WO2020067418A1 - Method for producing capsid protein of adeno-associated virus, and use thereof - Google Patents

Method for producing capsid protein of adeno-associated virus, and use thereof Download PDF

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WO2020067418A1
WO2020067418A1 PCT/JP2019/038137 JP2019038137W WO2020067418A1 WO 2020067418 A1 WO2020067418 A1 WO 2020067418A1 JP 2019038137 W JP2019038137 W JP 2019038137W WO 2020067418 A1 WO2020067418 A1 WO 2020067418A1
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adeno
associated virus
protein
antibody
capsid protein
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Japanese (ja)
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妃佐子 八浦
正克 西八條
佑介 平山
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株式会社カネカ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for producing a capsid protein of an adeno-associated virus.
  • the present invention further relates to a method for producing an anti-adeno-associated virus antibody using the adeno-associated virus capsid protein produced by the above-described production method.
  • Adeno-associated virus is a virus that does not have a helper-dependent envelope classified into the small parvoviridae family of dependent viruses that infect humans and primates.
  • the adeno-associated virus vector can introduce a gene into any of proliferating / non-proliferating cells, and can express a target gene for a long time in non-dividing cells.
  • AAV vectors are suitable for gene transfer into animal individuals because of their lower immunogenicity compared to adenovirus vectors and retrovirus vectors.
  • AAV vectors are widely used as safe and easy-to-handle virus vectors because they are non-pathogenic viruses. In recent years, research and development have been particularly advanced as viral vectors for gene therapy.
  • Non-Patent Document 1 describes that the association between the Rep protein of type 2 adeno-associated virus (hereinafter also referred to as AAV2) and the VP capsid protein occurs even in the absence of a packageable genome.
  • AAV2 adeno-associated virus
  • Non-Patent Document 2 describes that an AAV capsid protein is expressed and assembled in yeast.
  • Non-patent Document 2 examines the effect of adenovirus proteins on AAV single-stranded DNA formation in yeast.
  • Patent Document 1 describes that in gene therapy using a recombinant AAV vector, prior to administration of the recombinant AAV vector, the blood of the subject is circulated extracorporeally to deplete immunoglobulin by immunoadsorption. .
  • Anti-adeno-associated virus antibody (hereinafter also referred to as anti-AAV antibody) is useful as a reagent for detecting and quantifying AAV, and as an affinity ligand for AAV purification.
  • antigen for preparing an anti-AAV antibody a live virus, an inactivated virus, a peptide comprising a part of the capsid, a DNA encoding the capsid, and a recombinant capsid can be used.
  • the method using a live virus has a high probability that an antibody that binds to the virus can be obtained, but requires culturing of animal cells, the virus preparation is complicated and expensive, and it is a recombinant virus. There is a disadvantage that measures need to be taken.
  • the method using an inactivated virus there is a high probability that an antibody that binds to the virus can be obtained, and since it is inactivated, it is safe and no containment measures are required, but like a live virus, virus preparation is complicated and expensive.
  • virus preparation is complicated and expensive.
  • it is complicated to examine conditions for inactivation treatment and to confirm inactivation by cell assay.
  • the method using a peptide consisting of a part of the capsid is safe because it is not a virus, but it may be difficult to prepare depending on the amino acid sequence of the peptide, and the three-dimensional structure of the peptide and the capsid may be different. Antibodies may not bind to live virus.
  • the method using the DNA encoding the capsid is easy to prepare and low in cost, and is safe because it is not a virus.
  • the target DNA may not be transcribed and the antigen may not be produced in the immunized animal.
  • DNA immunization requires administration of a large amount of DNA, and a special device such as a gene gun may be used to improve transduction efficiency.
  • the method using a recombinant capsid is easy and low-cost because it uses a microorganism culture, and is safe because it is not a virus.
  • the obtained antibody may not bind to a live virus.
  • the present inventors have conducted intensive studies to solve the above-described problems, and as a result, produced a capsid protein of adeno-associated virus in bacteria, recovered the protein as an insoluble protein, and further solubilized the recovered insoluble protein. Have found that a capsid protein of an adeno-associated virus useful as an antigen for producing an anti-AAV antibody can be produced.
  • the present invention includes the following inventions.
  • ⁇ 1> (a) producing a capsid protein of an adeno-associated virus in bacteria; (B) recovering the protein as an insoluble protein; and (c) solubilizing the recovered insoluble protein;
  • a method for producing a capsid protein of an adeno-associated virus comprising: ⁇ 2> The method according to ⁇ 1>, wherein in step (a), the capsid protein of the adeno-associated virus is produced in a bacterium as a fusion protein with a tag protein.
  • the tag protein is a soluble protein.
  • ⁇ 4> The method according to any one of ⁇ 1> to ⁇ 3>, wherein the capsid protein of the adeno-associated virus is VP3.
  • step (b) After separating bacterial cells from the supernatant, the bacterial cells are crushed, and the precipitate after crushing is recovered to recover the protein as an insoluble protein.
  • step (c) The method according to any one of ⁇ 4>.
  • step (c) The method according to any one of ⁇ 1> to ⁇ 5>, wherein in step (c), the recovered insoluble protein is solubilized with a solubilizing agent.
  • ⁇ 7> The method according to ⁇ 6>, further comprising a step of removing the solubilizing agent after the step (c).
  • ⁇ 8> A step of producing a capsid protein of an adeno-associated virus by the method according to any one of ⁇ 1> to ⁇ 7>, and using the capsid protein of an adeno-associated virus obtained above as an antigen for a non-human immunized animal
  • ⁇ 10> a step of producing an adeno-associated virus capsid protein by the method according to any one of ⁇ 1> to ⁇ 7>, and administering the adeno-associated virus capsid protein obtained above to a naive B cell population
  • a method for producing a B cell population that produces an anti-adeno-associated virus antibody comprising the step of: ⁇ 11> A matrix for separating an anti-adeno-associated virus antibody, comprising a water-insoluble carrier to which an adeno-associated virus capsid protein obtained by the method according to any one of ⁇ 1> to ⁇ 7> is immobilized. .
  • ⁇ 12> A step of producing an adeno-associated virus capsid protein by the method according to any one of ⁇ 1> to ⁇ 7>, and immobilizing the adeno-associated virus capsid protein obtained above on a water-insoluble carrier.
  • a method for producing a matrix for separating an anti-adeno-associated virus antibody comprising the steps of: ⁇ 13> A method for removing anti-adeno-associated virus antibody from blood, comprising contacting the matrix according to ⁇ 11> with blood in vitro.
  • ⁇ 14> The method according to ⁇ 13>, wherein the anti-adeno-associated virus antibody is a neutralizing antibody.
  • ⁇ 15> A method for purifying an anti-adeno-associated virus antibody, comprising contacting the matrix according to ⁇ 11> with an antibody-containing sample.
  • the capsid protein of the adeno-associated virus can be produced more easily and in a larger amount than the production in yeast.
  • the capsid protein produced by the method of the present invention is useful as a virus-free antigen for immunization.
  • a chromatographic carrier that adsorbs an anti-AAV antibody can be produced.
  • FIG. 1 shows the results of evaluating the antibody titer of rabbit serum.
  • FIG. 2 shows the results of evaluating the antibody titer of alpaca serum.
  • the method for producing an adeno-associated virus capsid protein comprises: (A) producing the adeno-associated virus capsid protein in bacteria; (B) recovering the protein as an insoluble protein; and (c) solubilizing the recovered insoluble protein; including.
  • Adeno-associated virus is a virus belonging to the parvoviridae family that contains linear single-stranded DNA in the capsid.
  • Adeno-associated viruses include type 1 AAV (AAV1), type 2 AAV (AAV2), type 3 AAV (AAV3), type 4 AAV (AAV4), type 5 AAV (AAV5), type 6 AAV (AAV6), type 7 AAV (AAV7), type 8 AAV (AAV8), type 9 AAV (AAV9), type 10 AAV (AAV10), and the like are known, and any type is preferable, but type 2 AAV (AAV2) is preferable.
  • the capsid protein of the adeno-associated virus may be any of the three structural proteins VP1, VP2 or VP3, but VP3 having the highest ratio among the capsid constituent proteins is preferred.
  • the amino acid sequence of VP1 of AAV2 is registered under NCBI accession number YP_680426.1.
  • the amino acid sequence of VP2 of AAV2 is registered under NCBI accession number YP_680427.1.
  • the amino acid sequence of VP3 of AAV2 is registered under NCBI accession number YP_680428.1.
  • AAV1 is GenBank: AGA15926.1
  • AAV3 is GenBank: AAC55049.1
  • AAV4 is GenBank: AAC58045.1
  • AAV5 is GenBank: AAD137756.1.
  • the amino acid sequences registered in Reference Sequence: YP_077178.1, AAV8 in GenBank: AAN03857.1, and AAV9 in GenBank: AAS99264.1 can be used.
  • nucleotide sequence encoding each capsid protein a nucleotide sequence of a natural virus can be used.
  • the nucleotide sequence of AAV2 may be the nucleotide sequence of NCBI accession number NC_001401.
  • artificially synthesized genes optimized for codons that are frequently used by the host can also be used.
  • amino acid sequence of VP3 of AAV2 is shown in SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence of the artificially synthesized gene encoding VP3 of AAV2 is shown in SEQ ID NO: 2 in the sequence listing.
  • the amino acid sequence is not limited to the amino acid sequence of the adeno-associated virus described above, but may be one to several (preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5) in the amino acid sequence.
  • Mutant capsid proteins in which one, for example one, two, three or four) amino acids have been substituted, added and / or deleted may be used.
  • a mutation having an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence of the capsid protein Type capsid proteins may be used.
  • the mutation described above may be introduced, for example, for the purpose of improving tissue specificity (such as brain or liver) or improving blood stability.
  • the amino acid sequence identity can be determined by a program called BLASTX or BLASTP (Altschul SF, based on the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993)). et al: J Mol Biol 215: 403, 1990).
  • the default parameters of each program are used. Specific techniques for these analysis methods are well known to those skilled in the art.
  • the capsid protein of the adeno-associated virus is produced in bacteria.
  • the adeno-associated virus capsid protein may be produced in bacteria, preferably as a fusion protein with a tag protein.
  • a tag protein for example, a soluble protein can be used.
  • the soluble protein refers to a protein that has a very high solubility (hydrophilicity) by itself, and thus has a high possibility of expressing the target protein fused thereto in a soluble fraction.
  • Soluble proteins include glutathione-S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), cellulose binding domain (CBD), molecular chaperones (DNAJ, DNAK), protein A (SpA) or the like can be used, but is not particularly limited.
  • a polynucleotide encoding a capsid protein or a fusion protein of a capsid protein and a tag protein is introduced into an expression vector and expressed in a host cell bacterium. Good.
  • the expression vector is not particularly limited as long as it can be replicated in bacteria, but a plasmid vector, a phage vector, or the like can be used.
  • the vector may contain an origin of replication, a selection marker and a promoter, and if necessary, may contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like.
  • the expression vector may be any ordinary bacterial vector, and a commercially available bacterial vector can be used.
  • a commercially available bacterial vector can be used.
  • Plasmid vectors such as pET, pGEM-3Z, pGEX, pMAL, etc., bacteriophage vectors such as ⁇ EMBL3 (Stratagene), ⁇ DASHII (Funakoshi), Charomid DNA (Wako Pure Chemical Industries, Ltd.), Lorist6 (Wako Pure) And cosmid vectors manufactured by Yakuhin Kogyo Co., Ltd.).
  • E. coli-derived plasmids eg, pTrc99A, @ pKK223, @ pET3a
  • pA1-11 pXT1, pRc / CMV, pRc / RSV, pcDNA @ I / Neo, p3.times.FLAG-CMV-14, pCAT3, pcDNA3.1. , PCMV and the like.
  • the introduction of the polynucleotide into the vector can be performed by a conventional method.
  • a specific restriction enzyme site in the vector can be cut with a specific restriction enzyme, and the polynucleotide can be inserted into the cleavage site.
  • bacteria serving as hosts include Escherichia coli, Escherichia coli, Bacillus bacteria such as B. subtilis, B. megaterium, B. brevis, B. borstelenis, Staphylococcus, and Lactococcus lacla. Lactobacillus, Lactobacillus, Streptomyces, Brevibacillus, Pseudomonas, Corynebacterium, Serratia, Serratia Bacteria (Brevobacterium), Rhodococcus bacteria (Rhodoco cus), and the like can be given, but it is not particularly limited.
  • the bacterium is preferably E. coli.
  • Escherichia coli When Escherichia coli is used as a host, commonly used strains such as JM109, HB101, MC4100, MG1655, W3110, and BL21 can be used.
  • Bacterial transformation with the expression vector can be performed by a conventional method.
  • it can be performed by calcium chloride, calcium phosphate, DEAE-dextran-mediated transfection, electroporation, lipofection, and the like.
  • the capsid protein of adeno-associated virus can be produced in bacteria.
  • the method for culturing the transformant is not particularly limited, and a general method for culturing bacteria can be used.
  • the temperature, the pH of the medium, and the culture time can also be set as appropriate.
  • the medium used for culturing the transformant can be appropriately selected depending on the type of the host. For example, when culturing Escherichia coli, an LB medium or the like is used.
  • An antibiotic depending on the type of the selection marker may be added to the medium.
  • An expression inducing substance such as isopropyl- ⁇ -thiogalactopyranoside (IPTG) can also be used.
  • the protein produced in the step (a) is recovered as an insoluble protein.
  • the protein in the step (b), can be recovered as an insoluble protein by separating the bacterial cells and the supernatant, crushing the bacterial cells, and collecting the crushed precipitate.
  • bacterial cells are collected by a method such as centrifugation, filtration or membrane separation, and resuspended in an appropriate buffer (phosphate buffer (PBS) or the like).
  • PBS phosphate buffer
  • the cell wall and / or cell membrane of the recovered bacterial cells are destroyed by a method such as sonication, pressure crushing, osmotic pressure, freeze-thaw, surfactant treatment, or lysozyme treatment.
  • the protein produced in step (a) can be recovered as an insoluble protein by collecting the precipitate by a method such as centrifugation, filtration, or membrane separation.
  • the insoluble protein recovered in the step (b) is solubilized.
  • Solubilization of the insoluble protein can be performed, for example, using a solubilizing agent.
  • a solubilizing agent a chaotropic reagent, or various surfactants (nonionic, anionic, cationic, or zwitterionic surfactants) can be used.
  • the chaotropic reagent urea, thiourea, guanidine, guanidine hydrochloride, guanidine sulfate and the like can be used.
  • the surfactants include sodium dodecyl sulfate (SDS), n-methyltrimethylammonium hydrochloride (CTAB), and n-rauryl salcocinnato Lime can be used.
  • SDS sodium dodecyl sulfate
  • CAB n-methyltrimethylammonium hydrochloride
  • n-rauryl salcocinnato Lime can be used.
  • the capsid protein since the capsid protein has a cysteine residue, it can be solubilized by cleaving a disulfide bond in the presence of a reducing agent and then giving a positively charged compound to cysteine.
  • TAPS-sulfonate trimethylammoniopropylmethanethiosulfonate bromide
  • a step of removing the solubilizing agent after the step (c) may be further provided.
  • the solubilizing agent may be added to the insoluble protein recovered in step (b) to solubilize it, and then the bacteria (transformant) and cell residues thereof may be removed by filtration with a filter. Thereafter, the solubilizing agent may be removed by replacing the solubilized protein again with PBS using a dialysis membrane to obtain an insolubilized protein.
  • the protein obtained in the above steps (a) to (c) may be used as it is or may be subjected to further purification.
  • Purification methods include, for example, methods using solubility such as salting out and solvent precipitation, methods using molecular weight differences such as dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ion exchange chromatography.
  • Method using charge such as chromatography, method using specific affinity such as affinity chromatography, method using difference in hydrophobicity such as hydrophobic interaction chromatography, reverse phase high performance liquid chromatography, isoelectric point A method utilizing the difference between isoelectric points such as electrophoresis may be used.
  • the method for producing an anti-AAV antibody according to the present invention comprises the steps of producing an adeno-associated virus capsid protein by the above-described method for producing an adeno-associated virus capsid protein according to the present invention, and using the adeno-associated virus capsid protein obtained above. Administering a non-human immunized animal as an immunizing antigen.
  • the non-human immunized animal is preferably a mammal, a bird, or a cartilaginous fish.
  • mammals include dromedaries such as dromedaries, bactrian camels, llamas, alpacas, vicunas, guanagos, rodents such as mice, rats, rabbits, ruminants such as cattle, goats, sheep, monkeys, horses, and guinea pigs. be able to.
  • Birds include chickens, geese, ostriches, and the like.
  • Cartilage fish include sharks and rays.
  • the non-human immunized animal is preferably a camelid or a rodent, and more preferably a camelid.
  • a suspension or emulsion of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and an antigen is prepared, and the suspension or emulsion is administered several times to the vein, subcutaneous, intradermal, intraperitoneal, etc. of the animal to immunize the animal.
  • an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and an antigen
  • the serum of the immunized animal to which the capsid protein of the adeno-associated virus was administered was collected, and the serum was collected according to a conventional method (for example, ammonium sulfate fractionation, ion exchange chromatography, Protein A or Protein G affinity chromatography, gel filtration chromatography, etc.).
  • AAV antibodies can be obtained.
  • Antibody-producing cells can also be obtained from animals immunized with the capsid protein of the adeno-associated virus.
  • As antibody-producing cells spleen cells, lymph node cells, B lymphocytes and the like from immunized animals can be obtained. Monoclonal antibodies can be obtained using antibody-producing cells.
  • the cells producing the monoclonal antibody are not particularly limited, but can be obtained as a hybridoma by, for example, cell fusion between the antibody-producing cells and a myeloma cell line.
  • a hybridoma producing a monoclonal antibody can be obtained by a cell fusion method. That is, for example, spleen cells are obtained as antibody-producing cells from an immunized animal, and these cells and myeloma cells are obtained by a known method (G. Kohler et al., Nature, 256-495 (1975), Kohler.lerG. And Milstein, C., Methods Enzymol. (1981) 73: 3-46) to produce a hybridoma.
  • myeloma cell lines used for cell fusion include P3X63Ag8, P3U1 strain, and Sp2 / 0 strain in mice.
  • a fusion promoting agent such as polyethylene glycol or Sendai virus is used.
  • hypoxanthine-aminopterin-thymidine (HAT) medium can be used according to a conventional method.
  • the hybridoma obtained by cell fusion can be cloned by a limiting dilution method or the like.
  • Monoclonal antibody-producing cells may be produced by cell fusion as described above, or may be produced by other methods such as immortalization of B lymphocytes by introduction of oncogene DNA or infection with Epstein-Barr virus. Good.
  • cells that produce the desired antibody can be selected by screening the resulting antibody-producing cells such as hybridomas. Screening of antibody-producing cells can be performed using a method known in the art such as ELISA assay, Western blot analysis, radioimmunoassay, FACS, and the like. An antibody-producing cell that produces the desired antibody is cloned, cultured under appropriate conditions, and the secreted antibody is recovered and purified by a conventional method (eg, ion exchange chromatography, affinity chromatography, etc.).
  • a conventional method eg, ion exchange chromatography, affinity chromatography, etc.
  • a heavy chain antibody is preferable.
  • Camelids have antibodies composed of only heavy chains (heavy chain antibodies), and the variable regions thereof are called VHH antibodies (variable @ domain @ of @ heavy @ chain @ of @ heavy @ chain @ antibody).
  • Heavy chain antibodies VHH antibodies
  • VHH antibodies have the advantage of higher stability (stability to denaturants such as heat and alkalis and pH) compared to other antibodies such as IgG antibodies. Further, since the heavy chain antibody (VHH antibody) is a single domain antibody, there is an advantage that there is no combination of a light chain and a heavy chain, and it is easy to prepare.
  • the method for producing a B cell population producing an anti-AAV antibody according to the present invention comprises the steps of producing an adeno-associated virus capsid protein by the above-described method for producing an adeno-associated virus capsid protein according to the present invention, and the method obtained above. Administering the adeno-associated virus capsid protein to the naive B cell population.
  • International Publication WO2016 / 2760 discloses a method for producing an antigen-specific B cell population including IgG-positive B cells specific for a specific antigen, by using B cells with increased Bach2 gene expression as CD40 and CD40. And / or a method for producing a B cell population, comprising culturing in the presence of a means for acting on a BAFF receptor.
  • a B cell population that produces an antibody against the adeno-associated virus capsid protein can be selected.
  • a matrix for separating anti-AAV antibodies By immobilizing the adeno-associated virus capsid protein obtained by the method of the present invention on a water-insoluble carrier, a matrix for separating anti-AAV antibodies can be produced. That is, according to the present invention, there is provided a matrix for separating an anti-AAV antibody, comprising a water-insoluble carrier on which the capsid protein of adeno-associated virus is immobilized. The matrix is capable of adsorbing anti-AAV antibodies.
  • the carrier for immobilizing the capsid protein of the adeno-associated virus may be any water-insoluble carrier, and a carrier used in usual antigen-antibody reactions or affinity chromatography can be used.
  • Water-insoluble refers to a carrier that does not dissolve in water.
  • the carrier include inorganic carriers such as glass beads and silica gel; synthetic polymers such as polyvinyl alcohol, polyacrylate, polyacrylamide, and polystyrene; cross-linked synthetic polymers; crystalline cellulose, cellulose, agarose, and dextran.
  • Examples of the form of the carrier include a microplate, a tube, a membrane, a nonwoven fabric, a column, and beads.
  • the capsid protein of the adeno-associated virus may be immobilized on the carrier by physical adsorption or by a chemical bond.
  • the chemical bond may be any of a covalent bond, an ionic bond, a hydrophobic bond, and a hydrogen bond, but a covalent bond is preferable.
  • a reactive functional group is preferably present on the surface of the carrier.
  • Examples of the reactive functional group include an epoxy group, a thiol group, a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a silanol group, an amide group, a succinylimide group, and an acid anhydride group.
  • Anti-AAV antibodies can be removed from blood by bringing the matrix of the present invention into contact with blood in vitro.
  • an anti-adeno-associated virus neutralizing antibody (hereinafter, also referred to as an anti-AAV neutralizing antibody) can be removed from blood.
  • an anti-AAV neutralizing antibody in gene therapy using an AAV vector, administration of an AAV vector for gene therapy to a patient having an anti-AAV neutralizing antibody cannot provide a sufficient effect.
  • the anti-AAV neutralizing antibody from the blood of the patient by dialysis using the matrix of the present invention, the effect of the inhibitory effect of the anti-AAV neutralizing antibody when the AAV vector for gene therapy is administered to the patient Can be reduced.
  • the anti-AAV antibody can be purified by contacting the matrix of the present invention with an antibody-containing sample.
  • an anti-AAV antibody can be purified from an immune serum of an immunized animal immunized with an adeno-associated virus or a capsid protein.
  • Example 1 Breeding and cultivation of GST-VP3-expressing Escherichia coli XhoI at the N-terminal side of the gene (Escherichia coli optimized codon) encoding the amino acid sequence of VP3 of AAV2 (SEQ ID NO: 2 in the sequence listing), and An artificially synthesized gene (SEQ ID NO: 1) to which BamHI had been added was artificially synthesized (Eurofin Genomics).
  • This artificially synthesized gene was digested with XhoI and BamHI, and introduced into pGEX-6p-1 (GE Healthcare) digested with the same restriction enzymes to prepare a GST-VP3 expression plasmid pEX-K4J1-AAV-VP3.
  • the resulting plasmid was transformed into E. coli. coli JM109 (Takara Bio), left on ice for 30 minutes, and then heat-shocked at 42 ° C. for 45 seconds to transform. Thereafter, the transformant in which the introduction of the VP3 gene was confirmed was cultured in a Magic Media medium (invitrogen) in a flask at 30 ° C. overnight to obtain a culture solution of GST-VP3-expressing Escherichia coli.
  • a Magic Media medium invitrogen
  • Example 2 Purification of GST-VP3
  • the culture solution obtained in Example 1 was centrifuged at 9,000 rpm for 10 minutes, and the supernatant was removed.
  • the remaining cells were suspended in Dulbecco's phosphate-buffered saline (PBS), crushed with an ultrasonic crusher, and again centrifuged at 15,000 rpm for 5 minutes to precipitate the precipitate fraction containing the insolubilized GST-VP3.
  • PBS / 6M urea was added to the precipitate to solubilize GST-VP3, and then filtered with a 0.2 ⁇ m filter to remove any remaining recombinant bacteria.
  • the solubilized GST-VP3 was replaced again with PBS using a dialysis membrane (Slide-A-Lyzer TM G2Dialysis Cassettes, 10K MWCO, 3 mL) to obtain an insolubilized protein.
  • the protein concentration of the obtained protein suspension was measured using a 660 nm protein assay (Pierce TM 660 nm Protein Assay Reagent, Thermo Fisher Scientific) using BSA as a standard. As a result, 38.7 mg of the protein containing the target substance was obtained from 50 mL of the culture solution.
  • AAV2 empty particle is a virus particle having no nucleic acid inside.
  • HEK293T cells human embryonic kidney-derived cells
  • pRC2-mi342 vector and pHelper vector were transfected with pRC2-mi342 vector and pHelper vector, and after about 72 hours, the cells were disrupted, and the AAV2 empty particles were purified by cation chromatography.
  • Example 4 Rabbit immunization experiment using GST-VP3 as an antigen
  • the crude product of GST-VP3 obtained in Example 2 (1 mg / mL) was adjuvanted with an equal amount of TiterMax Gold (Funakoshi, Lot No .: G1216).
  • TiterMax Gold Feunakoshi, Lot No .: G12166
  • Std JW / CSK, female, 20 weeks old
  • Example 5 Evaluation of antibody titer of rabbit serum
  • the antibody titers of GST-VP3, which is an immunizing antigen, and empty particle (AAV2) prepared in Example 3 were enzyme-linked. Evaluation was performed by immunosorbent assay (ELISA). For dilution of serum or antibody, PBS containing 0.01% Tween 20 was used. AAV2 used for the evaluation was produced in HEK293 cells, and purified and prepared with AAVpro (registered trademark) Purification Kit (Takara Bio).
  • AAVpro registered trademark
  • GST-VP3 (antigen) or AAV2 was diluted with PBS (pH 7.4) and immobilized on a microplate (Immno Maxi-sorp, Nunc, Sigma-Aldrich), respectively. After the immobilized plate was washed with PBS containing 0.1% Tween 20, a serum antibody obtained from a rabbit was added thereto, and HRP-Goat Anti-Rabbit IgG (H + L) (Thermo Fisher Scientific, CatNo. 65-6120) and SureBlue TM TMB Microwell Peroxidase Substrate Kit (SeraCare Life Sciences, Inc.) as a chromogenic substrate, and the absorbance at 450 nm was confirmed. The results are shown in FIG.
  • antibodies obtained by immunization with GST-VP3 as an antigen include not only GST-VP3 but also antibodies that bind to AAV2. These results indicate that GST-VP3 is useful as an antigen for obtaining an anti-AAV antibody.
  • Example 6 Alpaca immunization experiment using GST-VP3 as an antigen A 5-week-old male alpaca was used as an immunized animal. Immunization was performed 5 times in total. Before the first immunization, blood was collected and used as a pre-immune control serum, followed by the first immunization. For the first immunization, Freund's Complete Adjuvant (FCA) was used as an adjuvant, 3.3 mg of an immunizing antigen (GST-VP3) was emulsified, and alpaca was immunized subcutaneously at multiple sites.
  • FCA Freund's Complete Adjuvant
  • GST-VP3 an immunizing antigen
  • FIA Freund's Complete Adjuvant
  • GST-VP3 an immunizing antigen
  • AAV2 empty particle
  • the collected peripheral blood was collected in a collection tube containing a separating agent, allowed to stand at room temperature for 3 hours or longer, and centrifuged (3500 rpm for 10 minutes) to obtain serum.
  • Alpaca peripheral blood lymphocytes and plasma fraction were separated from alpaca blood by density gradient centrifugation using Ficoll.
  • the alpaca heavy chain antibody was purified from the plasma fraction using a Protein G affinity column and a Protein A affinity column. Specifically, first, an alpaca plasma fraction was bound to a Protein G affinity column, and 0.15% NaCl and 0.58% acetic acid (pH 4.5) were eluted to obtain IgG2 (a heavy chain antibody with a short hinge region). did.
  • Example 7 Evaluation of antibody titer of alpaca serum
  • the antibody titers to GST-VP3, which is an immunizing antigen, and empty particle (AAV2) were determined by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • PBS containing 0.01% Tween 20
  • empty particles (AAV2) used for evaluation were produced in HEK293 cells, purified by cation chromatography, and prepared.
  • GST-VP3 (antigen) or empty particle (AAV2) was diluted with PBS (pH 7.4) and immobilized on a microplate (Immno Maxi-sorp, Nunc, Sigma-Aldrich), respectively.
  • Example 8 Preparation of GST-VP3 immobilized column
  • GST-VP3 prepared in the same manner as in Example 2 was used as a water-insoluble base material, and a commercially available activated carrier “NHS activated Sepharose 4 Fast Flow” (GE Healthcare) (Manufactured by the company).
  • This column is based on cross-linked agarose and has introduced N-hydroxysuccinimide (NHS) groups for immobilizing proteinaceous ligands.
  • NHS N-hydroxysuccinimide
  • a solution was prepared by diluting the final purified sample with a coupling buffer (0.2 M sodium carbonate, 0.5 M NaCl, pH 8.3) to a final concentration of about 10 mg / mL.
  • the carrier was washed three times with 1 mM HCl cooled in an ice bath, and the stock solution of the carrier was removed.
  • the suck-dried carrier was suspended in a sample diluting solution and mixed at 25 ° C. for 30 minutes to immobilize GST-VP3 on the carrier. Thereafter, the carrier suspension was filtered to recover an unreacted protein solution.
  • the carrier was washed with a coupling buffer
  • the carrier was washed three times with a blocking buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3), and the washing buffer (0.1 M acetic acid, 0.5 M NaCl) was used. , PH 4.0), and finally washed twice with a standard buffer (20 mM NaH2PO4-Na2HPO4, 150 mM NaCl, pH 7.4) to complete the preparation of the GST-VP3 immobilized carrier.
  • a control carrier a carrier which was not immobilized with GST-VP3 and was merely sealed with a blocking buffer (hereinafter, a control carrier) was also prepared.
  • Example 9 Adsorption of anti-AAV antibody by GST-VP3 immobilized column
  • Each 1 mL of the GST-VP3 immobilized carrier and the control carrier prepared in Example 7 was packed in a commercially available column (Tricorn 5/50), and the anti-AAV antibody was purified under the following conditions An AAV antibody adsorption experiment was performed. The antibody titer of the unadsorbed fraction obtained in the experiment was evaluated by the ELISA method of Example 4.

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Abstract

The present invention addresses the problem of providing a method for producing adeno-associated viral capsid protein useful as an antigen for the production of anti-AAV antibodies. The present invention provides a method for producing adeno-associated viral capsid protein, comprising (a) a step of producing adeno-associated viral capsid protein in bacteria; (b) a step of recovering the protein as an insoluble protein; and (c) a step of solubilizing the recovered insoluble protein.

Description

アデノ随伴ウイルスのカプシドタンパク質の製造方法及びその利用Method for producing capsid protein of adeno-associated virus and use thereof
　本発明は、アデノ随伴ウイルスのカプシドタンパク質の製造方法に関する。本発明はさらに、上記の製造方法で製造したアデノ随伴ウイルスのカプシドタンパク質を用いた、抗アデノ随伴ウイルス抗体の作製方法に関する。 << The present invention relates to a method for producing a capsid protein of an adeno-associated virus. The present invention further relates to a method for producing an anti-adeno-associated virus antibody using the adeno-associated virus capsid protein produced by the above-described production method.
　アデノ随伴ウイルス（Ａｄｅｎｏ－ａｓｓｏｃｉａｔｅｄ　ｖｉｒｕｓ：ＡＡＶ）はヒトや霊長目の動物に感染する小型のパルボウイルス科ディペンドウイルス属に分類されるヘルパー依存型のエンベロープを持たないウイルスである。アデノ随伴ウイルスベクター（ＡＡＶベクター）は増殖／非増殖のいずれの細胞にも遺伝子導入が可能であり、非分裂細胞においては目的遺伝子を長期間発現することができる。ＡＡＶベクターは、アデノウイルスベクターやレトロウイルスベクターと比較して、免疫原性が低いことから動物個体への遺伝子導入に適している。また、ＡＡＶベクターは、非病原性ウイルスであることから、安全で取扱いの容易なウイルスベクターとして広く使用されている。近年では特に遺伝子治療用のウイルスベクターとして研究開発が進んでいる。 Adeno-associated virus (AAV) is a virus that does not have a helper-dependent envelope classified into the small parvoviridae family of dependent viruses that infect humans and primates. The adeno-associated virus vector (AAV vector) can introduce a gene into any of proliferating / non-proliferating cells, and can express a target gene for a long time in non-dividing cells. AAV vectors are suitable for gene transfer into animal individuals because of their lower immunogenicity compared to adenovirus vectors and retrovirus vectors. AAV vectors are widely used as safe and easy-to-handle virus vectors because they are non-pathogenic viruses. In recent years, research and development have been particularly advanced as viral vectors for gene therapy.
　非特許文献１は、２型アデノ随伴ウイルス（以下、ＡＡＶ２とも言う）のＲｅｐタンパク質とＶＰカプシドタンパク質との会合が、パッケージ可能なゲノムの不在化においても生じることが記載されている。非特許文献１においては、Ｒｅｐタンパク質とＶＰカプシドタンパク質とを動物細胞において発現させて、Ｒｅｐタンパク質とＶＰカプシドタンパク質との相互作用を調べている。非特許文献２においては、ＡＡＶのカプシドタンパク質を酵母において発現させ、組み立てることが記載されている。非特許文献２においては、酵母において、ＡＡＶ一本鎖ＤＮＡ形成に及ぼすアデノウイルスタンパク質の影響が調べられている。 Non-Patent Document 1 describes that the association between the Rep protein of type 2 adeno-associated virus (hereinafter also referred to as AAV2) and the VP capsid protein occurs even in the absence of a packageable genome. In Non-Patent Document 1, the interaction between the Rep protein and the VP capsid protein is examined by expressing the Rep protein and the VP capsid protein in animal cells. Non-Patent Document 2 describes that an AAV capsid protein is expressed and assembled in yeast. Non-patent Document 2 examines the effect of adenovirus proteins on AAV single-stranded DNA formation in yeast.
　また、特許文献１には、組み換えＡＡＶベクターを用いた遺伝子治療において、組み換えＡＡＶベクターの投与に先立って、対象者の血液を体外循環させて免疫吸着により免疫グロブリンを枯渇させることが記載されている。 Patent Document 1 describes that in gene therapy using a recombinant AAV vector, prior to administration of the recombinant AAV vector, the blood of the subject is circulated extracorporeally to deplete immunoglobulin by immunoadsorption. .
　抗アデノ随伴ウイルス抗体（以下、抗ＡＡＶ抗体とも言う）は、ＡＡＶの検出や定量用の試薬として、並びにＡＡＶ精製用のアフィニティーリガンドとして有用である。抗ＡＡＶ抗体の作製のための抗原としては、生ウイルス、不活化ウイルス、カプシドの一部からなるペプチド、カプシドをコードするＤＮＡ、及び組換えカプシドを用いることが考えられる。 Anti-adeno-associated virus antibody (hereinafter also referred to as anti-AAV antibody) is useful as a reagent for detecting and quantifying AAV, and as an affinity ligand for AAV purification. As an antigen for preparing an anti-AAV antibody, a live virus, an inactivated virus, a peptide comprising a part of the capsid, a DNA encoding the capsid, and a recombinant capsid can be used.
　生ウイルスを用いる方法は、ウイルスに結合する抗体が取得できる確率は高いが、動物細胞の培養が必要であり、ウイルスの調製が煩雑かつ高コストであり、組換えウイルスであるため免疫時にも封じ込め対策が必要という欠点がある。不活化ウイルスを用いる方法は、ウイルスに結合する抗体が取得できる確率が高く、不活化しているので安全であり、封じ込め対策は不要であるが、生ウイルス同様、ウイルスの調製が煩雑かつ高コストであるという問題があり、また不活化処理の条件検討や細胞アッセイによる不活化の確認が煩雑であるという問題がある。 The method using a live virus has a high probability that an antibody that binds to the virus can be obtained, but requires culturing of animal cells, the virus preparation is complicated and expensive, and it is a recombinant virus. There is a disadvantage that measures need to be taken. In the method using an inactivated virus, there is a high probability that an antibody that binds to the virus can be obtained, and since it is inactivated, it is safe and no containment measures are required, but like a live virus, virus preparation is complicated and expensive. In addition, there is a problem that it is complicated to examine conditions for inactivation treatment and to confirm inactivation by cell assay.
　カプシドの一部からなるペプチドを用いる方法は、ウイルスではないので安全ではあるが、ペプチドのアミノ酸配列によっては調製が難しい場合があり、また、ペプチドとカプシドの立体構造が異なる場合もあるため、得られた抗体が生ウイルスに結合しない場合がある。カプシドをコードするＤＮＡを用いる方法は、調製が容易で低コストであり、またウイルスではないので安全ではあるが、目的ＤＮＡが転写されず、免疫動物中で抗原が生産されない場合がある。また、ＤＮＡ免疫には多量のＤＮＡの投与が必要であり、導入効率向上のためにジーンガンなど特殊な装置を使用する場合もあり、免疫動物の負担となるため好ましくない。 The method using a peptide consisting of a part of the capsid is safe because it is not a virus, but it may be difficult to prepare depending on the amino acid sequence of the peptide, and the three-dimensional structure of the peptide and the capsid may be different. Antibodies may not bind to live virus. The method using the DNA encoding the capsid is easy to prepare and low in cost, and is safe because it is not a virus. However, the target DNA may not be transcribed and the antigen may not be produced in the immunized animal. In addition, DNA immunization requires administration of a large amount of DNA, and a special device such as a gene gun may be used to improve transduction efficiency.
　組換えカプシドを用いる方法は、微生物培養を用いるため調製が容易かつ、低コストであり、ウイルスではないので安全ではあるが、得られた抗体が生ウイルスに結合しない場合がある。 (4) The method using a recombinant capsid is easy and low-cost because it uses a microorganism culture, and is safe because it is not a virus. However, the obtained antibody may not bind to a live virus.
国際公開ＷＯ２０１８／１０９２０７号International Publication WO2018 / 109207
　本発明の課題は、抗ＡＡＶ抗体を作製するための抗原としてアデノ随伴ウイルスのカプシドタンパク質を製造する方法を提供することである。本発明の別の課題は、上記の製造方法で製造したアデノ随伴ウイルスのカプシドタンパク質を用いた、抗ＡＡＶ抗体の作製方法を提供することである。 課題 An object of the present invention is to provide a method for producing an adeno-associated virus capsid protein as an antigen for producing an anti-AAV antibody. Another object of the present invention is to provide a method for producing an anti-AAV antibody using an adeno-associated virus capsid protein produced by the above-mentioned production method.
　本発明者らは、上記課題を解決するために鋭意検討した結果、アデノ随伴ウイルスのカプシドタンパク質を細菌において生産し、前記タンパク質を不溶性タンパク質として回収し、さらに回収した不溶性タンパク質を可溶化することによって、抗ＡＡＶ抗体を作製するための抗原として有用なアデノ随伴ウイルスのカプシドタンパク質を製造できることを見出した。 The present inventors have conducted intensive studies to solve the above-described problems, and as a result, produced a capsid protein of adeno-associated virus in bacteria, recovered the protein as an insoluble protein, and further solubilized the recovered insoluble protein. Have found that a capsid protein of an adeno-associated virus useful as an antigen for producing an anti-AAV antibody can be produced.
　即ち、本発明は、以下の発明を包含する。
＜１＞　（ａ）アデノ随伴ウイルスのカプシドタンパク質を細菌において生産する工程；
（ｂ）前記タンパク質を不溶性タンパク質として回収する工程；及び
（ｃ）回収した不溶性タンパク質を可溶化する工程；
を含む、アデノ随伴ウイルスのカプシドタンパク質の製造方法。
＜２＞　工程（ａ）において、アデノ随伴ウイルスのカプシドタンパク質を、タグタンパク質との融合タンパク質として、細菌において生産する、＜１＞に記載の方法。
＜３＞　タグタンパク質が、可溶性タンパク質である、＜２＞に記載の方法。
＜４＞　アデノ随伴ウイルスのカプシドタンパク質が、ＶＰ３である、＜１＞から＜３＞の何れか一に記載の方法。
＜５＞　工程（ｂ）において、細菌の菌体と上清を分離した後に、細菌菌体を破砕し、さらに破砕後の沈殿物を回収することにより、タンパク質を不溶性タンパク質として回収する、＜１＞から＜４＞の何れか一に記載の方法。
＜６＞　工程（ｃ）において、回収した不溶性タンパク質を可溶化剤により可溶化する、＜１＞から＜５＞の何れか一に記載の方法。
＜７＞　工程（ｃ）の後に、可溶化剤を除去する工程をさらに含む、＜６＞に記載の方法。 That is, the present invention includes the following inventions.
<1> (a) producing a capsid protein of an adeno-associated virus in bacteria;
(B) recovering the protein as an insoluble protein; and (c) solubilizing the recovered insoluble protein;
A method for producing a capsid protein of an adeno-associated virus, comprising:
<2> The method according to <1>, wherein in step (a), the capsid protein of the adeno-associated virus is produced in a bacterium as a fusion protein with a tag protein.
<3> The method according to <2>, wherein the tag protein is a soluble protein.
<4> The method according to any one of <1> to <3>, wherein the capsid protein of the adeno-associated virus is VP3.
<5> In step (b), after separating bacterial cells from the supernatant, the bacterial cells are crushed, and the precipitate after crushing is recovered to recover the protein as an insoluble protein. > The method according to any one of <4>.
<6> The method according to any one of <1> to <5>, wherein in step (c), the recovered insoluble protein is solubilized with a solubilizing agent.
<7> The method according to <6>, further comprising a step of removing the solubilizing agent after the step (c).
＜８＞　＜１＞から＜７＞の何れか一に記載の方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を抗原として非ヒト免疫動物に投与する工程を含む、抗アデノ随伴ウイルス抗体の作製方法。
＜９＞　抗アデノ随伴ウイルス抗体が重鎖抗体である、＜８＞に記載の方法。
＜１０＞　＜１＞から＜７＞の何れか一に記載の方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を、ナイーブＢ細胞集団に投与する工程を含む、抗アデノ随伴ウイルス抗体を産生するＢ細胞集団を製造する方法。
＜１１＞　＜１＞から＜７＞の何れか一に記載の方法により得られるアデノ随伴ウイルスのカプシドタンパク質が固定化されている水不溶性担体からなる、抗アデノ随伴ウイルス抗体を分離するためのマトリックス。
＜１２＞　＜１＞から＜７＞の何れか一に記載の方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を水不溶性担体に固定化する工程を含む、抗アデノ随伴ウイルス抗体を分離するためのマトリックスの製造方法。
＜１３＞　＜１１＞に記載のマトリックスと血液とを生体外で接触させることを含む、血液から抗アデノ随伴ウイルス抗体を除去する方法。
＜１４＞　抗アデノ随伴ウイルス抗体が、中和抗体である、＜１３＞に記載の方法。
＜１５＞　＜１１＞に記載のマトリックスと、抗体含有試料とを接触させることを含む、抗アデノ随伴ウイルス抗体を精製する方法。 <8> A step of producing a capsid protein of an adeno-associated virus by the method according to any one of <1> to <7>, and using the capsid protein of an adeno-associated virus obtained above as an antigen for a non-human immunized animal A method for producing an anti-adeno-associated virus antibody, comprising a step of administering.
<9> The method according to <8>, wherein the anti-adeno-associated virus antibody is a heavy chain antibody.
<10> a step of producing an adeno-associated virus capsid protein by the method according to any one of <1> to <7>, and administering the adeno-associated virus capsid protein obtained above to a naive B cell population A method for producing a B cell population that produces an anti-adeno-associated virus antibody, comprising the step of:
<11> A matrix for separating an anti-adeno-associated virus antibody, comprising a water-insoluble carrier to which an adeno-associated virus capsid protein obtained by the method according to any one of <1> to <7> is immobilized. .
<12> A step of producing an adeno-associated virus capsid protein by the method according to any one of <1> to <7>, and immobilizing the adeno-associated virus capsid protein obtained above on a water-insoluble carrier. A method for producing a matrix for separating an anti-adeno-associated virus antibody, comprising the steps of:
<13> A method for removing anti-adeno-associated virus antibody from blood, comprising contacting the matrix according to <11> with blood in vitro.
<14> The method according to <13>, wherein the anti-adeno-associated virus antibody is a neutralizing antibody.
<15> A method for purifying an anti-adeno-associated virus antibody, comprising contacting the matrix according to <11> with an antibody-containing sample.
　本発明によれば、酵母での生産に比べ、簡便かつ大量にアデノ随伴ウイルスのカプシドタンパク質を製造することができる。本発明の方法で製造したカプシドタンパク質は、ウイルスを含まない免疫用抗原として有用である。なお、本発明の方法で製造したカプシドタンパク質をリガンドとしたときは、抗ＡＡＶ抗体を吸着するクロマトグラフ担体を製造することができる。 According to the present invention, the capsid protein of the adeno-associated virus can be produced more easily and in a larger amount than the production in yeast. The capsid protein produced by the method of the present invention is useful as a virus-free antigen for immunization. When the capsid protein produced by the method of the present invention is used as a ligand, a chromatographic carrier that adsorbs an anti-AAV antibody can be produced.
図１は、ウサギ血清の抗体価を評価した結果を示す。FIG. 1 shows the results of evaluating the antibody titer of rabbit serum. 図２は、アルパカ血清の抗体価を評価した結果を示す。FIG. 2 shows the results of evaluating the antibody titer of alpaca serum.
　以下、本発明について詳細に記載する。
［アデノ随伴ウイルスのカプシドタンパク質の製造方法］
　本発明によるアデノ随伴ウイルスのカプシドタンパク質の製造方法は、
（ａ）アデノ随伴ウイルスのカプシドタンパク質を細菌において生産する工程；
（ｂ）前記タンパク質を不溶性タンパク質として回収する工程；及び
（ｃ）回収した不溶性タンパク質を可溶化する工程；
を含む。 Hereinafter, the present invention will be described in detail.
[Method for producing capsid protein of adeno-associated virus]
The method for producing an adeno-associated virus capsid protein according to the present invention comprises:
(A) producing the adeno-associated virus capsid protein in bacteria;
(B) recovering the protein as an insoluble protein; and (c) solubilizing the recovered insoluble protein;
including.
　アデノ随伴ウイルスは、カプシド中に直鎖状一本鎖ＤＮＡを含むパルボウイルス科に属するウイルスである。アデノ随伴ウイルスとしては、１型ＡＡＶ（ＡＡＶ１）、２型ＡＡＶ（ＡＡＶ２）、３型ＡＡＶ（ＡＡＶ３）、４型ＡＡＶ（ＡＡＶ４）、５型ＡＡＶ（ＡＡＶ５）、６型ＡＡＶ（ＡＡＶ６）、７型ＡＡＶ（ＡＡＶ７）、８型ＡＡＶ（ＡＡＶ８）、９型ＡＡＶ（ＡＡＶ９）及び１０型ＡＡＶ（ＡＡＶ１０）などが知られており、何れでもよいが、好ましくは２型ＡＡＶ（ＡＡＶ２）である。 Adeno-associated virus is a virus belonging to the parvoviridae family that contains linear single-stranded DNA in the capsid. Adeno-associated viruses include type 1 AAV (AAV1), type 2 AAV (AAV2), type 3 AAV (AAV3), type 4 AAV (AAV4), type 5 AAV (AAV5), type 6 AAV (AAV6), type 7 AAV (AAV7), type 8 AAV (AAV8), type 9 AAV (AAV9), type 10 AAV (AAV10), and the like are known, and any type is preferable, but type 2 AAV (AAV2) is preferable.
　アデノ随伴ウイルスのカプシドタンパク質としては、３種の構造タンパク質であるＶＰ１、ＶＰ２又はＶＰ３のうちの何れでもよいが、カプシドの構成タンパク質のうち、比率が最も高いＶＰ３が好ましい。 The capsid protein of the adeno-associated virus may be any of the three structural proteins VP1, VP2 or VP3, but VP3 having the highest ratio among the capsid constituent proteins is preferred.
　ＡＡＶ２のＶＰ１のアミノ酸配列は、ＮＣＢＩ登録番号ＹＰ＿６８０４２６．１に登録されている。
　ＡＡＶ２のＶＰ２のアミノ酸配列は、ＮＣＢＩ登録番号ＹＰ＿６８０４２７．１に登録されている。
　ＡＡＶ２のＶＰ３のアミノ酸配列は、ＮＣＢＩ登録番号ＹＰ＿６８０４２８．１に登録されている。
　その他、ＡＡＶ１はＧｅｎＢａｎｋ：ＡＧＡ１５９２６．１に、ＡＡＶ３はＧｅｎＢａｎｋ：ＡＡＣ５５０４９．１に、ＡＡＶ４はＧｅｎＢａｎｋ：ＡＡＣ５８０４５．１に、ＡＡＶ５はＧｅｎＢａｎｋ：ＡＡＤ１３７５６．１に、ＡＡＶ６はＧｅｎＢａｎｋ：ＡＡＢ９５４５０．１に、ＡＡＶ７はＮＣＢＩ　Ｒｅｆｅｒｅｎｃｅ　Ｓｅｑｕｅｎｃｅ：ＹＰ＿０７７１７８．１に、ＡＡＶ８はＧｅｎＢａｎｋ：ＡＡＮ０３８５７．１に、ＡＡＶ９はＧｅｎＢａｎｋ：ＡＡＳ９９２６４．１に、それぞれ登録されているアミノ酸配列を使用することができる。
　各カプシドタンパク質をコードする塩基配列としては、天然のウイルスの塩基配列を使用することができる。例えば、ＡＡＶ２の塩基配列はＮＣＢＩの登録番号ＮＣ＿００１４０１の塩基配列を使用することができる。また、宿主の使用頻度の高いコドンに最適化した人工合成遺伝子を使用することもできる。 The amino acid sequence of VP1 of AAV2 is registered under NCBI accession number YP_680426.1.
The amino acid sequence of VP2 of AAV2 is registered under NCBI accession number YP_680427.1.
The amino acid sequence of VP3 of AAV2 is registered under NCBI accession number YP_680428.1.
In addition, AAV1 is GenBank: AGA15926.1, AAV3 is GenBank: AAC55049.1, AAV4 is GenBank: AAC58045.1, AAV5 is GenBank: AAD137756.1. The amino acid sequences registered in Reference Sequence: YP_077178.1, AAV8 in GenBank: AAN03857.1, and AAV9 in GenBank: AAS99264.1 can be used.
As a nucleotide sequence encoding each capsid protein, a nucleotide sequence of a natural virus can be used. For example, the nucleotide sequence of AAV2 may be the nucleotide sequence of NCBI accession number NC_001401. In addition, artificially synthesized genes optimized for codons that are frequently used by the host can also be used.
　なお、ＡＡＶ２のＶＰ３のアミノ酸配列を、配列表の配列番号１に示し、ＡＡＶ２のＶＰ３をコードする人工合成遺伝子の塩基配列を、配列表の配列番号２に示す。 The amino acid sequence of VP3 of AAV2 is shown in SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence of the artificially synthesized gene encoding VP3 of AAV2 is shown in SEQ ID NO: 2 in the sequence listing.
　本発明においては、上記したアデノ随伴ウイルスのアミノ酸配列のみに限定されず、上記したアミノ酸配列において１～数個（好ましくは１～２０個、より好ましくは１～１０個、さらに好ましくは１～５個、例えば、１個、２個、３個、又は４個）のアミノ酸が置換、付加及び／又は欠失している変異型カプシドタンパクを使用してもよい。例えば、カプシドタンパク質のアミノ酸配列と８０％以上、８５％以上、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、又は９９％以上の同一性を有するアミノ酸配列を有する変異型カプシドタンパク質を使用してもよい。上記した変異は、例えば、組織特異性（脳又は肝臓など）の向上、または血中安定性の向上を目的として導入されたものでもよい。アミノ酸配列の同一性は、アルゴリズムＢＬＡＳＴ（Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990、Proc Natl Acad Sci USA 90: 5873, 1993）に基づくＢＬＡＳＴＸまたはＢＬＡＳＴＰと呼ばれるプログラム（Altschul SF, et al: J Mol Biol 215: 403, 1990）を利用して決定することができる。ＢＬＡＳＴＸを用いてアミノ酸配列を解析する場合は、パラメーターは、例えばｓｃｏｒｅ＝５０、ｗｏｒｄｌｅｎｇｔｈ＝３とすることができる。ＢＬＡＳＴとＧａｐｐｅｄ ＢＬＡＳＴプログラムを用いる場合は、各プログラムのデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は当業者にはよく知られている。 In the present invention, the amino acid sequence is not limited to the amino acid sequence of the adeno-associated virus described above, but may be one to several (preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5) in the amino acid sequence. Mutant capsid proteins in which one, for example one, two, three or four) amino acids have been substituted, added and / or deleted may be used. For example, a mutation having an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the amino acid sequence of the capsid protein Type capsid proteins may be used. The mutation described above may be introduced, for example, for the purpose of improving tissue specificity (such as brain or liver) or improving blood stability. The amino acid sequence identity can be determined by a program called BLASTX or BLASTP (Altschul SF, based on the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993)). et al: J Mol Biol 215: 403, 1990). When analyzing an amino acid sequence using BLASTX, parameters can be set to, for example, score = 50 and wordlength = 3. When using BLAST and Gapped @ BLAST programs, the default parameters of each program are used. Specific techniques for these analysis methods are well known to those skilled in the art.
　工程（ａ）においては、アデノ随伴ウイルスのカプシドタンパク質を細菌において生産する。
　アデノ随伴ウイルスのカプシドタンパク質は、好ましくは、タグタンパク質との融合タンパク質として、細菌において生産してもよい。タグタンパク質としては、例えば、可溶性タンパク質を使用することができる。可溶性タンパク質とは、それ自身の可溶性（親水性）が非常に高いため、それに融合している目的タンパク質を可溶画分に発現させられる可能性が高くなるタンパク質のことを言う。 In step (a), the capsid protein of the adeno-associated virus is produced in bacteria.
The adeno-associated virus capsid protein may be produced in bacteria, preferably as a fusion protein with a tag protein. As the tag protein, for example, a soluble protein can be used. The soluble protein refers to a protein that has a very high solubility (hydrophilicity) by itself, and thus has a high possibility of expressing the target protein fused thereto in a soluble fraction.
　可溶性タンパク質としては、グルタチオン－Ｓ－トランスフェラーゼ（ＧＳＴ）、マルトース結合タンパク質（ＭＢＰ）、チオレドキシン（Ｔｒｘ）、カルモジュリン結合ペプチド（ＣＢＰ）、セルロース結合ドメイン（ＣＢＤ）、分子シャペロン（ＤＮＡＪ，ＤＮＡＫ）、プロテインＡ（ＳｐＡ）等を使用することができるが、特に限定されない。 Soluble proteins include glutathione-S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), cellulose binding domain (CBD), molecular chaperones (DNAJ, DNAK), protein A (SpA) or the like can be used, but is not particularly limited.
　アデノ随伴ウイルスのカプシドタンパク質を細菌において生産するためには、カプシドタンパク質、又はカプシドタンパク質とタグタンパク質との融合タンパク質をコードするポリヌクレオチドを発現ベクターに導入し、宿主細胞である細菌において発現させればよい。 To produce a capsid protein of an adeno-associated virus in bacteria, a polynucleotide encoding a capsid protein or a fusion protein of a capsid protein and a tag protein is introduced into an expression vector and expressed in a host cell bacterium. Good.
　発現ベクターとしては、細菌において複製可能である限り特に限定されないが、プラスミドベクター、ファージベクター等を使用することができる。ベクターは、複製開始点、選択マーカー及びプロモーターを含んでいてもよく、必要に応じてエンハンサー、転写終結配列（ターミネーター）、リボソーム結合部位、ポリアデニル化シグナル等を含んでいてもよい。 The expression vector is not particularly limited as long as it can be replicated in bacteria, but a plasmid vector, a phage vector, or the like can be used. The vector may contain an origin of replication, a selection marker and a promoter, and if necessary, may contain an enhancer, a transcription termination sequence (terminator), a ribosome binding site, a polyadenylation signal, and the like.
　発現ベクターとしては、通常の細菌用のベクターであればよく、市販の細菌用ベクターを使用することができる。例えば、ｐＴｒｃＨｉｓ２ベクター、ｐｃＤＮＡ３．１／ｍｙｃ－Ｈｉｓベクター（Ｉｎｖｉｔｒｏｇｅｎ社製）、ｐＵＣ１１９（宝酒造社製）、ｐＢＲ３２２（宝酒造社製）、ｐＢｌｕｅｓｃｒｉｐｔ　ＩＩ　ＫＳ＋　Ｓｔｒａｔａｇｅｎｅ社製）、ｐＱＥ－Ｔｒｉ（Ｑｉａｇｅｎ社製）、ｐＥＴ、ｐＧＥＭ－３Ｚ、ｐＧＥＸ、ｐＭＡＬ等のプラスミドベクター、λＥＭＢＬ３（Ｓｔｒａｔａｇｅｎｅ社製）、λＤＡＳＨＩＩ（フナコシ社製）等のバクテリオファージベクター、Ｃｈａｒｏｍｉｄ　ＤＮＡ（和光純薬工業（株）製）、Ｌｏｒｉｓｔ６（和光純薬工業（株）製）等のコスミドベクター等が挙げられる。また、大腸菌由来のプラスミド（例えばｐＴｒｃ９９Ａ，　ｐＫＫ２２３，　ｐＥＴ３ａ）等の他、ｐＡ１－１１、ｐＸＴ１、ｐＲｃ／ＣＭＶ、ｐＲｃ／ＲＳＶ、ｐｃＤＮＡ　Ｉ／Ｎｅｏ、ｐ３×ＦＬＡＧ－ＣＭＶ－１４、ｐＣＡＴ３、ｐｃＤＮＡ３．１、ｐＣＭＶ等も挙げられる。 The expression vector may be any ordinary bacterial vector, and a commercially available bacterial vector can be used. For example, pTrcHis2 vector, pcDNA3.1 / myc-His vector (manufactured by Invitrogen), pUC119 (manufactured by Takara Shuzo), pBR322 (manufactured by Takara Shuzo), pBluescript II KS + Stratagene, and pQE-Tri (Qia) Plasmid vectors such as pET, pGEM-3Z, pGEX, pMAL, etc., bacteriophage vectors such as λEMBL3 (Stratagene), λDASHII (Funakoshi), Charomid DNA (Wako Pure Chemical Industries, Ltd.), Lorist6 (Wako Pure) And cosmid vectors manufactured by Yakuhin Kogyo Co., Ltd.). In addition to E. coli-derived plasmids (eg, pTrc99A, @ pKK223, @ pET3a), pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNA @ I / Neo, p3.times.FLAG-CMV-14, pCAT3, pcDNA3.1. , PCMV and the like.
  ポリヌクレオチドのベクターへの導入は、常法により行うことができる。ベクター中の特定の制限酵素部位を特定の制限酵素で切断し、その切断部位に上記ポリヌクレオチドを挿入することができる。 導入 The introduction of the polynucleotide into the vector can be performed by a conventional method. A specific restriction enzyme site in the vector can be cut with a specific restriction enzyme, and the polynucleotide can be inserted into the cleavage site.
　宿主である細菌としては、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ．）、バチルス属細菌（Ｂ．ｓｕｂｔｉｌｉｓ，　Ｂ．ｍｅｇａｔｅｒｉｕｍ，　Ｂ．ｂｒｅｖｉｓ，　Ｂ．ｂｏｒｓｔｅｌｅｎｉｓ等）、ブドウ球菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ）、乳酸連鎖球菌（Ｌａｃｔｏｃｏｃｃｕｓ　ｌａｃｔｉｓ）、乳酸桿菌（Ｌａｃｔｏｂａｃｉｌｌｕｓ）、ストレプトマイセス属細菌（Ｓｔｒｅｐｔｏｍｙｃｅｓ）、ブレビバチルス属細菌（Ｂｒｅｖｉｂａｃｉｌｌｕｓ）、シュードモナス属細菌（Ｐｓｅｕｄｏｍｏｎａｓ）、コリネバクテリウム属細菌（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ）、セラチア属細菌(Ｓｅｒｒａｔｉａ)、ブレビバクテリウム属細菌(Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ)、ロドコッカス属細菌(Ｒｈｏｄｏｃｏｃｃｕｓ)等を挙げることができるが、特に限定されない。細菌は、好ましくは大腸菌である。 Examples of bacteria serving as hosts include Escherichia coli, Escherichia coli, Bacillus bacteria such as B. subtilis, B. megaterium, B. brevis, B. borstelenis, Staphylococcus, and Lactococcus lacla. Lactobacillus, Lactobacillus, Streptomyces, Brevibacillus, Pseudomonas, Corynebacterium, Serratia, Serratia Bacteria (Brevobacterium), Rhodococcus bacteria (Rhodoco cus), and the like can be given, but it is not particularly limited. The bacterium is preferably E. coli.
　宿主として大腸菌を使用する場合、大腸菌としては、ＪＭ１０９、ＨＢ１０１、ＭＣ４１００、ＭＧ１６５５、Ｗ３１１０、ＢＬ２１等の一般的に用いられる株を用いることができる。 場合 When Escherichia coli is used as a host, commonly used strains such as JM109, HB101, MC4100, MG1655, W3110, and BL21 can be used.
　発現ベクターによる細菌の形質転換は、常法により行うことができる。例えば、塩化カルシウム、リン酸カルシウム、ＤＥＡＥ－デキストラン介在トランスフェクション、エレクトロポレーション、リポフェクション等で行うことができる。 細菌 Bacterial transformation with the expression vector can be performed by a conventional method. For example, it can be performed by calcium chloride, calcium phosphate, DEAE-dextran-mediated transfection, electroporation, lipofection, and the like.
　形質転換体を培養することにより、アデノ随伴ウイルスのカプシドタンパク質を細菌において生産することができる。形質転換体の培養方法は、特に限定されず、一般的な細菌の培養方法を使用することができる。温度、培地のｐＨ及び培養時間も適宜設定することができる。形質転換体の培養に用いられる培地は、宿主の種類に応じて適宜選択することができる。例えば、大腸菌を培養する場合には、ＬＢ培地等が用いられる。培地には、選択マーカーの種類に応じた抗生物質を添加してもよい。また、イソプロピル-β-チオガラクトピラノシド（ＩＰＴＧ）などの発現誘導物質を使用することもできる。 ア デ ノ By culturing the transformant, the capsid protein of adeno-associated virus can be produced in bacteria. The method for culturing the transformant is not particularly limited, and a general method for culturing bacteria can be used. The temperature, the pH of the medium, and the culture time can also be set as appropriate. The medium used for culturing the transformant can be appropriately selected depending on the type of the host. For example, when culturing Escherichia coli, an LB medium or the like is used. An antibiotic depending on the type of the selection marker may be added to the medium. An expression inducing substance such as isopropyl-β-thiogalactopyranoside (IPTG) can also be used.
　工程（ｂ）においては、工程（ａ）において生産したタンパク質を不溶性タンパク質として回収する。例えば、工程（ｂ）においては、細菌の菌体と上清を分離した後に、細菌菌体を破砕し、さらに破砕後の沈殿物を回収することにより、タンパク質を不溶性タンパク質として回収することができる。より具体的には、細菌菌体を遠心分離、濾過又は膜分離などの方法によって回収し、適当な緩衝液（リン酸緩衝液（ＰＢＳ）など）に再懸濁する。その後、例えば、超音波処理、圧力破砕、浸透圧法、凍結融解、界面活性剤処理、又はリゾチーム処理などの方法で、回収された細菌菌体の細胞壁及び／又は細胞膜を破壊する。その後、遠心分離、濾過又は膜分離などの方法により、沈殿を回収することにより、工程（ａ）において生産したタンパク質を、不溶性タンパク質として回収することができる。 In the step (b), the protein produced in the step (a) is recovered as an insoluble protein. For example, in the step (b), the protein can be recovered as an insoluble protein by separating the bacterial cells and the supernatant, crushing the bacterial cells, and collecting the crushed precipitate. . More specifically, bacterial cells are collected by a method such as centrifugation, filtration or membrane separation, and resuspended in an appropriate buffer (phosphate buffer (PBS) or the like). Thereafter, the cell wall and / or cell membrane of the recovered bacterial cells are destroyed by a method such as sonication, pressure crushing, osmotic pressure, freeze-thaw, surfactant treatment, or lysozyme treatment. Thereafter, the protein produced in step (a) can be recovered as an insoluble protein by collecting the precipitate by a method such as centrifugation, filtration, or membrane separation.
　工程（ｃ）においては、工程（ｂ）において回収した不溶性タンパク質を可溶化する。
　不溶性タンパク質の可溶化は、例えば、可溶化剤を用いて行うことができる。可溶化剤としては、カオトロピック試薬、）や各種界面活性剤（ノニオン性、アニオン性、カチオン性、又は双性イオン性の界面活性剤）を使用することができる。カオトロピック試薬としては、尿素、チオ尿素、グアニジン、塩酸グアニジン、硫酸グアニジンなどを使用することができる。また、界面活性剤としては硫酸ドデシルナトリウム（ＳＤＳ）、 n-セ チ ル ト リ メ チ ル ア ン モ ニ ウ ム 塩 酸塩 (ＣＴＡＢ)、ｎ－ラ ウ リ ル サ ル コ シ ン ナ ト リ ウ ムなどを使用することができる。
　また、カプシドタンパク質はシステイン残基を有するため、還元剤存在下でジスルフィド結合を切断した後、正電荷を有する化合物をシステインに付与することで、可溶化することも可能である。具体的には、ＴＡＰＳ－ｓｕｌｆｏｎａｔｅ（トリメチルアンモニオプロピルメタンチオスルホナート・ブロミド）などが使用できる。 In the step (c), the insoluble protein recovered in the step (b) is solubilized.
Solubilization of the insoluble protein can be performed, for example, using a solubilizing agent. As the solubilizing agent, a chaotropic reagent, or various surfactants (nonionic, anionic, cationic, or zwitterionic surfactants) can be used. As the chaotropic reagent, urea, thiourea, guanidine, guanidine hydrochloride, guanidine sulfate and the like can be used. The surfactants include sodium dodecyl sulfate (SDS), n-methyltrimethylammonium hydrochloride (CTAB), and n-rauryl salcocinnato Lime can be used.
Further, since the capsid protein has a cysteine residue, it can be solubilized by cleaving a disulfide bond in the presence of a reducing agent and then giving a positively charged compound to cysteine. Specifically, TAPS-sulfonate (trimethylammoniopropylmethanethiosulfonate bromide) or the like can be used.
　本発明においては、工程（ｃ）の後に、可溶化剤を除去する工程をさらに設けることができる。例えば、工程（ｂ）において回収した不溶性タンパク質に可溶化剤を添加して可溶化した後、フィルターろ過により細菌（形質転換体）やその細胞残渣を除去してもよい。その後、可溶化したタンパク質を、透析膜を用いてＰＢＳに再度置換することにより、可溶化剤を除去し、不溶化タンパク質を得てもよい。 In the present invention, a step of removing the solubilizing agent after the step (c) may be further provided. For example, the solubilizing agent may be added to the insoluble protein recovered in step (b) to solubilize it, and then the bacteria (transformant) and cell residues thereof may be removed by filtration with a filter. Thereafter, the solubilizing agent may be removed by replacing the solubilized protein again with PBS using a dialysis membrane to obtain an insolubilized protein.
　上記した工程（ａ）～（ｃ）において得たタンパク質は、そのまま使用してもよいし、あるいは更なる精製に供してもよい。精製方法としては、例えば塩析、溶媒沈殿法等の溶解度を利用する方法、透析、限外濾過、ゲル濾過、ドデシル硫酸ナトリウム－ポリアクリルアミドゲル電気泳動など分子量の差を利用する方法、イオン交換クロマトグラフィーなどの荷電を利用する方法、アフィニティークロマトグラフィーなどの特異的親和性を利用する方法、疎水性相互作用クロマトグラフィー、逆相高速液体クロマトグラフィーなどの疎水性の差を利用する方法、等電点電気泳動などの等電点の差を利用する方法などが挙げられる。 タ ン パ ク 質 The protein obtained in the above steps (a) to (c) may be used as it is or may be subjected to further purification. Purification methods include, for example, methods using solubility such as salting out and solvent precipitation, methods using molecular weight differences such as dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ion exchange chromatography. Method using charge such as chromatography, method using specific affinity such as affinity chromatography, method using difference in hydrophobicity such as hydrophobic interaction chromatography, reverse phase high performance liquid chromatography, isoelectric point A method utilizing the difference between isoelectric points such as electrophoresis may be used.
［抗ＡＡＶ抗体の作製方法］
　本発明による抗ＡＡＶ抗体の作製方法は、上記した本発明によるアデノ随伴ウイルスのカプシドタンパク質の製造方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を免疫抗原として非ヒト免疫動物に投与する工程を含む。 [Method for preparing anti-AAV antibody]
The method for producing an anti-AAV antibody according to the present invention comprises the steps of producing an adeno-associated virus capsid protein by the above-described method for producing an adeno-associated virus capsid protein according to the present invention, and using the adeno-associated virus capsid protein obtained above. Administering a non-human immunized animal as an immunizing antigen.
　非ヒト免疫動物としては、好ましくは哺乳類、鳥類、または軟骨魚類である。哺乳類としては、ヒトコブラクダ、フタコブラクダ、ラマ、アルパカ、ビクーニャ、グアナゴなどのラクダ科動物、マウス、ラット、ウサギなどのげっ歯類、ウシ、ヤギ、ヒツジなどの反芻類、サル、ウマ、モルモットなどを挙げることができる。鳥類としては、ニワトリ、ガチョウ、ダチョウなどを挙げることができる。軟骨魚類としてはサメ、エイが挙げられる。上記の中でも、非ヒト免疫動物としては、ラクダ科動物またはげっ歯類が好ましく、ラクダ科動物がより好ましい。 The non-human immunized animal is preferably a mammal, a bird, or a cartilaginous fish. Examples of mammals include dromedaries such as dromedaries, bactrian camels, llamas, alpacas, vicunas, guanagos, rodents such as mice, rats, rabbits, ruminants such as cattle, goats, sheep, monkeys, horses, and guinea pigs. be able to. Birds include chickens, geese, ostriches, and the like. Cartilage fish include sharks and rays. Among them, the non-human immunized animal is preferably a camelid or a rodent, and more preferably a camelid.
　アデノ随伴ウイルスのカプシドタンパク質の非ヒト免疫動物への投与は、通常の免疫方法に従って行うことができる。例えば完全フロイントアジュバント、不完全フロイントアジュバントなどのアジュバントと抗原との懸濁液もしくは乳化液を調製し、これを動物の静脈、皮下、皮内、腹腔内等に数回投与することによって動物を免疫化することができる。 (4) Administration of the capsid protein of the adeno-associated virus to a non-human immunized animal can be performed according to a usual immunization method. For example, a suspension or emulsion of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and an antigen is prepared, and the suspension or emulsion is administered several times to the vein, subcutaneous, intradermal, intraperitoneal, etc. of the animal to immunize the animal. Can be
　アデノ随伴ウイルスのカプシドタンパク質が投与された免疫動物の血清を採取し、常法（例えば、硫安分画、イオン交換クロマトグラフィー、Ｐｒｏｔｅｉｎ　Ａ又はＰｒｏｔｅｉｎ　Ｇアフィニティークロマトグラフィー、ゲルろ過クロマトグラフィーなど）に従って、抗ＡＡＶ抗体を取得することができる。 The serum of the immunized animal to which the capsid protein of the adeno-associated virus was administered was collected, and the serum was collected according to a conventional method (for example, ammonium sulfate fractionation, ion exchange chromatography, Protein A or Protein G affinity chromatography, gel filtration chromatography, etc.). AAV antibodies can be obtained.
　アデノ随伴ウイルスのカプシドタンパク質を免疫した動物から抗体産生細胞を取得することもできる。抗体産生細胞としては、免疫された動物からの脾細胞、リンパ節細胞、Ｂリンパ球等を取得することができる。抗体産生細胞を用いてモノクローナル抗体を得ることができる。 抗体 Antibody-producing cells can also be obtained from animals immunized with the capsid protein of the adeno-associated virus. As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals can be obtained. Monoclonal antibodies can be obtained using antibody-producing cells.
　モノクローナル抗体を産生する細胞は特に制限されないが、例えば、抗体産生細胞とミエローマ細胞株との細胞融合によりハイブリドーマとして得ることができる。モノクローナル抗体を産生するハイブリドーマは、細胞融合法によって得ることができる。即ち、免疫化した動物から抗体産生細胞として例えば脾細胞を取得し、これとミエローマ細胞とを公知の方法（G.Kohler et al .,Nature,256 495(1975)、Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46）により融合することにより、ハイブリドーマを作製することができる。 細胞 The cells producing the monoclonal antibody are not particularly limited, but can be obtained as a hybridoma by, for example, cell fusion between the antibody-producing cells and a myeloma cell line. A hybridoma producing a monoclonal antibody can be obtained by a cell fusion method. That is, for example, spleen cells are obtained as antibody-producing cells from an immunized animal, and these cells and myeloma cells are obtained by a known method (G. Kohler et al., Nature, 256-495 (1975), Kohler.lerG. And Milstein, C., Methods Enzymol. (1981) 73: 3-46) to produce a hybridoma.
　細胞融合に使用するミエローマ細胞株としては、例えばマウスではＰ３Ｘ６３Ａｇ８、Ｐ３Ｕ１株、Ｓｐ２／０株などが挙げられる。細胞融合を行なうに際しては、ポリエチレングリコール、センダイウイルスなどの融合促進剤を用い、細胞融合後のハイブリドーマの選抜にはヒポキサンチン・アミノプテリン・チミジン（ＨＡＴ）培地を常法に従って使用することができる。細胞融合により得られたハイブリドーマは限界希釈法等によりクローニングすることができる。モノクローナル抗体産生細胞は、上記したような細胞融合により生成してもよく、また癌遺伝子ＤＮＡの導入やＥｐｓｔｅｉｎ－Ｂａｒｒウイルスの感染によりＢリンパ球を不死化させるような他の方法で生成してもよい。 Examples of myeloma cell lines used for cell fusion include P3X63Ag8, P3U1 strain, and Sp2 / 0 strain in mice. When performing cell fusion, a fusion promoting agent such as polyethylene glycol or Sendai virus is used. For selection of hybridomas after cell fusion, hypoxanthine-aminopterin-thymidine (HAT) medium can be used according to a conventional method. The hybridoma obtained by cell fusion can be cloned by a limiting dilution method or the like. Monoclonal antibody-producing cells may be produced by cell fusion as described above, or may be produced by other methods such as immortalization of B lymphocytes by introduction of oncogene DNA or infection with Epstein-Barr virus. Good.
　次いで、得られたハイブリドーマなどの抗体産生細胞をスクリーニングすることによって、所望の抗体を産生する細胞を選別することができる。抗体産生細胞のスクリーニングは、ＥＬＩＳＡアッセイ、ウエスタンブロット分析、ラジオイムノアッセイ、ＦＡＣＳ等の当該技術分野において公知の方法を用いて行うことができる。所望の抗体を産生する抗体産生細胞をクローニングし、適切な条件下で培養し、分泌された抗体を回収し、常法（例えばイオン交換クロマトグラフィー、アフィニティークロマトグラフィー等）により精製することができる。 Next, cells that produce the desired antibody can be selected by screening the resulting antibody-producing cells such as hybridomas. Screening of antibody-producing cells can be performed using a method known in the art such as ELISA assay, Western blot analysis, radioimmunoassay, FACS, and the like. An antibody-producing cell that produces the desired antibody is cloned, cultured under appropriate conditions, and the secreted antibody is recovered and purified by a conventional method (eg, ion exchange chromatography, affinity chromatography, etc.).
　上記の方法により得られる、カプシドタンパク質に対する抗体は、アデノ随伴ウイルスの生ウイルスに反応することができることが確認されている。 抗体 It has been confirmed that the antibody against the capsid protein obtained by the above method can react with live virus of adeno-associated virus.
　本発明における抗ＡＡＶ抗体としては、重鎖抗体が好ましい。ラクダ科動物は、Ｈ鎖のみで構成される抗体（重鎖抗体）を有し、その可変領域はＶＨＨ抗体（variable domain of heavy chain of heavy chain antibody）と呼ばれている。重鎖抗体（ＶＨＨ抗体）はＩｇＧ抗体等他の抗体に比べて安定性（熱、アルカリなどの変性剤、ｐＨに対する安定性）が高いという利点がある。また、重鎖抗体（ＶＨＨ抗体）は、シングルドメイン抗体であるため、軽鎖・重鎖の組み合わせがなく、作製しやすいという利点がある。 重 As the anti-AAV antibody in the present invention, a heavy chain antibody is preferable. Camelids have antibodies composed of only heavy chains (heavy chain antibodies), and the variable regions thereof are called VHH antibodies (variable @ domain @ of @ heavy @ chain @ of @ heavy @ chain @ antibody). Heavy chain antibodies (VHH antibodies) have the advantage of higher stability (stability to denaturants such as heat and alkalis and pH) compared to other antibodies such as IgG antibodies. Further, since the heavy chain antibody (VHH antibody) is a single domain antibody, there is an advantage that there is no combination of a light chain and a heavy chain, and it is easy to prepare.
［抗ＡＡＶ抗体を産生するＢ細胞集団を製造する方法］
　本発明による抗ＡＡＶ抗体を産生するＢ細胞集団を製造する方法は、上記した本発明によるアデノ随伴ウイルスのカプシドタンパク質の製造方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を、ナイーブＢ細胞集団に投与する工程を含む。 [Method for Producing B Cell Population Producing Anti-AAV Antibody]
The method for producing a B cell population producing an anti-AAV antibody according to the present invention comprises the steps of producing an adeno-associated virus capsid protein by the above-described method for producing an adeno-associated virus capsid protein according to the present invention, and the method obtained above. Administering the adeno-associated virus capsid protein to the naive B cell population.
　国際公開ＷＯ２０１６／２７６０号には、特定抗原に対して特異的なＩｇＧ陽性Ｂ細胞を含む抗原特異的Ｂ細胞集団を製造する方法として、Ｂａｃｈ２遺伝子の発現が上昇しているＢ細胞を、ＣＤ４０及び／又は ＢＡＦＦ受容体に対する作用手段の存在下において培養する工程を含む、Ｂ細胞集団の製造方法が記載されている。本発明の方法により得られるアデノ随伴ウイルスのカプシドタンパク質を、ナイーブＢ細胞集団に投与することによって、アデノ随伴ウイルスのカプシドタンパク質に対する抗体を産生するＢ細胞集団を選別することができる。 International Publication WO2016 / 2760 discloses a method for producing an antigen-specific B cell population including IgG-positive B cells specific for a specific antigen, by using B cells with increased Bach2 gene expression as CD40 and CD40. And / or a method for producing a B cell population, comprising culturing in the presence of a means for acting on a BAFF receptor. By administering the adeno-associated virus capsid protein obtained by the method of the present invention to a naive B cell population, a B cell population that produces an antibody against the adeno-associated virus capsid protein can be selected.
［マトリックス、マトリックスの製造方法、およびマトリックスの利用］
　本発明の方法によりにより得られるアデノ随伴ウイルスのカプシドタンパク質を水不溶性担体に固定化することによって、抗ＡＡＶ抗体を分離するためのマトリックスを製造することができる。即ち、本発明によれば、アデノ随伴ウイルスのカプシドタンパク質が固定化されている水不溶性担体からなる、抗ＡＡＶ抗体を分離するためのマトリックスが提供される。上記マトリックスは、抗ＡＡＶ抗体を吸着することができる。 [Matrix, manufacturing method of matrix, and use of matrix]
By immobilizing the adeno-associated virus capsid protein obtained by the method of the present invention on a water-insoluble carrier, a matrix for separating anti-AAV antibodies can be produced. That is, according to the present invention, there is provided a matrix for separating an anti-AAV antibody, comprising a water-insoluble carrier on which the capsid protein of adeno-associated virus is immobilized. The matrix is capable of adsorbing anti-AAV antibodies.
　アデノ随伴ウイルスのカプシドタンパク質を固定化する担体としては、水不溶性担体であればよく、通常の抗原抗体反応やアフィニティークロマトグラフィーで用いられる担体を使用することができる。水不溶性とは、水に溶解しない担体をいう。担体としては、例えば、ガラスビーズ、シリカゲルなどの無機担体；ポリビニルアルコール、ポリアクリレート、ポリアクリルアミド、ポリスチレンなどの合成高分子、架橋された合成高分子、結晶性セルロース、セルロース、アガロース、デキストランなどの多糖類からなる有機担体；さらにはこれらの組み合わせによって得られる有機－有機、あるいは有機－無機などの複合担体などが挙げられる。担体の形態としては、マイクロプレート、チューブ、メンブレン、不織布、カラム、又はビーズなどを挙げることができる。 (4) The carrier for immobilizing the capsid protein of the adeno-associated virus may be any water-insoluble carrier, and a carrier used in usual antigen-antibody reactions or affinity chromatography can be used. Water-insoluble refers to a carrier that does not dissolve in water. Examples of the carrier include inorganic carriers such as glass beads and silica gel; synthetic polymers such as polyvinyl alcohol, polyacrylate, polyacrylamide, and polystyrene; cross-linked synthetic polymers; crystalline cellulose, cellulose, agarose, and dextran. Organic carriers comprising saccharides; and organic-organic or organic-inorganic composite carriers obtained by a combination of the above. Examples of the form of the carrier include a microplate, a tube, a membrane, a nonwoven fabric, a column, and beads.
　アデノ随伴ウイルスのカプシドタンパク質の担体への固定は、物理的吸着を用いてもよく、または化学結合を用いる方法でもよい。化学結合としては、共有結合、イオン結合、疎水結合、又は水素結合のいずれでもよいが、共有結合が好ましい。アデノ随伴ウイルスのカプシドタンパク質の担体への固定を共有結合により行う場合、担体の表面には、反応性官能基が存在していることが好ましい。反応性官能基としては、エポキシ基、チオール基、水酸基、アミノ基、アルデヒド基、カルボキシル基、シラノール基、アミド基、サクシニルイミド基、酸無水物基などが挙げられる。 固定 The capsid protein of the adeno-associated virus may be immobilized on the carrier by physical adsorption or by a chemical bond. The chemical bond may be any of a covalent bond, an ionic bond, a hydrophobic bond, and a hydrogen bond, but a covalent bond is preferable. When the capsid protein of the adeno-associated virus is fixed to the carrier by covalent bonding, a reactive functional group is preferably present on the surface of the carrier. Examples of the reactive functional group include an epoxy group, a thiol group, a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a silanol group, an amide group, a succinylimide group, and an acid anhydride group.
　本発明のマトリックスと血液とを生体外で接触させることによって、血液から抗ＡＡＶ抗体を除去することができる。好ましくは、血液から、抗アデノ随伴ウイルス中和抗体（以下、抗ＡＡＶ中和抗体とも言う）を除去することができる。ＡＡＶベクターを用いた遺伝子治療では、抗ＡＡＶ中和抗体を有する患者には、遺伝子治療用ＡＡＶベクターを投与しても、十分な効果が得られない。しかし、本発明のマトリックスを用いて透析により患者の血液中から抗ＡＡＶ中和抗体を除去することにより、当該患者に遺伝子治療用ＡＡＶベクターを投与した時の抗ＡＡＶ中和抗体による阻害作用の影響を減らすことができる。 (4) Anti-AAV antibodies can be removed from blood by bringing the matrix of the present invention into contact with blood in vitro. Preferably, an anti-adeno-associated virus neutralizing antibody (hereinafter, also referred to as an anti-AAV neutralizing antibody) can be removed from blood. In gene therapy using an AAV vector, administration of an AAV vector for gene therapy to a patient having an anti-AAV neutralizing antibody cannot provide a sufficient effect. However, by removing the anti-AAV neutralizing antibody from the blood of the patient by dialysis using the matrix of the present invention, the effect of the inhibitory effect of the anti-AAV neutralizing antibody when the AAV vector for gene therapy is administered to the patient Can be reduced.
　また、本発明のマトリックスと、抗体含有試料とを接触させることによって、抗ＡＡＶ抗体を精製することができる。例えば、本発明のマトリックスを用いることによって、アデノ随伴ウイルス又はカプシドタンパク質を免疫した免疫動物の免疫血清から、抗ＡＡＶ抗体を精製することができる。 抗 Also, the anti-AAV antibody can be purified by contacting the matrix of the present invention with an antibody-containing sample. For example, by using the matrix of the present invention, an anti-AAV antibody can be purified from an immune serum of an immunized animal immunized with an adeno-associated virus or a capsid protein.
　本願は、２０１８年９月２８日に出願された日本国特許出願第２０１８－１８４４１１号に基づく優先権の利益を主張するものである。２０１８年９月２８日に出願された日本国特許出願第２０１８－１８４４１１号の明細書の全内容が、本願に参考のため援用される。 This application claims the benefit of priority based on Japanese Patent Application No. 2018-184411 filed on Sep. 28, 2018. The entire contents of the specification of Japanese Patent Application No. 2018-184411 filed on September 28, 2018 are incorporated herein by reference.
　本発明を以下の実施例により更に具体的に説明するが、本発明はこれらに限定されるものではない。 The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.
（実施例１）ＧＳＴ－ＶＰ３発現大腸菌の育種と培養
　ＡＡＶ２のＶＰ３のアミノ酸配列（配列表の配列番号２）をコードする遺伝子（大腸菌最適化コドン）のＮ末端側にＸｈｏＩを、Ｃ末端側にＢａｍＨＩを付加した人工合成遺伝子（配列番号１）を人工合成した（ユーロフィンジェノミクス）。この人工合成遺伝子をＸｈｏＩ、ＢａｍＨＩで切断し、同じ制限酵素で切断したｐＧＥＸ－６ｐ－１（ＧＥヘルスケア）に導入してＧＳＴ－ＶＰ３発現プラスミドｐＥＸ－Ｋ４Ｊ１－ＡＡＶ－ＶＰ３を作製した。得られたプラスミドをＥ．ｃｏｌｉ　ＪＭ１０９（タカラバイオ）に添加し、氷上で３０分静置した後、４２℃で４５秒ヒートショックし形質転換した。その後、ＶＰ３遺伝子の導入が確認できた形質転換体を用いて、Ｍａｇｉｃ　Ｍｅｄｉａ培地（ｉｎｖｉｔｒｏｇｅｎ）を用いて３０℃一晩フラスコで培養することによりＧＳＴ－ＶＰ３発現大腸菌の培養液を得た。 (Example 1) Breeding and cultivation of GST-VP3-expressing Escherichia coli XhoI at the N-terminal side of the gene (Escherichia coli optimized codon) encoding the amino acid sequence of VP3 of AAV2 (SEQ ID NO: 2 in the sequence listing), and An artificially synthesized gene (SEQ ID NO: 1) to which BamHI had been added was artificially synthesized (Eurofin Genomics). This artificially synthesized gene was digested with XhoI and BamHI, and introduced into pGEX-6p-1 (GE Healthcare) digested with the same restriction enzymes to prepare a GST-VP3 expression plasmid pEX-K4J1-AAV-VP3. The resulting plasmid was transformed into E. coli. coli JM109 (Takara Bio), left on ice for 30 minutes, and then heat-shocked at 42 ° C. for 45 seconds to transform. Thereafter, the transformant in which the introduction of the VP3 gene was confirmed was cultured in a Magic Media medium (invitrogen) in a flask at 30 ° C. overnight to obtain a culture solution of GST-VP3-expressing Escherichia coli.
（実施例２）ＧＳＴ－ＶＰ３の精製
　実施例１で得られた培養液を９，０００ｒｐｍで１０分間遠心し、上清を除去した。残った菌体をダルベッコリン酸緩衝生理食塩水（ＰＢＳ）で懸濁し、超音波破砕機で菌体を破砕し、再度１５，０００ｒｐｍで５分間遠心し、不溶化したＧＳＴ－ＶＰ３を含む沈殿画分を得た。この沈殿にＰＢＳ／６Ｍ尿素を加え、ＧＳＴ－ＶＰ３を可溶化した後、０．２μｍのフィルターでろ過し、残存している可能性がある組換え菌を除去した。その後、可溶化したＧＳＴ－ＶＰ３を透析膜（Ｓｌｉｄｅ－Ａ－ＬｙｚｅｒＴＭ　Ｇ２Ｄｉａｌｙｓｉｓ　Ｃａｓｓｅｔｔｅｓ，１０Ｋ　ＭＷＣＯ，３ｍＬ）を用いて、ＰＢＳに再度置換し、不溶化タンパク質を得た。得られたタンパク質懸濁液のタンパク質濃度はＢＳＡを標準品として６６０ｎｍタンパク質アッセイ法（ＰｉｅｒｃｅＴＭ６６０ｎｍ　Ｐｒｏｔｅｉｎ　Ａｓｓａｙ　Ｒｅａｇｅｎｔ、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）を用いて、測定した。その結果、５０ｍＬの培養液から３８．７ｍｇの目的物を含むタンパク質を得た。 (Example 2) Purification of GST-VP3 The culture solution obtained in Example 1 was centrifuged at 9,000 rpm for 10 minutes, and the supernatant was removed. The remaining cells were suspended in Dulbecco's phosphate-buffered saline (PBS), crushed with an ultrasonic crusher, and again centrifuged at 15,000 rpm for 5 minutes to precipitate the precipitate fraction containing the insolubilized GST-VP3. I got PBS / 6M urea was added to the precipitate to solubilize GST-VP3, and then filtered with a 0.2 μm filter to remove any remaining recombinant bacteria. After that, the solubilized GST-VP3 was replaced again with PBS using a dialysis membrane (Slide-A-Lyzer ™ G2Dialysis Cassettes, 10K MWCO, 3 mL) to obtain an insolubilized protein. The protein concentration of the obtained protein suspension was measured using a 660 nm protein assay (Pierce ™ 660 nm Protein Assay Reagent, Thermo Fisher Scientific) using BSA as a standard. As a result, 38.7 mg of the protein containing the target substance was obtained from 50 mL of the culture solution.
（実施例３）ｅｍｐｔｙ　ｐａｒｔｉｃｌｅ（ＡＡＶ２）の調製
　ｅｍｐｔｙ　ｐａｒｔｉｃｌｅとは、内部に核酸を持たないウイルス粒子である。
　ＨＥＫ２９３Ｔ細胞（ヒト胎児腎由来細胞）をｐＲＣ２－ｍｉ３４２　ｖｅｃｔｏｒ，ｐＨｅｌｐｅｒ　ｖｅｃｔｏｒをトランスフェクトし、約７２時間後に細胞を破壊して、ＡＡＶ２のｅｍｐｔｙ　ｐａｒｔｉｃｌｅを陽イオンクロマトにて精製した。 (Example 3) Preparation of empty particle (AAV2) Empty particle is a virus particle having no nucleic acid inside.
HEK293T cells (human embryonic kidney-derived cells) were transfected with pRC2-mi342 vector and pHelper vector, and after about 72 hours, the cells were disrupted, and the AAV2 empty particles were purified by cation chromatography.
（実施例４）ＧＳＴ－ＶＰ３を抗原としたウサギ免疫実験
　実施例２で得たＧＳＴ－ＶＰ３の粗精製品（１ｍｇ／ｍＬ）をアジュバント：ＴｉｔｅｒＭａｘ　Ｇｏｌｄ（フナコシ、Ｌｏｔ　Ｎｏ．：Ｇ１２１６）を等量で混合し、エマルジョン溶液を調製した。このエマルジョン溶液をウサギ（Ｓｔｄ：ＪＷ／ＣＳＫ、雌、２０週齢）１匹あたり０．５ｍＬずつを数箇所に分散させながら、２週間毎に計４回免疫した。免疫後それぞれ１週間後に採血を行い、血清を得た。これを陰性対照（ＮＣ）とした。 (Example 4) Rabbit immunization experiment using GST-VP3 as an antigen The crude product of GST-VP3 obtained in Example 2 (1 mg / mL) was adjuvanted with an equal amount of TiterMax Gold (Funakoshi, Lot No .: G1216). To prepare an emulsion solution. This emulsion solution was immunized four times every two weeks while dispersing 0.5 mL per rabbit (Std: JW / CSK, female, 20 weeks old) in several places. One week after immunization, blood was collected to obtain serum. This was used as a negative control (NC).
（実施例５）ウサギ血清の抗体価の評価
　実施例４で得られたウサギ血清について、免疫抗原であるＧＳＴ－ＶＰ３と、実施例３で調製したｅｍｐｔｙ　ｐａｒｔｉｃｌｅ（ＡＡＶ２）に対する抗体価をそれぞれ酵素結合免疫吸着法（ＥＬＩＳＡ）にて評価した。血清や抗体の希釈には０．０１％　Ｔｗｅｅｎ　２０を含むＰＢＳを使用した。また、評価に使用したＡＡＶ２はＨＥＫ２９３細胞で産生し、ＡＡＶｐｒｏ（登録商標）Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（タカラバイオ）で精製して、調製した。ＧＳＴ－ＶＰ３（抗原）またはＡＡＶ２をＰＢＳ（ｐＨ７．４）で希釈し、マイクロプレート（Ｉｍｍｎｏ　Ｍａｘｉ－ｓｏｒｐ，Ｎｕｎｃ，Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）にそれぞれ固相化した。固相化プレートを０．１％　Ｔｗｅｅｎ　２０を含むＰＢＳで洗浄後、ウサギから得られた血清抗体を添加し、二次抗体にＨＲＰ－Ｇｏａｔ　Ａｎｔｉ－Ｒａｂｂｉｔ　ＩｇＧ（Ｈ＋Ｌ）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，ＣａｔＮｏ．６５－６１２０）と発色基質にＳｕｒｅＢｌｕｅＴＭ　ＴＭＢ　Ｍｉｃｒｏｗｅｌｌ　Ｐｅｒｏｘｉｄａｓｅ　Ｓｕｂｓｔｒａｔｅ　Ｋｉｔ（ＳｅｒａＣａｒｅ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅｓ，Ｉｎｃ．）を用いて発色し、４５０ｎｍの吸光度を確認した。結果を図１に示す。 (Example 5) Evaluation of antibody titer of rabbit serum For the rabbit serum obtained in Example 4, the antibody titers of GST-VP3, which is an immunizing antigen, and empty particle (AAV2) prepared in Example 3 were enzyme-linked. Evaluation was performed by immunosorbent assay (ELISA). For dilution of serum or antibody, PBS containing 0.01% Tween 20 was used. AAV2 used for the evaluation was produced in HEK293 cells, and purified and prepared with AAVpro (registered trademark) Purification Kit (Takara Bio). GST-VP3 (antigen) or AAV2 was diluted with PBS (pH 7.4) and immobilized on a microplate (Immno Maxi-sorp, Nunc, Sigma-Aldrich), respectively. After the immobilized plate was washed with PBS containing 0.1% Tween 20, a serum antibody obtained from a rabbit was added thereto, and HRP-Goat Anti-Rabbit IgG (H + L) (Thermo Fisher Scientific, CatNo. 65-6120) and SureBlue ™ TMB Microwell Peroxidase Substrate Kit (SeraCare Life Sciences, Inc.) as a chromogenic substrate, and the absorbance at 450 nm was confirmed. The results are shown in FIG.
　ＧＳＴ－ＶＰ３を抗原として免疫して得られた抗体の中にはＧＳＴ－ＶＰ３だけでなく、ＡＡＶ２にも結合する抗体も含まれることが確認できた。本結果より、ＧＳＴ－ＶＰ３は抗ＡＡＶ抗体の取得のための抗原として有用であることが示された。 Δ It was confirmed that antibodies obtained by immunization with GST-VP3 as an antigen include not only GST-VP3 but also antibodies that bind to AAV2. These results indicate that GST-VP3 is useful as an antigen for obtaining an anti-AAV antibody.
（実施例６）ＧＳＴ－ＶＰ３を抗原としたアルパカ免疫実験
　５週齢の雄のアルパカを免疫動物として用いた。免疫は、計５回実施した。初回免疫前に採血を行い免疫前コントロール血清とし、その後初回免疫を実施した。初回免疫は、アジュバントとしてＦｒｅｕｎｄ‘ｓ　Ｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（ＦＣＡ）を使用し、３．３ｍｇの免疫抗原（ＧＳＴ－ＶＰ３）をエマルジョンし、アルパカの皮下複数個所への免疫を実施した。２回目、３回目の免疫は、アジュバントとしてＦｒｅｕｎｄ‘ｓ　Ｉｎｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（ＦＩＡ）を使用し、それぞれ３．３ｍｇの免疫抗原（ＧＳＴ－ＶＰ３）とエマルジョンし、アルパカの皮下複数個所への免疫を実施した。４回目、５回目の免疫は、アジュバントとしてＦＩＡを使用し、それぞれ３．３ｍｇの免疫抗原（ＧＳＴ－ＶＰ３とｅｍｐｔｙ　ｐａｒｔｉｃｌｅ（ＡＡＶ２））とエマルジョンし、アルパカの皮下複数個所への免疫を実施した。３回目、５回目の免疫終了後に、アルパカ頚動脈より採血を実施し、アルパカ血液を取得した。採取した末梢血を分離剤入り採管に回収し、３時間以上室温で静置し、遠心（３５００ｒｐｍ　１０分）し、血清を得た。
　アルパカ血液からフィコールを用いた密度勾配遠心によってアルパカ末梢血リンパ球とプラズマ画分へ分離した。プラズマ画分から、ＰｒｏｔｅｉｎＧアフィニティーカラムとＰｒｏｔｅｉｎＡアフィニティーカラムを用いて、アルパカ重鎖抗体を精製した。具体的には、まずＰｒｏｔｅｉｎＧアフィニティーカラムにアルパカプラズマ画分を結合させ、０．１５％　ＮａＣｌ、０．５８％　酢酸（ｐＨ４．５）溶出を行い、ＩｇＧ２（ヒンジ領域が短い重鎖抗体）を取得した。 (Example 6) Alpaca immunization experiment using GST-VP3 as an antigen A 5-week-old male alpaca was used as an immunized animal. Immunization was performed 5 times in total. Before the first immunization, blood was collected and used as a pre-immune control serum, followed by the first immunization. For the first immunization, Freund's Complete Adjuvant (FCA) was used as an adjuvant, 3.3 mg of an immunizing antigen (GST-VP3) was emulsified, and alpaca was immunized subcutaneously at multiple sites. In the second and third immunizations, Freund's Complete Adjuvant (FIA) was used as an adjuvant, and each was emulsified with 3.3 mg of an immunizing antigen (GST-VP3), and immunization was performed at several subcutaneous locations of alpaca. . In the fourth and fifth immunizations, FIA was used as an adjuvant, and each was emulsified with 3.3 mg of an immunizing antigen (GST-VP3 and empty particle (AAV2)), and immunization was performed at several subcutaneous locations of alpaca. After the third and fifth immunizations, blood was collected from the alpaca carotid artery to obtain alpaca blood. The collected peripheral blood was collected in a collection tube containing a separating agent, allowed to stand at room temperature for 3 hours or longer, and centrifuged (3500 rpm for 10 minutes) to obtain serum.
Alpaca peripheral blood lymphocytes and plasma fraction were separated from alpaca blood by density gradient centrifugation using Ficoll. The alpaca heavy chain antibody was purified from the plasma fraction using a Protein G affinity column and a Protein A affinity column. Specifically, first, an alpaca plasma fraction was bound to a Protein G affinity column, and 0.15% NaCl and 0.58% acetic acid (pH 4.5) were eluted to obtain IgG2 (a heavy chain antibody with a short hinge region). did.
（実施例７）アルパカ血清の抗体価の評価
　実施例６で得られたアルパカ血清について、免疫抗原であるＧＳＴ－ＶＰ３と、ｅｍｐｔｙ　ｐａｒｔｉｃｌｅ（ＡＡＶ２）に対する抗体価をそれぞれ酵素結合免疫吸着法（ＥＬＩＳＡ）にて評価した。血清や抗体の希釈には０．０１％　Ｔｗｅｅｎ　２０を含むＰＢＳを使用した。また、評価に使用したｅｍｐｔｙ　ｐａｒｔｉｃｌｅ（ＡＡＶ２）はＨＥＫ２９３細胞で産生し、陽イオンクロマトで精製して、調製した。ＧＳＴ－ＶＰ３（抗原）またはｅｍｐｔｙ　ｐａｒｔｉｃｌｅ（ＡＡＶ２）をＰＢＳ（ｐＨ７．４）で希釈し、マイクロプレート（Ｉｍｍｎｏ　Ｍａｘｉ－ｓｏｒｐ，Ｎｕｎｃ，Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）にそれぞれ固相化した。固相化プレートを０．１％　Ｔｗｅｅｎ　２０を含むＰＢＳで洗浄後、アルパカから得られた血清抗体を添加し、二次抗体にＨＲＰ－rｒａｂｂｉｔ　Ａｎｔｉ－ａｌｐａｃａ　ＩｇＧ（Ｈ＋Ｌ）（アークリソース社）と発色基質にＳｕｒｅＢｌｕｅＴＭ　ＴＭＢ　Ｍｉｃｒｏｗｅｌｌ　Ｐｅｒｏｘｉｄａｓｅ　Ｓｕｂｓｔｒａｔｅ　Ｋｉｔ（ＳｅｒａＣａｒｅ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅｓ，Ｉｎｃ．）を用いて発色し、４５０ｎｍの吸光度を確認した。結果を図２に示す。精製した重鎖抗体を使用し、抗原固相化ＥＬＩＳＡを実施し、ＩｇＧ３の抗原に対する力価が有意に上昇していることを確認した。 (Example 7) Evaluation of antibody titer of alpaca serum For the alpaca serum obtained in Example 6, the antibody titers to GST-VP3, which is an immunizing antigen, and empty particle (AAV2) were determined by enzyme-linked immunosorbent assay (ELISA). Was evaluated. For dilution of serum or antibody, PBS containing 0.01% Tween 20 was used. In addition, empty particles (AAV2) used for evaluation were produced in HEK293 cells, purified by cation chromatography, and prepared. GST-VP3 (antigen) or empty particle (AAV2) was diluted with PBS (pH 7.4) and immobilized on a microplate (Immno Maxi-sorp, Nunc, Sigma-Aldrich), respectively. After washing the immobilized plate with PBS containing 0.1% Tween 20, serum antibody obtained from alpaca was added, and HRP-rrabbit Anti-alpaca IgG (H + L) (Arc Resources) was developed as a secondary antibody. The color was developed using SureBlue ™ TMB Microwell Peroxidase Substrate Kit (SeraCare Life Sciences, Inc.) as the substrate, and the absorbance at 450 nm was confirmed. The results are shown in FIG. Using the purified heavy chain antibody, an antigen-immobilized ELISA was performed, and it was confirmed that the titer of IgG3 against the antigen was significantly increased.
（実施例８）ＧＳＴ－ＶＰ３固定化カラムの作製
　実施例２と同様にして調製したＧＳＴ－ＶＰ３を、水不溶性基材として、市販の活性化担体「ＮＨＳ　ａｃｔｉｖａｔｅｄ　Ｓｅｐｈａｒｏｓｅ　４　Ｆａｓｔ　Ｆｌｏｗ」（ＧＥヘルスケア社製）を使用して固定化した。このカラムは、架橋アガロースをベースとし、タンパク性リガンド固定化用のＮ－ヒドロキシスクシンイミド（ＮＨＳ）基が導入されている。製品マニュアルに従い、ＧＳＴ－ＶＰ３を固定化し、ＧＳＴ－ＶＰ３固定化カラムを作製した。 (Example 8) Preparation of GST-VP3 immobilized column GST-VP3 prepared in the same manner as in Example 2 was used as a water-insoluble base material, and a commercially available activated carrier “NHS activated Sepharose 4 Fast Flow” (GE Healthcare) (Manufactured by the company). This column is based on cross-linked agarose and has introduced N-hydroxysuccinimide (NHS) groups for immobilizing proteinaceous ligands. According to the product manual, GST-VP3 was immobilized to prepare a GST-VP3 immobilized column.
　具体的には、最終精製サンプルをカップリング緩衝液（０．２Ｍ　炭酸ナトリウム、０．５Ｍ　ＮａＣｌ、ｐＨ８．３）で終濃度約１０ｍｇ／ｍＬに希釈した溶液を調製した。氷浴で冷やした１ｍＭ　ＨＣｌで担体を３回洗浄し、担体の保存液を除去した。その後すぐに、サックドライした担体をサンプル希釈溶液で懸濁し、２５℃で３０分間混合して、ＧＳＴ－ＶＰ３を担体に固定化した。その後、担体懸濁液をろ過し、未反応タンパク質溶液を回収した。カップリング緩衝液で担体を洗浄した後、ブロッキング用緩衝液（０．５Ｍ　エタノールアミン、０．５Ｍ　ＮａＣｌ、ｐＨ８．３）で３回洗浄、洗浄用緩衝液（０．１Ｍ　酢酸、０．５Ｍ　ＮａＣｌ、ｐＨ４．０）で３回洗浄した、最後に標準緩衝液（２０ｍＭ　ＮａＨ２ＰＯ４－Ｎａ２ＨＰＯ４、１５０ｍＭ　ＮａＣｌ、ｐＨ７．４）で２回洗浄してＧＳＴ－ＶＰ３固定化担体の作製を完了した。また、対照としてＧＳＴ－ＶＰ３を固定化せず、ブロッキング用緩衝液で封止しただけの担体（以下、対照担体）も作製した。 Specifically, a solution was prepared by diluting the final purified sample with a coupling buffer (0.2 M sodium carbonate, 0.5 M NaCl, pH 8.3) to a final concentration of about 10 mg / mL. The carrier was washed three times with 1 mM HCl cooled in an ice bath, and the stock solution of the carrier was removed. Immediately thereafter, the suck-dried carrier was suspended in a sample diluting solution and mixed at 25 ° C. for 30 minutes to immobilize GST-VP3 on the carrier. Thereafter, the carrier suspension was filtered to recover an unreacted protein solution. After the carrier was washed with a coupling buffer, the carrier was washed three times with a blocking buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3), and the washing buffer (0.1 M acetic acid, 0.5 M NaCl) was used. , PH 4.0), and finally washed twice with a standard buffer (20 mM NaH2PO4-Na2HPO4, 150 mM NaCl, pH 7.4) to complete the preparation of the GST-VP3 immobilized carrier. Further, as a control, a carrier which was not immobilized with GST-VP3 and was merely sealed with a blocking buffer (hereinafter, a control carrier) was also prepared.
（実施例９）ＧＳＴ－ＶＰ３固定化カラムによる抗ＡＡＶ抗体の吸着
　実施例７で作製したＧＳＴ－ＶＰ３固定化担体及び対照担体各１ｍＬを市販カラム（Ｔｒｉｃｏｒｎ５／５０）に充填し、下記条件で抗ＡＡＶ抗体の吸着実験を実施した。実験で得られた未吸着画分について、実施例４のＥＬＩＳＡ法により抗体価を評価した。 (Example 9) Adsorption of anti-AAV antibody by GST-VP3 immobilized column Each 1 mL of the GST-VP3 immobilized carrier and the control carrier prepared in Example 7 was packed in a commercially available column (Tricorn 5/50), and the anti-AAV antibody was purified under the following conditions An AAV antibody adsorption experiment was performed. The antibody titer of the unadsorbed fraction obtained in the experiment was evaluated by the ELISA method of Example 4.
＜クロマトグラフィー条件＞
カラム：ＧＳＴ－ＶＰ３固定化担体を充填したカラム
流速：０．２５ｍＬ／ｍｉｎ、接触時間４．０ｍｉｎ、
負荷液：実施例４で得られたウサギ血清を平衡化ｂｕｆｆｅｒで希釈した溶液
平衡化ｂｕｆｆｅｒ：ダルベッコリン酸緩衝生理食塩水（シグマ・アルドリッチ社製）
溶出条件：０．１Ｍグリシン塩酸（ｐＨ２．７） <Chromatography conditions>
Column: column filled with GST-VP3 immobilized carrier Flow rate: 0.25 mL / min, contact time 4.0 min,
Loading solution: A solution obtained by diluting the rabbit serum obtained in Example 4 with an equilibrating buffer. Equilibrated buffer: Dulbecco's phosphate buffered saline (manufactured by Sigma-Aldrich)
Elution conditions: 0.1 M glycine hydrochloride (pH 2.7)
　ウサギ血清を各担体で精製し、未吸着画分について実施例２と同様の方法でタンパク質濃度を測定し、実施例４と同様の方法で抗体価を評価した。それぞれの結果を表１に示す。未吸着画分に含まれる抗体のＧＳＴ－ＶＰ３に対する抗体価は精製前に比べて著しく減少していた。本結果より、ＧＳＴ－ＶＰ３固定化担体は、抗ＡＡＶ抗体を吸着することが可能であると考えられた。 Rabbit serum was purified on each carrier, the protein concentration of the unadsorbed fraction was measured in the same manner as in Example 2, and the antibody titer was evaluated in the same manner as in Example 4. Table 1 shows the results. The antibody titer against GST-VP3 of the antibody contained in the unadsorbed fraction was significantly reduced as compared to before the purification. From these results, it was considered that the GST-VP3 immobilized carrier can adsorb the anti-AAV antibody.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001

Claims (15)

  1. （ａ）アデノ随伴ウイルスのカプシドタンパク質を細菌において生産する工程；
    （ｂ）前記タンパク質を不溶性タンパク質として回収する工程；及び
    （ｃ）回収した不溶性タンパク質を可溶化する工程；
    を含む、アデノ随伴ウイルスのカプシドタンパク質の製造方法。     (A) producing the adeno-associated virus capsid protein in bacteria;
    (B) recovering the protein as an insoluble protein; and (c) solubilizing the recovered insoluble protein;
    A method for producing a capsid protein of an adeno-associated virus, comprising:
  2. 工程（ａ）において、アデノ随伴ウイルスのカプシドタンパク質を、タグタンパク質との融合タンパク質として、細菌において生産する、請求項１に記載の方法。 The method according to claim 1, wherein in step (a), the capsid protein of the adeno-associated virus is produced in a bacterium as a fusion protein with a tag protein.
  3. タグタンパク質が、可溶性タンパク質である、請求項２に記載の方法。 3. The method according to claim 2, wherein the tag protein is a soluble protein.
  4. アデノ随伴ウイルスのカプシドタンパク質が、ＶＰ３である、請求項１から３の何れか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein the capsid protein of the adeno-associated virus is VP3.
  5. 工程（ｂ）において、細菌の菌体と上清を分離した後に、細菌菌体を破砕し、さらに破砕後の沈殿物を回収することにより、タンパク質を不溶性タンパク質として回収する、請求項１から４の何れか一項に記載の方法。 In the step (b), after separating bacterial cells from the supernatant, the bacterial cells are crushed, and the precipitate after crushing is recovered, whereby the protein is recovered as an insoluble protein. The method according to claim 1.
  6. 工程（ｃ）において、回収した不溶性タンパク質を可溶化剤により可溶化する、請求項１から５の何れか一項に記載の方法。 The method according to any one of claims 1 to 5, wherein in step (c), the recovered insoluble protein is solubilized with a solubilizing agent.
  7. 工程（ｃ）の後に、可溶化剤を除去する工程をさらに含む、請求項６に記載の方法。 7. The method of claim 6, further comprising, after step (c), removing the solubilizing agent.
  8. 請求項１から７の何れか一項に記載の方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を抗原として非ヒト免疫動物に投与する工程を含む、抗アデノ随伴ウイルス抗体の作製方法。 A step of producing an adeno-associated virus capsid protein by the method according to any one of claims 1 to 7, and a step of administering the adeno-associated virus capsid protein obtained above as an antigen to a non-human immunized animal. A method for producing an anti-adeno-associated virus antibody.
  9. 抗アデノ随伴ウイルス抗体が重鎖抗体である、請求項８に記載の方法。 9. The method of claim 8, wherein the anti-adeno-associated virus antibody is a heavy chain antibody.
  10. 請求項１から７の何れか一項に記載の方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を、ナイーブＢ細胞集団に投与する工程を含む、抗アデノ随伴ウイルス抗体を産生するＢ細胞集団を製造する方法。 A method for producing an adeno-associated virus capsid protein by the method according to any one of claims 1 to 7, and a step for administering the obtained adeno-associated virus capsid protein to a naive B cell population. , A method of producing a B cell population that produces an anti-adeno-associated virus antibody.
  11. 請求項１から７の何れか一項に記載の方法により得られるアデノ随伴ウイルスのカプシドタンパク質が固定化されている水不溶性担体からなる、抗アデノ随伴ウイルス抗体を分離するためのマトリックス。 A matrix for separating an anti-adeno-associated virus antibody, comprising a water-insoluble carrier to which an adeno-associated virus capsid protein obtained by the method according to any one of claims 1 to 7 is immobilized.
  12. 請求項１から７の何れか一項に記載の方法によりアデノ随伴ウイルスのカプシドタンパク質を製造する工程、及び上記で得られたアデノ随伴ウイルスのカプシドタンパク質を水不溶性担体に固定化する工程を含む、抗アデノ随伴ウイルス抗体を分離するためのマトリックスの製造方法。 A method for producing an adeno-associated virus capsid protein by the method according to any one of claims 1 to 7, and a step for immobilizing the adeno-associated virus capsid protein obtained above on a water-insoluble carrier. A method for producing a matrix for separating an anti-adeno-associated virus antibody.
  13. 請求項１１に記載のマトリックスと血液とを生体外で接触させることを含む、血液から抗アデノ随伴ウイルス抗体を除去する方法。 A method for removing anti-adeno-associated virus antibodies from blood, comprising contacting the matrix according to claim 11 with blood in vitro.
  14. 抗アデノ随伴ウイルス抗体が、中和抗体である、請求項１３に記載の方法。 14. The method according to claim 13, wherein the anti-adeno-associated virus antibody is a neutralizing antibody.
  15. 請求項１１に記載のマトリックスと、抗体含有試料とを接触させることを含む、抗アデノ随伴ウイルス抗体を精製する方法。 A method for purifying an anti-adeno-associated virus antibody, comprising contacting the matrix according to claim 11 with a sample containing the antibody.
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