WO2024131982A1 - Conjugué protéine capsidique-anticorps, virus modifié, combinaison pharmaceutique et utilisation - Google Patents

Conjugué protéine capsidique-anticorps, virus modifié, combinaison pharmaceutique et utilisation Download PDF

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Publication number
WO2024131982A1
WO2024131982A1 PCT/CN2023/141417 CN2023141417W WO2024131982A1 WO 2024131982 A1 WO2024131982 A1 WO 2024131982A1 CN 2023141417 W CN2023141417 W CN 2023141417W WO 2024131982 A1 WO2024131982 A1 WO 2024131982A1
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antibody
cancer
capsid protein
amino acid
drug
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Chinese (zh)
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周德敏
张传领
张缘洁
纪德重
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Ningbo Institute Of Marine Medicine Peking University
Peking University
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Ningbo Institute Of Marine Medicine Peking University
Peking University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention belongs to the field of biomedicine and relates to a capsid protein-antibody conjugate, a modified virus, a drug combination and uses.
  • Gene therapy refers to the transfer of genetic material into the patient's cells to achieve the purpose of treating the disease.
  • gene therapy strategies can be divided into the following categories: 1 gene expression, 2 gene silencing, and 3 gene editing.
  • the vectors used for gene therapy in clinical practice are mainly viral vectors, mainly adenovirus, lentivirus, and recombinant adeno-associated virus vectors (rAAV).
  • rAAV recombinant adeno-associated virus vectors
  • rAAV has the advantages of non-integrated genome, long gene expression time, and weak immunogenicity, and is currently the most commonly used viral vector.
  • rAAV has poor infection selectivity for specific tissues, so the application range of rAAV vectors is narrow.
  • the current modification of rAAV vectors is mainly focused on the following four aspects: 1) Discovering new serotypes from nature to discover targeted viral vectors; 2) Discovering targeted viral capsids through multiple rounds of screening by directed evolution; 3) Predicting and modifying viral capsids through computer methods such as genetic algorithms; 4) Targeted modification of viral vectors through capsid modification strategies.
  • AAV is composed of three capsid proteins, VP1/VP2/VP3, with a ratio close to 1:1:10, and the icosahedron has a total of 60 protein units. That is, the number of capsid proteins is close to 5:5:10.
  • the three proteins VP1, VP2 and VP3 are produced from the same DNA reading frame through mRNA splicing and translation from different start codons, so the 533 amino acids at the C-terminus (i.e., the full length of VP3) of these three proteins are the same.
  • VP1 has a full length of 735 amino acids and a molecular weight of about 87KDa
  • VP2 has a full length of 598 amino acids, which is the same as the 138-735 amino acid sequence of the VP1 protein, and a molecular weight of about 72KDa
  • the VP3 protein has a full length of 533 amino acids, which is the same as the 203-735 amino acid sequence of the VP1 protein, and a molecular weight of about 62KDa.
  • the inventors After in-depth research and creative work, the inventors have prepared a capsid protein-antibody conjugate and a modified adeno-associated virus. The inventors surprisingly found that the capsid protein-antibody conjugate or the modified adeno-associated virus has a changed targeting property and has the potential to be used in the preparation of targeted drugs.
  • the following invention is provided:
  • One aspect of the present invention relates to a capsid protein-antibody conjugate, wherein the capsid protein is inserted with a non-natural amino acid, and the non-natural amino acid is connected to a target head molecule;
  • the targeting head molecule is an antibody.
  • the capsid protein-antibody conjugate wherein the capsid protein is a capsid protein of an adeno-associated virus, for example, one or more selected from VP1 protein, VP2 protein and VP3 protein.
  • the capsid protein in the capsid protein-antibody conjugate, is VP1 protein.
  • the capsid protein-antibody conjugate wherein the capsid protein is VP2 protein.
  • the capsid protein-antibody conjugate wherein the capsid protein is VP3 protein.
  • the capsid protein-antibody conjugate wherein the capsid protein is VP1 protein and VP2 protein.
  • the capsid protein-antibody conjugate wherein the capsid protein is VP1 protein and VP3 protein.
  • the capsid protein-antibody conjugate wherein the capsid protein is VP2 protein and VP3 protein.
  • the capsid protein-antibody conjugate wherein the capsid protein is VP1 protein, VP2 protein and VP3 protein.
  • the non-natural amino acid is one or more, for example, 1-60, 1-50, 1-40, 1-30, 1-20 or 1-10.
  • the non-natural amino acid in the capsid protein-antibody conjugate, is a non-natural amino acid with an azide group, preferably NAEK.
  • NAEK is shown in Formula I below:
  • each non-natural amino acid is connected to a target head molecule, such as an antibody.
  • each non-natural amino acid NAEK is connected to a target head molecule, such as an antibody.
  • the capsid protein-antibody conjugate wherein the insertion of the unnatural amino acid causes the virus to lose at least 80%, at least 90% of its ability to infect cells;
  • the ability to infect cells is completely or substantially lost
  • the cells are A431 cells.
  • the capsid protein-antibody conjugate wherein the position where the non-natural amino acid is inserted is S264+1, D327 or N587+1.
  • the first amino acid at the N-terminus of the VP1 protein is counted as 1;
  • S264+1 means inserting into the adjacent site after S264, that is, between the 264th and 265th amino acids;
  • D327 means mutating the amino acid at the 327th position to a non-natural amino acid;
  • N587+1 means inserting into the adjacent site after N587, that is, between the 587th and 588th amino acids.
  • the antibody in the capsid protein-antibody conjugate, is a nanobody or a single-chain antibody.
  • the antibody in the capsid protein-antibody conjugate, is an anti-tumor antibody.
  • the capsid protein-antibody conjugate wherein the antibody is a nanobody or a single-chain antibody targeting a target selected from the following:
  • EGFR EGFR
  • HER2 TfR
  • PD-1 PD-L1
  • CTLA-4 VEGFR
  • the capsid protein-antibody conjugate wherein the amino acids of the antibody are shown in any one of the sequences of SEQ ID NO:1 to SEQ ID NO:4.
  • the antibody in the capsid protein-antibody conjugate, is directly connected to the non-natural amino acid, or is connected through a coupling pair or a bifunctional linker.
  • the capsid protein-antibody conjugate wherein the conjugate pair is selected from: Spycatcher and DBCO-spy peptide, Biotin-DBCO and mono Streptavidin, Vidal-N-DBCO and Vidal-C;
  • DBCO is linked by reacting with the N3 group on the unnatural amino acid.
  • the capsid protein-antibody conjugate wherein the amino acid sequence of Spycatcher is as shown in SEQ ID NO:5; and/or the amino acid sequence of the spy peptide is as shown in SEQ ID NO:17.
  • the capsid protein-antibody conjugate wherein the coupling pair is Spycatcher and DBCO-VC-spy peptide, wherein DBCO is connected by reacting with the N3 group on NAEK.
  • the capsid protein-antibody conjugate wherein Spycatcher
  • the amino acid sequence is shown in SEQ ID NO:5; and/or the amino acid sequence of the DBCO-VC-spy peptide is shown in SEQ ID NO:19.
  • the capsid protein-antibody conjugate wherein the antibody is directly connected to the Spycatcher or connected through a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO:15.
  • the bifunctional linker in the capsid protein-antibody conjugate, is DBCO-PEG (n) -TCO, wherein n is a positive integer selected from 4 to 20, preferably n is 4; and
  • One or more amino acids in the antibody are substituted by a non-natural amino acid containing a tetrazine group; preferably, the glutamine (Q) at position 82 and/or the asparagine (N) at position 84 of the antibody shown in SEQ ID NO: 1 are substituted by a non-natural amino acid containing a tetrazine group;
  • the non-natural amino acid containing a tetrazine group is as shown in the following formula II:
  • the present invention also relates to a capsid protein-antibody conjugate, wherein the capsid protein is inserted with a non-natural amino acid (e.g., NAEK), and the non-natural amino acid is connected to an antibody, wherein:
  • a non-natural amino acid e.g., NAEK
  • the capsid protein is the capsid protein of adeno-associated virus
  • the non-natural amino acid is NAEK, and the insertion position is S264+1, D327 or N587+1;
  • the antibody is a nanobody or a single-chain antibody targeting a target selected from the following:
  • EGFR EGFR, HER2, TfR, PD-1, PD-L1, CTLA-4 and VEGFR;
  • the antibody and the non-natural amino acid are connected via a coupling pair or a bifunctional linker.
  • the capsid protein-antibody conjugate wherein the amino acids of the antibody are shown in any one of SEQ ID NO:1 to SEQ ID NO:4.
  • the capsid protein-antibody conjugate wherein the coupling pair is Spycatcher and DBCO-spy peptide, wherein DBCO is connected by reacting with the N3 group on NAEK.
  • the capsid protein-antibody conjugate wherein the amino acid sequence of Spycatcher is as shown in SEQ ID NO:5; and/or the amino acid sequence of the spy peptide is as shown in SEQ ID NO:17.
  • the capsid protein-antibody conjugate wherein the coupling pair is Spycatcher and DBCO-VC-spy peptide, wherein DBCO is connected by reacting with the N3 group on NAEK.
  • the capsid protein-antibody conjugate wherein the amino acid sequence of Spycatcher is as shown in SEQ ID NO:5; and/or the amino acid sequence of DBCO-VC-spy peptide is as shown in SEQ ID NO:19.
  • the capsid protein-antibody conjugate wherein the antibody is directly connected to the Spycatcher or connected through a linker; preferably, the amino acid sequence of the linker is as shown in SEQ ID NO:15.
  • the capsid protein-antibody conjugate wherein:
  • the bifunctional linker is DBCO-PEG (n) -TCO, wherein n is a positive integer selected from 4 to 20, preferably n is 4; and
  • One or more amino acids in the antibody are substituted by a non-natural amino acid containing a tetrazine group; preferably, the glutamine (Q) at position 82 and/or the asparagine (N) at position 84 of the antibody shown in SEQ ID NO: 1 are substituted by a non-natural amino acid containing a tetrazine group;
  • the non-natural amino acid containing a tetrazine group is as shown in the following formula II:
  • Another aspect of the present invention relates to a modified virus comprising the capsid protein-antibody conjugate described in any one of the present invention.
  • the modified virus comprises an expression plasmid; preferably, the expression plasmid comprises a multiple cloning site or is inserted with a target gene;
  • the target gene is the herpes simplex virus thymidine kinase gene or a reporter gene, such as a green fluorescent protein reporter gene GFP or a firefly luciferase reporter gene Luciferase.
  • a reporter gene such as a green fluorescent protein reporter gene GFP or a firefly luciferase reporter gene Luciferase.
  • the modified virus comprises pAAV-MCS plasmid, pAAV-RC plasmid and pHelper plasmid, wherein:
  • the pAAV-RC plasmid contains the following mutation sites:
  • the codon corresponding to S264+1, D327 or N587+1 is mutated to the stop codon TAG.
  • the S264+1 site indicates that a TAG codon is inserted between the 264th and 265th amino acids
  • the D327 site indicates that the codon corresponding to the 327th amino acid is mutated to TAG
  • the N587+1 site indicates that a TAG codon is inserted between the 587th and 588th amino acids.
  • the modified virus is also loaded with a target gene; preferably, the target gene is the herpes simplex virus thymidine kinase gene or a reporter gene, such as the green fluorescent protein reporter gene GFP or the firefly luciferase reporter gene Luciferase.
  • the target gene is the herpes simplex virus thymidine kinase gene or a reporter gene, such as the green fluorescent protein reporter gene GFP or the firefly luciferase reporter gene Luciferase.
  • Another aspect of the present invention relates to a pharmaceutical combination, comprising an effective amount of the capsid protein-antibody conjugate described in any one of the present invention or the modified virus described in any one of the present invention, and an effective amount of ganciclovir;
  • the pharmaceutical combination is a fixed combination, for example in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition; or
  • the drug combination is a non-fixed combination, for example, the capsid protein-antibody conjugate or the modified virus and ganciclovir are each in the form of a pharmaceutical composition.
  • Another aspect of the present invention relates to a method for preparing a targeted drug, comprising the step of loading a target gene (eg, a therapeutic gene) onto the modified virus described in any one of the present invention;
  • a target gene eg, a therapeutic gene
  • the targeted drug is a drug for treating or preventing tumors
  • the tumor is one or more selected from mesothelioma, breast cancer, sarcoma, lung cancer, liver cancer, head and neck squamous cell carcinoma, ovarian cancer, gastric cancer, renal cell carcinoma, colon cancer, rectal cancer, melanoma, prostate cancer, bladder cancer, leukemia, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma;
  • the tumor is a tumor that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting tumor tissue; preferably, the tumor tissue is a tumor tissue that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting diseased tissue
  • the targeted drug is a drug targeting nerves, muscles, lungs, eyes or ears.
  • Another aspect of the present invention relates to use of the capsid protein-antibody conjugate described in any one of the present invention or the modified virus described in any one of the present invention in the preparation of targeted drugs;
  • the targeted drug is a drug for treating or preventing tumors
  • the tumor is one or more selected from mesothelioma, breast cancer, sarcoma, lung cancer, liver cancer, head and neck squamous cell carcinoma, ovarian cancer, gastric cancer, renal cell carcinoma, colon cancer, rectal cancer, melanoma, prostate cancer, bladder cancer, leukemia, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma;
  • the tumor is a tumor that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting tumor tissue; preferably, the tumor tissue is a tumor tissue that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting diseased tissue
  • the targeted drug is a drug targeting nerves, muscles, lungs, eyes or ears.
  • the capsid protein-antibody conjugate or the modified virus according to the present invention is used as a carrier for delivering drugs, such as delivering therapeutic genes.
  • capsid protein-antibody conjugate according to any one of the present invention or the modified virus according to any one of the present invention, used for preparing a targeted drug
  • the targeted drug is a drug for treating or preventing tumors
  • the tumor is one or more selected from mesothelioma, breast cancer, sarcoma, lung cancer, liver cancer, head and neck squamous cell carcinoma, ovarian cancer, gastric cancer, renal cell carcinoma, colon cancer, rectal cancer, melanoma, prostate cancer, bladder cancer, leukemia, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma;
  • the tumor is a tumor that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting tumor tissue; preferably, the tumor tissue is a tumor tissue that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting diseased tissue
  • the targeted drug is a drug targeting nerves, muscles, lungs, eyes or ears.
  • Another aspect of the present invention relates to a method for treating or preventing a disease using a targeted drug, comprising the step of administering an effective amount of a targeted drug to a subject in need thereof, wherein the targeted drug has a modified virus of the present invention loaded with a target gene (e.g., a therapeutic gene) as a pharmaceutical ingredient;
  • a target gene e.g., a therapeutic gene
  • the targeted drug is a drug for treating or preventing tumors
  • the tumor is one or more selected from mesothelioma, breast cancer, sarcoma, lung cancer, liver cancer, head and neck squamous cell carcinoma, ovarian cancer, gastric cancer, renal cell carcinoma, colon cancer, rectal cancer, melanoma, prostate cancer, bladder cancer, leukemia, cervical cancer, endometrial cancer, lymphoma and nasopharyngeal carcinoma;
  • the tumor is a tumor that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting tumor tissue; preferably, the tumor tissue is a tumor tissue that highly expresses EGFR, HER2 or TfR;
  • the targeted drug is a drug targeting diseased tissue
  • the targeted drug is a drug targeting nerves, muscles, lungs, eyes or ears.
  • the adeno-associated virus is a recombinant adeno-associated virus (rAAV).
  • the adeno-associated virus is adeno-associated virus type 2 (AAV2 or rAAV2).
  • the term “retargeting” refers to destroying the affinity of the viral vector to its original receptor, and giving the virus new tissue targeting, organ targeting or cell targeting by coupling a new target head molecule.
  • "destroying the affinity of the viral vector to its original receptor” means that the electrostatic interaction between AAV and its initial adhesion receptor HSPG is destroyed by the insertion of non-natural amino acids, so that it loses its adhesion to cells, which is conducive to coupling antibodies (such as nanobodies or single-chain antibodies) at this site to achieve retargeting.
  • the term "targeting ligand” refers to a ligand molecule with targeting properties.
  • a ligand refers to a substance that can bind to a receptor to produce a certain physiological effect.
  • the ligand is usually a signal triggering molecule connected to a specific binding site of a target protein.
  • the ligand connected to the DNA double strand can generally be any small molecule or ion or even a protein (Teif VB, Rippe K. Statistical-mechanical lattice models for protein-DNA binding in chromatin. Journal of Physics: Condensed Matter. 2010, 22(41): 414105.).
  • the target head molecule is an antibody, such as a nanobody or a single-chain antibody.
  • the present invention achieves one or more of the following technical effects (1) to (8):
  • the modified virus of the present invention achieves a good retargeting effect
  • the unnatural amino acid-based method used in the present invention can avoid major changes to the capsid protein
  • the platform constructed by the present invention can facilitate the replacement of target molecules
  • the modified virus retargeting of the present invention is achieved independently of the target gene delivered by AAV;
  • the modified viruses of the present invention can effectively achieve tumor-specific gene delivery in vivo;
  • the modified virus of the present invention better maintains the affinity between the target head molecule and its receptor
  • the modified virus of the present invention has higher infection activity
  • the modified virus of the present invention has a higher packaging efficiency.
  • Figure 1A Schematic diagram of AAV2VP3 protein capsid and non-natural amino acid insertion sites.
  • Figure 1B Statistical graph of the infection activity of wild-type and various mutant AAV2s on A431 cells at equal genome titers.
  • Figure 2A A curve diagram of the affinity of 10E8-spycatcher to EGFR detected by SPR.
  • Figure 2B A curve diagram of the affinity of HB21-spycatcher to hTFR detected by SPR.
  • Figure 2C A curve diagram of the affinity of 5F7-spycatcher to HER2 detected by SPR.
  • Figure 2D Graph showing the affinity of 2RS-spycatcher to HER2 detected by SPR.
  • Figure 2E Flow cytometry analysis of the affinity of 10E8-spycatcher to A431 cells that highly express EGFR.
  • Figure 2F Flow cytometry analysis of the affinity of HB21-spycatcher to U87 cells that highly express hTFR picture.
  • Figure 2G Flow cytometry analysis of the affinity of 2RS-spycatcher to SKBr3 cells that highly express HER2.
  • Figure 2H Flow cytometry analysis of the affinity of 5F7-spycatcher to SKBr3 cells that highly express HER2.
  • FIG. 3A AAV2 N587+1 was validated by Western blot to verify the degree of nanoantibody coupling under different conditions.
  • Figure 3B Statistical graph of the proportion of capsids successfully coupled to nanoantibodies in AAV2 N587+1 in Figure 3A analyzed by Image J.
  • Figure 3C The results of characterization of AAV2 N587+1 unconjugated/conjugated nanoantibodies by immunoelectron microscopy.
  • Figure 4A Results of immunofluorescence confocal characterization of AAV WT and N587+1 unconjugated/conjugated nanoantibodies.
  • Figure 4B Statistical graph of changes in infection activity of S264+1/D327/N587+1 mutation site AAV2 coupled with 10E8-spycatcher at the cellular level.
  • Figure 4C The result of N587+1-GFP coupled nanoantibody specifically infecting A431 cells to express GFP observed under a microscope.
  • Figure 4D The results of Figure 4C were analyzed by fluorescence quantification using Image J.
  • Figure 4E Statistical graph showing the changes in infection activity of N587+1 spy/HB21 on U87 cells by detecting luciferase expression.
  • Figure 4F Statistical graph showing the changes in the infection activity of N587+1 spy/5F7 and N587+1 spy/HB21 on SKBr3 cells by detecting luciferase expression.
  • Figure 5A Western validation of AAV2 N587+1 to show the extent of nanoantibody coupling through vcspy linker under different conditions.
  • Figure 5B Statistical graph showing the changes in the infection activity of N587+1 spy/10E8 and N587+1 vcspy/10E8 on A431 cells by detecting luciferase expression.
  • Figure 6A Luciferase imaging detection results of N587+1 vcspy/10E8 in mice.
  • FIG. 6B Statistical results of in vivo luciferase expression detection in FIG. 5A .
  • FIG. 7A Graph showing the effect of N587+1 vcspy/10E8 -tk on GCV EC50 as measured by cell viability assay.
  • FIG7B Flow cytometry analysis of the effect of N587+1 vcspy/10E8 -HSV-tk on cell apoptosis.
  • FIG. 7C A graph showing the tumor size of mice after administration of N587+1 vcspy/10E8 -HSV-tk.
  • Figure 8A Western validation of the coupling degree between AAV2 N587+1 and different concentrations of nanoantibody-DBCO.
  • Figure 8B Statistical graph showing the changes in the infection activity of N587+1 10E8Q82 on A431 cells under different coupling concentrations by detecting luciferase expression.
  • Example 1 Construction of single-point mutant pAAV-RC plasmid, virus packaging and purification
  • AAV Helper-Free System is a mature three-plasmid recombinant AAV-2 packaging system developed by Stratagene, USA. In this system:
  • pAAV-MCS plasmid is a master plasmid in the AAV Helper Free system, which provides the ITR sequence necessary for AAV packaging and an MCS region with multiple restriction sites (multiple cloning sites that can be used for the insertion of target genes).
  • the pAAV-RC plasmid provides the cap gene for packaging capsid protein and the rep gene for expressing life cycle-related proteins;
  • the pHelper plasmid provides the auxiliary genes necessary for the AAV packaging process, namely the adenovirus VA, E4, and E2A genes.
  • the AAV293 cells specified for packaging in this system are modified from HEK293 and stably express another auxiliary gene - the adenovirus E1 gene.
  • the present invention inserts and subsequently modifies the non-natural amino acid NAEK on the surface of the AAV-2 capsid protein.
  • a point mutation is performed at the corresponding site of the cap gene containing the pAAV-RC plasmid to introduce the stop codon TAG, so as to construct a series of pAAV-RC plasmids capable of expressing different mutant capsid proteins.
  • AAV-2 virus AAV-2 produced by three-plasmid recombinant AAV-2 packaging system
  • a total of 20 sites were screened, designed and selected, namely: S264, S264+1, A266, Q325, Q325+1, D327, G328, R447, S452, S452+1, G453, G453+1, R459, E548, E548+1, S578, R585+1, N587, N587+1 and S662 (the first amino acid at the N-terminus of the VP1 protein is counted as 1).
  • the codons corresponding to the above sites were mutated to the stop codon TAG, where the site X+1 indicates the insertion of a TAG after the codon corresponding to the amino acid at position X.
  • the schematic positions of the original natural amino acid residues at the above sites in the VP1 protein structure are shown in Figure 1A.
  • mutant pAAV-RC plasmids corresponding to the above mutation sites were named pS264, pS264+1, pA266, pQ325, pQ325+1, pD327, pG328, pR447, pS452, pS452+1, pG453, pG453+1, pR459, pE548, pE548+1, pS578, pR585+1, pN587, pN587+1 and pS662, respectively.
  • the inventors used the pAAV-GFP and pAAV-luc plasmids previously constructed in the laboratory (the green fluorescent protein reporter gene GFP or the firefly luciferase reporter gene Luciferase were inserted into the multiple cloning site of pAAV-MCS, respectively).
  • AAV293 cells (Agilent) were plated at a density of 2.8*10 6 cells/dish; half of the solution was replaced before transfection.
  • Opti-MEM 400 ⁇ L/dish*10 dishes
  • the ratio of plasmid pSUPAR-YAV (pSUPAR-YAV, a plasmid expressing the enzyme/tRNA system required for the insertion of unnatural amino acids): pAAV-luc: pAAV-RC: phelper is 10 ⁇ g: 5 ⁇ g: 5 ⁇ g: 5 ⁇ g.
  • the wild type does not add pSUPAR-YAV plasmid, and the mutant pAAV-RC plasmid is the plasmid corresponding to the mutation site (the 20 mutant pAAV-RC plasmids constructed in step 1.1), such as pN587+1.
  • PEI polyethyleneimine, a transfection reagent
  • Mutant AAV2 virus DMEM+10%FBS+1%L-G+1%P-S+1mM NAEK;
  • Wild-type AAV2 virus DMEM+10% FBS+1% L-G+1% P-S;
  • P-S stands for penicillin-streptomycin dual antibody.
  • the AKTA purification system is based on a phosphate buffer system, and a 0.2 ⁇ m filter membrane is used to remove insoluble impurities.
  • Buffer A 20mM phosphate, 100mM NaCl.
  • Buffer B 20 mM phosphate, 1 M NaCl.
  • Buffer C 20 mM phosphate.
  • CsCl was added at a ratio of 10 g/20 ml of virus solution, followed by ultracentrifugation at 263,000 g, 20 ° C, 24 h in a horizontal rotor. After centrifugation, the area containing the virus band was aspirated with a syringe and a long flat needle. The collected virus solution was filtered through a 100 kDa ultrafiltration tube at 4000 g x 30 min, 4 ° C to remove residual CsCl. The ultrafiltration solution was PBS + 0.001% F-68 to reduce virus aggregation.
  • the purified viruses are wild-type AAV2 and 20 mutant AAV2 viruses, among which the mutant AAV2 viruses are named: S264 mutant AAV2, S264+1 mutant AAV2, A266 mutant AAV2, Q325 mutant AAV2, Q325+1 mutant AAV2, D327 mutant AAV2, G328 mutant AAV2, R447 mutant AAV2, S452 mutant AAV2, S452+1 mutant AAV2, G453 mutant AAV2, G453+1 mutant AAV2, R459 mutant AAV2, E548 mutant AAV2, E548+1 mutant AAV2, S578 mutant AAV2, R585+1 mutant AAV2, N587 mutant AAV2, N587+1 mutant AAV2 and S662 mutant AAV2.
  • AAV virus is a DNA virus.
  • DNase I is first used to remove excess DNA in the virus sample.
  • the genomic DNA in the virus is protected by the capsid protein and is not enzymatically hydrolyzed.
  • the high temperature condition in the first step can cause the viral capsid protein to cleave, thereby releasing the internal DNA can then be quantified by qPCR.
  • the inventors used an absolute quantitative method, using the luciferase gene in the AAV genome as a quantitative standard to calculate the number of genome copies successfully encapsidated.
  • the luciferase primer sequence used in this experiment was designed as follows:
  • A431 cells obtained from the National Cell Resource Platform Concord Cell Bank
  • A431 cells are human skin squamous cell carcinoma cells that highly express epidermal growth factor receptor (EGFR) on their cell surface.
  • EGFR epidermal growth factor receptor
  • the culture medium was discarded, and 50 ⁇ L of the luminescent substrate prepared in the Bright-GloTM Luciferase Assay System (Promega) was added to each well, and the chemiluminescence value was immediately detected under a chemiluminometer.
  • the results are shown in Figure 1B.
  • the results show that compared with wild-type AAV2, the infection activity of different mutation sites varies. Among them, after the insertion of non-natural amino acids near sites such as S264+1, D327, and N587+1 (for example, there are also G328, R585+1 and N587, but considering factors such as packaging efficiency, these three sites were not selected), the infection activity of A431 cells was basically lost. From the perspective of retargeting, these sites can be used as alternative sites for further modification of coupling.
  • Example 3 Construction and activity characterization of antibody-spycatcher fusion protein
  • nanobodies or single-chain antibodies targeting different receptors were selected, namely, nanobodies 10E8 targeting EGFR, nanobodies 2RS targeting HER2, nanobodies 5F7 targeting HER2, and single-chain antibodies HB21 targeting TfR.
  • Spycatcher sequences were fused to their C-termini through linkers (GGSGGT, SEQ ID NO: 15) to obtain four fusion proteins, which were named 10E8-spycatcher, 2RS-spycatcher, 5F7-spycatcher and HB21-spycatcher.
  • Spycatcher acts as a bridge to directly form a covalent interaction with the spy peptide of DBCO-spy peptide that can click on the surface of AAV with azide groups, thereby coupling the nanobody to the surface of AAV virus. Because the molecular weight of nanoantibodies is large, it is not easy to couple directly to the surface of AAV. For example, Biotin-DBCO and mono Streptavidin; Vidal-N-DBCO and Vidal-C, etc., the coupling pairs should be able to achieve similar coupling purposes.
  • the aforementioned DBCO represents dibenzocyclooctyne.
  • the nucleic acid sequences encoding the above fusion proteins (SEQ ID NO: 9 to SEQ ID NO: 12) were synthesized respectively, cloned into the pET-21a (+) (laboratory storage) Escherichia coli plasmid expression vector, and transfected into the TransB (DE3) expression competent medium (CD811-02, Beijing Quanshijin Biotechnology Co., Ltd.).
  • the transformed strain was inoculated into 2 ml LB medium containing 100 ⁇ g/ml ampicillin, and then activated overnight at 37°C and 220 rpm.
  • the activated bacterial solution was inoculated into 2 ⁇ YT medium for expansion culture until A600nm reached between 0.6 and 1.0.
  • IPTG Isopropyl ⁇ -d-thiogalactopyranoside
  • the supernatant was collected by centrifugation at 10000g and 4°C for 30 minutes, Ni-NTA His-Bind Resin (Invitrogen) was added, and the supernatant was enriched with His-tagged fusion proteins by shaking at 4°C for 2 hours.
  • washing buffer (20 mM phosphate, pH 7.8, 500 mM NaCl, 50 mM imidazole)
  • the target protein was eluted from His-Bind Resin with elution buffer (20 mM phosphate, pH 7.8, 500 mM NaCl, 500 mM imidazole) and collected and stored to obtain the crude product of the fusion protein.
  • cation exchange chromatography (Resource S, GE Healthcare) was used to further remove impurities.
  • the main elution peak was collected using a 10 KDa centrifugal filter Unit (Millipore), concentrated, and the buffer was replaced with PBS.
  • the affinity of the antibody fused with spycatcher and its receptor is evaluated by surface plasmon resonance technology (SPR).
  • SPR surface plasmon resonance technology
  • Human EGFR, human HER2 and human TfR receptors were respectively fixed on CM5 chips (Biacore) for analysis on the Biacore 8K system (GE Healthcare).
  • the extracellular domains of the three receptors were purchased from Sino-Biological.
  • the experiment was carried out at 25°C using 1 ⁇ PBS-P buffer.
  • the data were analyzed using Biacore 8K evaluation software.
  • the groups in which the injection segments could reach a steady state were analyzed using a 1:1 stable affinity model, while the kinetic model was used to fit the data for groups that showed sufficient curvature in both the binding and dissociation stages (slow binding and slow dissociation).
  • the protein concentrations of the proteins to be analyzed were measured using the BCA method (Pierce).
  • the affinity of the antibody-spycatcher fusion protein to its receptor at the cellular level was evaluated by flow cytometry (cytoflow).
  • A431 cells highly express EGFR, the target of 10E8-spycatcher, U87 cells highly express TfR, the target of HB21-spycatcher, and SKBr3 cells highly express HER2, the target of 2RS-spycatcher and 5F7-spycatcher (National Cell Resource Center Concordia Cell Bank or ATCC). Therefore, these three cells were used for affinity detection of the corresponding antibody-spycatcher fusion protein.
  • Antibody-spycatcher was diluted in series and added to the corresponding 1 ⁇ 10 5 cells (100 ⁇ l PBS) and incubated at room temperature for 30 min. Unbound proteins were then washed with PBS and centrifuged at 4°C, 400 g for 3 min, twice.
  • Example 4 Mutant AAV-2 coupled with antibody-spycatcher and validation
  • mutant AAV2 has been inserted with the non-natural amino acid NAEK with an azide group through the gene codon expansion technology.
  • mutant AAV2 (20 mutant AAV2 prepared in Example 1, here N587+1 mutant AAV2 is taken as an example) through the click reaction of the azide group, incubate at 4°C overnight, and couple with DBCO-spy (20 ⁇ M or 200 ⁇ M) small peptide to make the mutant AAV2 carry the spy tag.
  • the reactants were N587+1 mutant AAV2, DBCO-spy peptide, PBS buffer solution in the reaction system, shaking at 4°C overnight, 60 rpm, and ultrafiltration through a 100KDa ultrafiltration tube to remove impurities after the reaction, at 4000 rpm, 4°C, 30 min/time, for a total of 4 ultrafiltrations.
  • the DBCO-spy peptide is site-specifically coupled to the non-natural amino acid insertion site on the AAV2 capsid, such as N587+1, and the DBCO at the C-terminus of the peptide reacts with the N 3 group on the AAV, thereby connecting to AAV2.
  • DBCO-spy can be more accurately written as spy-DBCO.
  • the amino acid sequence of the spy peptide is AHIVMVDAYKPTKGGGGK (SEQ ID NO: 17). Its function is to click on the surface of the AAV2 capsid as a "handle” or anchor point so that the antibody-spycatcher can be covalently coupled to it.
  • ultrafiltration was performed through a 100KDa centrifugal filter Unit (Millipore) ultrafiltration tube to remove uncoupled small peptides. Then, different concentrations (5 ⁇ M and 100 ⁇ M) of antibody-spycatcher (the spycatcher at the C-terminus participated in the reaction) were added (taking 10E8-spycatcher as an example) and reacted in a 25°C water bath for 1 hour. After completion, 100KDa ultrafiltration was used to remove impurities, and the buffer system was replaced with PBS.
  • Millipore centrifugal filter Unit
  • the spy-spycatcher covalent reaction is based on the following principle: the lysine on the spycathcer protein, with the assistance of Glu at a nearby site, attacks the Asp on the spy tag, thereby forming a covalent bond.
  • the experimental results are shown in Figures 3A to 3B.
  • the results show that the antibody can be successfully coupled to the surface of the AAV capsid through the spy-spycatcher covalent binding pair.
  • the coupling group showed three protein bands corresponding to 30kDa migration (corresponding to the molecular weight of 10E8-spycatcher), indicating that AAV was successfully coupled to the nano antibody 10E8.
  • This example uses immunoelectron microscopy to characterize the successful coupling of mutant AAV2 to nanoantibodies.
  • the purified AAV2 N587+1 and N587+1 spy/10E8 viruses (virus samples prepared in 4.1 above) were added dropwise to the copper grid and incubated at room temperature for 20 minutes to allow the virus particles to adsorb on the copper grid. Then 1% BSA was added dropwise and blocked at room temperature for 10 minutes. After washing with PBS three times, rabbit monoclonal anti-DYKDDDDK (SEQ ID NO: 16) tag antibody (cat No. 14793, CST) was added and incubated at room temperature for 1 hour. Then washed twice with PBS, and then sheep anti-rabbit secondary antibody conjugated with 10nm gold particles (G3779, sigma) was added and incubated at room temperature for 30 minutes. After washing with PBS six times, 2% uranyl acetate was added and negatively stained for 1 minute. The sample was then air-dried and observed on a transmission electron microscope (JEM1400PLUS, Leica).
  • Example 5 Immunofluorescence experiments characterizing AAV-antibody conjugation and cell-level retargeting
  • This example uses immunofluorescence experiments to characterize the successful coupling of AAV-antibodies and to test cell-level retargeting.
  • A431 cells were plated in a confocal dish (NEST) at 1 ⁇ 10 5 cells/dish. After 24 hours, the purified AAV2WT, WT+10E8, N587+1 and N587+1 spy/10E8 virus liquids were added to the confocal dish and incubated at 4°C for 1 hour. After the virus binds to the cell membrane surface, the unbound virus is washed away with PBS. Then fix with 4% paraformaldehyde at room temperature for 20 minutes, and permeabilize with PBST (PBS+0.5% Triton) for 20 minutes. Then wash with PBS twice and block with 5% goat serum at room temperature for 1 hour.
  • PBST PBS+0.5% Triton
  • the virus adsorbed on the cell surface can be effectively detected, and the nanoantibody is also on the cell membrane (red), and the two show obvious co-localization (merge, yellow). This shows that the nanoantibody 10E8 is successfully coupled to the surface of the virus particle N587+1, and can achieve good retargeting.
  • the retargeting ability of the AAV-spy platform was evaluated by detecting the infection activity of AAV-nanoantibodies on target cells.
  • AAV2 mutants S264+1/D327/N587+1 can shield the initial infection activity to varying degrees.
  • these three mutant AAVs expressing luciferase were coupled to the nanoantibody 10E8 through the spy-spycatcher platform.
  • A431 cells were inoculated at 1 ⁇ 104 /well. The density was plated in a 96-well black plate. Virus particles of S264+1/D327/N587+1 without or with nanoantibodies were added to the cells at an MOI of 10K, and the expression of luciferase was measured 48 hours after infection.
  • the GFP-expressing N587+1 virus was coupled to the nanoantibody 10E8 and then infected with A431 cells. After 48 hours, the cells were observed and photographed under a fluorescence microscope. Subsequently, quantitative analysis was performed using Image J. The results showed ( Figure 4C and Figure 4D) that the AAV-spy platform can still effectively achieve retargeting. This indicates that the realization of AAV-spy retargeting is independent of the target gene delivered by AAV.
  • the AAV-spy retargeting platform strategy was further verified.
  • the expressed and purified HB21-spycatcher, 2RS-spycatcher and 5F7-spycatcher were coupled to N587+1 virus particles using the same strategy, and then the corresponding target cells U87 (high expression of TfR) and SKBr3 (high expression of HER2) were infected. Luciferase expression was measured after 48 hours to characterize the cell infection activity. The experimental results are shown in Figures 4E to 4F.
  • the results show that whether it is a single-chain antibody targeting TfR or a nanobody targeting HER2, it can be coupled to N587+1 virus particles using the AAV-spy platform strategy, which can restore the infection activity of the wild type to about 70%.
  • N587+1spy/2RS and N587+1spy/5F7 it can be seen that the effect of retargeting is positively correlated with the affinity of the coupled nanoantibody to the receptor, which further enriches the AAV-spy retargeting platform.
  • 6.1 VC-spy linker can successfully achieve site-specific coupling of AAV-NAEK with nanoantibodies
  • mutant AAV2 can be coupled to nanobodies through DBCO-VC-spy linker.
  • amino acid sequence of VC-spy is AHIVMVDAYKPTKGGGGVXK (SEQ ID NO: 19) As shown, wherein X represents citrulline (Cit).
  • the mutant AAV2 has been inserted with the non-natural amino acid NAEK with an azide group by gene codon insertion technology.
  • the reactants are N587+1 mutant AAV2, different concentrations of DBCO-VC-spy, PBS buffer in the reaction system, and the reaction conditions are shaking at 4°C overnight, 60rpm.
  • ultrafiltration is performed through a 100KDa ultrafiltration tube to remove impurities.
  • the ultrafiltration conditions are 4000rpm, 4°C, 30min/time, and a total of 4 ultrafiltrations.
  • antibody-spycatcher the spycatcher at the C-terminus participates in the reaction, taking 10E8-spycatcher as an example
  • react in a 25°C water bath for 1h by adding different concentrations (5 ⁇ M and 100 ⁇ M) of antibody-spycatcher (the spycatcher at the C-terminus participates in the reaction, taking 10E8-spycatcher as an example), react in a 25°C water bath for 1h.
  • impurities were removed using 100KDa ultrafiltration and the buffer system was replaced with PBS.
  • Example 4.1 After the reaction, the AAV vcspy/10E8 sample was taken and Western Blotting was performed in the same manner as in Example 4.1.
  • the experimental results are shown in Figure 5A.
  • the results show that the nanobody can also be successfully coupled to the AAV capsid surface through the DBCO-VC-spy covalent binding pair (named N587+1 vcspy/10E8 ).
  • the coupled group mainly showed a protein band with a migration amount of 30kDa for VP3 (corresponding to the molecular weight of vcspy-spycather-10E8), indicating that AAV was successfully coupled to the nanobody 10E8.
  • the concentration of DBCO-vcspy different degrees of coupling modification of nanoantibodies can be effectively achieved.
  • the target cell infection activity of N587+1 vcspy/10E8 was detected to determine whether it was improved compared to that of N587+1 spy/10E8 .
  • N587+1 mutant was still used as an example.
  • A431 cells were plated in a 96-well black plate at a density of 1 ⁇ 10 4 cells/well.
  • Virus particles of N587+1 spy/10E8 and N587+1 vcspy/10E8 were added to the cells at an MOI of 10K, and the expression of luciferase was measured 48 hours after infection.
  • Example 7 Detection of retargeting function of AAV-nanoantibodies at animal level
  • This example uses small animal imaging to detect the feasibility of the AAV-vcspy retargeting platform strategy at the animal level.
  • mice (6-8 weeks old) were implanted subcutaneously with A431 cells (1 ⁇ 10 5 cells per animal) in the right flank.
  • mice were injected intratumorally with luciferase-expressing N587+1 or N587+1 vcspy/10E8 (2 ⁇ 10 10 copies/mouse). Animals were imaged on days 5, 7, and 10 after injection. Mice were injected intraperitoneally with 100 ⁇ l of luciferase substrate (Promega) and imaged 10 min later using an IVIS SPECTRUM small animal live imager (PerkinElmer).
  • Example 8 Functional application of AAV-vcspy retargeting platform
  • HSV-tk/GCV Herpes simplex virus thymidine kinase/ganciclovir
  • tk The suicide gene HSV-tk (abbreviated as tk) is expressed to produce thymidine kinase, which can cause the prodrug GCV to undergo monophosphorylation, thereby converting the non-toxic GCV into toxic GCV triphosphate, i.e., a deoxyguanosine triphosphate analog, thereby inhibiting DNA polymerase and destroying DNA synthesis, inducing apoptosis in host cells.
  • GCV triphosphate i.e., a deoxyguanosine triphosphate analog
  • a bystander effect i.e., toxic metabolites produced by cells that can express the tk gene are transferred to neighboring cells, leading to cell death.
  • N587+1 vcspy/10E8 was used to deliver the suicide gene tk into tumor cells in a targeted manner, and then GCV was administered systemically (intraperitoneal injection, 100 mg/Kg) to achieve tumor treatment and expand the functional application of the AAV-vcspy retargeting platform.
  • the AAV-vcspy retargeting platform can effectively enable the N587+1 virus to specifically infect tumor cells, deliver the knockout suicide gene tk, phosphorylate the prodrug GCV, and induce tumor cell apoptosis, providing a new retargeting AAV platform for targeted gene therapy of tumors.
  • A431 cells were inoculated into the right axilla of Balb/C nude mice at 1.5 ⁇ 10 6 cells/mouse.
  • PBS, N587+1-tk or N587+1 vcspy/10E8 -tk were injected peritumorally at a dose of 2 ⁇ 10 10 copies/mouse.
  • GCV was injected intraperitoneally into the mice at a dose of 100 mg/kg for 5 consecutive days, and the tumor size of the mice was observed and measured every day.
  • Example 9 Upgrading and Optimizing the AAV-spy Retargeting Platform to AAV-PEG-nanobody
  • the AAV-spy retargeting platform introduces a spy-spycatcher protein covalent coupling pair, considering its possible immunogenicity is not conducive to the systemic administration of AAV, the coupling method of AAV and nanoantibodies was further studied.
  • the nanobody was optimized and modified in the next step.
  • Another non-natural amino acid with tetrazine (Formula II) was inserted into the nanobody at a fixed point, and then coupled with the AAV inserted into the NAEK at a fixed point through the DBCO-PEG (n) -TCO (n can be selected from a positive integer between 4 and 20, and n is 4 in this experiment) bifunctional linker. This enhances the practicality and flexibility of the AAV retargeting platform, in order to reduce its immunogenicity.
  • This example selects the EGFR-targeting nanobody 10E8 as an example, and the modification methods and conditions of other nanobodies or single-chain antibodies are the same or similar.
  • the mutation sites shown in Table 1 below are selected in the structurally conserved region of the 10E8 protein (SEQ ID NO: 1), wherein the amino acid positions are those in SEQ ID NO: 1.
  • the synthesized nucleic acid sequence containing the codon substitution was connected to the nucleic acid sequence encoding the His tag and cloned into the pET-21a(+) (addgene: #69740-3) Escherichia coli plasmid expression vector, which was co-transfected with the pUltra-MfpylRS plasmid into the TransB (DE3) strain (purchased from Quanshijin, catalog number: CD811-02).
  • the pUltra-MfpylRS plasmid encodes amber codon suppressor tRNA and tetrazine aminoacyl tRNA synthetase.
  • the unnatural amino acid tetrazine can be introduced at a specific site.
  • the transformed strain was inoculated into a medium containing 100 ⁇ g/ml ampicillin and 100 ⁇ g/ml spectinomycin. 2ml LB medium, then cultured at 37°C, 220rpm. The overnight culture was diluted to optical density in 2 ⁇ YT medium, and the culture was cultured at 37°C until A600nm reached about 1.5.
  • UAA tetrazine, synthesized in the laboratory
  • IPTG isopropyl ⁇ -d-thiogalactopyranoside
  • the cells were harvested by centrifugation and resuspended in His-Bind buffer (20mM phosphate, pH 8.0, 500mM NaCl, 20mM imidazole). Proteins were extracted by passing the cells through a Micofluidizer twice under 1200bar and cooling conditions.
  • the site-directed mutant 10E8 protein with non-natural amino acid substituted at position Q82 or N84 was obtained.
  • cation exchange chromatography Resource S, GE Healthcare
  • the main elution peak was collected using a 3KDa centrifugal filter Unit (Millipore), concentrated, and the buffer was replaced with PBS.
  • mutant AAV2 has been inserted with the non-natural amino acid NAEK with an azide group by gene codon insertion technology.
  • the reactants are N587+1 mutant AAV2, different concentrations of 10E8-Q82-DBCO, PBS buffer in the reaction system, shaking at 4°C overnight, 60rpm, and ultrafiltration through a 100KDa ultrafiltration tube to remove impurities after the reaction.
  • the ultrafiltration conditions are 4000rpm, 4°C, 30min/time, and a total of 4 ultrafiltrations.
  • the AAV 10E8Q82 sample was taken and electrophoresed under denaturing conditions by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a 0.45 ⁇ M PVDF membrane (Millipore). Blocked with 5% skim milk at room temperature for 1 hour. Then incubated with rabbit polyclonal anti-AAV2VP1/VP2/VP3 antibody at 4°C overnight. Wash the membrane three times with TBST buffer, and then incubate with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 1 hour, 80rpm. After washing the membrane three times, imaging analysis was performed in a high-sensitivity chemiluminescent gel imaging analysis system (ChemiDoc XRS System, Bio-rad). The coupling ratio was calculated by grayscale analysis using ImageJ software and then quantified.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the experimental results are shown in Figure 8A.
  • the results show that the antibody can be successfully coupled to the surface of the AAV capsid (named N587+1 10E8Q82 ) through the DBCO-PEG 4 -TCO covalent binding pair.
  • the coupled group mainly showed a protein band with a migration volume of 15kDa for VP3 (corresponding to the molecular weight of 10E8-Q82), indicating that AAV was successfully coupled to the nanoantibody 10E8.
  • concentration of 10E8-Q82-DBCO different degrees of coupling modification of nanoantibodies can be effectively achieved.
  • the retargeting ability of the AAV-PEG-nanobody platform was evaluated by detecting the infection activity of AAV-nanobody (N587+1 10E8Q82 prepared in 9.2 above) on target cells.
  • N587+1 mutant was still used as an example.
  • A431 cells were plated in a 96-well black plate at a density of 1 ⁇ 10 4 cells/well.

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Abstract

La présente invention appartient au domaine de la biomédecine, et concerne un conjugué protéine capsidique-anticorps, un virus modifié, une combinaison pharmaceutique et une utilisation. Plus particulièrement, la présente invention concerne un conjugué protéine capsidique-anticorps. Un acide aminé non naturel est inséré dans la protéine capsidique, et une molécule cible telle qu'un anticorps est reliée à l'acide aminé non naturel. Le virus modifié selon la présente invention a de bonnes perspectives d'application.
PCT/CN2023/141417 2022-12-23 2023-12-25 Conjugué protéine capsidique-anticorps, virus modifié, combinaison pharmaceutique et utilisation Ceased WO2024131982A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2024220714A1 (fr) * 2023-04-18 2024-10-24 Regents Of The University Of Minnesota Virus adéno-associés à capside modifiée

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CN104592364A (zh) * 2013-10-30 2015-05-06 北京大学 定点突变和定点修饰的腺相关病毒、其制备方法及应用
WO2017075615A1 (fr) * 2015-10-29 2017-05-04 Bullet Biotechnology, Inc. Intermédiaires de particule pseudo-virus, agents y étant fixés, leurs procédés de fabrication et leurs utilisations
CN110799524A (zh) * 2017-06-27 2020-02-14 瑞泽恩制药公司 向性修饰的重组病毒载体及其用于将遗传材料靶向引入人细胞内的用途
WO2022101363A1 (fr) * 2020-11-11 2022-05-19 European Molecular Biology Laboratory Particules virales modifiées destinées à la thérapie génique
WO2022253974A1 (fr) * 2021-06-02 2022-12-08 Uniqure Biopharma B.V. Vecteurs de virus adéno-associés modifiés pour se lier à une lipoprotéine de haute densité

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Publication number Priority date Publication date Assignee Title
CN104592364A (zh) * 2013-10-30 2015-05-06 北京大学 定点突变和定点修饰的腺相关病毒、其制备方法及应用
WO2017075615A1 (fr) * 2015-10-29 2017-05-04 Bullet Biotechnology, Inc. Intermédiaires de particule pseudo-virus, agents y étant fixés, leurs procédés de fabrication et leurs utilisations
CN110799524A (zh) * 2017-06-27 2020-02-14 瑞泽恩制药公司 向性修饰的重组病毒载体及其用于将遗传材料靶向引入人细胞内的用途
WO2022101363A1 (fr) * 2020-11-11 2022-05-19 European Molecular Biology Laboratory Particules virales modifiées destinées à la thérapie génique
WO2022253974A1 (fr) * 2021-06-02 2022-12-08 Uniqure Biopharma B.V. Vecteurs de virus adéno-associés modifiés pour se lier à une lipoprotéine de haute densité

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024220714A1 (fr) * 2023-04-18 2024-10-24 Regents Of The University Of Minnesota Virus adéno-associés à capside modifiée

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