Method for producing modified vector and method for modifying vector

Abstract

A method for preparing a modified vector and a method for modifying a vector, characterized by including: a step for bonding, to a functional group in a vector, a functional group (A) capable of bonding with said functional group in a linker compound having the functional group (A) and a functional group (B) capable of bonding with a ligand compound; and a step for bonding, to the functional group (B), a functional group (C) capable of bonding with said functional group (B) in a ligand compound having the functional group (C).

Description

Technical Field

[0001] The present invention relates to a method for producing a modified vector and a method for modifying a vector.

Background Art

[0002] Vectors are widely used in gene therapy, but problems such as insufficient migratability of the vector to the target tissue remain. As a method for solving this problem, modification of the protein constituting the vector has been carried out, but a sufficient effect for showing drug efficacy has not been obtained.

[0003] Therefore, in recent years, a method of chemically modifying a vector and introducing a ligand for enhancing the function into the vector has been developed (Non-Patent Document 1, etc.). However, in this method, since an integrated reagent of a site to be bonded to a functional group on the vector and a ligand for enhancing the function is used, there are limitations in the available reagents, and it is hard to say that it is an effective method.

[0004] As another method, a method of chemically modifying in two steps has been reported (Patent Document 1, etc.). However, this method uses a cysteine residue, and since it is necessary to release the cysteine residue by a reduction treatment before chemical modification, it is hard to say that it is a simple method. Further, there is a possibility that the vector may be decomposed by this reduction treatment.

[0005] As still another method, a method of chemically modifying after modifying the vector to introduce a cysteine residue mutation has been reported (Non-Patent Document 2, etc.), but since it takes time to introduce the mutation, it is hard to say that it is a simple method.

[0006] Therefore, a method for producing a modified vector and a method for modifying a vector that can easily introduce various ligands without modifying or pretreating the vector are not known at all, and there is a strong demand for providing these.

Prior Art Documents

Patent Document

[0007]

Patent Document 1

Non-Patent Document

[0008]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] An object of the present invention is to solve the above-described conventional problems and achieve the following objects. That is, an object of the present invention is to provide a method for producing a modified vector and a method for modifying a vector, which can easily introduce various ligands without modifying or pretreating the vector.

Means for Solving the Problems

[0010] As a result of intensive studies to achieve the above object, the present inventors have found that a linker compound having a functional group (A) capable of binding to the functional group in the vector and a functional group (B) capable of binding to the ligand compound is used. A method for producing a modified vector and a method for modifying a vector, which can easily introduce various ligands without modifying or pretreating the vector, by including a step of binding the functional group (A) and a step of binding the functional group (C) in the ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B).

[0011] The present invention is based on the above findings by the present inventors, and the means for solving the above problems are as follows. That is, <1> A method for producing a modified vector, comprising: a step of binding a functional group (A) in a linker compound having a functional group capable of binding to the functional group in the vector and a functional group (B) capable of binding to a ligand compound to the functional group; and a step of binding a functional group (C) in a ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B). <2> A method for modifying a vector, comprising: a step of binding a functional group (A) in a linker compound having a functional group capable of binding to the functional group in the vector and a functional group (B) capable of binding to a ligand compound to the functional group; and a step of binding a functional group (C) in a ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B).

Advantages of the Invention

[0012] According to the present invention, the above-mentioned various problems in the prior art can be solved, the above-mentioned object can be achieved, and a method for producing a modified vector and a method for modifying a vector can be provided, which can easily introduce various ligands without modifying or pretreating the vector.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

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Mode for Carrying Out the Invention

[0014] (Method for Producing Modified Vector) The method for producing the modified vector includes a first step of binding the functional group (A) in a linker compound having a functional group capable of binding to the functional group in the vector and a functional group (B) capable of binding to a ligand compound to the functional group in the vector, and a second step of binding the functional group (C) in a ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B), and may further have other steps.

[0015] There is no particular limitation on the order of performing the first step and the second step, and it can be appropriately selected according to the purpose. However, various ligands capable of binding to the linker compound can be easily introduced into the vector, and from the viewpoint of expanding the options of ligands, it is preferable to perform the second step after the first step.

[0016] <First Step> The first step is a step of binding the functional group (A) in a linker compound having a functional group capable of binding to the functional group in the vector and a functional group (B) capable of binding to a ligand compound to the functional group in the vector.

[0017] -Vector- There is no particular limitation on the vector, and it can be appropriately selected according to the purpose. For example, virus vectors, plasmid vectors, artificial chromosome vectors, cosmid vectors, fosmid vectors, etc. can be mentioned. Among these, virus vectors are preferable in that they are widely used in gene therapy.

[0018] The virus used as the virus vector is not particularly limited and can be appropriately selected according to the purpose. For example, adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes virus, poliovirus, papillomavirus, vaccinia virus, poxvirus, etc. can be mentioned. Among these, adeno-associated virus (AAV) is preferred in terms of low pathogenicity.

[0019] The serotypes of AAV used as the virus vector include AAV1 (type 1 AAV), AAV2 (type 2 AAV), AAV3 (type 3 AAV), AAV4 (type 4 AAV), AAV5 (type 5 AAV), AAV6 (type 6 AAV), AAV7 (type 7 AAV), AAV8 (type 8 AAV), AAV9 (type 9 AAV), AAV10 (type 10 AAV), AAV11 (type 11 AAV), AAV12 (type 12 AAV), AAV13 (type 13 AAV), AAV14 (type 14 AAV), and modified forms thereof, etc. There is no particular limitation, and it can be appropriately selected according to the purpose. The modified form is not particularly limited and can be appropriately selected according to the purpose. For example, AAV modified by genetic recombination (wild-type AAV) for improving the tissue specificity (target cell tropism) of the target cell, etc. can be mentioned.

[0020] --Functional groups in the vector-- The functional groups in the vector are not particularly limited and can be appropriately selected according to the purpose. For example, amino group, guanidino group, hydroxyl group, carboxyl group, and indole group, etc. can be mentioned. Among these, the amino group is preferred. The functional group in the vector may be a functional group in a lysine residue, arginine residue, tyrosine residue, serine residue, threonine residue, tryptophan residue. Among these, the functional group in the lysine residue is preferred. When the vector is the adeno-associated virus (AAV), the functional group in the vector is preferably a functional group constituting the capsid of the adeno-associated virus.

[0021] The capsid is composed of VP1, VP2, VP3, etc.

[0022] -Linker compound- The linker compound has a functional group (A) capable of binding to a functional group in the vector and a functional group (B) capable of binding to a ligand compound, and may further have a linking portion (D).

[0023] --Functional group (A) capable of binding to a functional group in the vector-- The functional group (A) capable of binding to a functional group in the vector is not particularly limited and can be appropriately selected according to the purpose. For example, a succinimidyl group, an isocyanate group, an aminomethoxyethyl group, a cyclohexenesulfonamide group, a carbonyl group, an aldehyde group, an unsaturated carbonyl group, diazonium terephthalate, a halogen atom, a maleimidyl group, a phthalimidyl group, a diazobenzene group, an unsaturated nitrile group, an arenyl group, a leaving group, etc. can be mentioned.

[0024] The leaving group is OSO2R' or OP(O)(OR') 2 (R' represents an alkyl group having 1 to 6 carbon atoms; an aryl group having 4 to 10 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include linear or branched alkyl groups, especially a methyl group; an ethyl group; (n-, i-)propyl groups; (n-, i-, t-)butyl groups. The aryl group having 4 to 10 carbon atoms refers to, for example, a phenyl group; (2-, 3-, 4-)tolyl groups; (1-, 2-)naphthyl groups; 2-pyrrolyl group; 2-furyl group; 3-thienyl group; 2-pyridyl group, which is an aromatic hydrocarbon group or a heterocyclic group composed of a 5- or 6-membered monocyclic or condensed ring. For example, a methanesulfonyl group, a toluenesulfonyl group, etc. can be mentioned. Among these, a succinimidyl group is preferable in terms of reactivity. The functional group (A) may be one or plural per molecule of the linker compound.

[0025] --Functional group (B) capable of binding to the ligand compound-- The functional group (B) capable of binding to the ligand compound is not particularly limited and can be appropriately selected according to the purpose. For example, an azide group, an alkynyl group, an alkenyl group, a carbonyl group, a phosphine group, a tetrazine group, a hydrazine group, and a hydroxylamine group can be mentioned. Among these, from the viewpoint of reactivity, an alkynyl group or an azide group is preferable, and an alkynyl group is more preferable. The functional group (B) may be one or plural per molecule of the linker compound. When there are plural, there is no particular limitation and it can be appropriately selected according to the purpose. For example, an azide group and an alkynyl group can be mentioned.

[0026] --Linking part (D)-- The linking part (D) can link the functional group (A) and the functional group (B), and there is no particular limitation as long as it does not react with the functional group (A), the functional group (B), and the ligand compound, and it can be appropriately selected according to the purpose.

[0027] The linking part (D) may have a linear structure or a branched structure, but the linear structure is preferable. The linking part (D) may be hydrophilic, hydrophobic, or amphiphilic, but the hydrophilicity is preferable. The length of the linking part (D) is not particularly limited and can be appropriately selected according to the purpose. For example, it can be adjusted by the number of carbon atoms constituting the linking part (D).

[0028] The chemical structure of the linking part (D) is not particularly limited and can be appropriately selected according to the purpose. For example, a structure having an alkylene group, a structure having a carbonyl group, a structure having an ether bond, a structure having a carboxylic acid ester bond, and combinations thereof can be mentioned. The structure having the ether bond may be chain-like (for example, a straight chain such as ethylene glycol, a branched chain such as propylene glycol), or may be cyclic (a structure including tetrahydropyran, tetrahydrofuran, 1,4-dioxane, etc.).

[0029] Specific examples of the connecting portion (D) include an alkylene group, an alkyleneoxy group, a poly(alkyleneoxy) group, etc. The alkylene group is not particularly limited and can be appropriately selected according to the purpose. For example, an alkylene group having 1 to 20 carbon atoms is preferable, an alkylene group having 1 to 10 carbon atoms is more preferable, and an alkylene group having 1 to 4 carbon atoms is even more preferable. The alkyleneoxy group is not particularly limited and can be appropriately selected according to the purpose. For example, an alkyleneoxy group having 1 to 100 carbon atoms is preferable, an alkyleneoxy group having 4 to 100 carbon atoms is more preferable, and an alkyleneoxy group having 5 to 50 carbon atoms is even more preferable. The poly(alkyleneoxy) group is not particularly limited and can be appropriately selected according to the purpose. For example, a poly(alkyleneoxy) group having 1 to 100 carbon atoms is preferable, a poly(alkyleneoxy) group having 5 to 50 carbon atoms is more preferable, and a poly(alkyleneoxy) group having 6 to 40 carbon atoms is even more preferable. The alkylene oxide unit in the poly(alkyleneoxy) group is not particularly limited and can be appropriately selected according to the purpose. For example, ethylene oxide and propylene oxide are preferable, and ethylene oxide is more preferable.

[0030] Specific examples of the linker compound are not particularly limited and can be appropriately selected according to the purpose. For example, DIBENZ[b,f]azocine-5(6H)-hexanoic acid, 11,12-didehydro-ε-oxo-,2,5-dioxo-3-sulfo-1-pyrrolidinyl ester, sodium salt, 4,7,10,13,16-Pentaoxa-20-azatricosanoic acid, 23-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-19,23-dioxo-,2,5-dioxo-1-pyrrolidinyl ester, etc. can be mentioned.

[0031] -Binding of the functional group (A) to the functional group in the vector- The binding of the functional group (A) to the functional group in the vector is not particularly limited and can be appropriately selected according to the purpose. For example, a method of mixing and reacting a solution containing the linker compound and the vector, a method of mixing and reacting a solution containing the linker compound to which the ligand compound is bound and the vector, etc. can be mentioned. After these methods, a method of adding a glycine solution and further reacting may also be used.

[0032] The solvent of the solution may be an aqueous solvent, an organic solvent, or a mixed solvent of an aqueous solvent and an organic solvent. The aqueous solvent is not particularly limited and can be appropriately selected according to the purpose. For example, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, phosphate buffer, distilled water, etc. can be mentioned. The pH of the HEPES buffer or phosphate buffer is not particularly limited and can be appropriately selected according to the purpose. However, a pH of 4 or higher and 12 or lower is preferable, a pH of 5 or higher and 11 or lower is more preferable, a pH of 6 or higher and 10 or lower is further preferable, and a pH of 7 or higher and 9 or lower is particularly preferable. The organic solvent is not particularly limited and can be appropriately selected according to the purpose, but it is preferably compatible with water. For example, nitrile solvents such as acetonitrile, alcoholic solvents such as methanol, ethanol, and propanol, ether solvents such as tetrahydrofuran and 1,4-dioxane, sulfur solvents such as dimethyl sulfoxide, and amide solvents such as dimethylformamide can be mentioned. There is no particular limitation on the mixing ratio of the aqueous solvent and the organic solvent, as long as no insoluble components are significantly formed.

[0033] There is no particular limitation on the final concentration (before adding the glycine solution) of the linker compound, and it can be appropriately selected according to the purpose. However, it is preferably 0.1 μM or more and 200 mM or less, more preferably 0.5 μM or more and 100 mM or less, still more preferably 5 μM or more and 80 mM or less, and particularly preferably 10 μM or more and 50 mM or less.

[0034] There is no particular limitation on the titer of the vector, and it can be appropriately selected according to the purpose. However, it is preferably 1×10 4 vg / μL or more and 1×10 100 vg / μL or less, more preferably 1×10 5 vg / μL or more and 5×10 80 vg / μL or less, still more preferably 1×10 6 vg / μL or more and 1×10 50 vg / μL or less.

[0035] There is no particular limitation on the concentration of the glycine solution, and it can be appropriately selected according to the purpose. However, it is preferably 0.1 μM or more and 20 M or less, more preferably 0.5 μM or more and 10 M or less, still more preferably 5 μM or more and 5 M or less, and particularly preferably 10 μM or more and 4 M or less.

[0036] There is no particular limitation on the temperature of the reaction, and it can be appropriately selected according to the purpose. However, it is preferably 0°C or more and 70°C or less, and more preferably 40°C or less. The reaction time before adding the glycine solution is not particularly limited and can be appropriately selected according to the purpose. However, it is preferably 5 minutes or more and 120 hours or less, more preferably 30 minutes or more and 96 hours or less, and even more preferably 1 hour or more and 72 hours or less. The reaction time after adding the glycine solution is not particularly limited and can be appropriately selected according to the purpose. However, it is preferably 5 minutes or more and 120 hours or less, more preferably 30 minutes or more and 96 hours or less, and even more preferably 1 hour or more and 72 hours or less.

[0037] <Second step> The second step is a step of bonding the functional group (C) in the ligand compound having a functional group (C) capable of bonding to the functional group (B) to the functional group (B).

[0038] -Ligand compound- The ligand compound has a functional group (C) capable of bonding to the functional group (B) and a ligand moiety, and may further have a linking moiety (E). The linking moiety (E) can link the functional group (C) and the ligand moiety, and is not particularly limited as long as it does not react with the linker compound, the functional group (C), and the ligand moiety, and can be appropriately selected according to the purpose. The structure and specific examples of the linking moiety (E) are the same as those of the linking moiety (D) in the aforementioned linker compound. The ligand moiety in the ligand compound is a substance that has an affinity or the like with tissues, cells, proteins, etc. in organisms, or is labeled using the affinity, and is not particularly limited and can be appropriately selected according to the purpose. The labeling means adding functions such as membrane permeability and target cell specificity to the vector, and adding the functions means modifying. In a broad sense, it can be said that the introduction of a linker and the addition of a scaffold function that facilitates the introduction of other functional substances are also modifications.

[0039] The ligand part is not particularly limited and can be appropriately selected according to the purpose. Examples include sugar compounds, peptides, low-molecular-weight compounds, immunoglobulins, and the like. These may be one or a plurality per molecule of the ligand compound. In the case of a plurality, there is no particular limitation and they can be appropriately selected according to the purpose. Examples include sugar compounds and peptides.

[0040] The sugar compound is not particularly limited and can be appropriately selected according to the purpose. Examples include monosaccharides, oligosaccharides, and polysaccharides. The monosaccharides mentioned here may have asymmetric carbons that are enantiomers. Oligosaccharides consist of 2 to 6 molecules of monosaccharides, and polysaccharides are polymers in which monosaccharides are linked linearly or branched. From the perspective of availability, monosaccharides, disaccharides, and trisaccharides are preferred. Also, the sugar compound is not particularly limited and can be appropriately selected according to the purpose. Examples include allose, arabinose, erythrose, fructose, fucose, galactose, galactosamine, glucose, glucosamine, gulose, glucuronic acid, idose, inositol, lyxose, mannose, mannosamine, psicose, rhamnose, ribose, sialic acid, sorbose, tagatose, talose, xylose, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, cyclodextrin, 2-Azidoethyl 2-acetamido-2-deoxy-β-D-galactopyranoside, 2-Azidoethyl β-D-Glucopyranoside, unnatural sugars, and the like. Furthermore, those with a polyethylene glycol group or a protecting group added thereto may also be included.

[0041] The protecting group is a substituent that protects hydroxyl groups and amino groups contained in sugar compounds, and can be appropriately selected according to the purpose. For example, protecting groups for hydroxyl groups include acyl groups, carbonate groups, carbamate groups, cyclic acetal groups, ether groups, etc. There are no particular restrictions on the acyl group, and it can be appropriately selected according to the purpose. For example, acetyl group, phenylacetyl group, halogenated acetyl group, methoxyacetyl group, phenoxyacetyl group, pivaloyl group, benzoyl group, etc. can be mentioned. There are no particular restrictions on the carbonate group, and it can be appropriately selected according to the purpose. For example, t-butyl carbonate group, benzyl carbonate group, etc. can be mentioned. There are no particular restrictions on the carbamate group, and it can be appropriately selected according to the purpose. For example, phenylcarbamate group, etc. can be mentioned. There are no particular restrictions on the cyclic acetal group, and it can be appropriately selected according to the purpose. For example, methylene acetal group, ethylidene acetal group, acetonide group, benzylidene acetal group, etc. can be mentioned. There are no particular restrictions on the ether group, and it can be appropriately selected according to the purpose. For example, benzyl ether group, etc. can be mentioned. For example, protecting groups for amino groups include acyl groups, carbamate groups, etc. There are no particular restrictions on the acyl group, and it can be appropriately selected according to the purpose. For example, acetyl group, phenylacetyl group, halogenated acetyl group, methoxyacetyl group, phenoxyacetyl group, pivaloyl group, benzoyl group, etc. can be mentioned. There are no particular restrictions on the carbamate group, and it can be appropriately selected according to the purpose. For example, 9-fluorenylmethyl carbamate group, phenylcarbamate group, t-butyl carbamate group, etc. can be mentioned. Among these protecting groups, the acetyl group is preferred due to its low toxicity and availability.

[0042] The peptide compound is not particularly limited and can be appropriately selected according to the purpose. For example, LRVRLASHLRKLRKRLLRDAKKKKKKKKKKKKKKKK (SEQ ID NO: 1), KKKKKKKKKKKKKKKKLRVRLASHLRKLRKRLLRDA (SEQ ID NO: 2), LRVRLASHLRKLRKRLLRDA (SEQ ID NO: 3), GRKKRRQRRRPPQ (SEQ ID NO: 4), RQIKIWFQNRRMKWK (SEQ ID NO: 5), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 6), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 7), RRRRRRRR (SEQ ID NO: 8), RRRRRRRRR (SEQ ID NO: 9), RRRRRRRRRR (SEQ ID NO: 10), HHHHHHHHHHHHHHHH (SEQ ID NO: 11), LLIILRRRIRKQAHAHSK (SEQ ID NO: 12), KLALKLALKALKAALKA (SEQ ID NO: 13), LLIILRRRIRKQAHAHSK (SEQ ID NO: 14), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 15), etc. may be mentioned. It may have a cyclic structure. Further, each amino acid may be in the D form, and it may be substituted with non-natural amino acids and / or non-natural amino acids may be inserted. It may contain salts, and those with a polyethylene glycol group added thereto may also be used.

[0043] The low-molecular compound is not particularly limited and can be appropriately selected according to the purpose. For example, it may be a substance that exhibits a medicinal effect in vivo, such as Paclitaxel or a low-molecular antibody. It may also be a substance for evaluating the affinity with a substance in vivo, such as biotin or a dye. Further, those with a polyethylene glycol group added thereto may also be used. There is no particular limitation on the pigment, and examples thereof include fluorescein, HiLyte Fluor 555, HiLyte Fluor 647, HiLyte Fluor 750, DyLight 350, DyLight 405, DyLight 550, DyLight 633, DyLight 755, Aleza Fluor 350, Aleza Fluor 405, Aleza Fluor 488, Aleza Fluor 532, Aleza Fluor 546, Aleza Fluor 555, Aleza Fluor 568, Aleza Fluor 594, Aleza Fluor 647, Aleza Fluor 680, Aleza Fluor 750, Cy 5, Cy 3, etc. Further, those with a polyethylene glycol group added thereto may also be used. There is no particular limitation on the immunoglobulin, and it can be appropriately selected according to the purpose. Examples thereof include IgG, IgM, IgA, IgD, IgE, etc.

[0044] There is no particular limitation on the upper limit value of the weight average molecular weight of the ligand compound, and it can be appropriately selected according to the purpose. However, it is preferably 500,000 or less, more preferably 300,000 or less, still more preferably 200,000 or less, and particularly preferably 100,000 or less. There is no particular limitation on the lower limit value of the weight average molecular weight of the ligand compound, and it can be appropriately selected according to the purpose. However, it is preferably 200 or more.

[0045] There is no particular limitation on the specific example of the ligand compound, and it can be appropriately selected according to the purpose. Examples thereof include 2-Azidoethyl 2-acetamido-2-deoxy-β-D-galactopyranoside, 2-Azidoethyl β-D-Glucopyranoside, etc.

[0046] --Functional group (C) capable of binding to functional group (B)-- The functional group (C) capable of binding to the functional group (B) is not particularly limited and can be appropriately selected according to the purpose. When the functional group (B) is an azide group, a phosphine group or an alkynyl group is preferable. When the functional group (B) is an alkynyl group, an azide group is preferable. When the functional group (B) is an alkenyl group, a tetrazine group or an alkenyl group is preferable. When the functional group (B) is a carbonyl group, a hydrazine group or a hydroxylamine group is preferable. When the functional group (B) is a phosphine group, an azide group is preferable. When the functional group (B) is a tetrazine group, an alkenyl group is preferable. When the functional group (B) is a hydrazine group, a carbonyl group is preferable. When the functional group (B) is a hydroxylamine group, a carbonyl group is preferable. The functional group (C) may be one or a plurality per molecule of the ligand compound. When there are a plurality, there is no particular limitation and they can be appropriately selected according to the purpose. Examples include an azide group and an alkynyl group.

[0047] -Binding of the functional group (C) to the functional group (B):- The binding of the functional group (C) to the functional group (B) is not particularly limited and can be appropriately selected according to the purpose. For example, a method of mixing and reacting a solution containing the reactant obtained by binding the functional group (A) to the functional group in the vector and the ligand compound, a method of mixing and reacting a solution containing the linker compound and the ligand compound, etc. can be mentioned.

[0048] The solvent of the solution may be an aqueous solvent, an organic solvent, or a mixed solvent of an aqueous solvent and an organic solvent. The aqueous solvent is not particularly limited and can be appropriately selected according to the purpose. Examples include HEPES buffer, phosphate buffer, distilled water, etc. The pH of the HEPES buffer or phosphate buffer is not particularly limited and can be appropriately selected according to the purpose. However, a pH of 1 or more and 12 or less is preferable, a pH of 2 or more and 12 or less is more preferable, a pH of 3 or more and 11 or less is further preferable, and a pH of 4 or more and 10 or less is particularly preferable. There are no particular restrictions on the organic solvent, and it can be appropriately selected according to the purpose, but it is preferably compatible with water. For example, nitrile solvents such as acetonitrile, alcoholic solvents such as methanol, ethanol, and propanol, ether solvents such as tetrahydrofuran and 1,4-dioxane, sulfur solvents such as dimethyl sulfoxide, and amide solvents such as dimethylformamide can be mentioned. There are no particular restrictions on the mixing ratio of the aqueous solvent and the organic solvent, as long as no insoluble components are significantly generated.

[0049] There are no particular restrictions on the final concentration of the ligand compound, and it can be appropriately selected according to the purpose, but it is preferably 0.01 mM or more and 100 mM or less, more preferably 0.1 mM or more and 80 mM or less, and even more preferably 0.2 mM or more and 50 mM or less.

[0050] There are no particular restrictions on the temperature of the reaction, and it can be appropriately selected according to the purpose, but it is preferably 0°C or more and 70°C or less, and more preferably 40°C or less. There are no particular restrictions on the reaction time, and it can be appropriately selected according to the purpose, but it is preferably 5 minutes or more and 120 hours or less, more preferably 30 minutes or more and 96 hours or less, and even more preferably 1 hour or more and 72 hours or less.

[0051] <Other steps> There are no particular restrictions on the other steps, and they can be appropriately selected according to the purpose. For example, a vector preparation step, a vector purification step, a reduction step, a modified vector purification step, etc. can be mentioned.

[0052] - Vector preparation step - There are no particular restrictions on the vector preparation step, and it can be appropriately selected according to the purpose. For example, methods such as transfecting a gene encoding a vector into cultured cells or the like and preparing a vector from a cell lysate or a cell supernatant can be mentioned.

[0053] - Vector purification step - The purification process of the vector is not particularly limited and can be appropriately selected according to the purpose. For example, methods of purification using commercially available kits, methods of purifying cell lysates using cation chromatography, anion chromatography, size exclusion chromatography, filters, ultrafiltration membranes, etc. can be mentioned.

[0054] - Reduction process - The reduction process is not particularly limited and can be appropriately selected according to the purpose. For example, after the step of binding the functional group (A) in the linker compound to the functional group in the vector, or after the step of binding the functional group (C) in the ligand compound to the functional group (B), a reducing agent such as DTT (dithiothreitol) is added to the reaction solution, or a reducing agent such as DTT is added to the modified vector after the purification process.

[0055] - Purification process of the modified vector - The purification process of the modified vector is not particularly limited and can be appropriately selected according to the purpose. For example, methods of purification using cation exchange column chromatography, anion chromatography, size exclusion chromatography, filters, ultrafiltration membranes, etc. can be mentioned. Purification may be performed between the first step and the second step of the modification reaction, or after the second step.

[0056] (Method for modifying a vector) The method for modifying the vector includes a first step of binding the functional group (A) in a linker compound having a functional group (A) capable of binding to the functional group in the vector and a functional group (B) capable of binding to a ligand compound to the functional group in the vector, and a second step of binding the functional group (C) in a ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B), and may further include other steps.

[0057] In the method for modifying a vector, the first step, the second step, and other steps are as described in the method for producing the modified vector.

Example

[0058] Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples at all.

[0059] <Reference Example 1: Production of AAV2> To HEK293T cells (ATCC), a medium solution of PEI (Polysciences) and a medium solution of pAAV2 (CELL BioLABs, Inc) modified so that GFP, a fluorescent protein, expresses VENUS (GenBank: ACQ43955.1), pRC-mi342 (Takara Bio Inc), and pHelper (Takara Bio Inc) were transfected, and cultured at 37 °C under 5% CO 2 for 4 days to produce AAV2. After adding EDTA (Etylenediaminetetraacetic acid) to detach the cells, the cells were centrifuged to recover the cell pellet and the centrifugal supernatant.

[0060] <Reference Example 2: Purification of AAV2> A vector was purified from the cell pellet obtained in Reference Example 1 using AAVpro (registered trademark) Purification Kit (AAV2) (TaKaRa 6232). As a result of measuring the titer of the obtained purified AAV2 using RT-PCR (Quantstudio, SYBR Green method), it was 4×10 9 vg / μL.

[0061] <Reference Example 3: Purification of AAV2> To the centrifugal supernatant obtained in Reference Example 1, a mixed solution of 40 wt% PEG8000 (Sigma) and 2.5 M aqueous NaCl solution was added, and then left standing at 4 °C for 16 hours. After centrifugation, the precipitate layer was washed with Dulbecco’s Phosphate-Buffered Saline containing 0.1 v / v% TritonX-100, 1 M MgCl 2The aqueous solution was further treated with endonuclease. After stopping the reaction with EDTA, centrifugation was performed to collect the supernatant. Also, the cell pellet obtained in Reference Example 1 was treated with Dulbecco's Phosphate-Buffered Saline containing 0.1 v / v% Triton X-100 and 1 M MgCl 2 The aqueous solution was further treated with endonuclease. After stopping the reaction with EDTA, centrifugation was performed to collect the supernatant. The obtained supernatants were mixed and purified by cation chromatography (Thermo, POROS 50HS) and anion chromatography (Thermo, POROS 50HQ). After concentration, AAV2 was obtained.

[0062] <Example 1: Modification of AAV2> The AAV2 solution obtained in Reference Example 2 was used.

[0063] (Preparation of Buffer) An aqueous solution of 1 M HEPES (gibco), an aqueous solution of 5 M NaCl (Nacalai Tesque), and Tween 20 (Sigma) were each diluted with water, and then an aqueous solution of 1 M NaOH was added to prepare a 50 mM HEPES aqueous solution at pH 8.3, a 165 mM NaCl aqueous solution, and a 0.1 v / v% Tween 20 aqueous solution.

[0064] (Preparation of Reaction Reagents 1A, 1B, 1C, 1D, 1E, 1F, 1G) Using the HEPES buffer prepared by the above method, reaction reagent 1A, which is a 0.125 mM solution of DIBENZ[b,f]azocine-5(6H)-hexanoic acid, 11,12-didehydro-ε-oxo-, 2,5-dioxo-3-sulfo-1-pyrrolidinyl ester, sodium salt (Aldrich chemistry), was prepared. Further, it was serially diluted to prepare reaction reagent 1B at 0.063 mM, reaction reagent 1C at 0.032 mM, reaction reagent 1D at 0.016 mM, reaction reagent 1E at 0.008 mM, reaction reagent 1F at 0.004 mM, and reaction reagent 1G at 0.002 mM of DIBENZ[b,f]azocine-5(6H)-hexanoic acid, 11,12-didehydro-ε-oxo-, 2,5-dioxo-3-sulfo-1-pyrrolidinyl ester, sodium salt solutions.

[0065] (Preparation of Reaction Reagents 2A, 2B, 2C, 2D) Using the HEPES buffer prepared by the above method, reaction reagent 2A, which is a 16 mM solution of 2-Azidoethyl 2-acetamido-2-deoxy-β-D-galactopyranoside (Sigma-Aldrich), was prepared. Further, it was serially diluted four-fold to prepare reaction reagent 2B at 4 mM, reaction reagent 2C at 1 mM, and reaction reagent 2D at 0.25 mM of 2-Azidoethyl 2-acetamido-2-deoxy-β-D-galactopyranoside solutions.

[0066] (The First Step) 9 μL of reaction reagents 1A, 1B, 1C, 1D, 1E, 1F, 1G prepared by the above method was mixed with 1 μL of purified AAV2 solution and reacted in an incubator at 37°C for 16 hours. Further, 2 μL of 2M glycine solution (Wako) prepared with the above buffer was added and reacted in an incubator at 37°C for 5 hours to obtain the first-step product 1A, the first-step product 1B, the first-step product 1C, the first-step product 1D, the first-step product 1E, the first-step product 1F, and the first-step product 1G. After reducing the reaction solution with DTT-containing sample buffer, SDS-PAGE (ATTO) was performed, and the progress of the reaction was confirmed by silver staining (Invitrogen). The results of silver staining are shown in Figure 1. The band of VP1 before the reaction was about 82 kDa, and after the reaction was about 88 kDa, indicating that a compound of 8 kDa was added.

[0067] Lane 1 in Figure 1 is a sample of the first-step product 1A, lane 2 is a sample of the first-step product 1B, lane 3 is a sample of the first-step product 1C, lane 4 is a sample of the first-step product 1D, lane 5 is a sample of the first-step product 1E, lane 6 is a sample of the first-step product 1F, lane 7 is a sample of the first-step product 1G, lane 8 is a sample of unmodified AAV2, and lane 9 is a marker.

[0068] (The second step) 24 μL of reaction reagents 2A, 2B, 2C, 2D prepared by the above method was mixed with 12 μL of the reaction solution of the first-step product 1A above and reacted in an incubator at 37°C for 16 hours to obtain modified vectors 2A, 2B, 2C, and 2D. After reducing the reaction solution with DTT (DITHIOTHREITOL)-containing sample buffer, SDS-PAGE (ATTO) was performed, and the progress of the reaction was confirmed by silver staining (Invitrogen). The results of silver staining are shown in Figure 2. The band of VP1 before the reaction was about 88 kDa, and after the reaction was about 92 kDa, indicating that a compound of 4 kDa was added.

[0069] Lane 1 in Figure 2 is a sample of modified vector 2A, lane 2 is a sample of modified vector 2B, lane 3 is a sample of modified vector 2C, lane 4 is a sample of modified vector 2D, lane 5 is a sample using buffer instead of reaction reagent 2, lane 6 is unmodified AAV2, and lane 7 is a marker.

[0070] <Example 2: Modification of AAV2> The purified AAV2 solution obtained in Reference Example 2 was used.

[0071] (Preparation of Phosphate Buffer) 200 mM sodium dihydrogen phosphate dihydrate (Wako) and 200 mM disodium hydrogen phosphate dodecahydrate (Wako) were mixed and diluted to prepare a 20 mM phosphate buffer at pH 8.5.

[0072] (Preparation of Reaction Reagent 1H) Using the phosphate buffer prepared by the above method, a 2 mM solution of DIBENZ[b,f]azocine-5(6H)-hexanoic acid, 11,12-didehydro-ε-oxo-,2,5-dioxo-3-sulfo-1-pyrrolidinyl ester, sodium salt (Aldrich) (reaction reagent 1H) was prepared.

[0073] (Preparation of Reaction Reagent 3) Using the phosphate buffer prepared by the above method, a 16 mM solution of 2-Azidoethyl β-D-Glucopyranoside (TCI) (reaction reagent 3) was prepared.

[0074] (First Step) 9 μL of reaction reagent 1H prepared by the above method was mixed with 1 μL of the purified AAV2 solution and reacted in an incubator at 37°C for 16 hours. Then, 2 μL of a 2 M glycine solution (Wako) prepared with the above phosphate buffer was added, and the reaction was stopped by allowing it to stand in an incubator at 37°C for 6 hours to obtain the first-step product 1H.

[0075] (Second step) By reacting reaction reagent 3 in the same manner as in Example 1, a modified vector 3A was obtained. SDS-PAGE (ATTO E-R7.5L) was performed, and silver staining (Invitrogen) was used to confirm the progress of the reaction. The results of silver staining are shown in Figure 3. The band of VP3 before the reaction was approximately 60 kDa, and after the reaction it was approximately 62 kDa, indicating that a compound of 2 kDa was added.

[0076] Lane 1 in Figure 3 is a sample of the product 1H from the first step of Example 2 (reaction reagent 1H), lane 2 is a sample of the modified vector 3A of Example 2 (reaction reagent 1H), lane 5 is a sample of unmodified AAV2, and lane 6 is the marker.

[0077] <Example 3: Modification of AAV2> The purified AAV2 solution obtained in Reference Example 2 was used.

[0078] (Preparation of reaction reagent 4) Using the phosphate buffer prepared by the above method, a 2 mM solution of 4,7,10,13,16-Pentaoxa-20-azatricosanoic acid, 23-(11,12-didehydrodibenz[b,f]azocin-5(6H)-yl)-19,23-dioxo-,2,5-dioxo-1-pyrrolidinyl ester (BROADPHARM) (reaction reagent 4) was prepared.

[0079] (First step) 9 μL of the reaction reagent 4 prepared by the above method was mixed with 1 μL of the purified AAV2 solution, and the mixture was reacted in an incubator at 37°C for 16 hours. Then, 2 μL of a 2 M glycine solution (Wako) prepared with the above phosphate buffer was added, and the reaction was stopped by allowing it to stand in an incubator at 37°C for 6 hours to obtain the product 4 of the first step. After reducing the reaction solution with a DTT-containing sample buffer, SDS-PAGE (ATTO) was performed, and the progress of the reaction was confirmed by silver staining (Invitrogen). The results of silver staining are shown in Fig. 3. The band of VP3 before the reaction was about 60 kDa, and after the reaction it was about 64 kDa, indicating that a compound of 4 kDa was added.

[0080] (Second step) By reacting the reaction reagent 3 in the same manner as in Example 1, the modified vector 3B was obtained. SDS-PAGE (ATTO E-R7.5L) was performed, and the progress of the reaction was confirmed by silver staining (Invitrogen). The results of silver staining are shown in Fig. 3. The band of VP3 before the reaction was about 64 kDa, and after the reaction it was about 68 kDa, indicating that a compound of 4 kDa was added.

[0081] Lane 3 in Fig. 3 is a sample of the product 4 of the first step of Example 3 (reaction reagent 4), lane 4 is a sample of the modified vector 3B of Example 3 (reaction reagent 4), lane 5 is a sample of unmodified AAV2, and lane 6 is a marker.

[0082] <Example 4: Modification of AAV2> The AAV2 solution (6.6×10 9 vg / μL, 500 μL) obtained in Reference Example 3 was used.

[0083] (Preparation of reaction reagents 1I and 1J) Using the same HEPES buffer as in Example 1, a 1 mM solution of 11,12-didehydro-ε-oxo-2,5-dioxo-3-sulfo-1-pyrrolidinyl ester, sodium salt of dibenz[b,f]azocine-5(6H)-hexanoic acid (Aldrich chemistry) was prepared and made up to 1 L. Further, 1 L was diluted to prepare 1 J, a 0.5 mM solution.

[0084] (Preparation of reaction reagent 2E) Using the same HEPES buffer as in Example 1, a 2 mM solution of 2-Azidoethyl 2-acetamido-2-deoxy-β-D-galactopyranoside (Sigma-Aldrich) was prepared and designated as 2E.

[0085] (First step) The reaction reagents 1 (4.5 mL) of 1I, 1J, and 1A prepared above and in Example 1 were mixed with the AAV2 solution (0.5 mL) and reacted in an incubator at 37 °C for 16 hours. Then, after adding 1 mL of a 2 M glycine solution (Wako) prepared with the same HEPES buffer as in Example 1 to the reaction solution, the mixture was reacted in an incubator at 37 °C for 7 hours to obtain the first-step product 1I, the first-step product 1J, and the first-step product 1A. After reducing the reaction solution with a DTT-containing sample buffer, SDS-PAGE (ATTO E-R7.5L) was performed, and silver staining (Invitrogen 45-1001) was used to confirm the progress of the reaction. The results of the silver staining are shown in Figure 4. The band of VP3 before the reaction was approximately 60 kDa, and after the reaction it was approximately 62 kDa, indicating that a compound of 2 kDa was added.

[0086] Lanes 1 and 8 in Figure 4 are unmodified AAV2, lane 2 is a sample of the first-step product 1A of Example 4 (reaction reagent 1A: 0.125 mM), lane 4 is a sample of the first-step product 1J of Example 4 (reaction reagent 1J 0.5 mM), lane 6 is a sample of the first-step product 1I of Example 4 (reaction reagent 1I: 1 mM), and lane 9 is a marker.

[0087] (Purification of Modified AAV2) In Example 4, the first-step products 1I, 1J, and 1A obtained by reacting with reaction reagents 1I, 1J, and 1A were purified by cation exchange column chromatography (carrier: POROS 50HS, eluent: 20 mM phosphate buffer (pH 7.4), gradient conditions: 100 - 370 mM aqueous NaCl solution). The results are shown in Figure 5. After purification, the titer was measured using RT-PCR (Quantstudio, SYBR Green method). As a result, the first-step product 1I was 5.8×10 11 vg / μL, the first-step product 1J was 1.8×10 12 vg / μL, and the first-step product 1A was 1.2×10 12 vg / μL.

[0088] In Figure 5, 1 is unmodified AAV2, 2 is a sample of the first-step product 1A in Example 4 (reaction reagent 1A: 0.125 mM), 3 is a sample of the first-step product 1J in Example 4 (reaction reagent 1J: 0.5 mM), 4 is a sample of the first-step product 1I in Example 4 (reaction reagent 1I: 1 mM). The vertical axis in Figure 5 represents the absorbance at 280 nm, and the horizontal axis represents the elution volume.

[0089] (Second Step) To 3 mL of the reaction solution after the above reaction, 6 mL each of reaction reagents 2E, 2C, and 2D were added and then mixed. By reacting in an incubator at 37°C for 16 hours, modified vectors 2E, 2F, and 2G were obtained. After reducing the reaction solution with DTT-containing sample buffer, SDS-PAGE (ATTO) was performed, and silver staining (Invitrogen) was used to confirm the progress of the reaction. The results of silver staining are shown in Figure 4. The band of VP3 before the reaction was approximately 62 kDa, and after the reaction it was approximately 63 kDa, indicating that a compound of 1 kDa was added.

[0090] Lanes 1 and 8 in Figure 4 are unmodified AAV2, lane 3 is a sample of the modified vector 2G of Example 4 (reaction reagent 1A: 0.125 mM and reaction reagent 2D: 0.25 mM), lane 5 is a sample of the modified vector 2F of Example 4 (reaction reagent 1J: 0.5 mM and reaction reagent 2C: 1 mM), lane 7 is a sample of the modified vector 2E of Example 4 (reaction reagent 1I: 1 mM and reaction reagent 2E: 2 mM), and lane 9 is a marker.

[0091] (Purification of modified vectors 2G and 2F) In Example 4, the modified vector 2F and the modified vector 2G obtained by the reaction of reaction reagents 2C and 2D were purified by cation exchange column chromatography (carrier: POROS 50HS, eluent: 20 mM phosphate buffer (pH 7.4), gradient condition: 100 - 370 mM aqueous NaCl solution). The results are shown in Figure 6. After purification, the titer was measured by RT-PCR (Quantstudio, SYBR Green method). As a result, the modified vector 2F was 2.6×10 12 vg / μL, and the modified vector 2G was 2.6×10 12 vg / μL.

[0092] In Figure 6, 1 is unmodified AAV2, 2 is a sample of the modified vector 2G of Example 4 (reaction reagent 1A: 0.125 mM and reaction reagent 2D: 0.25 mM), 3 is a sample of the modified vector 2F of Example 4 (reaction reagent 1J: 0.5 mM and reaction reagent 2C: 1 mM). The vertical axis in Figure 6 represents the absorbance at 280 nm, and the horizontal axis represents the elution volume.

[0093] (Purification of modified vector 2E) In the modified AAV2 obtained by the reaction with reaction reagent 2E in Example 4, ultrafiltration membranes (Vivaspin20 - 100 kDa) manufactured by GE Healthcare were used to repeat centrifugal concentration 5 times, and finally purified by buffer replacement with 20 mM phosphate buffer (pH 7.4, containing 250 mM NaCl). After purification, the titer was measured by RT-PCR (Quantstudio, SYBR Green method). As a result, the modified vector 2E was 1.8×10 12It was vg / μL.

[0094] Examples of aspects of the present invention include, for example, the following. <1> A step of bonding the functional group (A) in a linker compound having a functional group (A) capable of binding to the functional group in the vector and a functional group (B) capable of binding to a ligand compound to the functional group in the vector; and a step of bonding the functional group (C) in a ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B). A method for producing a modified vector, characterized by including the above. <2> The production method according to <1>, wherein the functional group in the vector is any one of an amino group, a guanidino group, a hydroxyl group, a carboxyl group, and an indole group. <3> The production method according to any one of <1> to <2>, wherein the functional group in the vector is a functional group in a lysine residue, an arginine residue, a tyrosine residue, a serine residue, a threonine residue, or a tryptophan residue. <4> The production method according to any one of <1> to <3>, wherein the functional group (A) is a succinimidyl group. <5> The production method according to any one of <1> to <4>, wherein the functional group (B) is any one of an azide group, an alkynyl group, an alkenyl group, a carbonyl group, a phosphine group, a tetrazine group, a hydrazine group, and a hydroxylamine group. <6> The production method according to any one of <1> to <5>, wherein the weight average molecular weight of the ligand compound is 100,000 or less. <7> The production method according to any one of <1> to <6>, wherein the ligand compound is a sugar compound. <8> The production method according to <7>, wherein the sugar compound has a polyethylene glycol group. <9> The production method according to any one of <1> to <6>, wherein the ligand compound is a peptide. <10> The production method according to any one of <1> to <9>, wherein the linker compound has a polyethylene glycol group. <11> The production method according to any one of <1> to <10>, wherein the vector is an adeno-associated virus vector. <12> The production method according to <11>, wherein the functional group in the vector is a functional group constituting the capsid of the adeno-associated virus vector. <13> The production method according to any one of <1> to <12>, wherein the functional group in the vector is an amino group, the functional group (A) is a succinimidyl group, the functional group (B) or (C) is an azide group and / or an alkynyl group, and the ligand compound is a sugar compound and / or a peptide. <14> The production method according to any one of <1> to <12>, wherein the functional group in the vector is a functional group in a lysine residue, the functional group (B) or (C) is an azide group and / or an alkynyl group, and the ligand compound is a sugar compound and / or a peptide. <15> A method for modifying a vector, comprising: a step of binding the functional group (A) in a linker compound having a functional group (A) capable of binding to the functional group and a functional group (B) capable of binding to a ligand compound to the functional group in the vector; and a step of binding the functional group (C) in a ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B).

Claims

1. In a functional group in a vector, a step of bonding the functional group (A) to the linker compound having a functional group (A) capable of bonding to the functional group and a functional group (B) capable of bonding to a ligand compound; to the functional group (B), a step of bonding the functional group (C) to the ligand compound having a functional group (C) capable of bonding to the functional group (B); comprising A method for producing a modified vector, wherein the functional group (B) is any one of an azide group, an alkynyl group, an alkenyl group, a carbonyl group, a phosphine group, a tetrazine group, a hydrazine group, and a hydroxylamine group.

2. The production method according to claim 1, wherein the functional group in the vector is any one of an amino group, a guanidino group, a hydroxyl group, a carboxyl group, and an indole group.

3. The production method according to any one of claims 1 to 2, wherein the functional group in the vector is a functional group in a lysine residue, an arginine residue, a tyrosine residue, a serine residue, a threonine residue, or a tryptophan residue.

4. further having a connecting portion (D) connecting the functional group (A) and the functional group (B), The production method according to any one of claims 1 to 3, wherein the connecting portion (D) is any one of an alkylene group, an alkyleneoxy group, and a poly(alkyleneoxy) group.

5. Further having a connecting portion (E) connecting the functional group (C) and the ligand compound, The production method according to any one of claims 1 to 4, wherein the connecting portion (E) is any one of an alkylene group, an alkyleneoxy group, and a poly(alkyleneoxy) group.

6. The production method according to any one of claims 1 to 5, wherein the weight average molecular weight of the ligand compound is 100,000 or less.

7. The production method according to any one of claims 1 to 6, wherein the ligand compound is a sugar compound.

8. The production method according to claim 7, wherein the sugar compound has a polyethylene glycol group.

9. The production method according to any one of claims 1 to 6, wherein the ligand compound is a peptide.

10. The production method according to any one of claims 1 to 9, wherein the linker compound has a polyethylene glycol group.

11. The production method according to any one of claims 1 to 10, wherein the vector is an adeno-associated virus vector.

12. The production method according to claim 11, wherein the functional group in the vector is a functional group constituting the capsid of the adeno-associated virus vector.

13. The production method according to any one of claims 1 to 12, wherein the functional group in the vector is an amino group, the functional group (A) is a succinimidyl group, the functional group (B) or (C) is an azide group and / or an alkynyl group, and the ligand compound is a sugar compound and / or a peptide.

14. The production method according to any one of claims 1 to 12, wherein the functional group in the vector is a functional group in a lysine residue, the functional group (B) or (C) is an azide group and / or an alkynyl group, and the ligand compound is a sugar compound and / or a peptide.

15. To a functional group in a vector, A step of binding the functional group (A) in a linker compound having a functional group (A) capable of binding to the functional group and a functional group (B) capable of binding to a ligand compound to the functional group; To the functional group (B), A step of binding the functional group (C) in a ligand compound having a functional group (C) capable of binding to the functional group (B) to the functional group (B); Including, A method for modifying a vector, characterized in that the functional group (B) is any one of an azide group, an alkynyl group, an alkenyl group, a carbonyl group, a phosphine group, a tetrazine group, a hydrazine group, and a hydroxylamine group.

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