WO2021153659A1 - Procédé de quantification de virus adéno-associé (vaa) - Google Patents

Procédé de quantification de virus adéno-associé (vaa) Download PDF

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WO2021153659A1
WO2021153659A1 PCT/JP2021/002994 JP2021002994W WO2021153659A1 WO 2021153659 A1 WO2021153659 A1 WO 2021153659A1 JP 2021002994 W JP2021002994 W JP 2021002994W WO 2021153659 A1 WO2021153659 A1 WO 2021153659A1
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antibody
aav
recognizes
carrier
same
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Japanese (ja)
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進 久保田
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Ａｇｃ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to a method for quantifying adeno-associated virus (AAV), a method for producing AAV, and a kit for quantifying AAV.
  • AAV adeno-associated virus
  • Adeno-associated virus is a particulate non-enveloped virus belonging to the Parvoviridae family that contains linear single-stranded DNA with capsids having three capsid proteins (VP1, VP2, and VP3).
  • AAV has the ability to infect a wide variety of cells including humans, and it is known that there are more than a dozen serotypes in primates.
  • Non-Patent Documents 1 to 3 AAV has at least 15 serotypes AAV (AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12, AAV13. ) has been reported.
  • AAV is being actively developed as a therapeutic gene transfer vector tailored to the purpose because each serotype has different cell directivity, is not pathogenic to humans, and is physicochemically stable. (Non-Patent Document 4).
  • AAV is a particle containing DNA and is not only used as a vector for gene therapy, but also developed as a DDS in which a target molecule such as a low molecular weight compound or nucleic acid is contained in a hollow particle of AAV. (Patent Document 1). Therefore, quantification of AAV prepared regardless of the contents in the capsid is essential.
  • Enzyme-linked immunosorbent assay (ELISA), Western blotting, and real-time PCR methods are known as AAV quantification methods (Patent Document 2 and Non-Patent Document 5).
  • AAV quantification methods Patent Document 2 and Non-Patent Document 5.
  • the real-time PCR method is widely used as a titer measurement of AAV, but since it is a method using a primer having a sequence complementary to the target DNA contained in AAV, AAV hollow particles cannot be quantified. ..
  • the real-time PCR method even if the DNA contained in AAV is fragmented and does not completely contain the target sequence, if the complementary sequence with the primer remains, the genome having the target sequence is included. Detected indistinguishable from AAV.
  • Western blotting can detect AAV hollow particles and AAV particles containing DNA without distinguishing them if an antibody using the target molecule as an epitope can be used.
  • this method has the disadvantages that the number of samples that can be processed at one time is limited, the analysis time is long, and the operation is complicated, and it is not quantitative.
  • ELISA which is widely used as a protein quantification system, is also used for quantification of viruses covered with protein shells.
  • ELISA is not an effective method for use in process development and is mostly used because of its low throughput, high cost, complicated operation, and large sample volume for measurement. do not have.
  • the Alpha LISA method is known as a method for detecting a new protein.
  • the AlphaLISA method is based on the technique (Non-Patent Document 6) reported by Ulman et al. As the LOCI (Luminescent Oxygen Channeling Immunoassay) method in 1994. However, the AlphaLISA method is not known as a method for quantifying AAV.
  • An object of the present invention is to provide a new AAV quantification method that solves the problems of the existing AAV quantification method.
  • AlphaLISA is a technology that enables the detection of target molecules under various solvent conditions, and has been used to detect target molecules contained in cultured cells and serum.
  • AlphaLISA utilizes two types of carriers (eg, beads) called donors and acceptors. It is a mechanism that can detect the luminescence signal when the molecule bound to the donor interacts with the molecule bound to the acceptor and the two carriers are in close proximity.
  • the photosensitizer in the donor excited by the laser converts the surrounding oxygen into excited singlet oxygen, the produced singlet oxygen diffuses around the donor and reaches the luminescent material in the nearby acceptor.
  • a chemiluminescent reaction is triggered in the carrier and light is emitted. By detecting the emitted light, it is possible to detect the target molecule.
  • This Alpha LISA has high throughput and can detect the target molecule with a small amount of sample.
  • AAV AAV using two antibodies that recognize the capsid protein of AAV, an acceptor carrier and a donor carrier, which results in a smaller amount of sample solution compared to conventional quantification methods.
  • the present invention has been completed by finding that the number of steps can be reduced and the amount can be quantified in a shorter time, and as a result, the throughput can be increased and the cost can be reduced. That is, the present invention is as follows.
  • the first antibody that recognizes the capsid protein of adeno-associated virus (AAV) and to which an affinity tag is bound A conjugate of a second antibody that recognizes the same AAV capsid protein and a carrier with a luminescent substance, A method for quantifying AAV using a carrier having a photosensitizer and a measurement sample solution.
  • a method for quantifying AAV using a conjugate of a second antibody that recognizes the same AAV capsid protein and a carrier having a luminescent substance, and a measurement sample solution At least one selected from the group consisting of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12 and AAV13 as the first antibody.
  • [4] At least one selected from the group consisting of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12 and AAV13 as the first antibody.
  • the first antibody is any of the antibodies listed in Table 1, and the second antibody is the same antibody as the first antibody or an antibody that recognizes the same capsid protein. [1] Or the method according to [2].
  • the first antibody is any of the antibodies listed in Table 1, and the second antibody is the same antibody as the first antibody or an antibody that recognizes the same conformational epitope [6]. 1] or the method according to [2]. [7] The method according to [1] or [2] above, wherein the first antibody and the second antibody are antibodies that recognize the same conformational epitope as any one antibody shown in Table 1. [8] The method according to [1] or [2] above, wherein the first antibody and the second antibody are antibodies that compete with any one of the antibodies shown in Table 1. [9] The method according to the above [1] or [2], wherein the first antibody and the second antibody are the same and are ADK1a, ADK5a or A20. [10] The method according to any one of [1] to [9] above, wherein the antibody is a monoclonal antibody.
  • the affinity tag is any one of biotin, glutathione-S-transferase, histidine peptide, FLAG peptide, Strep (II), Digoxigenin, HRP, Hapten, green fluorescent protein, and Fluorescein Isothiocyanate.
  • the photosensitizer is phthalocyanine.
  • a method for producing AAV which comprises a step of preparing AAV and a step of quantifying the prepared AAV using the method according to any one of the above [1] to [18].
  • Carriers with luminescent substances (iii) A second antibody that recognizes the same AAV capsid protein as AAV in (i) above, and (iv) Kit for AAV quantification, including carriers with photosensitizers.
  • a conjugate of the antibody which is the first antibody that recognizes the capsid protein of adeno-associated virus (AAV) and has an affinity tag bound, and a carrier having a photosensitizer
  • a kit for AAV quantification that contains a conjugate of a second antibody that recognizes the same AAV capsid protein and a carrier that has a luminescent substance.
  • a method for quantifying adeno-associated virus including the following steps: (Step 1) The measurement sample solution, the first antibody that recognizes the capsid protein of AAV and has an affinity tag bound, and the second antibody that recognizes the same capsid protein as the capsid protein recognized by the first antibody. And the step of mixing the carrier with the luminescent substance, (Step 2) A step of mixing a carrier having a photosensitive substance with the mixture obtained in Step 1. (Step 3) A step of irradiating the mixture obtained in step 2 with excitation light, and (step 4) a step of measuring a luminescence signal derived from the mixture obtained in step 3.
  • a method for quantifying adeno-associated virus including the following steps: (Step 1') The measurement sample solution, the first antibody that recognizes the capsid protein of AAV and the carrier having an affinity tag-bound antibody and a photosensitizer, and the same capsid as the capsid protein recognized by the first antibody. The step of mixing a second antibody that recognizes a protein with a carrier having a luminescent substance, (Step 2') A step of irradiating the mixture obtained in step 1'with excitation light, and (Step 3') a step of measuring an emission signal derived from the mixture obtained in step 2'.
  • [26] At least one selected from the group consisting of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12 and AAV13 as the first antibody.
  • [27] At least one selected from the group consisting of AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12 and AAV13 as the first antibody.
  • AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12 and AAV13 as the first antibody.
  • the present invention it is possible to quantify AAV with a measurement sample solution in an amount of 1/10 to 1/50 as compared with a conventional quantification method and in a small number of steps.
  • the amount of sample actually used for measurement is about 1/20 to 1/500 as compared with the conventional quantification method. Therefore, it is possible to shorten the fixed quantity time and reduce the cost.
  • FIG. 1 is a diagram showing a pRC2-mi342 Vector plasmid map.
  • FIG. 2 is a diagram showing a pHelperVector plasmid map.
  • FIG. 3 is a diagram showing a pAAV-GFP Vector plasmid map.
  • FIG. 4 is a graph showing a dilution curve of the antibody concentration of the biotinylated anti-AAV2 antibody. The biotinylated antibody concentration (M) was plotted on the horizontal axis, and the AlphaLISA signal was plotted on the vertical axis.
  • FIG. 5 is a diagram showing the results of investigating the effects of components (medium components) that may be mixed during AAV preparation on AlphaLISA.
  • AAV adeno-associated virus
  • capsid envelopeless regular icosahedron outer shell
  • VP1 capsid proteins
  • AAV serotypes there are various reports on AAV serotypes, but at least AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10 are the serotypes of AAV that infect humans.
  • AAV11, AAV12 and AAV13 are known.
  • “Viral particles” means particles composed of capsid protein shells.
  • the "virus particle” is a hollow particle (for example, AAV hollow) which is a virus-like particle composed not only of a virus genome (nucleic acid shape) but also of a capsid protein containing no virus genome. Particles) are also included.
  • AAV refers to either a virus particle containing a virus genome or a hollow particle.
  • Hollow particles include AAV hollow particles.
  • AAV hollow particles may contain specific molecules that are desired to be delivered to a subject using an AAV or AAV vector. Specific examples of the molecule include nucleic acids, peptides, small molecule compounds, and combinations thereof. Examples of the above-mentioned nucleic acid include functional nucleic acids, mRNA or fragments thereof, or combinations thereof.
  • a "functional nucleic acid” is an intracellular or intracellular, preferably intracellular, specific biological function, such as an enzymatic function, a catalytic function or a biological inhibitory or enhancing function (eg, transcription, translation). Inhibition or enhancement).
  • RNA interferants include nucleic acid aptamers (including RNA aptamers and DNA aptamers), antisense DNA, ribozymes (including deoxyribozymes), U1 adapters, molecular beacons, riboswitches, or transcription factor binding regions. And so on. Two or more kinds of functional nucleic acids may be included.
  • the mRNA is an mRNA encoding any protein and may be either a pre-mRNA or a mature mRNA.
  • the above-mentioned peptides include both oligopeptides and polypeptides. Specific examples of oligopeptides include peptide hormones and polypeptide fragments.
  • the small molecule compound described above is a compound having a molecular weight of 5000 or less, preferably 2000 or less, more preferably 1000 or less, and corresponds to a substance other than the nucleic acid and peptide.
  • Specific examples of the small molecule compound may be one known as an active ingredient of a pharmaceutical product, a contrast agent used as a diagnostic agent, a fluorescent dye, or the like.
  • Examples of low molecular weight compounds include immunostimulatory agents, anticancer agents, signaling inhibitors, metabolic antagonists, analgesics, anti-inflammatory agents, antibiotics, antiallergic agents, prophylactic and therapeutic agents for central nervous system diseases, and circulation.
  • AAV hollow particles which are formed of capsids and do not contain the AAV virus genome inside, have initial viral infectious activity such as specific recognition of target cells, adsorption and invasion into cells, and unshelling, but are necessary for self-renewal. Since it does not have a gene of origin, it has no viral proliferative activity. Therefore, the hollow particles can specifically deliver the contained molecule to the target cell, and can be a vehicle of an ideal drug delivery system (DDS) having safety for the administered individual.
  • DDS drug delivery system
  • the present invention provides an AAV quantification method. Specifically, a method for quantifying AAV using an extract of host cells containing AAV particles or a culture supernatant, and general protein purification such as liquid chromatography for protein separation and density gradient ultracentrifugation from a preparation containing AAV. Provided are a method for quantifying AAV in the fractionation preparation during the process, a method for quantifying the final purified preparation of AAV, and the like. Hereinafter, these quantification methods are collectively referred to as "the quantification method of the present invention”.
  • the quantification method of the present invention The first antibody that recognizes the AAV capsid protein and has an affinity tag attached to it.
  • a conjugate of a second antibody that recognizes the same AAV capsid protein (as the AAV capsid protein recognized by the first antibody) and a carrier that has a luminescent substance (the carrier is also referred to as an acceptor carrier). It is characterized by using a carrier having a photosensitizer (the carrier is also referred to as a donor carrier) and a measurement sample solution. Further, as another form of the quantification method of the present invention, A conjugate of the antibody that is the first antibody that recognizes the capsid protein of AAV and has an affinity tag bound to it and a carrier that has a photosensitizer. It is characterized by using a conjugate of a second antibody that recognizes the same AAV capsid protein (as the AAV capsid protein recognized by the first antibody) and a carrier having a luminescent substance, and a measurement sample solution.
  • the first antibody and the second antibody used in the quantification method of the present invention are not particularly limited as long as they are antibodies that recognize the capsid protein of AAV, but are preferably AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7. , AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12 and AAV13 are antibodies that recognize at least one AAV capsid protein, more preferably AAV1, AAV2, AAV3a, AAV3b, AAV4. , AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.10, AAV11, AAV12 and AAV13. .. These antibodies are preferably monoclonal antibodies.
  • the antibodies of the present invention include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys, but also artificially modified gene recombination such as chimeric antibodies, humanized antibodies, and bispecific antibodies. Type antibodies are also included.
  • the immunoglobulin class of the antibody is not particularly limited, and any class such as IgG such as IgG1, IgG2, IgG3 and IgG4, IgA, IgD, IgE and IgM may be used, but IgG and IgM are preferable.
  • the antibody of the present invention includes not only whole antibodies, but also antibody fragments such as Fv, Fab, and F (ab) 2, and monovalent or divalent or higher valences in which the variable region of the antibody is bound with a linker such as a peptide linker.
  • linker such as a peptide linker.
  • Low molecular weight antibodies such as single chain Fv (Diabody such as scFv, sc (Fv) 2 and scFv dimer) are also included.
  • the antibody of the present invention described above can be produced by a method well known to those skilled in the art.
  • a hybridoma that produces a monoclonal antibody can be produced as follows, basically using a known technique. That is, a desired antigen or a cell expressing a desired antigen is used as a sensitizing antigen, this is immunized according to a normal immunization method, and the obtained immune cell is fused with a known parent cell by a normal cell fusion method. It can be produced by screening monoclonal antibody-producing cells (hybridoma) by a normal screening method.
  • the hybridoma can be produced, for example, according to the method of Milstein et al. (Kohler. G.
  • the immunogenicity of the target AAV antigen When the immunogenicity of the target AAV antigen is low, it may be immunized by binding to a macromolecule having immunogenicity such as albumin.
  • a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing it into a host, and producing it using a gene recombination technique can be used (for example, Carl, AK). Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).
  • V region variable region
  • cDNA in the variable region (V region) of the antibody is synthesized from the mRNA of the hybridoma using reverse transcriptase.
  • a DNA encoding the V region of the antibody of interest is obtained, it is ligated with the DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector.
  • the DNA encoding the V region of the antibody may be incorporated into an expression vector containing the DNA of the antibody C region. It is incorporated into an expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or a promoter.
  • the host cell can then be transformed with this expression vector to express the antibody.
  • the antibody of the present invention is preferably any of the antibodies (antibody clones) listed in Table 1 below, most preferably ADK1a when quantifying AAV1, and ADK5a and AAV2 when quantifying AAV5. If so, it is A20 (see references below for information on each antibody in Table 1).
  • ADK1a when quantifying AAV1
  • ADK5a when quantifying AAV5. If so, it is A20 (see references below for information on each antibody in Table 1).
  • epitope refers to a part of an antigen to which an antibody specifically binds.
  • Epitope determinants usually consist of chemically active surface groups of molecules (eg, amino acids or sugar side chains) and usually have specific three-dimensional structural properties as well as specific charge properties.
  • the epitope can be continuous or discontinuous (discontinuous).
  • stereoconformational epitope refers to a discontinuous epitope formed by the spatial relationship between amino acids of an antigen other than a continuous series of amino acids.
  • a conformational epitope is an epitope that is present in the conformation of an active protein but not in a denatured protein.
  • Antibodies that recognize the same epitope as the above-mentioned series of antibodies and competing antibodies are also antibodies that can be used in the quantification method of the present invention.
  • an antibody that recognizes the same epitope (preferably the same conformational epitope) as any of the antibodies shown in Table 1 or a competing antibody is also an antibody that can be used in the quantification method of the present invention.
  • the “competitive antibody” is an antibody that competes with an epitope (preferably a conformational epitope) of the capsid of AAV.
  • AAV1 antibodies that recognize the same epitope as ADK1a shown in Table 1 and antibodies that compete with each other can be mentioned.
  • the first antibody and the second antibody may be antibodies that can recognize (same) AAV at the same time, and the epitopes of both antibodies may be the same or different. Both antibodies preferably recognize the same epitope (preferably the same conformational epitope).
  • the preferred combination of the first antibody and the second antibody is as follows. (i) For AAV1, the first antibody and the second antibody are two selected from the group consisting of ADK1a, ADK1b, 4E4 and 5H7. The first antibody and the second antibody may be the same or different. It is preferably the same. (ii) For AAV2, the first antibody and the second antibody are two selected from the group consisting of A20, A1, C37B and D3. The first antibody and the second antibody may be the same or different.
  • the first antibody and the second antibody are ADK4.
  • the first antibody and the second antibody are two selected from the group consisting of ADK5a, ADK5b and 3C5.
  • the first antibody and the second antibody may be the same or different. It is preferably the same.
  • the first and second antibodies are ADK6.
  • the first antibody and the second antibody are ADK8.
  • the first and second antibodies are ADK8 / ADK9.
  • the first antibody and the second antibody are two selected from the group consisting of ADK9 and PAV9.1.
  • the first antibody and the second antibody may be the same or different.
  • first antibody and the second antibody are as follows. (i) For AAV1, the first antibody and the second antibody: ADK1a (ii) For AAV2, first antibody and second antibody: A20 (iii) For AAV5, the first antibody and the second antibody: ADK5a
  • an affinity tag is bound to the first antibody.
  • a first antibody to which a pre-prepared affinity tag is bound may be used, or a step of binding the affinity tag to the first antibody may be carried out if desired. It is convenient to use a first antibody to which a pre-prepared affinity tag is attached, and such antibodies may be commercially available.
  • the affinity tag is not particularly limited as long as it allows the first antibody to bind to the donor carrier, and specifically, biotin, glutathione-S-transferase, histidine peptide, FLAG peptide, Strep (II), digoxigenin ( DIG), HRP, Hapten, green fluorescent protein, Fluorescein Isothiocyanate and the like are exemplified.
  • biotin is used as the affinity tag.
  • the first antibody to which the affinity tag is bound binds to a carrier (donor carrier) having a photosensitizer.
  • a conjugate of the first antibody bound to the affinity tag and a carrier having a photosensitizer (donor carrier) may be used.
  • the carrier having a photosensitive substance (donor carrier) is an inert carrier such as glass, silica gel, or resin, and is a carrier on which a photosensitive substance and a molecule exhibiting specific affinity are immobilized. be.
  • the affinity tag when the affinity tag is biotin, it binds to the donor carrier via a biotin-binding substance (eg, streptavidin) contained in the donor carrier.
  • a biotin-binding substance eg, streptavidin
  • the affinity tag when the affinity tag is DIG, it binds to the donor carrier via the anti-DIG antibody contained in the donor carrier.
  • the "photosensitive substance” is not particularly limited as long as it is a substance capable of converting surrounding oxygen into excited singlet oxygen by being excited, and light of 600 to 700 nm is preferable (eg, light of 600 to 700 nm). It is a substance that generates singlet oxygen when irradiated with laser light).
  • phthalocyanines include porphyrins, phthalocyanines, chlorines, tetraphenylporphyrins, benzoporphyrin derivatives, purpurins, pheophorbides and metal complexes thereof, and phthalocyanines are preferably used.
  • a conjugate of a second antibody and a carrier having a luminescent substance is used.
  • the carrier having a luminescent substance is an inert carrier such as glass, silica gel, or resin, which has a luminescent substance and for immobilizing a second antibody. More preferably, the carrier has both a luminescent material and a fluorescent material.
  • the "luminescent substance” is not particularly limited as long as it is a substance that emits light by reacting with singlet oxygen. Specific examples thereof include thioxin derivatives.
  • the "fluorescent substance” is not particularly limited as long as it is a substance that emits light in the vicinity of 600 nm by receiving the luminescence energy generated from the luminescent substance that has reacted with the singlet oxygen molecule.
  • Specific examples include Europium.
  • Other substances that can specifically detect singlet oxygen include DCFH1 (Chem. Res. Toxicol., 1992, 5, 227-231) and DMAX (J. Am. Chem. Soc., 2001, 123, 2530- 2536), ATTA-Eu3 + (Chem. Commun., 2005, 3553-3555), MTTA-Eu3 + (J. Am. Chem. Soc., 2006, 128, 13442-13450) and the like are known.
  • the luminescent substance may be bound to the carrier in advance, or a step of binding the luminescent substance to the carrier may be carried out if desired.
  • the carrier having a luminescent substance there is a commercially available carrier (for example, manufactured by PerkinElmer Co., Ltd.), which is preferable because it is simple.
  • the fluorescent substance can be bound to the carrier in the same manner as the above-mentioned luminescent substance, and can be purchased.
  • the mode of binding between the acceptor carrier and the second antibody is not particularly limited, and a method usually practiced in the art is used, thus obtaining a bond between the second antibody and the carrier having a luminescent substance.
  • the weight ratio of the acceptor beads to the second antibody is usually 10: 1-50: 1, by mixing the two in the presence of NaBH 3 CN. conduct. Unreacted groups may be blocked with carboxymethylamine. Specifically, it can be carried out by the method described in Examples.
  • the carrier used for the acceptor carrier is not particularly limited as long as it contains a luminescent substance, and may be composed of a polymer or the like generally used in the art. .. Further, the carrier used for the donor carrier is not particularly limited as long as it contains a photosensitive substance, and may be composed of a polymer or the like generally used in the art.
  • the carrier is preferably beads. Therefore, the donor carrier and the acceptor carrier can preferably be donor beads and acceptor beads, respectively.
  • the sizes of the acceptor beads and the donor beads are not particularly limited as long as a luminescent signal can be generated from the luminescent substance in the acceptor beads by irradiating the photosensitive substance in the donor beads with excitation light.
  • the distance between the photosensitizer in the donor beads and the luminescent material in the acceptor beads is too great to cause interaction between the substances. If it is too large, the donor beads and acceptor beads may not be close to each other due to steric hindrance, and interaction between substances is unlikely to occur.
  • the quantification method of the present invention is carried out by the following steps.
  • Step 1 A step of mixing a measurement sample solution, a first antibody, and a carrier having a luminescent substance (acceptor carrier) with the second antibody.
  • Step 2 A step of mixing a carrier (donor carrier) having a photosensitizer with the mixture obtained in step 1.
  • Step 3 A step of irradiating the mixture obtained in step 2 with excitation light, and (step 4) a step of measuring a luminescence signal derived from the mixture obtained in step 3.
  • the quantification method of the present invention is specifically carried out by the following steps.
  • Step 1' A step of mixing a measurement sample solution, a carrier having a first antibody and a photosensitizer (donor carrier), and a carrier having a second antibody and a luminescent substance (acceptor carrier).
  • Step 2' A step of irradiating the mixture obtained in step 1'with excitation light, and
  • Step 3' a step of measuring an emission signal derived from the mixture obtained in step 2'.
  • the “first antibody”, “carrier”, and “second antibody” have the same meanings as described above.
  • the “measurement sample solution” refers to a solution directly used to measure the amount of AAV by the quantification method of the present invention.
  • Examples of the measurement sample solution used in step 1 (or step 1') include a sample solution in which AAV is present or the presence or absence of AAV needs to be confirmed, and includes negative control and positive control in measurement.
  • Examples of the above-mentioned measurement sample solution include an extract obtained through a step of extracting AAV produced in a host cell from a host cell, a culture supernatant of a cell producing AAV, and a diluted solution thereof. NS.
  • Extracts containing AAV obtained through the step of extracting AAV from AAV-producing cells, culture supernatants of AAV-producing cells, and diluted solutions thereof are also preferably exemplified as measurement sample solutions. Further, solutions obtained in a series of steps for producing and producing AAV can also be targeted. For example, fractionation preparations during general protein purification steps such as liquid chromatography for protein separation and density gradient ultracentrifugation from preparations containing AAV, final AAV purification preparations, and their diluted solutions. Can be a target.
  • a measurement sample solution before dilution such as a culture supernatant and cell extract of various cells producing AAV, a purified sample of AAV, etc.
  • the sample varies depending on its AAV content, but can be appropriately diluted to obtain a measurement sample solution.
  • the AAV concentration of the measurement sample solution in the present invention is preferably 1.0E + 07 to 1.0E + 12 particles / mL. If it is 1.0E + 07 or less, it cannot be measured at the limit of quantification. If it is 1.0E + 12 or higher, signal attenuation occurs due to the hook effect. It is preferably 1.0E + 08 to 5.0E + 11 particles / mL, and more preferably 1.0E + 09 to 1.0E + 11 particles / mL.
  • the measurement sample solution in the present invention is 0.5 to 10 ⁇ L, preferably 1.0 to 5.0 ⁇ L, more preferably 1.25 to 4.0 ⁇ L, particularly preferably 2.0 to 3.0 ⁇ L, and most preferably 2.5 ⁇ L.
  • the amount of the measurement sample solution may be smaller than that of the conventional ELISA method or the like. Specifically, 1/10 to 1/50 of the amount is sufficient compared to the conventional method (eg, ELISA method). Therefore, in the quantification method of the present invention, it is possible to reduce the amount of the sample used for quantification.
  • the amount of the measurement sample solution is 1/10 to 1/50, preferably 1/20 to 1/40 as compared with the ELISA method. Is enough.
  • the amount can be measured in an amount of 0.01 to 1 ⁇ L, preferably 0.05 to 0.5 ⁇ L, and more preferably 0.08 to 0.1 ⁇ L.
  • the present invention enables quantification with 1/10 to 1/50 of the amount of the measurement sample and in a smaller number of steps as compared with the conventional quantification method.
  • the amount of the sample actually used is about 1/20 to 1/500 as compared with the conventional quantification method. Therefore, it is possible to shorten the fixed quantity time and reduce the cost.
  • the measurement sample solution of the present invention has a final concentration of medium components of 0.89% or less, preferably 0.30% or less. This is because when the medium component exceeds 0.89%, the Alpha LISA signal is greatly attenuated, and when it is 0.30% or less, it is hardly attenuated.
  • the medium component refers to a component used to form a medium for culturing AAV-producing cells, and is not particularly limited as long as it is a normally used component.
  • the medium component may contain fetal bovine serum (FBS) as a medium or in combination with the medium.
  • FBS fetal bovine serum
  • the excitation light in step 3 (or step 2') and the luminescence signal in step 4 (or step 3') are appropriately selected depending on the light-sensitive substance or luminescent substance used.
  • phthalocyanine is used as the photosensitive substance
  • thioxin is used as the luminescent substance
  • the excitation light is irradiated at 680 nm
  • the photosensitive substance in the donor beads causes the surrounding oxygen to be in a singlet excited state and a chemical reaction occurs in the acceptor beads.
  • 615 nm emission signal can be detected.
  • the luminescence signal is proportional to the amount of AAV in the test sample, it is quantified by analysis using a standard curve. More specifically, the quantification method of the present invention can be carried out based on the examples described later or according to the "ELISA to AlphaLISA ImmunoAssay Conversion Guide" of PerkinElmer.
  • Method for Producing AAV the step of preparing AAV and the prepared AAV are described in the above 1.
  • a method for producing AAV which comprises a step of quantifying using the method described in the method for quantifying AAV.
  • the steps of preparing AAV can be performed according to methods commonly practiced in the art. For example, extraction of AAV from host cells, extraction from AAV vector-producing cells, and the like can be mentioned. Examples of the extraction method include a freeze-thaw method, an ultrasonic crushing method, and a solution extraction method. Examples of the solution extraction method include a method of contacting a host cell with an acidic solution.
  • the method of contacting the host cells with an acidic solution is to suspend the host cells recovered by centrifugation or filtration in an acidic solution, or to add a component that can make the culture solution acidic to a culture medium containing the host cells.
  • the pH of the acidic solution include pH 3.0 to 6.9, preferably pH 3.0 to 6.0, and more preferably pH 3.0 to 5.0.
  • the acidic solution is not particularly limited to the present invention, but citric acid, acetic acid, malic acid, phosphoric acid, hydrochloric acid, sulfuric acid, nitrate, lactic acid, propionic acid, butyric acid, oxalic acid, malonic acid, and succinic acid.
  • the containing solution is exemplified.
  • citric acid, acetic acid, phosphoric acid, and salts thereof are preferably exemplified in the present invention, and citric acid is more preferably exemplified.
  • the time of contact between the host cell and the acidic solution is not particularly limited to the present invention, and examples thereof include 1 minute to 48 hours, preferably 5 minutes to 24 hours.
  • Examples of the temperature condition at the time of contact between the host cell and the acidic solution include 0 to 40 ° C., preferably 4 to 37 ° C.
  • the AAV extract extracted from the host cell may contain DNA derived from the host cell.
  • the step of quantifying AAV is carried out according to the method described in "1. Quantifying method of AAV" above.
  • kits for AAV quantification including carriers with luminescent material.
  • the present invention (i') A conjugate of the antibody, which is the first antibody that recognizes the capsid protein of adeno-associated virus (AAV) and has an affinity tag bound, and a carrier having a photosensitizer, and (ii') Provided is a kit for AAV quantification, which comprises a conjugate of a second antibody that recognizes the same AAV capsid protein and a carrier having a luminescent substance.
  • AAV adeno-associated virus
  • a kit for AAV quantification which comprises a conjugate of a second antibody that recognizes the same AAV capsid protein and a carrier having a luminescent substance.
  • Each component may be included in the kit in part or in whole as a mixture, or each component may be included in the kit as a single component.
  • the above (i) and (ii) may be provided in the kit of the present invention in a pre-combined state, and similarly, the above (iii) and (iv) may be provided in the kit of the present invention in a pre-combined state. May be done.
  • the kit of the present invention may further include a reaction buffer, a reagent for dilution, a positive control for preparing a calibration curve, and the like. Further, in the case of quantification of AAV carried out in the method for producing AAV of the present invention, a reagent for preparing AAV (eg, a reagent for extracting AAV) may be included.
  • a reagent for preparing AAV eg, a reagent for extracting AAV
  • Example 1 Expression of AAV2 (1) Seeding of cells for AAV2 production HEK293T cells (manufactured by Takara Bio, manufactured by AAVpro) suspended in DMEM (manufactured by Sigma) containing 10% FBS (manufactured by Gibco) in a T75 flask for cell culture (manufactured by Corning). 293T Cell Line) was sown. After that, the culture was continued in a CO 2 incubator at 37 ° C, and it was confirmed that the culture was about 70 to 80% confluent.
  • the Falcon tube was vibrated to loosen the cell pellet.
  • the cell pellet was suspended in fresh medium and the number of cells was counted with a cell counter.
  • Cell density were seeded in T75 flasks or T225 flasks so that 4,000cells / cm 2 or 8,000cells / cm 2.
  • Incubation was carried out at 37 ° C. and 5% CO 2 for 4 days when seeded at 4,000 cells / ml or for 3 days when seeded at 8,000 cells / mL.
  • the culture medium in the flask was removed, DPBS was added, and the mixture was spread throughout, and then the supernatant was removed.
  • a plasmid containing the encoding plasmid (Takara Bio, pRC2-mi342 Vector, Fig. 1) and the adenovirus E2A sequence (SEQ ID NO: 3), VA sequence (SEQ ID NO: 4), and E4 sequence (SEQ ID NO: 5) (Takara).
  • a plasmid containing an expression cassette of the fluorescent protein GFP (CELL BIOLABS, pAAV-GFP, FIG.
  • Example 2 Collection of culture supernatant sample of AAV2-producing cells After completion of the culture of Example 1 (3), add 562.5 ⁇ L (1/80 volume) of 0.5 M EDTA (pH 8.0) to the culture solution and mix well. did. After reacting at room temperature for 10 minutes, the cells were detached by tapping the T225 flask. The detached cells were collected as a solution and centrifuged (1,750 x g, 10 minutes, 4 ° C). The supernatant after centrifugation was a culture supernatant sample.
  • 0.5 M EDTA pH EDTA
  • Example 3 Recovery of cell extract of AAV2-producing cells 2 mL of cell eluate (AAV Extraction Solution A, manufactured by Takara Bio Co., Ltd.) was added to the cell pellet after collecting the culture supernatant of Example 2. The cells were added and vortexed to suspend the cells for 15 seconds. After standing at room temperature for 5 minutes, the cells were suspended by shaking with a vortex for another 15 seconds. The cell suspension was centrifuged (9,000 x g, 10 minutes, 4 ° C). The cells were further suspended by shaking with a vortex for 15 seconds. After standing at room temperature for 5 minutes, the cells were suspended by shaking with a vortex for another 15 seconds. After centrifugation (9,000 x g, 10 minutes, 4 ° C), the supernatant was collected, and AAV Extraction Solution B (manufactured by Takara Bio Inc.) was added thereto, which was used as a cell extract.
  • AAV Extraction Solution B manufactured by Takara Bio Inc.
  • Example 4 Purification of AAV2
  • AAVpro Purification Kit manufactured by Takara Bio Inc.
  • rAAV2 purify according to the protocol of AAVpro Purification Kit (manufactured by Takara Bio Inc.) and purify rAAV2.
  • Got HiTrap AVB Sepharose HP GE Healthcare
  • Example 5 Quantification of AAV2 by ELISA AAV was quantified using the AAV Titration ELISA kit series (manufactured by PROGEN Biotechnik).
  • the samples used for quantification were each step of the AAV2 production process, that is, the culture supernatant of Example 2, the cell extract of Example 3, and the purified sample of Example 4.
  • 20 ⁇ L of the sample was diluted 50-fold to serially with Assay Buffer to prepare a measurement sample solution.
  • 10 ⁇ L of the sample was diluted 10-fold to serially with Assay Buffer to prepare a measurement sample solution.
  • Anti-AAV5 antibody PROGEN Biotechnik, Anti-AAV-5, Mouse-Mono (ADK5a), Catalog No.610148
  • Biotinylated anti-AAV5 antibody PROGEN Biotechnik, Anti-AAV-5, Mouse-Mono (ADK5a), Biotin, Catalog No.615148
  • the AbSelect BSA Removal Kit (Expedeon, 820-0100) was used to remove the components affecting Alpha LISA from the antibody solution, and the procedure was followed.
  • the precipitate (antibody) from each antibody solution obtained in the final step of the kit was dissolved in PBS.
  • a 1 mg / mL antibody solution (anti-AAV1 antibody, anti-AAV2 antibody or anti-AAV5 antibody), 0.625 ⁇ L of 10% Tween-20, and 5 ⁇ L of 400 mM NaBH 3 CN were added. Incubation was carried out at 37 ° C. for 24-48 hours while slowly stirring with a rotating shaker (6-10 rpm). These were designated as antibody-labeled AlphaLISA acceptor beads.
  • a 65 mg / mL carboxylmethylamine (CMO) solution was prepared with 800 mM NaOH. 5 ⁇ L of the prepared CMO solution was added to antibody-labeled AlphaLISA acceptor beads to block unreacted groups. After reacting at 37 ° C.
  • CMO carboxylmethylamine
  • Precipitated antibody-labeled AlphaLISA acceptor beads were suspended in 100 ⁇ L storage buffer (PBS + 0.05% Proclin-300) (final concentration 5 mg / mL antibody-labeled AlphaLISA acceptor beads). After shaking with a vortex and spinning down lightly, the bead solution was sonicated in an ultrasonic cleaner for 30 seconds. Antibody-labeled AlphaLISA acceptor beads were stored in opaque vials at 4 ° C.
  • AAV2 (purified sample) prepared in Example 4 was used as a sample of AlphaLISA.
  • measurement sample solutions with different dilution ratios were prepared using AlphaLISA Immunoassay buffer (manufactured by PerkinElmer) (500-fold dilution, 2500-fold dilution).
  • the biotinylated antibody was prepared with AlphaLISA Immunoassay buffer so that the dilution series and the final concentration were 10nM, 3nM, 1nM, 0.3nM, 0.1nM, 0.03nM, 0.01nM.
  • the plate reader chose the setting condition that the emission light of 615 nm is detected for the excitation light of 680 nm.
  • a dilution curve of the biotinylated antibody concentration was obtained (Fig. 4). From FIG. 4, the hook point was set to 0.03 nM. The biotinylated antibody concentration below this hook point was set as the optimum concentration. For the evaluation, the average value of n3 was used for the measurement sample. Subsequent examples were tested at the optimum biotinylated antibody concentration.
  • n3 The average value of n3 was used for each evaluation. As a result, it was found that the signal of AlphaLISA was particularly attenuated when the medium component exceeded 0.89% in the measurement sample solution. It was also found that the Alpha LISA signal was not attenuated when the medium component was 0.3% or less. From these facts, it was found that the measurement sample solution needs to be set so that the final concentration of the medium component is 0.89% or less, preferably 0.3% or less. In the following examples, when preparing the sample, the final concentration of the medium component was set to 0.3% or less, and the experiment was carried out.
  • the concentration of the solution component did not affect AlphaLISA or less and that the hook point was not exceeded.
  • the concentration was calculated from the Alpha LISA signal value by the average value of points below the hook point and above the quantification limit. In the control, the value was obtained from the average of n3.
  • AAV was quantified by ELISA using the same sample in the same manner as in Example 5. In each quantification method, the dilution ratio of the sample is as follows.
  • ELISA When the culture supernatant is used as a sample, 20 ⁇ L of the sample is diluted 50-fold with Assay Buffer, and when the cell extract and purified sample are used as a sample, 10 ⁇ L of the sample is diluted 10-fold with Assay Buffer for measurement. A sample solution was prepared. Based on this, three or more serially diluted solutions were prepared using Assay Buffer and used as the measurement sample solution.
  • AlphaLISA When the culture supernatant was used as a sample, it was diluted 30-fold with Immunoassay buffer, and when the cell extract and purified sample were used as a sample, it was diluted 100-fold with Immunoassay buffer to prepare a measurement sample solution.
  • the present invention it is possible to quantify with a measurement sample in an amount of 1/10 to 1/50 as compared with a conventional quantification method and in a small number of steps.
  • the amount of the sample actually used is about 1/20 to 1/500 as compared with the conventional quantification method. Therefore, it is possible to shorten the fixed quantity time and reduce the cost.

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Abstract

L'invention a pour objet de fournir un nouveau procédé de quantification qui résout les problèmes des procédés de quantification de virus adéno-associé (VAA) déjà existants. Selon l'invention, il est possible de raccourcir la durée de quantification et d'abaisseur le coût au moyen d'un premier anticorps reconnaissant la protéine de capside du virus adéno-associé (VAA), lequel anticorps est lié à un marqueur d'affinité, au moyen d'un agrégat d'un second anticorps reconnaissant la protéine de capside du même virus adéno-associé (VAA), et d'un support possédant une substance luminescente, et au moyen du procédé de quantification de virus adéno-associé (VAA) de l'invention qui met en œuvre un support possédant un photosensibilisateur, et une solution d'échantillon de mesure.
PCT/JP2021/002994 2020-01-31 2021-01-28 Procédé de quantification de virus adéno-associé (vaa) WO2021153659A1 (fr)

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