Composition for enhancing immunogenicity

Abstract

The present invention pertains to a composition for enhancing immunogenicity that contains, as an active ingredient, particles containing a modified amphiphilic polymer, said modified amphiphilic polymer being an amphiphilic polymer in which the hydrophobic segment is a poly(hydroxy acid) and the hydrophilic segment is a polysaccharide and to which an immune activator is bound, and an antigen.

Description

Immunogenicity enhancing composition

[0001] The present invention relates to a composition for enhancing immunogenicity, which comprises, as active ingredients, particles containing a modified amphiphilic polymer in which an immunostimulator is bound to an amphiphilic polymer, and an antigen.

[0002] Because administering an antigen alone is insufficient to fully induce an immune response to an antigen, an adjuvant (immune activator) is used in combination. However, substances that have been reported to have a high adjuvant effect have not been approved for human use due to safety concerns. While only a limited number of adjuvants, such as aluminum hydroxide and MF59, are used in pharmaceuticals, there is a need to develop adjuvants that can induce a stronger immune response while ensuring safety.

[0003] To solve the above-mentioned problems, the development of particulate adjuvants is underway. By encapsulating antigens or immune activators in particulate form, it is expected that safety and delivery efficiency to target cells will be improved. Particulate adjuvants have been proposed using a wide range of materials, including fatty acids, biodegradable polymers, and viral proteins (Non-Patent Documents 1 and 2).

[0004] Recently, a technology has been reported for antigen-adjuvant microparticle complexes in which an antigen is encapsulated in adjuvant microparticles composed of an amphiphilic polymer whose hydrophobic segment is a poly(hydroxy acid) and whose hydrophilic segment is a polysaccharide containing β-glucan (Patent Documents 1 and 2). This technology has been successful in inducing a strong immune response to an antigen with a small amount of antigen and a small number of administrations. However, despite the long-awaited development of an effective adjuvant with performance far superior to conventional adjuvants using existing microparticle technology, such development has not been realized to date.

[0005] WO2010 / 098432 WO2015 / 053354

[0006] Immunology, 2006, No. 117, pp. 78-88 Nature Materials, 2011, No. 10, pp. 243-251

[0007] The object of the present invention is to provide a composition for enhancing immunogenicity that has strong immunostimulatory activity by enhancing the immunostimulatory effect of particles composed of an amphiphilic polymer in which the hydrophobic segment is a poly(hydroxy acid) and the hydrophilic segment is a polysaccharide.

[0008] In order to overcome the above problems, the present inventor discovered that a composition for enhancing immunogenicity comprising a modified amphiphilic polymer in which an immunoactivator is bound to the amphiphilic polymer and particles containing an antigen is effective, and thus completed the present invention.

[0009] That is, the present invention has the following configurations (1) to (19): (1) A composition for immunogenicity enhancement, comprising particles containing, as active ingredients, a modified amphiphilic polymer, which is an amphiphilic polymer to which an immunoactivator is bound, wherein the hydrophobic segment is poly(hydroxy acid) and the hydrophilic segment is polysaccharide, and an antigen. (2) The composition for immunogenicity enhancement according to (1), which does not contain lipid as a component other than the antigen constituting the particles. (3) The composition for immunogenicity enhancement according to (1) or (2), wherein the particles further comprise an unmodified amphiphilic polymer, which is an amphiphilic polymer to which an immunoactivator is not bound, wherein the hydrophobic segment is poly(hydroxy acid) and the hydrophilic segment is polysaccharide. (4) The composition for immunogenicity enhancement according to any of (1) to (3), wherein the polysaccharide is dextran, β-glucan, mannan, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, or pullulan. (5) The composition for enhancing immunogenicity according to (4), wherein the β-glucan is a polymer of glucose linked by one or more β-1,3 bonds and / or one or more β-1,6 bonds. (6) The composition for enhancing immunogenicity according to (4) or (5), wherein the β-glucan is black yeast glucan, curdlan, pachyman, laminaran, lichenan, schizophyllan, lentinan, scleroglucan, or pachymaran. (7) The composition for enhancing immunogenicity according to any of (1) to (6), wherein the poly(hydroxy acid) is poly(lactic acid-glycolic acid), polylactic acid, or polyglycolic acid. (8) The composition for enhancing immunogenicity according to any of (1) to (7), wherein the bond between the amphiphilic polymer and the immunoactivator in the modified amphiphilic polymer is a covalent bond. (9) The composition for immunogenicity enhancement according to any one of (1) to (8), wherein the binding site of the immunoactivator in the modified amphiphilic polymer is the hydrophilic segment. (10) The composition for immunogenicity enhancement according to any one of (1) to (9), wherein the immunoactivator is a ligand or agonist that binds to a Toll-like receptor (TLR), a NOD-like receptor (NLR), a RIG-like receptor, a C-type lectin receptor (CLR), or a stimulator of interferon genes (STING).(11) The composition for enhancing immunogenicity according to (10), wherein the ligand or agonist that binds to a Toll-like receptor (TLR) is a ligand or agonist that binds to TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, or TLR11. (12) The composition for enhancing immunogenicity according to (10) or (11), wherein the ligand or agonist that binds to a Toll-like receptor (TLR) is any one of the following (i) to (vii): (i) a ligand or agonist that binds to TLR2 selected from the group consisting of peptidoglycan, lipoprotein, lipopolysaccharide, and zymosan; (ii) a ligand or agonist that binds to TLR3 selected from the group consisting of Poly(I:C) and poly(A:U); (iii) a ligand or agonist that binds to TLR4 selected from the group consisting of lipopolysaccharide (LPS), HSP60, RS09, and MPLA; (iv) flagellin, which is a ligand or agonist that binds to TLR5; (v) a ligand or agonist that binds to TLR7 or 8 selected from the group consisting of imidazoquinoline compounds and single-stranded RNA; and (vi) a ligand or agonist that binds to TLR9 selected from the group consisting of bacterial DNA, unmethylated CpG DNA, hemozorin, ODN1585, ODN1668, and ODN1826. (vii) A ligand or agonist that binds to TLR11 selected from the group consisting of profilin and uropathogenic bacteria. (13) The composition for immunogenicity enhancement according to any one of (1) to (12), wherein the number of molecules of the immune activator bound to one molecule of the modified amphiphilic polymer is 1 to 100. (14) The composition for immunogenicity enhancement according to any one of (1) to (13), wherein the average particle size of the particles is 0.1 to 50 μm. (15) A medicine comprising, as an active ingredient, the composition for immunogenicity enhancement according to any one of (1) to (14). (16) A vaccine comprising, as an active ingredient, the composition for immunogenicity enhancement according to any one of (1) to (14). (17) A vaccine for the treatment and / or prevention of cancer, comprising, as an active ingredient, the composition for immunogenicity enhancement according to any one of (1) to (14).(18) A method for enhancing immunogenicity, comprising administering to a subject the composition for enhancing immunity according to any one of (1) to (14). (19) A method for treating and / or preventing cancer, comprising administering to a subject the composition for enhancing immunogenicity according to any one of (1) to (14), the medicament according to (15), or the vaccine according to (16) or (17).

[0010] This specification includes the disclosure of Japanese Patent Application No. 2021-139683, which is a priority document of the present application.

[0011] The present invention provides a composition for enhancing immunogenicity that enables stronger immune activation than conventional compositions.

[0012] Dextran-NH ２ 1 shows the results of GPC measurement of R848-dextran-PLGA. １ The results of H-NMR measurement are shown below. ２ 8 shows the results of GPC measurement of black yeast glucan-PLGA. １ 1 shows the results of H-NMR measurement. 1 shows the results of DLS measurement of OVA-containing R848-modified dextran-PLGA particles. 1 shows the results of DLS measurement of OVA-containing R848-modified black yeast glucan-PLGA particles. 1 shows the results of DLS measurement of TRP2-containing R848-modified black yeast glucan-PLGA particles. 1 shows the results of activation of mouse bone marrow-derived dendritic cells by a composition for enhancing immunogenicity.

[0013] The present invention relates to an immunogenic composition (particularly a composition for enhancing immunogenicity) containing, as an active ingredient, particles comprising a modified amphiphilic polymer, which is an amphiphilic polymer having an immunoactivator bound thereto, and an antigen, wherein the hydrophobic segment is a poly(hydroxy acid) and the hydrophilic segment is a polysaccharide, preferably particles comprising a modified amphiphilic polymer, an antigen, and an unmodified amphiphilic polymer, which is an amphiphilic polymer having no immunoactivator bound thereto, wherein the hydrophobic segment is a poly(hydroxy acid) and the hydrophilic segment is a polysaccharide.

[0014] The amphiphilic polymer that constitutes the particles together with the antigen will now be described. "Amphiphilic" means having both hydrophilic and hydrophobic properties. When a certain portion (segment) has higher solubility in water than other portions, that portion (segment) is said to be hydrophilic. It is desirable for the hydrophilic portion to be soluble in water, but even if it is sparingly soluble, it is acceptable as long as it has higher solubility in water than other portions. Furthermore, when the solubility in water of a certain portion (segment) is lower than other portions, that portion (segment) is said to be hydrophobic. It is desirable for the hydrophobic portion to be insoluble in water, but even if it is soluble, it is acceptable as long as it has lower solubility in water than other portions.

[0015] An "amphiphilic polymer" is a polymer that has the above-described amphiphilic properties as a whole molecule. An amphiphilic "polymer" refers to a molecular structure in which the hydrophilic segment, the hydrophobic segment, or both in the amphiphilic molecule are composed of repeating structures of minimum units (monomers). The structure of the amphiphilic polymer in the present invention is not particularly limited, and specific examples include linear block polymers in which polysaccharides and poly(hydroxy acids) are linked together, branched polymers with multiple branches containing polysaccharides or poly(hydroxy acids), graft polymers consisting of a polysaccharide main chain and a poly(hydroxy acid) side chain, and graft polymers consisting of a poly(hydroxy acid) main chain and a polysaccharide side chain. However, linear block polymers in which polysaccharides and poly(hydroxy acids) are linked together are preferred.

[0016] In the present invention, the hydrophilic segment of the amphiphilic polymer is characterized by being a polysaccharide. While there are no particular limitations on the polysaccharide, specific examples include dextran, β-glucan, mannan, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid, and pullulan, and dextran or β-glucan is preferred.

[0017] Glucan is a glucose-containing polysaccharide, and β-glucan contains one or more β-bonds between glucose subunits. That is, the β-glucan used in the present invention contains β-bonds, or may contain only β-bonds. Furthermore, the β-glucan used in the present invention may be branched or linear. Preferred β-glucans include those containing one or more β-1,3 bonds and / or one or more β-1,6 bonds, and those containing one or more β-1,2 bonds and / or β-1,4 bonds. However, those containing one or more β-1,3 bonds and / or one or more β-1,6 bonds are more preferred, and those containing one or more β-1,3 bonds are even more preferred. Specific examples of β-glucans containing one or more β-1,3 bonds include curdlan, pachyman, laminaran, lichenan, schizophyllan, lentinan, scleroglucan, black yeast glucan (preferably, β-1,3 glucan or β-1,6 glucan derived from black yeast), or pachymaran, and preferred examples include curdlan, pachyman, laminaran, schizophyllan, scleroglucan, black yeast glucan, or pachymaran.

[0018] Examples of linear β-glucans containing one or more β-1,3 bonds include β-glucans consisting mainly of β-1,3 bonds (e.g., curdlan and pachyman) and β-glucans consisting of β-1,3 bonds and β-bonds other than β-1,3 bonds (e.g., laminaran and lichenan).

[0019] Examples of branched β-glucans containing one or more β-1,3 bonds include β-glucans consisting of β-1,3 and β-1,6 bonds (for example, schizophyllan, lentinan, scleroglucan, and black yeast glucan).

[0020] The β-glucan used in the present invention may be a derivatized β-glucan. Examples of derivatization include an addition reaction of a carboxymethyl group and an oxidative cleavage reaction. Examples of derivatized β-glucans include carboxymethylcurdlan, in which a carboxymethyl group is added to curdlan, and pachymaran, in which pachyman is cleaved.

[0021] The number average molecular weight of the polysaccharide is not particularly limited, but is preferably 500 to 100,000, more preferably 500 to 50,000, and even more preferably 1,000 to 10,000, for example, 1,000 to 8,000, 1,000 to 6,000, or 1,000 to 4,000. The number average molecular weight is an average molecular weight calculated by a method that does not take into account weighting of molecular size, and the number average molecular weight of the polysaccharide can be determined by gel permeation chromatography (GPC).

[0022] In the present invention, the hydrophobic segment of the amphiphilic polymer is characterized by being a poly(hydroxy acid). While the poly(hydroxy acid) is not particularly limited, it is preferably a biocompatible polymer that does not cause significant adverse effects when administered to a living body. Biocompatibility here refers to a polymer with an LD50 of 2,000 mg / kg or greater when orally administered to rats. The poly(hydroxy acid) may also be a copolymer of multiple types of hydroxy acids, but is preferably a polymer of two or fewer types of hydroxy acids.

[0023] Specific preferred examples of the poly(hydroxy acid) include polyglycolic acid, polylactic acid, poly(2-hydroxybutyric acid), poly(2-hydroxyvaleric acid), poly(2-hydroxycaproic acid), poly(2-hydroxycapric acid), poly(malic acid), and derivatives and copolymers of these polymeric compounds. Poly(lactic acid-glycolic acid), polylactic acid, or polyglycolic acid is preferred, and poly(lactic acid-glycolic acid) is more preferred. Furthermore, when the poly(hydroxy acid) is poly(lactic acid-glycolic acid), the composition ratio (lactic acid / glycolic acid) (mol / mol) of the poly(lactic acid-glycolic acid) is not particularly limited as long as the object of the present invention is achieved, but is preferably 99 / 1 to 1 / 99, and more preferably 80 / 20 to 20 / 80, e.g., 60 / 40 to 40 / 60, or 50 / 50.

[0024] The number average molecular weight of the poly(hydroxy acid) is not particularly limited, but is preferably 500 to 1,000,000, more preferably 500 to 100,000, and even more preferably 500 to 50,000, for example, 500 to 40,000, 500 to 30,000, 500 to 20,000, or 500 to 15,000. The number average molecular weight of the poly(hydroxy acid) is determined from the difference between the number average molecular weight of an amphiphilic polymer in which the hydrophobic segment is a poly(hydroxy acid) and the hydrophilic segment is a polysaccharide and the number average molecular weight of the polysaccharide.

[0025] The number average molecular weight of the amphiphilic polymer (unmodified amphiphilic polymer not bound to an immunostimulatory factor) constituting the particles is not particularly limited, but is preferably 1,000 to 1,000,000, more preferably 1,000 to 100,000, and even more preferably 9,000 to 50,000, for example, 9,000 to 40,000, 9,000 to 30,000, 9,000 to 20,000, or 9,000 to 15,000. The number average molecular weight of the amphiphilic polymer is determined by gel permeation chromatography (GPC).

[0026] The amphiphilic polymer may be produced by a known method, specifically, a method of adding a poly(hydroxy acid) to a polysaccharide and carrying out a condensation reaction, or a method of adding a hydroxy acid-activated monomer to a polysaccharide and carrying out a polymerization reaction, can be mentioned as examples.

[0027] Furthermore, when the amphiphilic polymer is a linear block polymer in which a polysaccharide and a poly(hydroxy acid) are linked together, it may be produced by a known method, specifically, a production method in which a poly(hydroxy acid) copolymer is subjected to a condensation reaction with the reducing end of the polysaccharide of the amphiphilic polymer using an activator for the terminal functional group (Macromol. Rapid Commun., 31, pp. 1664-1684 (2010)) can be mentioned as an example.

[0028] When the amphiphilic polymer is a graft polymer having a polysaccharide main chain and a poly(hydroxy acid) side chain, it can be produced as follows: (1), (2) or (3).

[0029] (1) A method of producing a graft polymer by adding a hydroxy acid-activated monomer to a polysaccharide in the presence of a tin catalyst to carry out a polymerization reaction and introducing poly(hydroxy acid) (Macromolecules, 31, pp. 1032-1039 (1998)).

[0030] (2) A method for producing a graft polymer by activating the partially unprotected hydroxyl groups of a polysaccharide, most of whose hydroxyl groups are protected with substituents, with a base, adding a hydroxy acid-activated monomer to introduce a graft chain consisting of poly(hydroxy acid), and finally removing the protecting groups (Polymer, 44, pp. 3927-3933, (2003)).

[0031] (3) A method for producing a graft polymer by subjecting a copolymer of poly(hydroxy acid) to a condensation reaction with a polysaccharide using a dehydrating agent and / or a functional group activator (Macromolecules, 33, pp. 3680-3685 (2000)).

[0032] A "modified amphiphilic polymer" is a polymer in which an immunostimulatory factor is bound to the amphiphilic polymer. The bond referred to here may be a non-covalent bond or a covalent bond, but is preferably a covalent bond. The non-covalent bond is preferably a hydrophobic interaction, but may also be an ionic bond (electrostatic interaction), a hydrogen bond, a coordinate bond, a van der Waals bond, physical adsorption, or a combination of these.

[0033] The term "immune activator" as used herein refers to a substance that activates one or more types of immune cells and can maintain or enhance the immune function of the cells. The term "immune cells" as used herein refers to, for example, T lymphocytes, B lymphocytes, NK cells, monocytes, dendritic cells, granulocytes, macrophages, myeloid-derived suppressor cells, Langerhans cells and their precursor cells, and the immune cell groups present in tumors.

[0034] The immune activator used in the present invention is not particularly limited, and specific examples include ligands or agonists that bind to Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-like receptors, C-type lectin receptors (CLRs), or stimulatory factor for interferon genes (STINGs), with preference given to ligands or agonists that bind to TLRs.

[0035] Specific examples of TLRs include TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, and TLR11, and specific examples of ligands or agonists that bind to them include the following (i) to (vii):

[0036] (i) A ligand or agonist that binds to TLR2 selected from the group consisting of peptidoglycan, lipoprotein, lipopolysaccharide, and zymosan.

[0037] (ii) A ligand or agonist that binds to TLR3 selected from the group consisting of Poly(I:C) and Poly(A:U).

[0038] (iii) a ligand or agonist that binds to TLR4 selected from the group consisting of lipopolysaccharide (LPS), HSP60, RS09, and MPLA.

[0039] (iv) Flagellin, a ligand or agonist that binds to TLR5.

[0040] (v) A ligand or agonist that binds to TLR7 or 8 selected from the group consisting of imidazoquinoline compounds and single-stranded RNA.

[0041] (vi) A ligand or agonist that binds to TLR9 selected from the group consisting of bacterial DNA, unmethylated CpG DNA, hemozorin, ODN 1585, ODN 1668, and ODN 1826. (vii) A ligand or agonist that binds to TLR11 selected from the group consisting of profilin and uropathogenic bacteria.

[0042] In the present invention, the immunoactivator is preferably a ligand or agonist that binds to TLR7 or 8 (TLR7 / 8 ligand or agonist), more preferably the ligand or agonist described in (v) above.

[0043] Preferable specific examples of the imidazoquinoline compounds (v) include, for example, compounds described in U.S. Patent No. 8,951,528 and compounds described in WO2015 / 103989, such as 4-amino-2-(ethoxymethyl)-a,a-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanol (Resquimod (R848)), 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine (Resquimod (R848)), ), 1-(4-amino-2-ethylaminomethylimidazo-[4,5-c]quinolin-1-yl)- 2-methylpropan-2-ol (Gardiquimod), N-[4-(4-amino-2-ethyl-1H-imi dazo[4,5-c]quinolin-1-yl)butyl-]methanesulfonamide (PF-4878691 ), 4-amino-aa-dimethyl-2-methoxyethyl-1H-imidazo[4,5-c]quinolin e-1-ethanol, 1-(2-(3-(benzyloxy)propoxy)ethyl)-2-(ethoxymethyl )-1H-imidazo[4,5-c]quinolin-4-amine, 4-amino-2-ethoxymethyl-aa -dimethyl-6,7,8,9-tetrahydro-1H-imidazo[4,5-c]quinoline-1-eth anol, N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazo[4,5-c]quin olin-l-yl]eyhoxy}ethyl)-n'-phenylurea, 1-2-amino-2-methylpropy l)-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine, 1-{4-[(3 ,5-dichlorophenyl)sulfonyl]butyl}-2-ethyl-1H-imidazo[4,5-c]qu inolin-4-amine, N-(2-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]ethoxy}ethyl)-n'-cyclohexylurea, N-{3-[4-amino-2-(ethoxymethyl)-1H-imi dazo[4,5-c]quinolin-1-yl]propyl}-n'-(3-cyanophenyl)thiourea, N-[3-(4-amino-2-butyl-1H-im) Examples of such compounds include 2-butyl-1-[3-(methylsulfonyl)propyl]-1H-imidazo[4,5-c]quinolin-4-amine, 2-butyl-1-[3-(methylsulfonyl)propyl]-1H-imidazo[4,5-c]quinolin-4-amine, and derivatives thereof, but are not particularly limited as long as they bind to TLR7 or TLR8.

[0044] Specific examples of ligands or agonists that bind to NOD-like receptors (NLRs) include M-TriDAP and PGN. Other examples include ligands or agonists for NOD1, such as Tri-DAP, iE-DAP, and C12-iE. Furthermore, other examples include ligands or agonists for NOD2, such as MDP, N-glycosyl-MDP, Murabutide, M-TriLyS-D-ASN, M-TriLYS, and L18-MDP.

[0045] Specific examples of ligands or agonists that bind to RIG-like receptors include 5'ppp-dsRNA, poly(dA:dT), poly(dG:dC), and poly(I:C).

[0046] Specific examples of ligands or agonists that bind to C-type lectin receptors (CLR) include trehalose 6,6-dibehenate, zymosan, WGP, HKSC, HKCA, and curdlan AL.

[0047] Ligands or agonists that bind to stimulator of interferon genes (STING) include c-di-GMP, c-di-AMP, 2'3'-cGAMP, 3'3'-cGAMP, or 2'2'-cGAMP.

[0048] The binding site of the immunoactivator in the modified amphiphilic polymer is not particularly limited, but is preferably a polysaccharide segment. The molar ratio of the immunoactivator to the amphiphilic polymer (the number of molecules of the immunoactivator bound to one molecule of the modified amphiphilic polymer) is not particularly limited as long as the object of the present invention is achieved, but is preferably 1 to 100, more preferably 1 to 50, even more preferably 1 to 30, and particularly preferably 1 to 10, relative to the modified amphiphilic polymer.

[0049] The number average molecular weight of the modified amphiphilic polymer is not particularly limited, but is preferably 1,000 to 1,000,000, more preferably 1,000 to 100,000, and even more preferably 2,000 to 50,000, for example, 2,000 to 40,000, 2,000 to 30,000, 2,000 to 20,000, or 2,000 to 15,000. The number average molecular weight of the modified amphiphilic polymer is determined by gel permeation chromatography (GPC).

[0050] The modified amphiphilic polymer may be produced by a known method, specifically, by the following method (1) or (2).

[0051] (1) A method of binding an immunostimulator to the polysaccharide segment of an amphiphilic polymer Examples of production methods include a method in which the hydroxyl groups of a polysaccharide main chain are quantitatively activated using a reaction reagent such as, but not limited to, 1,1'-carbonyldiimidazole (CDI) or disuccinimidyl carbonate (DSC), and then a reactive functional group, such as an amino group, which is separately introduced into the immunoactivator and which covalently bonds to the activated hydroxyl groups, is subjected to a condensation reaction with the activated hydroxyl groups; a method in which the hydroxyl groups of the polysaccharide are converted into other functional groups with higher reactivity, such as, but not limited to, an amino group, a carboxyl group, a maleimide group, a thiol group, and then a functional group that bonds to these reactive functional groups is separately introduced into the immunoactivator and then the immunoactivator is subjected to a condensation reaction with the polysaccharide; and a method in which the hydroxyl groups of the polysaccharide are converted into cationic amino groups or anionic carboxyl groups, and then the amino or carboxyl group is ionic-bonded by electrostatic interaction to a carboxyl or amino group of opposite charge that is separately introduced into the immunoactivator.

[0052] (2) A method of binding an immunostimulatory factor to a poly(hydroxy acid) segment of an amphiphilic polymer The α-terminus of a poly(hydroxy acid) can be linked to a polysaccharide to form a copolymer, and various reactive functional groups, including but not limited to, an amino group, a carboxyl group, a maleimide group, a thiol group, etc., can be introduced into the ω-terminus. Examples of such methods include a production method in which an immunostimulatory factor to which a functional group that binds to the reactive functional group introduced into the ω-terminus of the poly(hydroxy acid) has been separately introduced is subjected to a condensation reaction with the poly(hydroxy acid), or a production method in which the ω-terminus of the poly(hydroxy acid) is converted into a cationic amino group or an anionic carboxyl group, and then the amino group or carboxyl group is ionic-bonded via electrostatic interaction with a carboxyl group or amino group of opposite charge that has been separately introduced into the immunostimulatory factor.

[0053] In order to maintain the immunostimulatory effect over a long period of time, the modified amphiphilic polymer is preferably water-insoluble as a whole so that it is not immediately excreted from the body. "Water-insoluble" here means that the solubility in water is 1 g (amphiphilic polymer) / 100 ml (water) or less.

[0054] The particles, which are the active ingredient of the immunogenicity enhancing composition of the present invention, are characterized by containing an antigen and the modified amphiphilic polymer as constituent components, and preferably further containing an unmodified amphiphilic polymer to which no immune activator is bound. The particles of the present invention can have significantly enhanced immune activation ability compared to particles containing an antigen and an unmodified amphiphilic polymer but not a modified amphiphilic polymer, a mixture of the particles and an immune activator, or an antigen alone.

[0055] When a combination of a modified amphiphilic polymer and an unmodified amphiphilic polymer is included as a component other than the antigen constituting the particle, the combination is not particularly limited as long as the particle has immunostimulatory activity. The hydrophilic segments of the modified amphiphilic polymer and the unmodified amphiphilic polymer may be different polymers or the same polymer, and similarly, the hydrophobic segments of the modified amphiphilic polymer and the unmodified amphiphilic polymer may be different polymers or the same polymer. Furthermore, when the segments of the modified amphiphilic polymer and the unmodified amphiphilic polymer are different polymers, there may be three or more types, and the linkage modes of the hydrophilic segments and hydrophobic segments of the modified amphiphilic polymer and the unmodified amphiphilic polymer may be different or the same. For example, the modified amphiphilic polymer may be a block polymer and the unmodified amphiphilic polymer may be a graft polymer. Preferably, the segments of the modified amphiphilic polymer and the unmodified amphiphilic polymer are composed of the same polymer.

[0056] When a combination of a modified amphiphilic polymer and an unmodified amphiphilic polymer is contained as components other than the antigen that constitute the particles, the ratio of the weight of the modified amphiphilic polymer to the total weight of the modified amphiphilic polymer and the unmodified amphiphilic polymer is preferably 0.01 to 99.99 wt %, more preferably 0.1 to 99.9 wt %, even more preferably 1 to 99 wt %, still more preferably 5 to 95 wt %, and particularly preferably 5 to 80 wt %.

[0057] Components other than the antigen that constitute the particles may include components other than amphipathic polymers. As a specific example, well-known lipids may be included as components of liposomes or lipid nanoparticles, which are particles that can encapsulate antigens. However, as is clear from the examples, the expected effects of the present invention can be sufficiently obtained without using lipids, and therefore it is preferable that the particles do not include lipids as components other than the antigen.

[0058] The particle structure is not particularly limited, but a structure in which the hydrophilic segment of the amphiphilic polymer constituting the particle is located inside the particle and the hydrophobic segment is located in the outer layer of the particle in order to form a particle-antigen complex is preferred, because the antigen is encapsulated in the particle and can be more stably retained.

[0059] The method for producing the particles is not particularly limited, and examples thereof include a submerged drying method, a spray drying method, and a pulverization method. The particles of the present invention are preferably produced by a submerged drying method.

[0060] Examples of methods for producing particles by the submerged drying method include an O / W emulsion method, a W / O / W emulsion method, and an S / O / W emulsion method.

[0061] For example, when particles are produced by the O / W emulsion method, they can be produced by mixing a water-immiscible organic solvent in which a powder of an amphiphilic polymer constituting the particles has been dissolved with an aqueous solution in which a surface modifier and an antigen have been dissolved to prepare an O / W emulsion solution, and then removing the water-immiscible organic solvent from the O / W emulsion solution to obtain the particles.

[0062] An example of producing particles by the W / O / W emulsion method includes the steps of: mixing an aqueous solvent in which an antigen has been dissolved with a water-immiscible organic solvent in which an amphiphilic polymer powder constituting the particles has been dissolved to prepare a W / O emulsion solution; mixing the W / O emulsion solution with an aqueous solution of a surface modifier to prepare a W / O / W emulsion solution; and removing the water-immiscible organic solvent from the W / O / W emulsion solution to obtain particles.

[0063] An example of producing particles by the S / O / W emulsion method includes the steps of: mixing an aqueous solvent in which an antigen has been dissolved with a water-immiscible organic solvent in which an amphipathic polymer powder constituting the particles has been dissolved to prepare a W / O emulsion solution; removing the solvent from the W / O emulsion solution to obtain a solid; dispersing the solid in a water-immiscible organic solvent to obtain an S / O suspension solution; mixing the S / O suspension solution with an aqueous solution of a surface modifier to prepare an S / O / W emulsion solution; and removing the water-immiscible organic solvent from the S / O / W emulsion solution to obtain particles.

[0064] The antigen content in the particles (antigen / particle) is preferably 0.01 to 20% by weight, more preferably 0.1 to 10% by weight, for example, 0.5 to 10% by weight. The antigen content can be determined by extracting the antigen from the particles using an organic solvent and quantifying the antigen by gel electrophoresis or liquid chromatography.

[0065] The surface modifier used for particle preparation is preferably a water-soluble polymer or a surfactant. The water-soluble polymer here refers to a polymer compound having a solubility in water of 1 g (water-soluble polymer) / 100 ml (water) or more.

[0066] Examples of water-soluble polymers that can be used as surface modifiers include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneimine, polyacrylic acid, polymethacrylic acid, poly-1,3-dioxolane, 2-methacryloyloxyethyl phosphorylcholine polymer, poly-1,3,6-trioxane, polyamino acids, peptides, proteins, and carbohydrates (monosaccharides, oligosaccharides, polysaccharides, etc.), with polyvinyl alcohol being more preferred.

[0067] Examples of surfactants that can be used as surface modifiers include nonionic surfactants such as polyoxyethylene polyoxypropylene glycol, sucrose fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan mono-fatty acid esters, polyoxyethylene sorbitan di-fatty acid esters, polyoxyethylene glycerin mono-fatty acid esters, polyoxyethylene glycerin di-fatty acid esters, polyglycerin fatty acid esters, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil; alkyl sulfates such as sodium lauryl sulfate, ammonium lauryl sulfate, and sodium stearyl sulfate; and lecithin, with polyoxyethylene polyoxypropylene glycol being more preferred.

[0068] The water-immiscible organic solvent used for particle preparation is preferably one in which the amphiphilic polymer and the modified amphiphilic polymer are soluble and the polysaccharide is poorly soluble or insoluble. The solubility of the water-immiscible organic solvent in water is preferably 30 g (water-immiscible organic solvent) / 100 ml (water) or less. Specific examples of water-immiscible organic solvents include ethyl acetate, isopropyl acetate, butyl acetate, dimethyl carbonate, diethyl carbonate, methylene chloride, and chloroform.

[0069] The aqueous solvent used for particle preparation is an aqueous solution containing water and, optionally, water-soluble components, such as inorganic salts, sugars, organic salts, amino acids, peptides, proteins, and nucleic acids.

[0070] The particle surface may be bonded to a surface modifier used in the manufacturing process. The bond here may be a non-covalent bond or a covalent bond. The non-covalent bond is preferably a hydrophobic interaction, but may also be an ionic bond (electrostatic interaction), a hydrogen bond, a coordinate bond, a van der Waals bond, a physical adsorption, or a combination thereof.

[0071] The average particle size of the particles is preferably 0.1 to 50 μm, more preferably 0.1 to 25 μm, even more preferably 0.1 to 10 μm, and particularly preferably 0.1 to 1 μm, for example, 0.2 to 1 μm or 0.3 to 1 μm. The average particle size here can be determined by the cumulant method using a dynamic light scattering device (DLS: for example, ELS-Z, manufactured by Otsuka Electronics Co., Ltd.).

[0072] The "immunogenicity enhancing composition" of the present invention is a composition capable of enhancing immune responses to antigens in vivo, comprising as active ingredients particles containing a modified amphiphilic polymer in which an immune activator is bound to the amphiphilic polymer, and an antigen. The type of immune response induced by the immunogenicity enhancing composition is not limited. The types of immune responses induced include Th1-type immune responses and Th2-type immune responses, and it is known that one type of immune response predominates depending on the type of antigen, immune activator, administration site, and administration method. However, the present invention can induce both Th1-type and Th2-type immune responses.

[0073] In the present invention, an "antigen" refers to a substance that induces immunity in the body and can be used as a vaccine for the treatment and / or prevention of disease. By using particles containing an antigen and a modified amphiphilic polymer to which an immunoactivator of the present invention is bound as an active ingredient, the immune response elicited by the antigen can be enhanced.

[0074] Examples of antigens include peptides, proteins, glycoproteins, glycolipids, lipids, carbohydrates, nucleic acids, polysaccharides, and viruses, bacteria, allergy-causing substances, tissues, cells, etc. Specific examples include pollen-derived antigens, hepatitis A virus-derived antigens, hepatitis B virus-derived antigens, hepatitis C virus-derived antigens, hepatitis D virus-derived antigens, hepatitis E virus-derived antigens, hepatitis F virus-derived antigens, HIV virus-derived antigens, influenza virus-derived antigens, herpesvirus (HSV-1, HSV-2)-derived antigens, anthrax-derived antigens, chlamydia-derived antigens, pneumococcus-derived antigens, Japanese encephalitis virus-derived antigens, measles virus-derived antigens, rubella virus-derived antigens, tetanus bacillus-derived antigens, varicella virus-derived antigens, SARS virus-derived antigens, Epstein-Barr virus-derived antigens, Examples of antigens include papillomavirus-derived antigens, Helicobacter pylori-derived antigens, rabies virus-derived antigens, West Nile virus-derived antigens, hantavirus-derived antigens, streptococcus-derived antigens, staphylococcus-derived antigens, Bordetella pertussis-derived antigens, Mycobacterium tuberculosis-derived antigens, malaria parasite (Plasmodium)-derived antigens, poliovirus-derived antigens, various zoonotic infectious disease-derived antigens, various food allergy-derived antigens, and autoantigens.

[0075] Another preferred example of an antigen is a cancer antigen, which is a substance derived from a protein specifically expressed in cancer cells and has the effect of treating and / or preventing cancer through an immune response when administered from an ex vivo source to the living body.

[0076] The immunogenicity-enhancing composition of the present invention can be used for the treatment and / or prevention of diseases such as cancer, infectious diseases, or allergies. Examples of cancers for which the immunogenicity-enhancing composition of the present invention can be used include basal cell carcinoma, Paget's disease, brain tumor, bladder cancer, esophageal cancer, leukemia, lymphoma, liver cancer, gallbladder cancer, sarcoma, mast cell tumor, adrenocortical carcinoma, Ewing's tumor, Hodgkin's lymphoma, mesothelioma, multiple myeloma, thyroid cancer, skin cancer (melanoma, etc.), lung cancer, pharyngeal cancer, gastric cancer, pancreatic cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, and head and neck cancer. Infectious diseases include infections caused by hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis F virus, HIV virus, influenza virus, herpes virus (HSV-1, HSV-2), anthrax, chlamydia, pneumococcus, Japanese encephalitis virus, measles virus, rubella virus, tetanus bacillus, varicella virus, SARS virus, Epstein-Barr virus, papillomavirus, Helicobacter pylori, rabies virus, West Nile virus, hantavirus, streptococcus, staphylococcus, pertussis bacillus, tuberculosis bacillus, malaria parasite, or poliovirus. Allergies include pollen allergies, various food allergies, and allergies to autoantigens.

[0077] The immunogenicity enhancing composition of the present invention can be used as an active ingredient in a vaccine for treating and / or preventing cancer when a cancer antigen is used as the antigen. Thus, the present invention provides a vaccine containing the immunogenicity enhancing composition of the present invention as an active ingredient.

[0078] According to a preferred embodiment of the present invention, there is provided a method for immune activation (particularly, a method for immunogenicity enhancement) comprising administering the immunogenicity-enhancing composition, medicament, or vaccine of the present invention to a (living) subject. Also provided is a method for treating and / or preventing diseases such as cancer, infectious diseases, or allergies, comprising administering the immunogenicity-enhancing composition, medicament, or vaccine of the present invention to a (living) subject. The method for activating an immune response or treating and / or preventing a disease using the immunogenicity-enhancing composition, medicament, or vaccine of the present invention is not limited, and the immunogenicity-enhancing composition, medicament, or vaccine may be administered to a living body (subject) or contacted with immunocompetent cells extracted from the living body (subject). The method of administration to a living body (subject) is not particularly limited, and examples include subcutaneous administration, intradermal administration, intramuscular administration, nasal administration, pulmonary administration, oral administration, transdermal administration, sublingual administration, intravaginal administration, intraperitoneal administration, and lymph node administration, with intradermal or subcutaneous administration being preferred. The living body (subject) to which the compound is administered may be a human or a non-human animal, but is preferably a human, or a pig, cow, bird, sheep, horse, donkey, goat, camel, dog, cat, ferret, rabbit, monkey, rat, mouse, or guinea pig that is kept as a livestock, pet, or laboratory animal. The subject may be a subject with a disease such as cancer, infectious disease, or allergy, or a subject that does not have a disease such as cancer, infectious disease, or allergy but is at high risk of developing the disease.

[0079] When the immunogenicity enhancing composition of the present invention is used as a medicine (including a vaccine), it may be formulated by incorporating various pharmaceutically useful additives, and specific examples of additives include buffers, antioxidants, salts, polymers, or sugars.

[0080] When the immunogenicity enhancing composition of the present invention is used as a pharmaceutical, the dosage of particles is appropriately determined depending on the administration method and number of administrations. For example, when the immunogenicity enhancing composition of the present invention is administered subcutaneously to a human, the amount of particles to which an immune activator is bound is 0.01 to 1,000 mg per administration.

[0081] Examples are shown below, but the present invention is not limited to these examples.

[0082] Example 1 Synthesis of R848-dextran-PLGA In this example, a linear block polymer was synthesized by adding poly(lactic acid-glycolic acid) (poly(lactic-co-glycolic acid); PLGA) to dextran and carrying out a condensation reaction. The resulting amphiphilic polymer, dextran-poly(lactic acid-glycolic acid) (dextran-PLGA), was covalently bonded to resiquimod (R848), an imidazoquinoline compound serving as an immunoactivator and known as a TLR7 / 8 agonist, to synthesize R848-dextran-poly(lactic acid-glycolic acid) (R848-dextran-PLGA).

[0083] <Dextran having a primary amino group at the reducing end (dextran-NH ２ Synthesis of Dextran-NHBoc> Sodium triacetoxyborohydride (203.5 mg) and N-Boc-ethylenediamine (76.1 μl) were added to a dimethyl sulfoxide solution of dextran (number average molecular weight 2,100, PHARMACOSMOS) at a concentration of 500 mg / 2 ml, and the reaction was carried out with stirring at 60°C for 91 hours. Next, unreacted N-Boc-ethylenediamine was removed by dialysis using water as the external solution, and the mixture was freeze-dried to synthesize dextran-NHBoc. The Boc terminal group of the obtained dextran-NHBoc (450 mg) was deprotected (35% aqueous hydrochloric acid solution (5 ml) / dimethyl sulfoxide (5 ml), stirred at room temperature for 35 hours), purified by water dialysis, and freeze-dried to obtain a polymer powder (dextran-NH ２ ) was recovered.

[0084] Dextran-NH ２ The number average molecular weight of dextran-NH was determined by GPC measurement (column: TSK-gel α-5000 × 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan) (Table 1, Figure 1: Dextran-NH ２ ). １ H-NMR measurement confirmed that a primary amino group had been introduced into the reducing end of dextran.

[0085] <Synthesis of dextran-PLGA> Poly(lactic acid-glycolic acid) with heterobifunctional terminal groups (COOH-PLGA-OH, Fujifilm Wako Pure Chemical Industries, Ltd., PLGA-5020, number average molecular weight 8,900) (350 mg) was mixed with a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (35.5 mg) and N-hydroxysuccinimide (NHS) (21.3 mg) in dimethyl sulfoxide (0.58 ml), and the mixture was allowed to react at 50°C for 2 hours to convert the carboxyl group at one end to an active ester (NHS). A dextran with a primary amino group introduced at the reducing end (dextran-NH ２ , number average molecular weight 2,700) (100 mg) was added, and the active ester of NHS-PLGA-OH (NHS) and dextran-NH ２ The condensation reaction with the primary amino group of the dextran-NH was carried out for 110.5 hours. After the reaction, the unreacted dextran-NH was removed by water dialysis. ２ After removing the residue, unreacted NHS-PLGA-OH was removed by ultracentrifugation purification to obtain amphiphilic polymer (1-1).

[0086] The number-average molecular weight of dextran-PLGA was determined by GPC measurement (column: TSK-gel α-5000 × 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan) (Table 1, Figure 2: dextran-PLGA). １ H-NMR measurement confirmed that the condensation reaction had progressed.

[0087] Modification Reaction of Dextran-PLGA with R848 (Synthesis of CASSPy-Dextran-PLGA) A solution of CDI (21.7 mg) in dimethyl sulfoxide (0.1 ml) was added dropwise to a solution of dextran-PLGA (100 mg) in dimethyl sulfoxide (0.4 ml), followed by the dropwise addition of a solution of heterocrosslinker (cysteamine SS pyridyl; CASSPy) (4.9 mg) prepared by reacting 2,2'-dipyridyl disulfide with cysteamine hydrochloride in dimethyl sulfoxide (0.1 ml), followed by stirring at room temperature for 2.5 hours. The reaction solution was placed in a dialysis membrane and subjected to dimethyl sulfoxide dialysis and then water dialysis. The contents were centrifuged (9,000 rpm, 4°C, 30 minutes) to remove the supernatant, which was then lyophilized to obtain a polymer powder.

[0088] The number average molecular weight of CASSPy-dextran-PLGA was determined by GPC measurement (column: TSK-gel α-5000 × 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan). The CASSPy introduction rate (%) was １ This was confirmed by H-NMR measurement.

[0089] (Synthesis of SH-dextran-PLGA) A solution of dithiothreitol (DTT, 15.5 mg) in dimethyl sulfoxide (0.05 ml) was added as a reagent for reducing disulfide groups (SS bonds) to SH groups to a solution of CASSPy-dextran-PLGA (108.8 mg) in dimethyl sulfoxide (1.1 ml), and the mixture was allowed to react at room temperature for 4.5 hours. Subsequently, water dialysis was performed as a purification method to remove excess DTT, followed by centrifugation (9000 rpm, 30 minutes, 4°C) and lyophilization to recover the polymer powder. １ H-NMR measurement confirmed that the pyridyl had been deprotected and a thiol group had been introduced.

[0090] (Synthesis of R848-dextran-PLGA) To a solution of SH-dextran-PLGA (40 mg) in dimethyl sulfoxide (1 ml), maleimide-containing R848 (Mal-R848; a resiquimod derivative synthesized by adding an amino group to the tertiary hydroxyl group of resiquimod via two carbon atoms and then coupling with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), synthesized by NARD Laboratory Co., Ltd.) (0.008 mmol) was added and reacted at room temperature for 118 hours. To remove unreacted Mal-R848, the mixture was purified by dialysis with dimethyl sulfoxide and water as the external solution, followed by centrifugation (9000 rpm, 30 minutes, 4°C) and lyophilization to recover the modified amphiphilic polymer (1-2) in powder form.

[0091] In addition, the dextran-NH ２ Dextran-NH having a number average molecular weight of 1,800 was synthesized according to the synthesis method of ２ was synthesized, and dextran-NH ２ (number average molecular weight 1,800) and PLGA-5020 (number average molecular weight 8,900) were used to obtain amphiphilic polymer (2-1), which was then synthesized in the same manner as for modified amphiphilic polymer (1-2), to obtain modified amphiphilic polymer (2-2). The evaluation results for each polymer are shown in Table 1.

[0092] The number-average molecular weights of dextran-PLGA and R848-dextran-PLGA were determined by GPC (column: TSK-gel α-5000 x 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan) (Table 1). The R848 introduction rate (%) of R848-dextran-PLGA was １ This was confirmed by H-NMR measurement (Table 1, Figure 3: R848-dextran-PLGA).

[0093] Example 2 Synthesis of R848-black yeast glucan-PLGA In this example, a linear block polymer was synthesized by adding poly(lactic acid-glycolic acid) (PLGA) to black yeast glucan and carrying out a condensation reaction. The resulting amphiphilic polymer, black yeast glucan-poly(lactic acid-glycolic acid) (black yeast glucan-PLGA), was covalently bound with R848 as an immunoactivator to synthesize R848-black yeast glucan-poly(lactic acid-glycolic acid) (R848-black yeast glucan-PLGA).

[0094] <Black yeast glucan having a primary amino group at the reducing end (Black yeast glucan-NH ２ 5 g of black yeast glucan (Daiso Co., Ltd.) was dissolved in 150 ml of dimethyl sulfoxide, and then 5 ml of 35% aqueous hydrochloric acid solution was added and stirred for 20 minutes at 105° C. The reaction solution was transferred to a dialysis membrane and dialyzed in water, followed by freeze-drying to obtain a black yeast glucan hydrolysate (number average molecular weight 2,400) as a powder.

[0095] Sodium triacetoxyborohydride (413.3 mg) and N-Boc-ethylenediamine (246.9 μl) were added to a 2.5 g / 8 ml dimethyl sulfoxide solution of black yeast glucan hydrolysate (number average molecular weight 2,400), and the reaction was carried out at 55°C for 169.5 hours with stirring. Unreacted N-Boc-ethylenediamine was then removed by dialysis against water as the external solution, yielding black yeast glucan-NHBoc. The Boc terminal group of black yeast glucan-NHBoc (1.19 g) was then deprotected (35% aqueous hydrochloric acid solution (14.3 ml) / dimethyl sulfoxide (24 ml), stirred at room temperature for 2 hours), purified by water dialysis, and freeze-dried to obtain a polymer powder (black yeast glucan-NHBoc). ２ ) was recovered.

[0096] Black yeast glucan-NH ２ The number average molecular weight of black yeast glucan-NH was determined by GPC measurement (column: TSK-gel α-5000 × 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan) (Table 1, Figure 4: Black yeast glucan-NH ２ ). １H-NMR measurement confirmed that a primary amino group had been introduced into the reducing end of the black yeast glucan.

[0097] <Synthesis of Black Yeast Glucan-PLGA> Poly(lactic acid-glycolic acid) having heterobifunctional terminal groups (COOH-PLGA-OH, Fujifilm Wako Pure Chemical Industries, Ltd., PLGA-5020, number average molecular weight 8,900) (4.462 g) was mixed with a solution of EDC (479.25 mg) and NHS (287.73 mg) in dimethyl sulfoxide (2.5 ml), and the mixture was allowed to react at 50°C for approximately 18 hours to convert the carboxyl group at the α-terminus to an active ester (NHS). Black yeast glucan having a primary amino group at the ω-terminus (Black Yeast Glucan-NH ２ , number average molecular weight 1,500) (749 mg) was added, and the active ester of NHS-PLGA-OH (NHS) and black yeast glucan-NH ２ After the reaction, unreacted black yeast glucan-NH was removed by water dialysis. ２ After removing the unreacted NHS-PLGA-OH, the unmodified amphiphilic polymer (3-1) was obtained by ultracentrifugation purification.

[0098] The number-average molecular weight of black yeast glucan-PLGA was determined by GPC measurement (column: TSK-gel α-5000 x 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan) (Table 1, Figure 5: black yeast glucan-PLGA). １ H-NMR measurement confirmed that the condensation reaction had progressed.

[0099] <Modification Reaction of Black Yeast Glucan-PLGA with R848> (Synthesis of CASSPy-Black Yeast Glucan-PLGA) A solution of CDI (26 mg) in dimethyl sulfoxide (0.1 ml) was added dropwise to a solution of black yeast glucan-PLGA (polymer (3-1), 200 mg) in dimethyl sulfoxide (0.5 ml), and the mixture was stirred at room temperature for 22 hours. Next, a solution of a heterocrosslinker (cysteamine SS pyridyl; CASSPy) (29.1 mg) prepared by reacting 2,2'-dipyridyl disulfide with cysteamine hydrochloride in dimethyl sulfoxide (0.15 ml) was added dropwise, and the mixture was stirred at room temperature for 65.5 hours. The reaction solution was placed in a dialysis membrane and dialyzed against dimethyl sulfoxide and then water. The contents were centrifuged (9,000 rpm, 4°C, 30 minutes) to remove the supernatant, and the mixture was lyophilized to obtain a polymer powder.

[0100] The number average molecular weight of CASSPy-black yeast glucan-PLGA was determined by GPC measurement (column: TSK-gel α-5000 × 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan). The CASSPy introduction rate (%) was １ This was confirmed by H-NMR measurement.

[0101] (Synthesis of SH-black yeast glucan-PLGA) A solution of dithiothreitol (DTT, 40 mg) in dimethyl sulfoxide (0.1 ml) was added to a solution of CASSPy-black yeast glucan-PLGA (150 mg) in dimethyl sulfoxide (1 ml), which serves as a reagent for reducing disulfide groups (SS bonds) to SH groups, and the mixture was allowed to react at room temperature for 4 hours. Subsequently, water dialysis was performed as a purification method to remove excess DTT, followed by centrifugation (9000 rpm, 30 minutes, 4°C) and lyophilization to recover the polymer powder. １ H-NMR measurement confirmed that the pyridyl had been deprotected and a thiol group had been introduced.

[0102] (Synthesis of R848-Yeast Glucan-PLGA) To a solution of SH-Yeast Glucan-PLGA (109.5 mg) in dimethyl sulfoxide (0.75 ml), R848 (Mal-R848, synthesized by NARD Laboratory Co., Ltd.) having a maleimide group (0.024 mmol) was added, and the mixture was allowed to react at room temperature for approximately 69 hours. Next, to remove unreacted Mal-R848, the mixture was purified by dialysis using dimethyl sulfoxide and water as the external solution, followed by centrifugation (9000 rpm, 30 minutes, 4°C) and lyophilization to recover the modified amphiphilic polymer (3-2) in powder form.

[0103] The number-average molecular weight of R848-black yeast glucan-PLGA was determined by GPC measurement (column: TSK-gel α-5000 x 2 manufactured by Tosoh Corporation, DMF solvent, detector: RI, standard: pullulan) (Table 1). The R848 introduction rate (%) of R848-black yeast glucan-PLGA was １ This was confirmed by H-NMR measurement (Table 1, Figure 6: R848-Yeast Glucan-PLGA).

[0104] Modified amphiphilic polymers with different amounts of R848 per amphiphilic polymer molecule were synthesized by quantitatively controlling the amount of CASSPy introduced into the hydroxyl groups of the polysaccharide backbone using a similar method. For example, while the amount of R848 introduced into one molecule of modified amphiphilic polymer (3-2) was 1.5 molecules, modified amphiphilic polymer (3-4) was obtained by controlling the amount of CASSPy introduced to be high using the same amphiphilic polymer (3-1), and modified amphiphilic polymer (3-3) was obtained by controlling the amount of CASSPy introduced to be low.

[0105] In addition, the black yeast glucan-NH ２ Black yeast glucan-NH having a number average molecular weight of 1,200 and 2,300 was synthesized according to the synthesis method of ２ , and curdlan-NH having a number average molecular weight of 3,500 ２ was synthesized, and black yeast glucan-NH was synthesized according to the same synthesis method as for the amphiphilic polymer (3-1) and the modified amphiphilic polymer (3-2). ２(number average molecular weight 1,200) and PLGA-5020 (number average molecular weight 8,900) were used to synthesize amphiphilic polymer (4-1), modified amphiphilic polymer (4-2), black yeast glucan-NH ２ (number average molecular weight 2,300) and PLGA-5020 (number average molecular weight 8,900) were used to prepare amphiphilic polymer (5-1), curdlan-NH ２ Amphiphilic polymer (6-1) and modified amphiphilic polymer (6-2) were obtained using PLGA-5020 (number average molecular weight 3,500) and PLGA-5020 (number average molecular weight 8,900). The evaluation results of each polymer are shown in Table 1.

[0106]

[0107] Example 3 Preparation of Particles (OVA-Containing R848-Modified Dextran-PLGA Particles (1), OVA-Containing R848-Modified Black Yeast Glucan-PLGA Particles (2) to (9), OVA-Containing R848-Modified Curdlan-PLGA Particles (10), Comparative Particles: OVA-Containing Dextran-PLGA Particles (11), OVA-Containing Black Yeast Glucan-PLGA Particles (12)) Using the S / O / W Emulsion Method <Preparation of OVA-Containing R848-Modified Dextran-PLGA Particles> A polymer mixed powder (30 mg) of R848-dextran-PLGA (Table 1, amphiphilic polymer (1-2)) (6 mg) and dextran-PLGA (Table 1, amphiphilic polymer (1-1)) (24 mg) was dissolved in 1.2 ml of dimethyl carbonate and 133 μl of tert-butanol to prepare a polymer solution. To the polymer solution, 0.3 ml of a 0.5% (w / v) aqueous solution of OVA (egg white albumin, Sigma) was added dropwise, and the mixture was stirred for 1 minute at 11,000 rpm using a mixer (Polytron, PT2100S) to produce a W / O emulsion. The W / O emulsion was pre-frozen with liquid nitrogen and then freeze-dried for 4 hours using a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1000) at a trap cooling temperature of -45°C and a vacuum of 20 Pa. The resulting solid was dispersed in 3 ml of ethyl acetate to prepare an S / O suspension. The S / O suspension was added dropwise to 12 ml of a 1% (w / v) aqueous solution of polyvinyl alcohol, and the mixture was stirred for 3 minutes at 5,000 rpm using a mixer (Silverson, L5M-A) to prepare an S / O / W emulsion. The ethyl acetate was removed from the S / O / W emulsion solution by submerged drying to obtain a particle suspension. The suspension was transferred to a 50 ml tube and centrifuged at 3,000 g for 30 minutes to precipitate the particles. After removing the supernatant, the particles were resuspended in 50 ml of distilled water and washed by centrifuging under the same conditions to precipitate the particles again. This washing procedure was repeated once more, and after removing the supernatant, the particles were suspended in 2.4 ml of an aqueous solution containing 5% (w / v) mannitol and 0.1% (w / v) polysorbate 80.The suspension was filtered through a mesh filter (1 μm), pre-frozen with liquid nitrogen, and then freeze-dried using a freeze dryer at a trap cooling temperature of −45° C. and a vacuum of 20 Pa for 12 hours to obtain OVA-containing R848-modified dextran-PLGA particles (1).

[0108] <Preparation of OVA-containing R848-modified black yeast glucan-PLGA particles> A polymer mixed powder (50 mg) of R848-black yeast glucan-PLGA (Table 1, modified amphiphilic polymer (3-2)) (10 mg) and black yeast glucan-PLGA (Table 1, unmodified amphiphilic polymer (3-1)) (40 mg) was dissolved in a mixed solution of 0.9 ml of dimethyl carbonate and 100 μl of tert-butanol to prepare a polymer solution. 0.5 ml of a 0.5% (w / v) aqueous solution of OVA (ovalbumin, Sigma) was added dropwise to the polymer solution, and a W / O emulsion solution was produced by stirring for 1 minute at 11,000 rpm using a mixer (Polytron, PT2100S). The W / O emulsion solution was pre-frozen with liquid nitrogen and then freeze-dried for 12 hours using a freeze dryer (Tokyo Rikakikai Co., Ltd., FD-1000) at a trap cooling temperature of -45°C and a vacuum of 20 Pa. The resulting solid was dispersed in 5 ml of ethyl acetate to prepare an S / O suspension solution. The S / O suspension solution was added dropwise to 20 ml of 1% (w / v) aqueous polyvinyl alcohol solution and stirred for 5 minutes at 6,000 rpm using a mixer (Silverson, L5M-A) to prepare an S / O / W emulsion solution. The ethyl acetate was removed from the S / O / W emulsion solution by submerged drying to yield a particle suspension. The suspension was transferred to a 50 ml tube and centrifuged at 8,000 rpm for 10 minutes to precipitate the particles. After removing the supernatant, the particles were resuspended in 25 ml of distilled water and washed by centrifuging under the same conditions as above to precipitate the particles again. This washing procedure was repeated once more, and after removing the supernatant, the particles were suspended in 4 ml of an aqueous solution containing 5% (w / v) mannitol and 0.1% (w / v) polysorbate 80. The suspension was pre-frozen in liquid nitrogen and then freeze-dried for 12 hours using a freeze dryer at a trap cooling temperature of −45°C and a vacuum of 20 Pa to obtain OVA-containing R848-modified black yeast glucan-PLGA particles (2). Using a similar method, OVA-containing R848-modified black yeast glucan-PLGA particles (3) to (9) were obtained using R848-black yeast glucan-PLGA and black yeast glucan-PLGA (Table 1, amphiphilic polymers (3-1) to (3-4), (4-1), and (4-2)) as base materials at various ratios.

[0109] <Preparation of OVA-containing R848-modified curdlan-PLGA particles> OVA-containing R848-modified curdlan-PLGA particles (10) were obtained in the same manner as above using a polymer mixed powder of R848-curdlan-PLGA (Table 1, modified amphiphilic polymer (6-2)) and curdlan-PLGA (Table 1, unmodified amphiphilic polymer (6-1)).

[0110] <Preparation of Comparative Particles: OVA-Loaded Dextran-PLGA Particles, OVA-Loaded Black Yeast Glucan-PLGA Particles> OVA-loaded dextran-PLGA particles (11) were obtained in the same manner as above using a powder of dextran-PLGA (Table 1, unmodified amphiphilic polymer (1-1)) alone.

[0111] OVA-containing black yeast glucan-PLGA particles (12) were obtained in the same manner as above using a powder of black yeast glucan-PLGA (Table 1, unmodified amphiphilic polymer (3-1)) alone.

[0112] The evaluation results for each particle are shown in Table 2. The average particle size was calculated by the cumulant method using a dynamic light scattering device (ELS-Z, Otsuka Electronics Co., Ltd.) (Table 2, Figure 7: OVA-containing R848-modified dextran-PLGA particles, Figure 8: OVA-containing R848-modified black yeast glucan-PLGA particles). The OVA antigen content (w / w) was determined by extracting the antigen from the particles using an organic solvent, subjecting the extracted antigen to gel electrophoresis using a gel electrophoresis device (TEFCO), and then staining with a colloidal CBB staining kit (TEFCO).

[0113]

[0114] Example 4 Preparation of Particles (TRP2-Containing R848-Modified Black Yeast Glucan-PLGA Particles (13) to (16), Comparative Particle: TRP2-Containing Black Yeast Glucan-PLGA Particle (17)) Using the S / O / W Emulsion Method The following particles were prepared using an epitope peptide of tyrosinase-related protein 2 (TRP2), which is known as a cancer antigen, particularly a melanoma antigen. Specifically, the epitope peptide common to the amino acid sequence of human-derived TRP2 (UniProt Accession No.: P40126) and mouse-derived TRP2 (UniProt Accession No.: P29812) was derived from polypeptides corresponding to the regions of SEQ ID NOs: 180 to 188 of these amino acid sequences.

[0115] Preparation of TRP2-Containing R848-Modified Black Yeast Glucan-PLGA Particles: 100 mg of a polymer powder mixture consisting of R848-Black Yeast Glucan-PLGA (Table 1, modified amphiphilic polymer (4-2)) and Black Yeast Glucan-PLGA (Table 1, unmodified amphiphilic polymer (4-1)) was added with 4 ml of dimethyl carbonate (containing 20% ​​t-butanol and 0.05% Span 80) and dissolved in a small mixer (11,000 rpm, 1 minute). To the resulting polymer solution, 1 ml of an aqueous acetonitrile solution (75%, 0.1% trifluoroacetic acid (TFA); TRP2 peptide: 1 mg / ml) containing the TRP2 epitope peptide (Greiner) was added, followed by 1 ml of distilled water. The mixture was emulsified in a small mixer (19,000 rpm, 1 minute). The emulsion was freeze-dried, and 10 ml of ethyl acetate was added to the resulting powder. The mixture was dispersed by stirring with a stirrer (300 rpm, 1 minute). 40 ml of a polyvinyl alcohol (PVA) aqueous solution (Nippon Synthetic Chemical Industry Co., Ltd., 1% EG-05P) was then added, and the mixture was roughly emulsified by stirring with a stirrer (500 rpm, 1 minute). This solution was further stirred with a mixer (5,000 rpm, 10 minutes) to emulsify. The mixture was then degassed by stirring with a stirrer (100 rpm, 10 minutes), and the organic solvent was removed using an evaporator. The remaining solution was filtered through a cell strainer (40 μm), ice-cooled for 30 minutes, and then centrifuged (3,000 g, 4°C, 30 minutes) to remove the supernatant. 50 ml of a 0.1% Tween 80 aqueous solution was then added, dispersed using a vortex mixer, and centrifuged (4,000 g, 4°C, 30 minutes) to remove the supernatant. Eight ml of a mannitol solution (5% mannitol, 0.1% PVA aqueous solution) was added as an excipient to the mixture, which was dispersed by vortexing. The mixture was then filtered under reduced pressure using a 1 μm mesh filter and lyophilized overnight to obtain TRP2-containing R848-modified black yeast glucan-PLGA particles (14). TRP2-containing R848-modified black yeast glucan-PLGA particles (13), (15), and (16) were obtained in a similar manner, except that R848-modified black yeast glucan-PLGA (Table 1, amphiphilic polymer 4-2) and black yeast glucan-PLGA (Table 1, amphiphilic polymer 4-1) were used in different weight ratios.

[0116] <Preparation of Comparative Particles: TRP2-Containing Black Yeast Glucan-PLGA Particles> Using a powder of black yeast glucan-PLGA (Table 1, unmodified amphiphilic polymer (5-1)) alone, TRP2-containing black yeast glucan-PLGA particles (17) were obtained in the same manner as above.

[0117] The evaluation results for each particle are shown in Table 3. The average particle size was calculated by the cumulant method using a dynamic light scattering device (Otsuka Electronics Co., Ltd., ELS-Z) (Table 3, Figure 9: TRP2-containing R848-modified black yeast glucan-PLGA particles).

[0118] The TRP2 peptide (antigen) content (w / w) was evaluated by HPLC. 1 ml of acetone was added to 5 mg of particle powder, and the mixture was dispersed by vortexing for 30 seconds and ultrasonication for 5 minutes. After centrifugation (13,000 rpm, 4°C, 5 minutes), the supernatant was removed. 1 ml of acetone was added to the precipitate, and the mixture was dispersed by vortexing for 30 seconds. After centrifugation (13,000 rpm, 4°C, 5 minutes), the supernatant was removed (twice in total). The mixture was dried in a centrifugal evaporator at 30°C for 30 minutes. 200 μl of 50% acetonitrile aqueous solution (containing 0.1% TFA) was added to the dried extract, and the mixture was dissolved by vortexing for 3 seconds and ultrasonication for 5 minutes. To this solution, 20 μl of acetylated tryptophan (10 μg / ml in 1% dimethyl sulfoxide, 0.1% TFA aqueous solution) was added as an internal standard, and the mixture was centrifuged (13,000 rpm, 4°C, 5 minutes) to recover the supernatant. The supernatant was injected into an HPLC (Shimadzu Corporation, SCL-10A, column: YMC-pack ODS-AM [AM12S05-1506WT]), and gradient elution was performed by gradually increasing the acetonitrile concentration in the eluent. Detection was performed by UV-visible absorption (220 nm), and the ratio of the peptide peak area to the internal standard peak area was calculated. The TRP2 peptide antigen content was determined using the results of a sample containing TRP2 peptide and mannitol powder treated in the same manner as above as the standard for 100% content.

[0119]

[0120] Reference Example 1: Induction of Murine Bone Marrow-Derived Dendritic Cells Female C57BL / 6 mice (Japan SLC, Inc.) were euthanized by cervical dislocation, and the femurs were harvested. Both ends of the femur were cut with scissors, and RPMI 1640 medium (hereinafter referred to as RPMI medium) containing 10% fetal bovine serum (FBS, Sigma), 100 units / ml penicillin, and 100 μg / ml streptomycin (Nacalai Tesque) was injected into the femur using a syringe to recover bone marrow cells. The cells were precipitated by centrifugation at 410 g for 5 minutes, and the supernatant was removed. The recovered cells were suspended in 1 ml of hemolysis buffer (BD Bioscience) and allowed to stand at room temperature for 5 minutes for hemolysis. After hemolysis, 10 ml of RPMI medium was added to the cell suspension, and the cells were precipitated by centrifugation at 1,500 rpm for 5 minutes, and the supernatant was removed. The cells were suspended in RPMI medium containing GM-CSF (hereinafter referred to as culture medium) and then seeded onto a 6-well plate (Corning, Flat Bottom Tissue Culture Treated, Polystyrene). The seeded plate was incubated at 5% CO ２ , 37°C, and 100% humidity. ２ The cells were cultured in an incubator (PHC). The culture medium was changed on days 2 and 5 of culture, and on day 6 of culture, the floating cells were aspirated and then strongly suspended using a micropipette to recover only cells that were lightly adherent to the plate, i.e., induced dendritic cells. The recovered dendritic cells were suspended in "CELLBANKER2" (Nippon Zenyaku Kogyo Co., Ltd.) and stored at -150°C until use.

[0121] Example 5 In vitro stimulation test using mouse bone marrow-derived dendritic cells (BMDC) (TRP2-containing R848-modified black yeast glucan-PLGA particles (14)) <Method> The dendritic cells obtained in Reference Example 1 were mixed with a culture medium at a concentration of 1 x 10 per well. ５ The TRP2-containing R848-modified black yeast glucan-PLGA particles (14) obtained in Example 4 were added to the plate at a concentration of 0.1 mg / ml. ２The cells were cultured in an incubator for 18 hours, and the supernatant was collected. The amount of TNF-α in the supernatant was measured using the "Mouse TNF-α ELISA developing kit" (MABTECH) according to the attached protocol. For comparison, 0.1 μg / ml LPS (positive control), antigen (TRP2 peptide), 0.1 mg / ml TRP2-containing black yeast glucan-PLGA particles (17), and a mixture of 0.1 mg / ml TRP2-containing black yeast glucan-PLGA particles (17) and R848 were used. The antigen and R848 in the comparative example were added in amounts equivalent to those in the 0.1 mg / ml TRP2-containing R848-modified black yeast glucan-PLGA particles (14).

[0122] <Results> The amount of TNF-α in the supernatant is shown in Figure 10. When TRP2-containing R848-modified black yeast glucan-PLGA particles (14) were used, the amount of TNF-α produced was higher than when TRP2-containing black yeast glucan-PLGA particles (17) and a mixture of TRP2-containing black yeast glucan-PLGA particles (17) and R848 were used. This demonstrates that binding R848 to TRP2-containing black yeast glucan-PLGA particles more potently activates dendritic cells.

[0123] Example 6 In vitro stimulation test 2 using mouse bone marrow-derived dendritic cells (BMDC) (OVA-containing R848-modified dextran-PLGA particles (1), OVA-containing R848-modified black yeast glucan-PLGA particles (2)) <Method> The dendritic cells obtained in Reference Example 1 were cultured together with a culture medium at 2 × 10 per well. ５ The cells were seeded onto a 48-well plate so that each well contained 0.1 mg / ml of OVA-containing R848-modified black yeast glucan-PLGA particles (2) and OVA-containing R848-modified dextran-PLGA particles (1) obtained in Example 3. A comparative group was also prepared to receive only OVA in the same amount as the particles. The cells were then incubated in CO ２After culturing for 18 hours in an incubator, the supernatant was removed. Cells were then detached by adding 0.5 mM EDTA and treating for 5 minutes. The cells were then collected using a micropipette. The collected cell suspension was replaced with a phosphate buffer solution containing 1% FBS, and an APC-labeled anti-CD86 antibody, a FITC-labeled anti-CD11c antibody, and an APC-Cy7-labeled anti-MHCII antibody (all from BD Bioscience) were added. The antibody labeling reaction was carried out by incubating the cells at 4°C for 30 minutes. After completion of the antibody labeling reaction, the expression level of the activation marker (CD86) on dendritic cells (CD11c-positive, MHCII-positive cells) was evaluated by flow cytometry using the mean fluorescence intensity (MFI). As a comparative example, OVA alone (negative control) was added at the same concentration, and the expression level of the activation marker was similarly compared.

[0124] <Results> Analysis of MFI, an index of CD86 expression level, showed that OVA-containing R848-modified black yeast glucan-PLGA particles (2) showed 2.39-fold higher expression intensity than the OVA-only group. Furthermore, OVA-containing R848-modified dextran-PLGA particles (1) showed 3.11-fold higher fluorescence intensity than the OVA-only group. This demonstrates that, like black yeast glucan-PLGA particles, dextran-PLGA particles also have significantly higher dendritic cell activation ability due to R848 binding.

[0125] (Example 7) In vitro stimulation test 3 using mouse bone marrow-derived dendritic cells (BMDCs) (OVA-containing R848-modified black yeast glucan-PLGA particles (5), (7), (8), (9)) <Method> The OVA-containing R848-modified black yeast glucan-PLGA particles (5), (7), (8), and (9) obtained in Example 3 were evaluated using the dendritic cells obtained in Reference Example 1 in the same manner as in Example 6. The OVA-containing R848-modified black yeast glucan-PLGA particles (5), (7), (8), and (9) were added to a concentration of 0.1 mg / ml, and as a comparative example, OVA alone (negative control) was added to the same concentration.

[0126] <Results> Analysis of MFI, an index of CD86 expression level, was performed in the same manner as in Example 6. The OVA-containing R848-modified black yeast glucan-PLGA particles (5), (7), (8), and (9) exhibited fluorescence intensities that were 4.50-fold, 5.77-fold, 5.55-fold, and 5.35-fold higher, respectively, than the comparative OVA-only group. In other words, it was revealed that the particles had similarly high dendritic cell activation ability regardless of the ratio of the modified amphiphilic polymer weight to the total weight of the modified amphiphilic polymer and the unmodified amphiphilic polymer, regardless of whether the ratio was 10% to 100%.

[0127] The immunogenicity enhancing composition of the present invention can be used as a pharmaceutical, particularly as a vaccine for treating and / or preventing infectious diseases, cancer, etc.

[0128] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A composition for enhancing immunogenicity, comprising a modified amphiphilic polymer and an antigen, wherein the hydrophobic segment is a poly(hydroxy acid) and the hydrophilic segment is a polysaccharide, and the amphiphilic polymer is bound with an immunostimulatory factor.

2. The composition for enhancing immunogenicity according to claim 1, which does not contain a lipid as a component other than the antigen constituting the particle.

3. The composition for enhancing immunogenicity according to claim 1, further comprising an unmodified amphiphilic polymer, wherein the particle is an amphiphilic polymer in which the hydrophobic segment is a poly(hydroxy acid) and the hydrophilic segment is a polysaccharide, and the immunostimulatory factor is not bound.

4. The composition for enhancing immunogenicity according to claim 1, wherein the polysaccharide is dextran, β-glucan, mannan, chitin, chitosan, gellan gum, alginic acid, hyaluronic acid or pullulan.

5. The composition for enhancing immunogenicity according to claim 4, wherein the β-glucan is a polymer of glucose linked by one or more β-1,3 linkages and / or one or more β-1,6 linkages.

6. The composition for enhancing immunogenicity according to claim 4 or 5, wherein the β-glucan is Aureobasidium pullulans glucan, curdlan, pachyman, laminaran, lichenan, schizophyllan, lentinan, scleroglucan or pachymaran.

7. The composition for enhancing immunogenicity according to claim 1, wherein the poly(hydroxy acid) is poly(lactic-co-glycolic acid), polylactic acid or polyglycolic acid.

8. The composition for enhancing immunogenicity according to claim 1, wherein the binding between the amphiphilic polymer and the immunostimulatory factor in the modified amphiphilic polymer is a covalent bond.

9. The composition for enhancing immunogenicity according to claim 1, wherein the binding site of the immunostimulatory factor in the modified amphiphilic polymer is the hydrophilic segment.

10. The composition for enhancing immunogenicity according to claim 1, wherein the immunostimulatory factor is a ligand or agonist that binds to a Toll-like receptor (TLR), NOD-like receptor (NLR), RIG-like receptor or C-type lectin receptor (CLR) or stimulator of interferon genes (STING).

11. The composition for enhancing immunogenicity according to claim 10, wherein the ligand or agonist that binds to the Toll-like receptor (TLR) is a ligand or agonist that binds to TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9 or TLR11.

12. The composition for enhancing immunogenicity according to claim 10 or 11, wherein the ligand or agonist that binds to the Toll-like receptor (TLR) is any one of the following (i) to (vii). (i) A ligand or agonist that binds to TLR2 selected from the group consisting of peptidoglycan, lipoprotein, lipopolysaccharide and zymosan (ii) A ligand or agonist that binds to TLR3 selected from the group consisting of Poly(I:C) and poly(A:U) (iii) A ligand or agonist that binds to TLR4 selected from the group consisting of lipopolysaccharide (LPS), HSP60, RS09 and MPLA (iv) Flagellin, which is a ligand or agonist that binds to TLR5 (v) A ligand or agonist that binds to TLR7 or 8 selected from the group consisting of imidazoquinoline compounds and single-stranded RNA (vi) A ligand or agonist that binds to TLR9 selected from the group consisting of bacterial DNA, unmethylated CpG DNA, hemozoin, ODN1585, ODN1668 and ODN1826 (vii) A ligand or agonist that binds to TLR11 selected from the group consisting of profilin and uropathogenic bacteria

13. The composition for enhancing immunogenicity according to claim 1, wherein the number of molecules of the immunostimulatory factor that binds to one molecule of the modified amphiphilic polymer is 1 to 100.

14. The composition for enhancing immunogenicity according to claim 1, wherein the average particle size of the particles is 0.1 to 50 μm.

15. A medicament containing the composition for enhancing immunogenicity according to claim 1 as an active ingredient.

16. A vaccine containing the composition for enhancing immunogenicity according to claim 1 as an active ingredient.

17. A vaccine for the treatment and / or prevention of cancer, containing the composition for enhancing immunogenicity according to claim 1 as an active ingredient.