to different antigens, can function independently, with minimal crosstalk between the components. Binding to HSA does not affect the biological function of the effector molecules. Therefore, it is expected that the fusion constructs V1SA-3005Hz6-V1SA and 3005Hz6-2V1SA will function normally in blocking VEGFA signaling and inhibiting endothelial cell proliferation upon albumin binding in vivo.

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Abstract

L'invention concerne une construction de fusion anti-VEGFA. La construction de fusion est formée par liaison d'un anticorps anti-VEGFA ou d'un fragment de liaison à l'antigène de celui-ci avec un anticorps anti-albumine ou un fragment de liaison à l'antigène de celui-ci. La construction de fusion a une forte affinité de liaison VEGFA et une activité biologique pour inhiber la prolifération HUVEC stimulée par VEGFA, et les fonctions des domaines n'affectent pas l'une l'autre. L'invention concerne également un procédé de préparation de la construction de fusion, et son utilisation dans la préparation d'un produit pour le traitement et/ou la prévention de maladies. L'anticorps anti-VEGFA ou le fragment de liaison à l'antigène de celui-ci a une activité de traitement et/ou de diagnostic de maladies.
PCT/CN2025/073963 2024-03-13 2025-01-22 Construction de fusion anti-vegfa, son procédé de préparation et son utilisation Pending WO2025189977A1 (fr)

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CN101031588A (zh) * 2004-06-01 2007-09-05 多曼蒂斯有限公司 药物组合物，融合物和结合物
WO2011116181A1 (fr) * 2010-03-17 2011-09-22 Caris Life Sciences, Inc. Procédés théranostiques et diagnostiques utilisant sparc et hsp90
US20110172398A1 (en) * 2009-10-02 2011-07-14 Boehringer Ingelheim International Gmbh Bispecific binding molecules for anti-angiogenesis therapy
WO2011107507A1 (fr) * 2010-03-03 2011-09-09 Boehringer Ingelheim International Gmbh Polypeptides de liaison bêta-amyloïdes biparatopiques
US9527925B2 (en) * 2011-04-01 2016-12-27 Boehringer Ingelheim International Gmbh Bispecific binding molecules binding to VEGF and ANG2
MX2014001019A (es) * 2011-07-27 2014-05-13 Glaxo Group Ltd Dominios variables singulares anti-vgf fusionados con dominios de fc.
PE20141522A1 (es) * 2011-08-17 2014-11-17 Glaxo Group Ltd Proteinas y peptidos modificados
FR2979346B1 (fr) * 2011-08-23 2013-09-27 Univ Joseph Fourier Nanocorps anti-vcam-1
WO2014189303A1 (fr) * 2013-05-23 2014-11-27 아주대학교산학협력단 Peptide transtumoral spécifique de la neuropiline et protéine de fusion comprenant ce peptide fusionné
EP3630822A1 (fr) * 2017-06-02 2020-04-08 Boehringer Ingelheim International GmbH Polythérapie anticancéreuse
EP3717682A4 (fr) * 2017-11-30 2021-09-01 University of Delhi, South Campus Bibliothèque de fragments d'anticorps et ses utilisations
KR20210142638A (ko) * 2019-02-22 2021-11-25 우한 이지 바이오파마 씨오., 엘티디. Cd3 항원 결합 단편 및 이의 응용
CN114127106B (zh) * 2019-07-19 2024-01-12 神州细胞工程有限公司 人源化抗VEGF Fab抗体片段及其用途
WO2021013061A1 (fr) * 2019-07-19 2021-01-28 神州细胞工程有限公司 Anticorps anti-vegfr2 humanisé et son utilisation
CN114106190B (zh) * 2020-08-31 2025-04-08 普米斯生物技术(珠海)有限公司 一种抗vegf/pd-l1双特异性抗体及其用途
EP4386009A4 (fr) * 2021-08-09 2025-05-14 Yuanpu Biotechnology (Wuhan) Co., Ltd. Polypeptide de fusion bispécifique et son application
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