Composition for inducing antigen-specific immune response that contains polyrotaxane and antigen

Abstract

Provided is a pharmaceutical composition for use in inducing an antigen-specific immune response, the pharmaceutical composition comprising: a polyrotaxane having a plurality of macroring molecules, an axis molecule that penetrates the rings of the macroring molecules, and caps that bind to the ends of the axis molecule, in which at least some of the macroring molecules include an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH; and an antigen.

Description

Composition for inducing antigen-specific immune response comprising polyrotaxane and antigen

[0001] The present disclosure relates to a composition for inducing an antigen-specific immune response, comprising a polyrotaxane and an antigen, a method for producing the composition, and a vaccine composition for preventing or treating a target disease, comprising the composition.

[0002] The recent pandemic has reaffirmed the importance of developing vaccines against infectious diseases. Furthermore, the effectiveness of antibody drugs, such as Opdivo, which target immune checkpoint proteins, has recently attracted attention in cancer immunotherapy. One example is a method that activates cytotoxic T cells (CTLs) that specifically recognize cancer antigens presented on the surface of cancer cells and directly attack cancer cells. In vaccines and cancer immunotherapy, the use of antigens or molecules that produce these antigens, such as proteins and their partial peptides derived from infectious disease-causing viruses, pathogenic bacteria, or cancers, as well as DNA or mRNA encoding these proteins, has been explored to activate antibody-producing B cells and CTLs that attack cancer cells. However, activating B cells and CTLs requires multiple processes in vivo, including efficient antigen delivery to antigen-presenting cells, antigen uptake, antigen processing, and antigen presentation on MHC molecules. Furthermore, highly purified antigens have reduced immunogenicity, necessitating the use of adjuvants; aluminum adjuvants, such as aluminum hydroxide, are often used in human applications. However, although aluminum adjuvants induce inflammatory responses, they are not suitable for activating dendritic cells. Therefore, the use of TLR ligands such as PolyI:C is being considered for anti-tumor immunity, but these have side effects.

[0003] The present inventors have previously developed a polyrotaxane (PRX)-based intracellular delivery carrier that recognizes and transforms the shape and charge distribution of genome editing molecules, nucleic acids, proteins, etc., allowing for highly efficient interaction with these molecules. This carrier exhibits higher intracellular delivery efficiency and safety than Lipofectamine CRISPRMAX, the most commonly used carrier among commercially available Cas9RNP delivery reagents (Non-Patent Document 1). However, while this intracellular delivery carrier can deliver nucleic acids and proteins into cells, it remains unclear whether it can actually activate antigen-specific B cells and T cells in vivo through processes such as decomposition of the delivered antigen and antigen presentation.

[0004] Applied Materials Today, Toru Taharabaru et al., 27 (2022) 101488

[0005] Therefore, an object of the present disclosure is to provide a pharmaceutical composition for use in inducing an antigen-specific immune response, a method for producing said pharmaceutical composition, and a vaccine composition for preventing or treating a target disease, comprising said pharmaceutical composition.

[0006] To achieve the above-mentioned object, the present disclosure provides a pharmaceutical composition for use in inducing an antigen-specific immune response (pharmaceutical composition 1 of the present disclosure). Pharmaceutical composition 1 of the present disclosure comprises: a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has one proton at neutral pH and two protons at acidic pH; and an antigen.

[0007] The present disclosure also provides a pharmaceutical composition (pharmaceutical composition 2 of the present disclosure) for use in inducing an antigen-specific immune response. Pharmaceutical composition 2 of the present disclosure comprises: a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group having an amino group via an intracellularly degradable bond; and an antigen.

[0008] The present disclosure also provides a method for producing a pharmaceutical composition for use in inducing an antigen-specific immune response (method for producing the pharmaceutical composition of the present disclosure 1). The method for producing the pharmaceutical composition of the present disclosure 1 comprises mixing a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has one proton at neutral pH and two protons at acidic pH, with an antigen to form a polyion complex.

[0009] The present disclosure also provides a method for producing a pharmaceutical composition for use in inducing an antigen-specific immune response (method for producing a pharmaceutical composition of the present disclosure, 2). The method for producing a pharmaceutical composition of the present disclosure, 2, comprises mixing a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond, with an antigen to form a polyion complex.

[0010] The present disclosure also provides a vaccine composition for preventing or treating a target disease (the vaccine composition of the present disclosure), comprising the pharmaceutical composition of the present disclosure.

[0011] According to the present disclosure, it is possible to provide a pharmaceutical composition for use in inducing an antigen-specific immune response, a method for producing the pharmaceutical composition, and a vaccine composition for preventing or treating a target disease, which includes the pharmaceutical composition.

[0012] Figure 1 shows the structure of DET-PRX (PEG: 20 kDa) in DO (heavy water). 1 Figure 2 shows the H-NMR spectrum of Cys / DET-PRX (PEG: 20 kDa) in DO (heavy water). 1Figure 3 shows the H-NMR spectrum. Figure 3 shows the results of agarose gel electrophoresis of a mixture of DET-PRX and OVA. N / C indicates the cation (amino group of DET-PRX) / anion (carboxy group of OVA) charge ratio. Figure 4 shows the results of measuring the particle size of the DET-PRX and OVA complex. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to OVA alone. N / C indicates the cation (amino group of DET-PRX) / anion (carboxy group of OVA) charge ratio. Figure 5 shows the results of measuring the zeta potential of the DET-PRX and OVA complex. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to OVA alone. N / C indicates the cation (amino group of DET-PRX) / anion (carboxy group of OVA) charge ratio. Figure 6 shows the results of measuring the zeta potential of the Cys / DET-PRX and OVA complex. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to OVA alone. †: p<0.05 compared to Cys / DET-PRX. ‡: p<0.05 compared to N / C=1. N / C indicates the cation (amino group of Cys / DET-PRX) / anion (carboxy group of OVA) charge ratio. Figure 7(A) shows the results of measuring the zeta potential of the DET-PRX and OVA complex. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to DET-PRX / OVA (pH=7.4). Figure 7(B) shows the results of measuring the zeta potential of the Cys / DET-PRX and OVA complex. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to Cys / DET-PRX / OVA (pH=7.4). Figure 8 shows a fluorescence measurement test of BMDCs treated with FITC-OVA complex. Each bar value represents the mean ± SE of six experiments. *: p<0.05 compared to FITC-OVA alone. Figure 9 (A) shows a measurement test of a cell surface marker (CD80) on BMDCs treated with OVA complexes. Figure 9 (B) shows a measurement test of a cell surface marker (CD86) on BMDCs treated with OVA complexes. Each bar value represents the mean ± SE of six experiments. *: p<0.05 compared to OVA alone. †: p<0.05 compared to DET-PRX / OVA.Figure 10(A) shows the results of measuring the cell surface marker (MHCII) of DCs derived from mice immunized with OVA complexes. Figure 10(B) shows the results of measuring the cell surface marker (MHCII) of DCs derived from mice immunized with OVA complexes. Each bar value represents the mean ± SE of 4 to 9 experiments. *: p<0.05 compared to OVA alone. †: p<0.05 compared to Alum / OVA. ‡: p<0.05 compared to DET-PRX / OVA. Figure 11 shows the results of measuring the anti-OVA antibody titers of mice immunized with OVA complexes. Each bar value represents the mean ± SE of 5 to 9 experiments. *: p<0.05 compared to OVA alone. †: p<0.05 compared to Alum / OVA. ‡: p<0.05 compared to DET-PRX / OVA. Figure 12 shows the results of measuring the levels of inflammatory cytokines secreted by mice immunized with OVA complexes. Each bar value represents the mean ± SE of 5 to 9 experiments. *: p<0.05 compared to PBS. †: p<0.05 compared to OVA alone. ‡: p<0.05 compared to Alum / OVA. §: p<0.05 compared to DET-PRX / OVA. Figure 13 shows the results of a safety evaluation study of mice immunized with an OVA complex. Each bar value represents the mean ± SE of nine experiments. Figure 14 shows the results of a safety evaluation study of mice immunized with an OVA complex. Each bar value represents the mean ± SE of nine experiments. Figure 15 shows the levels of tetramer-positive CD8+ T cells in PBMCs from SARS-CoV-2 patients pulsed with DET-PRX / NF9. Figure 16 shows the results of agarose gel electrophoresis of a mixture of DET-PRX and mCherry mRNA. N / P indicates the charge ratio of cations (amino groups of DET-PRX) to anions (phosphate groups of mRNA). Figure 17 shows the results of agarose gel electrophoresis of a mixture of Cys / DET-PRX and mCherry mRNA. N / P indicates the charge ratio of cations (amino groups of DET-PRX) to anions (phosphate groups of mRNA). Figure 18 shows the results of measuring the zeta potential of the complex of DET-PRX and mCherry mRNA. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to mRNA alone. †: p<0.05 compared to DET-PRX.‡: p<0.05 compared to N / P=1. N / P indicates the charge ratio of cations (amino groups of DET-PRX) to anions (phosphate groups of mRNA). Figure 19 shows the particle size measurement results for the complex of DET-PRX and mCherry mRNA. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to mRNA alone. †: p<0.05 compared to DET-PRX. ‡: p<0.05 compared to N / P=1. N / P indicates the charge ratio of cations (amino groups of DET-PRX) to anions (phosphate groups of mRNA). Figure 20 shows the fluorescence measurement test for HeLa cells treated with Cy5-GFP mRNA complexes. Each bar value represents the mean ± SE of three experiments. *: p<0.05 compared to Cy5-GFP mRNA alone. Figure 21 shows the immunization schedule for HLA-A24 transgenic mice with DET-PRX / SARS-CoV-2 spike protein. Figure 22 shows the results of measuring the amount of IFN-γ secreted by splenocytes derived from HLA-A24 transgenic mice immunized with DET-PRX / SARS-CoV-2 spike protein. *: p<0.05 compared to DET-PRX alone. NS: No statistically significant difference.

[0013] 1. Pharmaceutical Composition of the Present Disclosure <Pharmaceutical Composition 1 of the Present Disclosure> The present disclosure provides a pharmaceutical composition (pharmaceutical composition 1 of the present disclosure) for use in inducing an antigen-specific immune response. Pharmaceutical composition 1 of the present disclosure comprises: a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates the rings of the macrocyclic molecules, and a cap attached to the end of the axle molecule, wherein at least some of the macrocyclic molecules include an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH; and an antigen.

[0014] <Pharmaceutical Composition 2 of the Present Disclosure> The present disclosure also provides a pharmaceutical composition (pharmaceutical composition 2 of the present disclosure) for use in inducing an antigen-specific immune response. Pharmaceutical composition 2 of the present disclosure comprises: a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap bonded to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond; and an antigen.

[0015] (Polyrotaxane) Polyrotaxane consists of a plurality of macrocyclic molecules and an axon molecule that penetrates the rings of the macrocyclic molecules and has caps on both ends. Polypseudorotaxane has a plurality of macrocyclic molecules and an axon molecule that penetrates the rings of the macrocyclic molecules, but does not have a cap structure.

[0016] (Macrocyclic Molecule) A macrocyclic molecule refers to a cyclic molecule having an internal opening (cavity) large enough for an axial molecule to pass through and having a modifiable group (e.g., a hydroxyl group). Examples of macrocyclic molecules include cyclic polyethers, cyclic polyesters, cyclic polyetheramines, cyclic polyamines, crown ethers, cucurbit[n]urils, calixarenes, cyclic amides, transition metal complexes, cyclodextrins, cyclodextrin derivatives, and any combination thereof. The cyclodextrins are cyclic oligosaccharide compounds, including, for example, α-cyclodextrin (hexasaccharide), β-cyclodextrin (heptasaccharide), and γ-cyclodextrin (octasaccharide). The macrocyclic molecule may be substituted with a substituent other than the amine-containing group and intracellularly degradable amino group described below, such as a methyl group, a hydroxyethyl group, a hydroxypropyl group, an acetyl group, a carboxymethyl group, a succinyl group, a glucosyl group, a carboxyethyl group, a sulfobutyl group, an amino group, a halogen atom, or any combination thereof.

[0017] (Axial Molecule) The axial molecule is usually a polymer, such as a polymer of a single monomer (homopolymer) or a copolymer of two or more types of monomers. A polymer refers to a macromolecule formed by repeating many of one or more monomers. A monomer is usually a molecule having one carbon-carbon double bond or a molecule having at least two functional groups per molecule. A copolymer may be a random copolymer, an alternating copolymer, and / or a block copolymer. When the axial molecule is a homopolymer, examples of the polymer include polyalkylene oxide, polyethylene glycol (PEG), polypropylene glycol, polyvinyl ether, polymethyl vinyl ether, polyethyleneimine, poly(trimethylene oxide), poly(ε-caprolactone), polylactic acid, polyamino acid, poly(ε-lysine), polyamide, poly(iminooligomethylene), ionene, poly(vinyldiene chloride), polypropylene, oligopropylene, polyethylene, oligoethylene, poly(alkylenebenzimidazole), polyurethane, poly(viologen), poly(N-dimethyldecamethyleneammonium), poly(dimethylsiloxane), polyaniline, polycarbonate, poly(methyl methacrylate), poly(N-acylethyleneimine), poly(4-vinylpyridine)-dodecylbenzenesulfonic acid complex, fullerene-polyethylene glycol conjugate, and hydrophobized polysaccharide. When the axial molecule is a copolymer, examples thereof include a copolymer of polyethylene glycol and polypropylene glycol, a copolymer of poly(ε-caprolactone) and polylactic acid, a copolymer of polylactic acid and polyethylene glycol, a polyethylene glycol-polysaccharide graft copolymer, and a polypropylene glycol-polysaccharide graft copolymer. The axial molecule may also be a branched multi-arm polymer, such as a star-shaped polyethylene glycol, a hyperbranched polyether, a hyperbranched oligoethylene glycol, or a hyperbranched oligopropylene glycol. These polymers may be modified or substituted with a substituent, as necessary.The degree of polymerization and molecular weight of the axial molecule may be any length that allows a required amount of macrocyclic molecules to penetrate depending on the target antigen (nucleic acid or protein) to be introduced into cells. For example, polymers with number-average molecular weights (Mn) of about 200 to 1,000,000 Da, about 400 to 50,000 Da, about 500 to 40,000 Da, or about 1,000 to 30,000 Da can be used.

[0018] In polyrotaxanes and polypseudorotaxanes, the axial molecules penetrate the macrocyclic molecules by penetrating through the openings of the macrocyclic molecules. Polyrotaxanes and polypseudorotaxanes have multiple macrocyclic molecules sewn onto the axial molecules in this way. The number of macrocyclic molecules in one polyrotaxane molecule can be set depending on the purpose, but can be, for example, an average of about 5 to about 200, or about 10 to about 100. The macrocyclic molecules are not covalently bonded to the axial molecule, and therefore can move in both the rotational and axial directions relative to the axial molecule.

[0019] (CAP) A cap (CAP) is a bulky substituent attached to the end of a polyrotaxane to prevent the macrocyclic molecule from detaching from the axis molecule. Examples of the cap include dinitrophenyl groups, cyclodextrins, adamantane groups, trityl groups, fluoresceins, silsesquioxanes, pyrenes, substituted benzenes (which may be substituted with one or more alkyl, alkyloxy, hydroxy, halogen, cyano, sulfonyl, carboxyl, amino, and phenyl groups), steroids, amino acids, oligopeptides, oligosaccharides, sugar derivatives, groups having one or more benzene rings (benzyloxycarbonyl (Z) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyl ester (OBz) group), and groups having one or more tertiary butyl groups (tertiary butylcarbonyl (Boc) group, amino acid tert-butyl ester (OBu) group). Preferably, the cap is a dinitrophenyl group, a cyclodextrin group, an adamantane group, a trityl group, a fluorescein group, a silsesquioxane group, or a pyrene group, and more preferably an adamantane group or a cyclodextrin group. The cap is attached to the end of the polypseudorotaxane to prevent the macrocyclic molecule sewn by the axis molecule from detaching from the axis molecule. Therefore, the cap has sufficient steric bulk to block the macrocyclic molecule from escaping from the axis molecule.

[0020] Preferably, the cap is attached to the stalk molecule via an intracellularly degradable bond. An intracellularly degradable bond refers to a bond that is easily degraded under conditions in the intracellular environment that differ from those outside the cell, such as physicochemical conditions such as pH or biological conditions such as intracellular enzymes. Preferably, an intracellularly degradable bond is a bond that is not easily degraded outside the cell. Here, the terms "degradable" and "not easily degraded" do not have an absolute meaning and do not mean complete degradability or complete indegradability, respectively. "Degradable" and "not easily degraded" refer to relative ease of degradability and relative indegradability, respectively, when compared with the extracellular and intracellular conditions. For example, the condition in the intracellular environment that differs from the extracellular environment may be the GSH concentration. In this case, an intracellularly degradable bond may be a bond that is not easily degraded at a GSH concentration of 0.2 mM or less (extracellular concentration) but is degradable at a GSH concentration of 2 to 10 mM (intracellular concentration). Examples of intracellularly degradable bonds include carbamate bonds (-NH(C=O)O-), ketal bonds (-OC(CH3)2O-), amide bonds (-NHCO-), disulfide bonds (-SS-), acetal bonds (-C(OH)O-), orthoester bonds, vinyl ether bonds (-CH2=CH-O-), hydrazide bonds, and ester bonds (-COO-).

[0021] Preferably, the cap is bonded to the stalk molecule via a carbamate bond. Bonding of the cap to the stalk molecule via a bond that can be degraded in cells can be performed with reference to methods described in, for example, Journal of Controlled Release 76 (2001) 11-25; Biomacromolecules 2003, 4, 1426-1432; Langmuir 2011, 27 (2), 612-617; Angew. Chem. Int. Ed. 2013, 52, 7300-7305; J. Mater. Chem. B, 2013, 1, 3535-3544; Biomaterials, 34, 2480-2491 (2013); Scientific Reports, 3, 2252 (2013), etc.

[0022] (Amine-Containing Group) In the pharmaceutical composition 1 of the present disclosure, at least a portion of the macrocyclic molecules in the polyrotaxane contain an amine-containing group (hereinafter, sometimes simply referred to as an "amine-containing group") that has a monovalent proton at neutral pH and a divalent proton at acidic pH (preferably, a weakly acidic pH (e.g., pH 5.5)) (hereinafter, also referred to as an "amine-containing group-containing polyrotaxane"). The amine-containing group is safe for biological components because it has a monovalent proton at neutral pH, while it has a divalent proton at acidic pH (preferably, a weakly acidic pH (e.g., pH 5.5)), which is the intraendosomal environment, and thus can disrupt the endosomal membrane. This can promote the release of the pharmaceutical composition of the present disclosure, which has been taken up into an intracellular endosome, by disrupting the endosome and into the cytoplasm.

[0023] The amine-containing group preferably contains a secondary amine and an amino group, typically -L 1 -NH-L 2 -NH2 (where L 1 and L 2 are the same or different and are linear or branched C1-6 alkylene groups).

[0024] In the amine-containing group, examples of the straight-chain or branched C1-6 alkylene group include -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -CH(CH3)CH2-, -CH2CH(CH3)-, -CH(CH2CH3)CH2-, -CH2CH(CH2CH3)-, -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)- and the like, preferably -(CH2)2-.

[0025] More specifically, the amine-containing group includes -(CH2)2-NH-(CH2)2-NH2 (diethylenetriamine group; hereinafter, also simply referred to as "DET").

[0026] (Intracellularly Degradable Amino Group) In another embodiment, in a polyrotaxane, at least some of the macrocyclic molecules contain a group having an amino group via an intracellularly degradable bond (hereinafter, may be referred to as an "intracellularly degradable amino group") (hereinafter, also referred to as an "intracellularly degradable amino group-containing polyrotaxane"). After the intracellularly degradable amino group is released into the cytoplasm from the endosome, the intracellularly degradable bond is cleaved by a degrading enzyme, thereby weakening the bond between the antigen and the polyrotaxane and promoting dissociation of the antigen from the polyrotaxane.

[0027] Intracellularly degradable amino groups are typically -L 3 -XL 4 -NH2 (where L 3 and L 4 are the same or different and are a linear or branched C1-6 alkylene group or are absent, and X is an intracellularly degradable bond. Here, the "intracellularly degradable bond" is as defined above for the cap.

[0028] In the intracellularly degradable amino group, a group having an amino group (in the above formula, -L 4 Examples of the alkyl group corresponding to -NH2 include -CH2-NH2, -(CH2)2-NH2, -(CH2)3-NH2, -(CH2)4-NH2, -(CH2)5-NH2, -(CH2)6-NH2, and -CH(CH3)CH2-NH2, and preferably -(CH2)2-NH2.

[0029] A more specific example of an intracellularly degradable amino group is -(CH2)2-SS-(CH2)2-NH2 (cystamine).

[0030] (Amine-Containing Group / Intracellularly Degradable Amino Group) In pharmaceutical composition 2 of the present disclosure, at least a portion of the macrocyclic molecules in the polyrotaxane contains an amine-containing group and further contains an intracellularly degradable amino group (hereinafter also referred to as a "polyrotaxane containing an amine-containing group / intracellularly degradable amino group"). In this case, the amine-containing group and the intracellularly degradable amino group are typically different groups. In addition, in this case, the amine-containing group is preferably bound to the macrocyclic molecule via a bond that is not degraded or is difficult to degrade intracellularly. As a result, even if some of the bonds of the intracellularly degradable amino groups are degraded and the amino groups dissociate from the macrocyclic molecules before the pharmaceutical composition of the present disclosure is released into the cells, the amino groups of the amine-containing groups can maintain the bond between the polyrotaxane and the antigen. Furthermore, after release into the cytoplasm, most of the bonds of the intracellularly degradable amino groups are cleaved, thereby weakening the bond between the antigen and the polyrotaxane and promoting dissociation of the antigen from the polyrotaxane.

[0031] In a polyrotaxane, when at least some macrocyclic molecules have an amine-containing group and / or an intracellularly degradable amino group, the amine-containing group and / or the intracellularly degradable amino group may be chemically bonded via a functional group of the macrocyclic molecule. For example, when the macrocyclic molecule is a cyclodextrin, a hydroxyl group of the macrocyclic molecule may be substituted with an amine-containing group and / or an intracellularly degradable amino group (collectively referred to as "macrocyclic molecule substituents" in this paragraph). The hydroxyl group of the cyclodextrin may be a hydroxyl group in the glucose constituting the cyclodextrin. In this case, the substituent of the macrocyclic molecule may be bonded to the oxygen atom constituting the hydroxyl group via -O-CO-NH-, -O-COO-, -O-OC-, -O-, -OC(OH)-, or -OC(═S)NH- (in all cases, the leftmost -O- represents the oxygen atom derived from the hydroxyl group). For example, the macrocyclic molecule is α-cyclodextrin, and the oxygen atom constituting the hydroxyl group of the α-cyclodextrin is connected to -L via -O-CO-NH-. 1 -NH-L 2 When a substituent represented by -NH2 is bonded, it can be represented by the following structural formula.

[0032]

[0033] Polyrotaxanes can be produced by methods well known to those skilled in the art, such as those described in Production Examples 1 to 10 of paragraphs 0046 to 0067 of WO 2022 / 163729.

[0034] Polyrotaxanes do not induce fever in a subject upon administration.

[0035] (Polyrotaxane Composition) The pharmaceutical composition of the present disclosure may comprise, as the polyrotaxane, a polyrotaxane composition comprising a polyrotaxane comprising an amine-containing group and a polyrotaxane comprising an intracellularly degradable amino group, instead of a single polyrotaxane selected from the group consisting of a polyrotaxane comprising an amine-containing group, a polyrotaxane comprising an intracellularly degradable amino group, and a polyrotaxane comprising an amine-containing group / an intracellularly degradable amino group.

[0036] The molar ratio of the polyrotaxane having an amine-containing group to the polyrotaxane having an intracellularly degradable amino group in the polyrotaxane composition is not particularly limited, as long as the polyrotaxane having an amine-containing group that has been taken up into an intracellular endosome in the polyrotaxane composition disrupts the endosome and promotes release of the polyrotaxane composition into the cytoplasm, and the intracellularly degradable bond of the released polyrotaxane having an intracellularly degradable amino group in the polyrotaxane composition is cleaved by a degrading enzyme, thereby weakening the bond between the antigen and the polyrotaxane and promoting dissociation of the antigen from the polyrotaxane; for example, the molar ratio may be 3:1 to 1:3.

[0037] (Antigen) An antigen refers to a natural or artificial biological material that can induce an antigen-specific immune response as an active ingredient, and any antigen can be used.

[0038] In the pharmaceutical composition of the present disclosure, the polyrotaxane (or the polyrotaxane composition) and the antigen may be in any state, but preferably form a polyion complex. A polyion complex refers to a complex formed or something formed between polymers having multiple charges on their surfaces due to electrostatic interactions in an aqueous solution, buffer solution, or medium. In the pharmaceutical composition of the present disclosure, the molar ratio of the polyrotaxane (or the polyrotaxane composition) to the antigen is not particularly limited, but may be 1:0.25 to 0.25:1, and preferably 1:0.5 to 0.5:1. The polyion complex can be formed by mixing the polyrotaxane (or the polyrotaxane composition) and the antigen. Mixing can be performed by stirring in an aqueous solution, buffer solution, or medium at room temperature. The mixing time can be appropriately set depending on the polyrotaxane (or the polyrotaxane composition) and the antigen, as well as their concentrations, and the like, but is usually about 5 minutes to overnight, 5 minutes to 6 hours, 5 to 180 minutes, 5 to 120 minutes, 5 to 60 minutes, or 5 to 30 minutes.

[0039] The pharmaceutical composition of the present disclosure obtained as described above can induce a stronger antigen-specific immune response in a subject administered with the composition or in cells treated with the composition, compared to administration of or treatment with an antigen alone. Therefore, the pharmaceutical composition of the present disclosure can be used as a pharmaceutical composition for inducing an antigen-specific immune response.

[0040] Examples of the induction of an antigen-specific immune response include, but are not limited to, the induction of antigen-presenting cells that express costimulatory molecules, the induction of antigen-presenting cells that express major histocompatibility complex (MHC), the induction of the production of antibodies that specifically recognize the antigen, the induction of the production of inflammatory cytokines, and the induction of antigen-specific T cells (CD8-positive T cells, CD4-positive T cells).

[0041] Examples of the induction of antigen-presenting cells that express costimulatory molecules include the induction of dendritic cells that express CD80 or CD86. Dendritic cells treated with the pharmaceutical composition of the present disclosure have a significantly higher percentage of dendritic cells that express CD80 or CD86 than dendritic cells treated with an antigen alone. In particular, when the polyrotaxane contained in the pharmaceutical composition of the present disclosure is a polyrotaxane containing an amine-containing group / intracellularly degradable amino group, the percentage of dendritic cells that express CD80 or CD86 is significantly increased. The percentage of dendritic cells that express CD80 or CD86 can be measured according to the procedures described in the Examples below.

[0042] Examples of the induction of antigen-presenting cells expressing MHC include the induction of dendritic cells expressing MHC class I (MHC1) or MHC class II (MHCII). Subjects administered with the pharmaceutical composition of the present disclosure have a significantly higher proportion of dendritic cells expressing MHC1 or MHCII compared to subjects administered with an antigen alone. In particular, when the polyrotaxane contained in the pharmaceutical composition of the present disclosure is a polyrotaxane containing an amine-containing group, the proportion of dendritic cells expressing MHC1 is significantly increased. Furthermore, when the pharmaceutical composition of the present disclosure containing a polyrotaxane containing an amine-containing group is used to induce dendritic cells expressing MHC1, it is preferable that the pharmaceutical composition of the present disclosure does not contain an adjuvant. The proportion of dendritic cells expressing MHC1 or MHCII can be measured according to the procedures described in the Examples below.

[0043] Examples of the induction of production of antibodies that specifically recognize an antigen include the induction of production of IgG that specifically recognizes the antigen. Subjects administered with the pharmaceutical composition of the present disclosure produce significantly higher amounts of IgG than subjects administered with the antigen alone or together with an adjuvant. In particular, when the polyrotaxane contained in the pharmaceutical composition of the present disclosure is a polyrotaxane containing an amine-containing group, the amount of IgG produced is significantly increased. The amount of IgG produced can be measured according to the procedures described in the Examples below.

[0044] Examples of the induction of inflammatory cytokine production include the induction of IL-6 production. Subjects administered with the pharmaceutical composition of the present disclosure exhibit significantly higher IgG production than subjects administered with an antigen alone or together with an adjuvant. In particular, when the polyrotaxane contained in the pharmaceutical composition of the present disclosure is a polyrotaxane containing an amine-containing group, the IgG production is significantly increased. Furthermore, when the pharmaceutical composition of the present disclosure uses a polyrotaxane containing an amine-containing group, no adjuvant is required to induce IgG production. The IgG production can be measured according to the procedures described in the Examples below.

[0045] Examples of induction of antigen-specific T cells (CD8+ T cells, CD4+ T cells) include induction of T cells (CD8+ T cells) that specifically recognize cells presenting an antigen on MHC class I (MHC1) or MHC class II (MHCII) and exhibit cytotoxic activity, and T cells (CD4+ T cells) that activate CD8+ T cells and B cells. When PBMCs derived from a subject once exposed to an antigen are stimulated with the pharmaceutical composition of the present disclosure, the proportion of antigen-specific CD8+ T cells among the CD8+ T cells is significantly higher than when stimulated with the antigen alone. The proportion of antigen-specific CD8+ T cells among the CD8+ T cells can be measured according to the methods described in the Examples below.

[0046] Subjects to which the pharmaceutical compositions of the present disclosure may be administered include mammals (e.g., humans and non-human animals such as mice, rats, hamsters, rabbits, goats, cows, horses, dogs, cats, pigs, monkeys, dolphins, and sea lions, with humans being preferred).

[0047] Cells to be treated with the pharmaceutical compositions of the present disclosure include antigen-presenting cells (dendritic cells, macrophages, B cells, etc.).

[0048] The pharmaceutical composition of the present disclosure may be a pharmaceutical composition for oral or parenteral administration, such as an injection for intravenous injection, subcutaneous injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracerebral injection, or infusion. Alternatively, the pharmaceutical composition of the present disclosure may be a cell treatment agent for cell therapy. When administered to a subject such as a mammal (including model animals such as mice and humans), the pharmaceutical composition of the present disclosure may be administered orally, or an injection or infusion may be administered into the blood (into a vein or artery).

[0049] The pharmaceutical composition of the present disclosure may or may not contain a pharmacologically acceptable carrier (formulation additive). The type of formulation additive used in the preparation of the pharmaceutical composition of the present disclosure, the ratio of the formulation additive to the active ingredient, and the method of preparation of the pharmaceutical composition of the present disclosure can be appropriately selected by those skilled in the art depending on the form of the composition. Formulation additives can generally be incorporated at a concentration of 1% to 99% by weight of the active ingredient. Various organic or inorganic carrier substances commonly used as formulation materials are used as formulation additives, and are incorporated as solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid formulations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as needed. Suitable examples of solvents include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil, etc. Suitable examples of solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate. Suitable examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates, polyoxyethylene hydrogenated castor oil, and the like. Suitable examples of isotonic agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, and glucose. Suitable examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Suitable examples of soothing agents include benzyl alcohol.

[0050] Suitable examples of preservatives include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Suitable examples of antioxidants include sulfites, ascorbic acid, etc. Suitable examples of coloring agents include water-soluble food tar dyes (e.g., food dyes such as Food Red No. 2 and No. 3, Food Yellow No. 4 and No. 5, Food Blue No. 1 and No. 2, water-insoluble lake dyes (e.g., aluminum salts of the above-mentioned water-soluble food tar dyes), natural dyes (e.g., β-carotene, chlorophyll, red iron oxide, etc.), etc. Suitable examples of sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia, etc.

[0051] Dosage forms of the pharmaceutical composition of the present disclosure include oral preparations such as syrups, emulsions, and suspensions; and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intratumoral injections, intraperitoneal injections, etc.), infusions, drip infusions, and sustained-release preparations, each of which can be safely administered orally or parenterally. The pharmaceutical composition of the present disclosure can be manufactured by methods commonly used in the formulation technology field, such as the methods described in the Japanese Pharmacopoeia. Specific manufacturing methods for the formulations are described in detail below.

[0052] For example, injections are produced by dissolving, suspending, or emulsifying the antigen as an active ingredient in an aqueous solvent (e.g., distilled water, physiological saline, Ringer's solution, etc.) or an oily solvent (e.g., vegetable oils such as olive oil, sesame oil, cottonseed oil, and corn oil, propylene glycol, etc.) together with dispersing agents (e.g., polysorbate 80, polyoxyethylene hydrogenated castor oil 60, etc.), polyethylene glycol, carboxymethylcellulose, sodium alginate, etc.), preservatives (e.g., methylparaben, propylparaben, benzyl alcohol, chlorobutanol, phenol, etc.), isotonicity agents (e.g., sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, etc.). In this case, additives such as solubilizing agents (e.g., sodium salicylate, sodium acetate, etc.), stabilizers (e.g., human serum albumin, etc.), and soothing agents (e.g., benzyl alcohol, etc.) may be used, if desired.

[0053] The dosage and frequency of administration of the pharmaceutical composition of the present disclosure vary depending on the subject, route of administration, target disease, symptoms, etc., but those skilled in the art can appropriately select an appropriate dosage. For example, when administered into human blood, the daily dose can be 0.001 to 100 g, or 0.01 to 1000 mg / kg. The daily dose may also be divided and administered several times. The administration frequency can be daily, weekly, every two weeks, monthly, or once every several months. The administration period can be appropriately determined based on the improvement of symptoms, and can be one month, several months, six months, one year, several years, five years, or ten years.

[0054] 2. Method for Producing a Pharmaceutical Composition of the Present Disclosure <Method for Producing a Pharmaceutical Composition of the Present Disclosure 1> The present disclosure also provides a method for producing a pharmaceutical composition of the present disclosure (Method for Producing a Pharmaceutical Composition of the Present Disclosure 1). Method for Producing a Pharmaceutical Composition of the Present Disclosure 1 includes mixing a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating rings of the macrocyclic molecules, and a cap bonded to an end of the axle molecule, wherein at least some of the macrocyclic molecules have an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, with an antigen to form a polyion complex.

[0055] <Method for producing a pharmaceutical composition according to the present disclosure, step 2> The present disclosure also provides a method for producing a pharmaceutical composition according to the present disclosure (method for producing a pharmaceutical composition according to the present disclosure, step 2). The method for producing a pharmaceutical composition according to the present disclosure, step 2, comprises mixing a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating rings of the macrocyclic molecules, and a cap bonded to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond, with an antigen to form a polyion complex.

[0056] In the manufacturing methods 1 and 2 of the pharmaceutical composition of the present disclosure, the "macrocyclic molecule", the "axle molecule", the "cap", the "polyrotaxane", the "amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH", the "group having an amino group via an intracellularly degradable bond", the "antigen", and the "polyion complex" may be the same as those described in the pharmaceutical composition of the present disclosure.

[0057] The pharmaceutical composition of the present disclosure can be prepared by mixing the polyrotaxane (or the polyrotaxane composition) with the antigen to form a polyion complex. The mixing ratio of the polyrotaxane (or the polyrotaxane composition) to the antigen is not particularly limited, but may be 1:0.25 to 0.25:1, and preferably 1:0.5 to 0.5:1. Mixing can be performed by stirring in an aqueous solution, buffer solution, or medium at room temperature. The mixing time can be appropriately set depending on the polyrotaxane (or the polyrotaxane composition) and the antigen, their concentrations, etc., but is typically about 5 minutes to overnight, 5 minutes to 6 hours, 5 to 180 minutes, 5 to 120 minutes, 5 to 60 minutes, or 5 to 30 minutes.

[0058] 3. Vaccine Composition of the Present Disclosure The antigen contained in the pharmaceutical composition of the present disclosure may be an antigen of a target disease (hereinafter also referred to as a "target antigen"). A pharmaceutical composition of the present disclosure containing a target antigen can strongly induce a target antigen-specific immune response in a subject to which it is administered. Therefore, the pharmaceutical composition of the present disclosure can be used as a vaccine composition (vaccine composition of the present disclosure) for preventing or treating the target disease.

[0059] Examples of the target antigen include, but are not limited to, tumor antigens in cancer, viral antigens in viral infections, bacterial antigens in bacterial infections, fungal antigens in fungal infections, and parasitic antigens in parasitic infections.

[0060] Examples of the tumor antigen include 5T4, α5β1-integrin, activated integrin β7, 707-AP, α-fetoprotein (AFP), lectin-reactive AFP, ART-4, AURKA (AURORA A), B7H4, BAGE, β-catenin, BCMA, Bcr-abl, BTAA, MN / CA IX antigen, CA125, CA19-9, CA72-4, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD47, CD52, CD56, CD80, CD96, CD123, CDK4, carcinoembryonic antigen (CEA), CLL1, CT, and Cyclin A1, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE ​​(GAGE-1, GAGE-2, etc.), GD2 (Ganglioside G2), GnT-V, Gp100, HAGE, β-human chorionic gonadotropin (HCG), HER2 / neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, iCE, insulin growth factor (IGF)-1, IGF-2, IGF-1R, IL-2R, IL-5, KIAA0205, K-Ras, LAGE, LDLR / FUT, MAGE (MAGE-3, MAGE-4, MAGE-5, MAGE-6, etc.), MART-1 / melan-A, MART-2 / Ski, MC1R, mesothelin (MSLN), myosin, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, prostatic acid phosphatase (PAP), proteinase-3, PRAME (Melanoma antigen preferentially expressed in tumors), p53, p190 minor, bcr-abl, Pml / RARα, prostate tumor antigen-1 (PCTA-1), PRAME, prostate-specific antigen (PSA), PSM, PSMA, RAGE, RAS, RHAMM (CD168), RU1, RU2, SAGE, SART-1, SART-3, thyroglobulin, survivin, telomerase reverse transcriptase (TERT or TRT), TEL / AML1, TGFβ, TIM3, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, NY-Eso-1, NY-Eso-B, etc.

[0061] Cancers associated with the tumor antigens include, but are not limited to, malignant melanoma, lung cancer, synovial sarcoma, breast cancer, esophageal cancer, ovarian cancer, bladder cancer, prostate cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, rectal cancer, skin cancer, neuroblastoma, myeloma, B-cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, and multiple myeloma.

[0062] Examples of the viral antigen include viruses from the Adenoviridae family, such as adenovirus; viruses from the Coronaviridae family, such as coronavirus; viruses from the Filoviridae family, such as Ebolavirus; viruses from the Flaviviridae family, such as hepatitis C virus (HCV), dengue virus, Japanese encephalitis virus, West Nile virus, and yellow fever virus; viruses from the Hepadnaviridae family, such as hepatitis B virus (HVB); viruses from herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), and varicella zoster virus (VZV). Herpesviridae, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV); Orthomyxoviridae, such as influenza virus type A (Influenzavirus A), influenza virus type B (Influenzavirus B), and influenza virus type C (Influenzavirus C); Paramyxoviridae, such as measles virus, human parainfluenza virus types 1-4, mumps virus, and respiratory syncytial virus (RSV);Parvoviridae family, such as Parvovirus B19; Picornaviridae family, such as Enterovirus, Poliovirus, Human Rhinovirus A-B, Hepatitis A Virus, Coxsackievirus, and Echovirus; Poxviridae family, such as Variola Virus and Vaccinia Virus; Retroviridae family, such as Human Immunodeficiency Virus (HIV)-1, 2, and Human T-lymphocytropic Virus (HTLV)-I, II; Rabies Virus and Vesicular Stomatitis Virus; Examples include antigens derived from viruses such as Rhabdoviridae, including rubella virus and chikungunya virus; and Togaviridae, including rubella virus and chikungunya virus.

[0063] Examples of viral infections involving the viral antigens include, but are not limited to, conjunctivitis, cystitis, pneumonia, encephalitis, gastroenteritis, esophagitis, hepatitis, nephritis, pancreatitis, dermatitis, stomatitis, Ebola virus disease, chickenpox, rubella, acquired immunodeficiency syndrome, SARS, dengue fever, influenza, West Nile fever, and yellow fever.

[0064] Examples of the bacterial antigen include bacteria of the genus Clostridium such as Clostridium tetani; bacteria of the genus Escherichia such as Escherichia coli; bacteria of the genus Helicobacter such as Helicobacter pyloris; bacteria of the genus Legionella such as Legionella pneumophila; bacteria of the genus Listeria such as Listeria monocytogenes; bacteria of the genus Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and the like. Mycobacterium species such as Neisseria gonorrhoeae and Neisseria meningitidis; Pseudomonas species such as Pseudomonas aeruginosa; Salmonella species such as Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A; Staphylococcus species such as Staphylococcus aureus; Streptococcus pneumoniae and Streptococcus pyogenes. Examples of antigens include antigens derived from bacteria such as Streptococcus, such as Streptococcus pyogenes.

[0065] Examples of bacterial infections involving the bacterial antigens include, but are not limited to, tetanus, gastric ulcer, Legionnaires' disease, listeriosis, tuberculosis, leprosy, gonorrhea, meningitis, Pseudomonas aeruginosa infection, typhoid fever, food poisoning, and the like.

[0066] Examples of the fungal antigen include those of the genus Aspergillus such as Aspergillus fumigatus; those of the genus Blastomyces such as Blastomyces dermatitidis; those of the genus Candida such as Candida albicans; those of the genus Coccidioides such as Coccidioides immitis and Coccidioides posadasii; those of the genus Cryptococcus such as Cryptococcus neoformans and Cryptococcus gattii; those of the genus Histoplasma capsulatum; Examples of antigens include those derived from fungi such as Histoplasma species such as Paracoccidioides brasiliensis, Paracoccidioides species such as Paracoccidioides brasiliensis, and Sporothrix species such as Sporothrix schenckii.

[0067] Fungal infections involving the fungal antigens include, but are not limited to, aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, and the like.

[0068] Examples of the parasite antigen include antigens derived from parasites such as liver fluke (Clonorchis sinensis), Japanese blood fluke (Schistosoma japonicum), human roundworm (Ascaris lumbricoides), human pinworm (Enterobius vermicularis), cysticercus cellulosae, fish tapeworm (Diphyllobothrium latum), Echinococcus, Entamoeba histolytica, and malaria parasite (Plasmodium).

[0069] Parasitic infections involving the parasitic antigens include, but are not limited to, Clonorchiasis, Schistosomiasis japonicum, Ascariasis, Enterobiasis, Cysticercosis, Diphyllobothriasis, Echinococcosis, Dysentery, Malaria, etc.

[0070] Specific examples of the target antigen include, but are not limited to, proteins or partial peptides thereof, nucleic acids, and sugar chains.

[0071] When the target antigen is a protein or a partial peptide thereof (protein antigen), the protein antigen can be prepared by any method. For example, when the protein antigen is a tumor antigen for cancer, the tumor antigen may be a protein or a partial peptide thereof isolated and purified from cancer cells of a mammal (including model animals such as mice and humans) or tissues containing cancer cells. When the protein antigen is a viral antigen for viral infections, a bacterial antigen for bacterial infections, or a parasitic antigen for parasitic infections, the viral antigen or the like may be a protein or a partial peptide thereof isolated and purified from a virus or the like. The protein antigen may be a protein or a partial peptide thereof chemically synthesized or biochemically synthesized in a cell-free translation system, or a recombinant protein or a partial peptide thereof produced from a transformant into which a nucleic acid having a nucleotide sequence encoding the protein or the partial peptide has been introduced.

[0072] Protein antigens can be produced from mammalian cancer cells or tissues containing cancer cells, viruses, bacteria, or parasites by publicly known protein purification methods. Specifically, cancer cells or tissues containing cancer cells, virus-infected cells, bacteria, or parasites are homogenized, debris is removed by low-speed centrifugation, and the supernatant is centrifuged at high speed to precipitate a fraction containing cell or bacterial cell membranes or parasite epidermis (if necessary, the fraction containing the cell membrane equivalent is purified by density gradient centrifugation or the like), and the fraction is then subjected to chromatography such as reverse-phase chromatography, ion-exchange chromatography, or affinity chromatography to prepare the protein antigen.

[0073] When the protein antigen is a partial peptide, the partial peptide can be produced according to a known peptide synthesis method. The peptide synthesis method may be, for example, either solid-phase synthesis or liquid-phase synthesis. A partial peptide constituting the protein antigen can be produced based on the amino acid information of the antigen protein. The partial peptide thus obtained can be purified and isolated by a known purification method. Examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.

[0074] Furthermore, a protein antigen can also be produced by culturing a transformant containing a nucleic acid encoding it and isolating and purifying the protein antigen from the resulting culture. The nucleic acid encoding the protein antigen may be DNA or RNA, or may be a DNA / RNA chimera. DNA is preferred. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. If single-stranded, it may be the sense strand (i.e., coding strand) or the antisense strand (i.e., non-coding strand).

[0075] When the protein antigen is a tumor antigen, DNA encoding the protein antigen can be cDNA derived from mammalian cancer cells or any tissue containing cancer cells (including model animals such as mice and humans). When the protein antigen is a viral, bacterial, or parasitic antigen, DNA derived from viruses can be used. The cDNA encoding the protein antigen can also be directly amplified by polymerase chain reaction (hereinafter referred to as "PCR") or reverse transcriptase-PCR (hereinafter referred to as "RT-PCR") using total RNA or mRNA (mRNA encoding the protein antigen) prepared from the cancer cells or tissue containing cancer cells, virus, bacteria, or parasite as a template. Alternatively, the cDNA encoding the protein antigen can be cloned by colony or plaque hybridization or PCR from a cDNA library prepared by inserting cDNA fragments prepared from total RNA or mRNA prepared from the cancer cells or tissue, virus, bacteria, or parasite into an appropriate vector. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid, etc.

[0076] The DNA encoding the protein antigen obtained as described above can be ligated downstream of a promoter in an appropriate expression vector, either directly or after digestion with a restriction enzyme or the addition of a linker, depending on the purpose. The DNA may have a translation initiation codon ATG at its 5'-end and a translation termination codon TAA, TGA, or TAG at its 3'-end. These translation initiation and termination codons can be added using an appropriate synthetic DNA adapter.

[0077] An expression vector containing DNA encoding a protein antigen is introduced into appropriate cells, and the introduced cells are cultured, whereby the protein antigen can be isolated and purified.

[0078] When the antigen is a nucleic acid (nucleic acid antigen) encoding the protein antigen, the nucleic acid antigen may be DNA or RNA. When the nucleic acid antigen is DNA, a specific example is an expression vector containing DNA encoding the protein antigen, which is used when preparing the above-mentioned protein antigen. When the nucleic acid antigen is RNA, a specific example is mRNA encoding the protein antigen, which is used when preparing the protein antigen.

[0079] When the antigen is a glycan (glycan antigen), the glycan antigen can be prepared by any method, for example, by homogenizing cells, removing cell debris by low-speed centrifugation, and then centrifuging the supernatant at high speed to precipitate a cell membrane-containing fraction (if necessary, further purifying the cell membrane fraction by density gradient centrifugation or the like).

[0080] The vaccine composition of the present disclosure can be a pharmaceutical composition for oral or parenteral administration, and the formulation additives that can be contained, dosage form, administration target, dosage, administration frequency, etc. may be in accordance with the descriptions for the pharmaceutical composition of the present disclosure.

[0081] 4. Method for preventing or treating a target disease according to the present disclosure <Method 1 for preventing or treating a target disease according to the present disclosure> The present disclosure also provides a method for preventing or treating a target disease (Method 1 for preventing or treating a target disease according to the present disclosure). Method 1 for preventing or treating a target disease according to the present disclosure comprises administering to a subject a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates rings of the macrocyclic molecules, and a cap bonded to an end of the axle molecule, wherein at least some of the macrocyclic molecules include an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and an antigen for the target disease.

[0082] <Method 2 for preventing or treating a target disease according to the present disclosure> The present disclosure also provides a method for preventing or treating a target disease (method 2 for preventing or treating a target disease according to the present disclosure). Method 2 for preventing or treating a target disease according to the present disclosure comprises administering to a subject a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates rings of the macrocyclic molecules, and a cap bonded to an end of the axle molecule, wherein at least some of the macrocyclic molecules include an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and an antigen for the target disease.

[0083] In the methods 1 and 2 for preventing or treating a target disease of the present disclosure, the "macrocyclic molecule," the "axle molecule," the "cap," the "polyrotaxane," the "amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH," the "group having an amino group via an intracellularly degradable bond," the "antigen," and the "polyion complex" may be the same as those described for the pharmaceutical composition of the present disclosure. The formulation additives that can be contained, the dosage form, the target of administration, the dosage, the administration frequency, and the like may also be the same as those described for the pharmaceutical composition of the present disclosure.

[0084] 5. Use of the Present Disclosure in the Prevention or Treatment of a Target Disease <Use 1 of the Present Disclosure in the Prevention or Treatment of a Target Disease> The present disclosure also provides a polyrotaxane for use in the prevention or treatment of a target disease, the polyrotaxane having a plurality of macrocyclic molecules, an axle molecule passing through rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules include an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH; and an antigen for the target disease (Use 1 of the Present Disclosure in the Prevention or Treatment of a Target Disease).

[0085] <Use 2 of the Present Disclosure in the Prevention or Treatment of a Target Disease> The present disclosure also provides a polyrotaxane for use in the prevention or treatment of a target disease, the polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond; and an antigen for the target disease (Use 2 of the Present Disclosure in the Prevention or Treatment of a Target Disease).

[0086] In Uses 1 and 2 for the prevention or treatment of target diseases of the present disclosure, the "macrocyclic molecule," the "axle molecule," the "cap," the "polyrotaxane," the "amine-containing group having one proton at neutral pH and two protons at acidic pH," the "group having an amino group via an intracellularly degradable bond," the "antigen," and the "polyion complex" may be the same as those described for the pharmaceutical composition of the present disclosure. The possible formulation additives, dosage form, administration target, dosage, administration frequency, etc. may also be as described for the pharmaceutical composition of the present disclosure.

[0087] 6. Use for producing a vaccine composition for preventing or treating a target disease according to the present disclosure <Use 1 for producing a vaccine composition for preventing or treating a target disease according to the present disclosure> The present disclosure also provides use of a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules include an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and an antigen for the target disease (Use 1 for producing a vaccine composition for preventing or treating a target disease according to the present disclosure).

[0088] <Use 2 for producing a vaccine composition for preventing or treating a target disease of the present disclosure> The present disclosure also provides use of a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond, and an antigen for the target disease (Use 2 for producing a vaccine composition for preventing or treating a target disease of the present disclosure).

[0089] In Uses 1 and 2 of the present disclosure for producing a vaccine composition for preventing or treating a target disease, the "macrocyclic molecule," the "axle molecule," the "cap," the "polyrotaxane," the "amine-containing group having one proton at neutral pH and two protons at acidic pH," the "group having an amino group via an intracellularly degradable bond," the "antigen," and the "polyion complex" may be the same as those described for the pharmaceutical composition of the present disclosure. The formulation additives that can be contained, the dosage form, the target of administration, the dosage, the administration frequency, and the like may also be the same as those described for the pharmaceutical composition of the present disclosure.

[0090] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents, kits, etc. were used according to their protocols.

[0091] (Preparation Example 1) Preparation of polyrotaxane (Ad-cap-PRX) having multiple cyclodextrins (CyD), polyethylene glycol (PEG) penetrating the rings of the CyD, and caps (adamantane groups) bound to both ends of the PEG via carbamate bonds. Ad-cap-PRX was prepared according to the following method, with some modifications to the method reported by Araki et al. (Araki, J. et al., Macromolecules 38, 7524-7527 (2005)).

[0092] Preparation Example 1-1: Preparation of PEG (20 kDa) with Amino Groups Protected at Both Ends (PEG-DAT) PEG (20 kDa) (56 g, 2.8 mmol) and N,N-carbonyldiimidazole (CDI) (2.0 g, 12.4 mmol) were dissolved in tetrahydrofuran (THF) (200 mL) and stirred at 50°C for 18 hours under nitrogen purging. The reaction mixture was added dropwise to ethylenediamine (6.0 mL, 88.0 mmol) and stirred at 50°C for 2 hours. Ethanol (200 mL) was added, and the mixture was allowed to stand at -20°C for 2 hours. The precipitate was collected by centrifugation, washed several times with cold ethanol, and dried under reduced pressure to obtain PEG-DAT. Yield: 52.7 g, 94%.

[0093] Preparation Example 1-2: Preparation of polypseudorotaxane (PEG-DAT / α-CyD PPRX) having multiple CyDs and PEG penetrating the CyD rings. PEG-DAT (3.0 g) was added to a 12% (w / v) aqueous α-CyD solution (100 mL). After stirring overnight at 4°C, the precipitate was collected by centrifugation and lyophilized to obtain PEG-DAT / α-CyD PPRX.

[0094] (Preparation Example 1-3) Preparation of Ad-cap-PRX. 1-Adamantaneacetic acid (2.45 g, 12.6 mmol), BOP reagent (5.25 g, 11.8 mmol), 1-hydroxybenzotriazole (HOBt) (1.75 g, 11.4 mmol), and N-ethyldiisopropylamine (EDIPA) (2.28 mL, 13.2 mmol) were dissolved in dimethylformamide (DMF) (100 mL) and PEG-DAT / α-CyD PPRX (14 g) was added. After stirring for 48 hours at 4°C under a nitrogen atmosphere, the precipitate was collected by centrifugation and washed twice with methanol / DMF (1:1 v / v) and methanol, respectively. The resulting product was dissolved in dimethyl sulfoxide (DMSO) and precipitated by adding excess water. The same procedure was repeated three times, and the resulting precipitate was lyophilized to obtain Ad-cap-PRX. Yield: 10.23 g, 82% (based on PEG).

[0095] (Preparation Example 2) Preparation of polyrotaxane (DET-PRX: 2G) in which multiple CyDs have diethylenetriamine. Ad-cap-PRX (3 g, 38.82 mmol) from Preparation Example 1 was dissolved in DMSO (90 mL), CDI (3.54 g, 21.72 mmol) was added, and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. The reaction solution was added dropwise to diethylenetriamine (DET) (23.52 mL, 21.72 mmol), and the mixture was stirred overnight at room temperature under a nitrogen atmosphere. The product was dialyzed against water (Spectra / Por TM The membrane (MWCO: 10 kDa) was dried by freeze-drying to obtain DET-PRX.

[0096] Preparation Example 3: Preparation of Polyrotaxane (Cys / DET-PRX: 5G) with Multiple CyDs Containing Diethylenetriamine and Cystamine. Ad-cap-PRX (100 mg, 1.35 μmol) from Preparation Example 1 was dissolved in DMSO (6 mL), CDI (106 mg, 0.656 mmol) was added, and the mixture was stirred at 25°C under a nitrogen atmosphere for 24 hours. Cystamine (Cys) dihydrochloride (1.165 g, 5.17 mmol) was dissolved in DMSO (17 mL), triethylamine (TEA) (1.43 mL, 10.33 mmol) was added, and the mixture was desalted by stirring at room temperature for 30 minutes. DET (710 μL, 3.28 mmol) was added to the desalted cystamine solution, and the mixture was stirred at room temperature for 30 minutes. The Ad-cap-PRX solution was added dropwise, and the mixture was further stirred overnight at 25°C under a nitrogen atmosphere. The reaction mixture was then dropped into ethanol under stirring to precipitate. After centrifugation and removal of the supernatant, the precipitate was washed five times with ethanol and the ethanol was removed using an evaporator. The precipitate was then redissolved in water and lyophilized to obtain Cys / DET-PRX.

[0097] Example 1 1 Structural analysis by H-NMR DET-PRX of Production Example 2 and Cys / DET-PRX of Production Example 3 were 1Structural analysis was performed using H-NMR. Both DET-PRX and Cys / DET-PRX were confirmed to be polyrotaxanes with approximately 55 CyD rings threaded with 20 kDa PEG (Figs. 1 and 2). Furthermore, in the case of DET-PRX, approximately 2.8 DETs were attached per CyD (Fig. 1). In the case of Cys / DET-PRX, approximately 1.1 DETs and 0.9 Cyss were attached per CyD (Fig. 2).

[0098] (Example 2) Polyion complex formation test with protein (OVA)

[0099] Example 2-1: Electrophoresis: Samples (7.5 mg / sample) containing ovalbumin (OVA) (2-4 mg) in DET-PRX solution (2 μL; N / C = 0, 1, 5, 10, 15) and HBSS buffer (pH = 7.4, 4-7 μL) were mixed by inversion and incubated at room temperature for 15 minutes to obtain samples. 6x Loading dye (2 μL) was added to each sample, and the samples were separated by electrophoresis on a 2.5% agarose gel (100 V, 30 minutes). After electrophoresis, the agarose gel was stained with CBB-G250 (room temperature, 30 minutes). The agarose gel was then destained (30 minutes) and photographed (FUJIFILM Fluoroimage Analyzer, FLA-9000). The results are shown in Figure 3. In the agarose gel electrophoresis, the addition of DET-PRX shifted the band of free OVA in a concentration-dependent manner, suggesting that DET-PRX formed a complex with OVA.

[0100] Example 2-2: Measurement of particle size and zeta potential. HBSS buffer (pH = 7.4), DET-PRX or Cys / DET-PRX solutions at various concentrations (N / C = 1, 10, 50), and OVA (OVA: 1 μg / μL, 3 μL) were mixed by pipetting and incubated at room temperature for 15 minutes to obtain samples. HBSS buffer (700 μL) was added to each sample (300 μL), and particle size and zeta potential were measured by dynamic light scattering (Malver Zetasizer Nano). The particle size results are shown in Figure 4. DET-PRX (N / C = 50) formed particles with OVA with a diameter of approximately 25 nm. The zeta potential results are shown in Figures 5 and 6. The zeta potential of each sample to which DET-PRX or Cys / DET-PRX had been added was higher than that of OVA.

[0101] These results suggest that DET-PRX or Cys / DET-PRX can form a polyion complex with OVA.

[0102] Example 3: Measurement of Environmentally Responsive Deformability under Acidic Conditions HBSS buffer (pH = 5.5 or 7.4), DET-PRX solution (N / C = 50), or Cys / DET-PRX solution (N / C = 50), and OVA (OVA: 1 μg / μL, 3 μL) were mixed by pipetting and incubated at room temperature for 15 minutes to obtain each sample. HBSS buffer (pH = 5.5 or 7.4; 700 μL) was added to each sample (300 μL), and the ζ potential was measured by dynamic light scattering (Malver Zetasizer Nano). The ζ potential results are shown in Figure 7 . The ζ potential of each sample under acidic conditions (pH = 5.5) was higher than that under neutral conditions (pH = 7.4). This suggests that in the endosomal environment (pH = 5.5), DET-PRX and Cys / DET-PRX exhibit strong cell membrane disruption activity due to the enhanced ζ potential, and efficiently induce endosomal escape of OVA.

[0103] (Example 4) Test of OVA complex uptake into mouse bone marrow-derived dendritic cells (BMDCs) BMDCs were seeded on a 24-well plate (2.0 × 10 5 To the plate, 400 μL of cells / well was added, followed by 100 μL of sample solution (FITC-OVA 1.5 μg / well and DET-PRX (N / C = 50) or FITC-OVA 1.5 μg / well and Cys / DET-PRX (N / C = 50)) and incubation at 37°C for 4 hours. The cells were harvested, centrifuged at 500 × g for 3 minutes, and the supernatant was removed. This process was repeated twice: 500 μL of PBS containing 0.5% FBS, centrifuged at 500 × g for 3 minutes, and the supernatant was removed. 1000 μL of PBS containing 0.5% FBS was added to the cells, and the cell fluorescence was monitored using a flow cytometer. The results are shown in Figure 8. DET-PRX / OVA and Cys / DET-PRX / OVA were taken up by BMDCs to a greater extent than OVA alone.

[0104] (Example 5) Measurement of cell surface markers (CD80 and CD86) of BMDCs that have incorporated OVA complexes BMDCs were seeded (2.0 × 10 5To the plate, 400 μL of cells / well was added, and 100 μL of sample solution (FITC-OVA 1.5 μg / well and DET-PRX (N / C=50) or FITC-OVA 1.5 μg / well and Cys / DET-PRX (N / C=50)) was added and incubated at 37°C for 4 hours. The cells were detached, centrifuged at 500 × g for 3 minutes, and the supernatant was removed. This process was repeated twice: adding 500 mL of PBS containing 0.5% FBS to the cells, centrifuging at 500 × g for 3 minutes, and removing the supernatant. 1 mL of PBS containing 0.5% FBS was added to the cells, and 1 μL of anti-mouse CD16 / 32 antibody was added and incubated for 10 minutes on ice. 1 μL of a cocktail antibody (CD11c, CD86, CD80: each) was added to the cells, and the cells were incubated for 10 minutes on ice. 900 μL of PBS containing 0.5% FBS was added to the cells, followed by centrifugation (500 × g, 3 minutes), and the supernatant was removed. 1000 μL of PBS containing 0.5% FBS was added again, and the maturation of BMDCs was assessed by measuring the percentage of CD80- and CD86-positive cells using a flow cytometer. The results are shown in Figure 9. Treatment of BMDCs with DET-PRX / OVA and Cys / DET-PRX / OVA increased the percentage of CD80- and CD86-positive cells, indicating BMDC maturation.

[0105] Example 6 Measurement of Cell Surface Markers (MHC1 and MHCII) of DCs Derived from Mice Immunized with OVA Complexes PBS, OVA, Alum / OVA, and various complexes (DET-PRX / OVA, DET-PRX / Alum / OVA, Cys / DET-PRX / OVA) solutions (200 μL; N / C=50 (N / C indicates the charge ratio of cation (amino group of DET-PRX) to anion (carboxy group of OVA)), OVA 10 μg / mouse) were subcutaneously administered (prime) to the right inguinal region of C57BL / 6J mice (6-week-old, female). One week after administration, the mice were subcutaneously administered (boost) with 200 μL of the above solution (prime). One week after the boost administration, the right inguinal lymph nodes of the mice were collected. Cells were collected from the lymph nodes and centrifuged (400 × g, 3 minutes), and the supernatant was removed. PBS containing 0.5% FBS (100 μL) was added to the cells, and the cells were diluted to 1.0 x 10 6 The cell concentration was adjusted to 100 cells / mL. Anti-mouse CD16 / 32 antibody (1 μL) was added to the cells, followed by pipetting and incubation on ice for 10 minutes. CD11c (Per / CP5.5) antibody, MHC1 (PE) antibody, and MHCII (APC) antibody (1 μL each) were added to the cells, followed by pipetting and incubation on ice for 10 minutes. PBS (1 mL) was added to the cells, followed by pipetting and centrifugation (400 x g, 3 minutes), and the supernatant was removed. PBS containing 0.5% FBS (700 μL) was added to the cells, and the maturation of DCs was assessed by measuring the percentage of MHC1- and MHCII-positive cells using a flow cytometer. The results are shown in Figure 10. Immunization of mice with DET-PRX / OVA or Cys / DET-PRX / OVA increased the percentage of MHC1- and MHCII-positive cells, suggesting DC maturation. In particular, DET-PRX / OVA or Cys / DET-PRX / OVA significantly increased the percentage of MHC1-positive cells compared to the complex of Alum and OVA, which is known as a common adjuvant.

[0106] Example 7: Measurement of anti-OVA antibody titers in mice immunized with OVA conjugates. PBS, OVA, Alum / OVA, and various conjugates (DET-PRX / OVA, Cys / DET-PRX / OVA) solutions (200 μL; N / P = 50, OVA 10 μg / mouse) were subcutaneously administered (prime) to the right groin of C57BL / 6J mice (6-week-old, female). One week after the administration, the mice were subcutaneously administered (boost) with 200 μL of the above solution (boost) to the right groin. One week after the boost administration, blood was collected from the inferior vena cava of the mice. OVA (10 mg / mL) was then diluted to 10 μg / mL with 0.1 N carbonate buffer. OVA (10 μg / mL) solution (50 μL) was added to a 96-well ELISA half plate and incubated overnight at 4°C. The wells were washed three times with PBST (150 μL), 1% BSA-PBS (50 μL) was added, and incubated for 1 hour at room temperature. The wells were then washed three times with PBST (150 μL). 50 μL of each sample (9 samples, including the 10-fold diluted blood sample as the highest concentration, followed by eight 5-fold serial dilutions) was added to the wells and incubated for 2 hours at room temperature. The wells were washed three times with PBST (150 μL), and Horse Radish Peroxidase-conjugated goat anti-mouse IgG antibody (50 μL) diluted 4000-fold in BSA-PBS was added to the wells and incubated for 1 hour at room temperature. The wells were washed three times with PBST (150 μL), and TMB reagent (50 μL) was added to the wells and incubated for 20 minutes at room temperature. The reaction was stopped by adding 1N sulfuric acid solution (50 μL) to the wells, and the absorbance at 450 nm was measured using a microplate reader. The absorbance at 550 nm was measured as the plate background, and this was subtracted from the absorbance at 450 nm to determine the anti-OVA antibody titer. The results are shown in Figure 11. Antibody titers were observed after administration of DET-PRX / OVA or Cys / DET-PRX / OVA to mice, and the antibody titers were found to be higher than those after administration of OVA alone. Notably, mice treated with DET-PRX / OVA exhibited higher antibody titers than those treated with the Alum / OVA complex.

[0107] Example 8: Measurement of inflammatory cytokine secretion in mice immunized with OVA complexes. PBS, OVA, Alum / OVA, and various complexes (DET-PRX / OVA, DET-PRX / Alum / OVA, Cys / DET-PRX / OVA) solutions (200 μL; N / P = 50, OVA 10 μg / mouse) were subcutaneously administered (prime) to the right groin of C57BL / 6J mice (6-week-old, female). One week after the administration, the mice were subcutaneously administered (boost) with 200 μL of the above solutions in the right groin. One day after the boost administration, blood was collected from the inferior vena cava of the mice. Next, a 200-fold dilution of the capture antibody was made with 1× coating buffer, and 50 μL was added to a 96-well half plate for ELISA. The plate was sealed and incubated (4°C overnight). The wells were washed three times with PBST (150 μL). After diluting the detection antibody 200-fold in 1x assay diluent A, 50 μL was added to the plate and incubated at 4°C for 1 hour. The wells were washed four times with PBST (150 μL). TMB reagent (50 μL) was added to the wells and incubated in the dark for 10, 20, or 30 minutes at room temperature. The reaction was stopped by adding 1N sulfuric acid solution (50 μL). The absorbance at 450 nm was measured using a microplate reader. The absorbance at 550 nm was measured as the plate background and subtracted from the absorbance at 450 nm to determine the amount of inflammatory cytokine secretion. The results are shown in Figure 12. Administration of DET-PRX / OVA increased the secretion of the inflammatory marker IL-6.

[0108] Example 9: Safety Evaluation Test of Mice Immunized with OVA Conjugates Solutions (200 μL; N / P=50, OVA 10 μg / mouse) of PBS, OVA, Alum / OVA, and various conjugates (DET-PRX / OVA, DET-PRX / Alum / OVA, Cys / DET-PRX / OVA) were subcutaneously administered (prime) to the right groin of C57BL / 6J mice (6-week-old, female). One week after administration, the mice were subcutaneously administered (boost) with 200 μL of the above solutions in the right groin. 24 hours after the boost administration, blood was collected from the inferior vena cava of the mice, and blood biochemistry tests were performed. The results are shown in Figures 13 and 14. Administration of any of the OVA conjugates had no significant effect on the test values.

[0109] Example 10: Measurement of the Level of Tetramer-Positive CD8+ T Cells in PBMCs from SARS-CoV-2 Patients Pulsed with DET-PRX / NF9 PBMCs from SARS-CoV-2-Infected and Recovered Humans were pulsed overnight in RPMI 1640 medium (Thermo Fisher Scientific, Cat# 11875101) containing the NF9 peptide (NYNYLYRLF (SEQ ID NO: 1); amino acid residues 448-456 of the SARS-CoV-2 spike protein) alone (100 nM) or DET-PRX / NF9 (molar ratio = 1:1). After washing with RPMI 1640 medium, they were maintained in RPMI 1640 medium containing 10% FBS and 30 U / ml recombinant human IL-2 (Peprotec, Cat# 200-02) for 14 days. MHCI tetramers loaded with SARS-CoV-2-derived peptides were analyzed using a QuickSwitch™ Quant HLA-A * The antibody was prepared using the 24:02 Tetramer Kit-PE (MBL International Corporation, Cat# TB-7302-K1) according to the manufacturer's protocol. HLA-A was used as a negative control. *The 24:02-restricted KW9 peptide (KYKLKHIVW (SEQ ID NO: 2)) was included. Peptide exchange rates were quantified by flow cytometry, and over 90% of the tetramer was used to stain PBMCs. PBMCs were stained with the tetramer for 30 minutes on ice. After tetramer staining, cells were counterstained with the following antibodies: CD3 AF537 (UCHT1, 1 / 25 dilution, Cat# 58-0038-42, eBioscience), CD8 APCcy7 (HIT8a, 1 / 100 dilution, Cat# 300926), CD14 PerCP / Cy5.5 (HCD14, 1 / 100 dilution, Cat# 325622), CD4 BV750 (SK3, 1 / 100 dilution, Cat# 344644), CD45RA APC (HI100, 1 / 100 dilution, Cat# 304112), and CCR7 BV510 (G043H7, 1 / 25 dilution, Cat# 353232, Biolegend). Dead cells were stained with 7-aminoactinomycin D (Biolegend). After 20 minutes of incubation on ice, cells were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat# 09154-85), and the levels of tetramer-positive CD8+ T cells were analyzed by flow cytometry using a Cytek Northern Lights (Cytek Japan). Flow cytometry data were analyzed using FACS Diva v9.0 (BD) and FlowJo software v10 (Tree Star). The results are shown in Figure 15. When SARS-CoV-2 patient-derived PBMCs were pulsed with DET-PRX / NF9, the frequency of NF9-specific CD8+ T cells increased 2.7-fold compared to NF9-only pulsing. Furthermore, the binding ability of CD8+ T cells to tetramers also increased 1.2-fold, suggesting that a CD8+ T cell population with high tetramer-binding affinity was induced, suggesting that a different CD8+ T cell type was induced compared to NF9-only pulsing. No significant changes in NF9-specific CD8+ T cell differentiation were observed.

[0110] (Example 11) Polyion complex formation test with mRNA

[0111] Example 11-1: Electrophoresis. Samples containing mCherry mRNA (0.25 μg / μL, 1 μL), DET-PRX or Cys / DET-PRX solutions (2 μL) at various concentrations (N / P = 0, 0.25, 0.5, 0.75, 1, 5), and HBSS buffer (pH = 7.4, 7 μL) were vortexed (room temperature, 10 seconds) and incubated (room temperature, 15 minutes). 6x Loading dye (2 μL) was added to each sample, and the samples were separated by electrophoresis on a 2% agarose gel (100 V, 30 minutes). After electrophoresis, the agarose gel was stained with EtBr (room temperature, 30 minutes). The agarose gel was then photographed (FUJIFILM Fluoroimage Analyzer, FLA-9000). The results are shown in Figures 16 and 17. After electrophoresis, the addition of DET-PRX or Cys / DET-PRX induced a concentration-dependent shift in the mRNA band in agarose gels, suggesting that DET-PRX or Cys / DET-PRX formed a complex with mRNA, resulting in the band shift.

[0112] Example 11-2: Measurement of particle size and zeta potential. HBSS buffer (pH = 7.4), DET-PRX solutions at various concentrations (N / P = 1, 10, 50), and mCherry mRNA (0.25 μg / μL, 12 μL) were mixed by pipetting and incubated at room temperature for 15 minutes to obtain samples. HBSS buffer (700 μL) was added to each sample (300 μL), and particle size and zeta potential were measured by dynamic light scattering (Malver Zetasizer Nano). The results for zeta potential are shown in Figure 18. The zeta potential of each sample containing DET-PRX increased with increasing DET-PRX concentration. The results for particle size are shown in Figure 19. Each sample containing DET-PRX (N / P = 1, 10, 50) formed larger particles than either mRNA or DET-PRX alone. These results suggest that DET-PRX can form a polyion complex with mRNA.

[0113] (Example 12) mRNA complex uptake test into Hela cells Hela cells were seeded on a 24-well plate (3.75 × 104 The cells were then washed twice with serum-free medium, and Cy5-GFP mRNA and various conjugate solutions (N / P = 50, Cy5-GFP mRNA 0.5 μg / well; 500 μL) were added and incubated at 37°C for 4 hours. The cells were then washed twice with serum-free medium, and serum-free medium (FBS(+); 500 μL) was added and incubated at 37°C for 20 and 44 hours. The cells were then collected, centrifuged at 3000 rpm for 3 minutes, and the supernatant was removed. PBS containing 0.5% FBS (500 μL) was then added to the cells, and the cells were centrifuged at 400 x g for 3 minutes. The supernatant was removed. PBS containing 0.5% FBS (500-1000 μL) was added to the cells, and the cell fluorescence was monitored using a flow cytometer. The results are shown in Figure 20. DET-PRX / mRNA and Cys / DET-PRX / mRNA were taken up into HeLa cells to a greater extent than mRNA alone.

[0114] 5 T cells were seeded in each well and pulsed with overlapping peptides of the SARS-CoV-2 spike protein (a collection of 316 partial peptides of the amino acid sequence represented by SEQ ID NO: 3, each partial peptide being represented by a 15-amino acid sequence shifted by 4 amino acids from the N-terminus of the amino acid sequence represented by SEQ ID NO: 3 toward the C-terminus) (2 μg / ml), followed by 18 hours of culture. T cell responses were evaluated using ImmunoSpot (Cellular Technology Limited) as a measure of spot-forming cell counts (SFC) / 2x10. 5 The threshold for a positive reaction was defined as two or more spots. If the negative control (no peptide) had zero spots, the threshold for a positive reaction was defined as two or more spots. If the negative control had one or more spots, a positive reaction was defined as two times or more the number of spots. The results are shown in Figure 22. Administration of DET-PRX / SARS-CoV-2 spike protein increased IFN-γ secretion from T cells.

[0115] (Example 14) Fever-inducing test of DET-PRX In mice administered with lipid nanoparticles (LNP), a significant increase in skin surface temperature was observed after administration compared to before administration, but in mice administered with DET-PRX, no significant change in skin surface temperature was observed before and after administration.

[0116] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0117] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0118] This application claims priority based on Japanese Patent Application No. 2024-152734, filed September 4, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0119] <Appendix> Some or all of the above embodiments and examples can be described as in the appendix below, but are not limited to the following. <Pharmaceutical Composition 1> (Appendix 1) A pharmaceutical composition for use in inducing an antigen-specific immune response, comprising: a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap bonded to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH; and an antigen. (Appendix 2) The pharmaceutical composition according to Appendix 1, wherein the amine-containing group includes a secondary amine and an amino group. (Appendix 3) The pharmaceutical composition according to Appendix 2, wherein the amine-containing group is a diethylenetriamine group. <Pharmaceutical Composition 2> (Appendix 4) A pharmaceutical composition for use in inducing an antigen-specific immune response, comprising: a polyrotaxane having a plurality of macrocyclic molecules, an axon molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axon molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group having an amino group via an intracellularly degradable bond; and an antigen. (Appendix 5) The pharmaceutical composition according to Appendix 4, wherein the intracellularly degradable bond is a bond selected from carbamate, ketal, amide, ester, and disulfide. (Appendix 6) The pharmaceutical composition according to Appendix 5, wherein the intracellularly degradable bond is a disulfide bond. (Appendix 7) The pharmaceutical composition according to Appendix 6, wherein the group having an amino group via an intracellularly degradable bond is -(CH2)2-SS-(CH2)2-NH2. (Appendix 8) The pharmaceutical composition of any one of Appendices 1 to 7, wherein the cap is attached to the stalk molecule via an intracellularly degradable bond. (Appendix 9) The pharmaceutical composition of Appendices 8, wherein the intracellularly degradable bond is a bond selected from carbamate, ketal, amide, ester, and disulfide. (Appendix 10) The pharmaceutical composition of Appendices 9, wherein the intracellularly degradable bond is a carbamate bond.(Appendix 11) The pharmaceutical composition according to any one of Appendices 1 to 10, wherein the macrocyclic molecule is α-cyclodextrin. (Appendix 12) The pharmaceutical composition according to any one of Appendices 1 to 11, wherein the axis molecule is PEG. <Vaccine Composition> (Appendix 13) The pharmaceutical composition according to any one of Appendices 1 to 12, wherein the antigen is an antigen in a target disease. (Appendix 14) The pharmaceutical composition according to Appendices 13, wherein the antigen in the target disease is a tumor antigen in cancer, a viral antigen in a viral infection, a bacterial antigen in a bacterial infection, a fungal antigen in a fungal infection, or a parasitic antigen in a parasitic infection. (Appendix 15) The pharmaceutical composition according to Appendices 13 or 14, wherein the antigen in the target disease is a protein or a partial peptide thereof, a nucleic acid, or a sugar chain. (Appendix 16) The pharmaceutical composition according to Appendices 15, wherein the nucleic acid is an expression vector containing DNA or mRNA. (Appendix 17) A vaccine composition for preventing or treating the target disease, comprising the composition according to any one of Appendices 13 to 16. <Method for producing pharmaceutical composition 1> (Appendix 18) A method for producing a pharmaceutical composition for use in inducing an antigen-specific immune response, comprising mixing the polyrotaxane having a plurality of macrocyclic molecules, an axial molecule that penetrates rings of the macrocyclic molecules, and a cap that is bonded to an end of the axial molecule, wherein at least some of the macrocyclic molecules have an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, with an antigen to form a polyion complex. (Appendix 19) <Method for producing pharmaceutical composition 2> A method for producing a pharmaceutical composition for use in inducing an antigen-specific immune response, the method comprising mixing the polyrotaxane, which has a plurality of macrocyclic molecules, an axial molecule that penetrates rings of the macrocyclic molecules, and a cap that is bonded to an end of the axial molecule, and at least a portion of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least a portion of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond, with an antigen to form a polyion complex.<Method 1 for preventing or treating a target disease> (Appendix 20) A method for preventing or treating a target disease, comprising administering to a subject a polyrotaxane having a plurality of macrocyclic molecules, an axial molecule penetrating rings of the macrocyclic molecules, and a cap bonded to an end of the axial molecule, wherein at least a portion of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and an antigen for the target disease. <Method 2 for preventing or treating a target disease> (Appendix 21) A method for preventing or treating a target disease, comprising administering to a subject a polyrotaxane having a plurality of macrocyclic molecules, an axon molecule that penetrates the rings of the macrocyclic molecules, and a cap attached to an end of the axon molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond, and an antigen for the target disease. <Use 1 in preventing or treating a target disease> (Appendix 22) A polyrotaxane for use in the prevention or treatment of a target disease, having a plurality of macrocyclic molecules, an axon molecule that penetrates the rings of the macrocyclic molecules, and a cap attached to an end of the axon molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and an antigen for the target disease. <Use 2 in the prevention or treatment of a target disease> (Supplementary Note 23) A polyrotaxane for use in the prevention or treatment of a target disease, comprising a plurality of macrocyclic molecules, an axial molecule that passes through rings of the macrocyclic molecules, and a cap that is bonded to an end of the axial molecule, wherein at least some of the macrocyclic molecules comprise an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules comprise a group that has an amino group via an intracellularly degradable bond; and an antigen in the target disease.<Use 1 for producing a vaccine composition for preventing or treating a target disease> (Appendix 24) Use of a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates rings of the macrocyclic molecules, and a cap bonded to an end of the axle molecule, wherein at least a portion of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and an antigen for the target disease, for the production of a vaccine composition for preventing or treating a target disease. <Use 2 for producing a vaccine composition for preventing or treating a target disease> (Appendix 25) Use of a polyrotaxane having a plurality of macrocyclic molecules, an axial molecule that penetrates rings of the macrocyclic molecules, and a cap bonded to an end of the axial molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond, and an antigen for the target disease, for the production of a vaccine composition for preventing or treating a target disease.

[0120] As described above, the present disclosure provides a pharmaceutical composition for use in inducing an antigen-specific immune response, a method for producing the pharmaceutical composition, and a vaccine composition for preventing or treating a target disease, comprising the pharmaceutical composition. Therefore, the present disclosure is extremely useful, for example, in the pharmaceutical field.

Claims

1. A pharmaceutical composition for use in inducing an antigen-specific immune response, comprising: a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates the rings of the macrocyclic molecules, and a cap attached to the end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has one proton at neutral pH and two protons at acidic pH; and an antigen.

2. The pharmaceutical composition of claim 1, wherein the amine-containing group comprises a secondary amine and an amino group.

3. The pharmaceutical composition of claim 2, wherein the amine-containing group is a diethylenetriamine group.

4. A pharmaceutical composition for use in inducing an antigen-specific immune response, comprising: a polyrotaxane having a plurality of macrocyclic molecules, an axon molecule that penetrates the rings of the macrocyclic molecules, and a cap attached to the end of the axon molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond; and an antigen.

5. The pharmaceutical composition of claim 4, wherein the intracellularly degradable bond is a bond selected from carbamates, ketals, amides, esters, and disulfides.

6. The pharmaceutical composition according to claim 5, wherein the intracellularly degradable bond is a disulfide bond.

7. The pharmaceutical composition according to claim 6, wherein the group having an amino group via an intracellularly degradable bond is -(CH2)2-SS-(CH2)2-NH2.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the cap is attached to the stalk molecule via an intracellularly degradable bond.

9. The pharmaceutical composition of claim 8, wherein the intracellularly degradable bond is a bond selected from carbamates, ketals, amides, esters, and disulfides.

10. The pharmaceutical composition of claim 9, wherein the intracellularly degradable bond is a carbamate bond.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the macrocyclic molecule is α-cyclodextrin.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the stalk molecule is PEG.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the antigen is an antigen in a target disease.

14. The pharmaceutical composition according to claim 13, wherein the antigen in the target disease is a tumor antigen in cancer, a viral antigen in viral infection, a bacterial antigen in bacterial infection, a fungal antigen in fungal infection, or a parasitic antigen in parasitic infection.

15. The pharmaceutical composition according to claim 13 or 14, wherein the antigen in the target disease is a protein or a partial peptide thereof, a nucleic acid, or a sugar chain.

16. The pharmaceutical composition of claim 15, wherein the nucleic acid is an expression vector comprising DNA or mRNA.

17. A vaccine composition for preventing or treating the target disease, comprising the composition according to any one of claims 13 to 16.

18. A method for producing a pharmaceutical composition for use in inducing an antigen-specific immune response, the method comprising mixing the polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates the rings of the macrocyclic molecules, and a cap that is bonded to the end of the axle molecule, wherein at least some of the macrocyclic molecules have amine-containing groups that have a monovalent proton at neutral pH and a divalent proton at acidic pH, with an antigen to form a polyion complex.

19. A method for producing a pharmaceutical composition for use in inducing an antigen-specific immune response, the method comprising mixing the polyrotaxane having a plurality of macrocyclic molecules, an axial molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axial molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group having an amino group via an intracellularly degradable bond, with an antigen to form a polyion complex.

20. A method for preventing or treating a target disease, comprising administering to a subject a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule that penetrates the rings of the macrocyclic molecules, and a cap bonded to the end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and an antigen for the target disease.

21. A method for preventing or treating a target disease, the method comprising administering to a subject a polyrotaxane having a plurality of macrocyclic molecules, an axial molecule that penetrates the rings of the macrocyclic molecules, and a cap bonded to an end of the axial molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group that has a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group that has an amino group via an intracellularly degradable bond, and an antigen for the target disease.

22. A polyrotaxane for use in the prevention or treatment of a target disease, comprising a plurality of macrocyclic molecules, an axle molecule passing through the rings of the macrocyclic molecules, and a cap attached to the end of the axle molecule, wherein at least some of the macrocyclic molecules comprise an amine-containing group having one proton at neutral pH and two protons at acidic pH; and an antigen in the target disease.

23. A polyrotaxane for use in the prevention or treatment of a target disease, comprising a plurality of macrocyclic molecules, an axle molecule passing through the rings of the macrocyclic molecules, and a cap attached to the end of the axle molecule, wherein at least some of the macrocyclic molecules comprise an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules comprise a group having an amino group via a bond that is degradable within a cell; and an antigen in the target disease.

24. Use of a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group having one proton at neutral pH and two protons at acidic pH, for the manufacture of a vaccine composition for the prevention or treatment of a target disease; and an antigen for the target disease.

25. Use of a polyrotaxane having a plurality of macrocyclic molecules, an axle molecule penetrating the rings of the macrocyclic molecules, and a cap attached to an end of the axle molecule, wherein at least some of the macrocyclic molecules contain an amine-containing group having a monovalent proton at neutral pH and a divalent proton at acidic pH, and at least some of the macrocyclic molecules contain a group having an amino group via an intracellularly degradable bond, for the production of a vaccine composition for the prevention or treatment of a target disease; and an antigen for the target disease.

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