Method for producing dendritic cell vaccine

Abstract

To provide a production method of a dendritic cell vaccine which improves intake of an antigen into dendritic cells.SOLUTION: The production method of a dendritic cell vaccine comprises mixing dendritic cells, a polymer compound having in a side chain a group represented by the general formula (1) in the figure, and an antigen. (In the formula, X1 represents a residue obtained by removing a terminal amino group and a terminal carboxyl group from a cell-penetrating peptide; and X2 represents a hydroxy group, an amino group, a C1-4 alkoxyl group, or a benzyloxy group.)SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing a dendritic cell vaccine, etc. [Background technology]

[0002] The three main methods of cancer treatment are surgery, radiation therapy, and chemotherapy. Chemotherapy includes chemotherapy using anticancer drugs, hormone therapy (endocrine therapy) using hormones, and molecular targeted therapy using molecularly targeted drugs.

[0003] In recent years, immunotherapy has been attracting attention as a new cancer treatment method (Patent Document 1). Among immunotherapies, dendritic cell vaccine therapy, which uses dendritic cells that specifically recognize cancer cells, is expected to be the fourth cancer treatment method because it has few side effects. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2018-083849 Summary of the Invention [Problem to be solved by the invention]

[0005] When producing dendritic cells that specifically recognize cancer cells (dendritic cell vaccines), cancer antigens must be taken up by dendritic cells, but one of the challenges with dendritic cell vaccine therapy is the low permeability (uptake) of the cancer antigens into dendritic cells. The objective of the present disclosure is to improve the uptake of antigens into dendritic cells in the production of dendritic cell vaccines. [Means for solving the problem]

[0006] The present inventors have found that the use of a polymer compound having a membrane-permeable peptide in its side chain improves the uptake of antigens into dendritic cells, and have made further improvements.

[0007] The present disclosure encompasses, for example, the subject matter described in the following sections: Section 1. A method for producing a dendritic cell vaccine, comprising mixing dendritic cells, a polymer compound having a group represented by the following general formula (1) in its side chain, and an antigen: [ka] (In the formula, X 1 indicates the residues obtained by removing the terminal amino group and terminal carboxyl group from the membrane-permeable peptide, and X 2 represents a hydroxyl group, an amino group, an alkoxyl group having 1 to 4 carbon atoms, or a benzyloxy group. Section 2. The membrane-permeable peptide is a peptide having an arginine oligomer in which 7 to 30 arginines are peptide-bonded, a peptide having an amino acid sequence of GRKKRRQRRRPPQ, a peptide having an amino acid sequence of TRQARRNRRRRWRERQR, a peptide having an amino acid sequence of RRRRNRTRRNRRRVR, a peptide having an amino acid sequence of TRRQRTRRARRNR, a peptide having an amino acid sequence of KLTRAQRRAAARKNKRNTR, a peptide having an amino acid sequence of RQIKIWFQNRRMKWKK, a peptide having an amino acid sequence of KMTRAQRRAAARRNRWTAR Item 2. A method for producing a dendritic cell vaccine according to Item 1, wherein the membrane-permeable peptide is at least one selected from the group consisting of a peptide having the amino acid sequence RQIKIWFQNRRMKWKK, a peptide having the amino acid sequence NAKTRRHERRRKLAIER, a peptide having the amino acid sequence DAATATRGRSAASRPTERPRAPARSASRPDDPVD, a peptide having the amino acid sequence GWTLNSAGYLLGKINLKALAALAKKIL, and a peptide having the amino acid sequence AGYLLGKINLKALAALAKKIL. Section 3. Item 3. The method for producing a dendritic cell vaccine according to Item 1 or 2, wherein the trunk polymer of the polymer compound having a group represented by general formula (1) in a side chain is a vinyl hydrophilic polymer. Section 4. Item 4. The method for producing a dendritic cell vaccine according to any one of Items 1 to 3, wherein the antigen is a cancer antigen and the dendritic cell vaccine is a cancer vaccine. Section 5. A dendritic cell vaccine comprising a dendritic cell, a polymer compound having a group represented by the following general formula (1) in its side chain, and an antigen: [ka] (In the formula, X 1 indicates the residues obtained by removing the terminal amino group and terminal carboxyl group from the membrane-permeable peptide, and X 2 represents a hydroxyl group, an amino group, an alkoxyl group having 1 to 4 carbon atoms, or a benzyloxy group. Section 6. An adjuvant for dendritic cell vaccine production, comprising a polymer compound having a group represented by the following general formula (1) in its side chain: [ka] (In the formula, X 1 indicates the residues obtained by removing the terminal amino group and terminal carboxyl group from the membrane-permeable peptide, and X 2 represents a hydroxyl group, an amino group, an alkoxyl group having 1 to 4 carbon atoms, or a benzyloxy group. [Effects of the Invention]

[0008] A method for producing a dendritic cell vaccine that improves antigen uptake into dendritic cells is provided. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the results of a test of antigen uptake by dendritic cells. [Figure 2] 1 shows the results of comparing the effects of dendritic cell vaccine administration on tumor volume. [Figure 3] 1 shows the results of comparing the effects of dendritic cell vaccine administration on the number of IFN-γ positive cells. DETAILED DESCRIPTION OF THE INVENTION

[0010] Each embodiment included in the present disclosure will be described in further detail below. The present disclosure encompasses a method for producing a dendritic cell vaccine, which comprises mixing dendritic cells, a polymer compound having a group represented by general formula (1) described below in its side chain, and an antigen. In this specification, the method for producing the dendritic cell vaccine may be referred to as the "method for producing the dendritic cell vaccine of the present disclosure." Furthermore, in this specification, the dendritic cell vaccine produced by the method for producing the dendritic cell vaccine may be referred to as the "dendritic cell vaccine of the present disclosure."

[0011] The dendritic cells used in the present disclosure are not particularly limited and can be selected appropriately depending on the subject of administration, etc. For example, the dendritic cells used in the present disclosure are preferably mammalian-derived dendritic cells. Examples of mammals include humans and non-human mammals such as rats, mice, rabbits, cows, pigs, dogs, cats, sheep, and monkeys. Furthermore, the dendritic cells used in the present disclosure may be, for example, monocyte-derived dendritic cells obtained from a subject to be administered with the dendritic cell vaccine of the present disclosure, or may be commercially available dendritic cells.

[0012] The polymer compound used in the present disclosure has a group represented by the following general formula (1) on the side chain.

[0013] [ka]

[0014] In general formula (1), X 1 indicates residues remaining after removing the terminal amino group and terminal carboxyl group from the membrane-permeable peptide.

[0015] At least one of the amino acids constituting the membrane-permeable peptide residue is preferably a basic amino acid, and the basic amino acid may be either L- or D-isomer.

[0016] Examples of basic amino acids include arginine, ornithine, lysine, hydroxylysine, and histidine. Among these, guanidino group-containing amino acids are preferred, and arginine is more preferred. The ratio of basic amino acids to all amino acids constituting the membrane-permeable peptide residue is preferably 50% or more, more preferably 70% or more, on a molar basis. Of the amino acids constituting the membrane-permeable peptide residue, amino acids other than basic amino acids are preferably neutral amino acids. In this specification, when referring to amino acids, it means α-amino acids unless otherwise specified.

[0017] The number of amino acids constituting the membrane-permeable peptide residue can be, for example, about 5 to 40. The upper or lower limit of this range may be, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39. More specifically, it may be, for example, 6 to 39.

[0018] Examples of membrane-permeable peptides include hydrophilic basic peptides such as peptides having an arginine oligomer in which 7 to 30 arginines are peptide-bonded, peptides having an amino acid sequence of GRKKRRQRRRPPQ (e.g., HIV-1 Tat: SEQ ID NO: 1), peptides having an amino acid sequence of TRQARRNRRRRWRERQR (e.g., HIV-1 Rev: SEQ ID NO: 2), peptides having an amino acid sequence of RRRRNRTRRNRRRVR (e.g., FHV Coat: SEQ ID NO: 3), peptides having an amino acid sequence of TRRQRTRRARRNR (e.g., HTLV-II Rex: SEQ ID NO: 4), and peptides having an amino acid sequence of KLTRAQRRAAARKNKRNTR (e.g., CCMV Gag: SEQ ID NO: 5); peptides having an amino acid sequence of RQIKIWFQNRRMKWKK (e.g., Antennapedia: SEQ ID NO: 6), and peptides having an amino acid sequence of KMTRAQRRAAARRNRWTAR (e.g., BMW Examples of such peptides include amphipathic basic peptides such as Gag (SEQ ID NO: 7), a peptide having the amino acid sequence RQIKIWFQNRRMKWKK (e.g., penetratin (SEQ ID NO: 8)), a peptide having the amino acid sequence NAKTRRHERRRKLAIER (e.g., P22N (SEQ ID NO: 9)), and a peptide having the amino acid sequence DAATATRGRSAASRPTERPRAPARSASRPDDPVD (e.g., VP22 (SEQ ID NO: 10)); and hydrophobic basic peptides such as a peptide having the amino acid sequence GWTLNSAGYLLGKINLKALAALAKKIL (e.g., transportan (SEQ ID NO: 11)) and a peptide having the amino acid sequence AGYLLGKINLKALAALAKKIL (e.g., TP-10 (SEQ ID NO: 12)). The membrane-permeable peptide may be the above-mentioned arginine oligomer or a peptide consisting of the amino acid sequence shown in SEQ ID NOs: 1 to 12. Among them, the membrane-permeable peptide is preferably a hydrophilic basic peptide, more preferably a peptide having an arginine oligomer, and even more preferably a peptide consisting of an arginine oligomer. The number of repeating arginine residues in the arginine oligomer is preferably 7 to 20, more preferably 7 to 15, and even more preferably 7 to 10.

[0019] In general formula (1), X 2 represents a hydroxyl group, an amino group, an alkoxyl group having 1 to 4 carbon atoms, or a benzyloxy group. Examples of the alkoxyl group having 1 to 4 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, a 1-methylpropoxy group, and a t-butoxy group. X 2 As the alkyl group, a hydroxyl group, an amino group, a t-butoxy group, or a benzyloxy group is preferred, a hydroxyl group or an amino group is more preferred, and an amino group is most preferred. In the polymer compound having a group represented by general formula (1) in a side chain, when a plurality of groups represented by general formula (1) are present, X 1 and X 2 may be the same or different.

[0020] In this specification, the main chain portion of a polymer compound having a group represented by general formula (1) in its side chain is referred to as a backbone polymer.

[0021] The trunk polymer is not particularly limited, but is preferably a hydrophilic polymer. Here, a hydrophilic polymer refers to a water-soluble polymer or a polymer that swells in water. In this specification, a water-soluble polymer refers to a polymer that is uniformly dissolved in water at 25°C under normal pressure in an amount of 0.1% by mass or more.

[0022] Examples of hydrophilic polymers include polysaccharides or modified polysaccharides such as guar gum, agarose, mannan, glucomannan, polydextrose, lignin, chitin, chitosan, carrageenan, pullulan, chondroitin sulfate, cellulose, hemicellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, starch, cationic starch, and dextrin; water-soluble proteins or water-soluble polypeptides such as albumin, casein, gelatin, polyglutamic acid, and polylysine; poly(meth)acrylic acid, poly(hydroxyethyl acrylate), poly(meth)acrylamide, and poly(N-vinyl acetate). Examples of suitable vinyl hydrophilic polymers include vinyl-based hydrophilic polymers such as acrylate, polyvinylpyrrolidone, polyvinyl alcohol, poly(2-aminoethyl (meth)acrylate), (meth)acrylic acid / acrylamide copolymer, (meth)acrylic acid / N-isopropylacrylamide copolymer, (meth)acrylic acid / N-vinylacetamide copolymer, (meth)acrylic acid / maleic acid copolymer, (meth)acrylic acid / fumaric acid copolymer, ethylene / maleic acid copolymer, isobutylene / maleic acid copolymer, styrene / maleic acid copolymer, alkyl vinyl ether / maleic acid copolymer, and alkyl vinyl ether / fumaric acid copolymer; and water-soluble polyurethanes. In this specification, (meth)acrylic acid means acrylic acid and / or methacrylic acid.

[0023] As the trunk polymer, a polymer having a carboxyl group is preferred, a hydrophilic polymer having a carboxyl group is more preferred, a copolymer of a monomer having a carboxyl group and a monomer not having a carboxyl group is even more preferred, and a (meth)acrylic acid / N-vinylacetamide copolymer is most preferred, because this facilitates grafting of a membrane-permeable peptide group onto the trunk polymer.

[0024] The ratio of the number of monomer units having a carboxyl group to the number of monomer units constituting the trunk polymer is, for example, preferably about 5 to 80%, more preferably about 10 to 60%, and may be, for example, about 20 to 40%.

[0025] The number of membrane-permeable peptide residues in a polymer compound having a group represented by general formula (1) in its side chain is preferably about 0.001 to 0.9, more preferably about 0.005 to 0.8, and most preferably about 0.01 to 0.7, relative to the number of monomer units (monosaccharide units in the case of polysaccharides or modified polysaccharides, and amino acid units in the case of water-soluble proteins or water-soluble polypeptides) constituting the trunk polymer. For example, it may be about 0.05 to 0.5.

[0026] The weight-average molecular weight of the trunk polymer is, for example, preferably 10 kDa to 10,000 kDa, more preferably 30 kDa to 5,000 kDa, and most preferably 100 kDa to 3,000 kDa. In this specification, the term "weight-average molecular weight" refers to the weight-average molecular weight determined by GPC analysis using an aqueous solvent, and refers to the pullulan-equivalent weight-average molecular weight when the trunk polymer is a polysaccharide, a modified polysaccharide, or a water-soluble protein, or to the polyethylene glycol (PEG) or polyethylene oxide (PEO)-equivalent weight-average molecular weight when the trunk polymer is a vinyl-based hydrophilic polymer.

[0027] The method for producing a polymer compound having a group represented by general formula (1) in its side chain is not particularly limited, and various methods widely known to those skilled in the art can be applied. For example, the polymer compound may be produced by polymerizing a polymerizable monomer having a group represented by general formula (1), or by introducing a group represented by general formula (1) into a backbone polymer. For example, the polymer compound can be obtained by peptide reaction of the amino group of a membrane-permeable peptide with the carboxyl group of the backbone polymer. The reaction between the carboxyl group and the amino group can be carried out by a known method, such as succinimide esterification of the carboxyl group with N-hydroxysuccinimide followed by reaction with the amino group.

[0028] The antigen used in the present disclosure is preferably, for example, a cancer antigen. The cancer antigen is not particularly limited as long as it is cancer cell-specific, and examples thereof include proteins, peptides, DNA, and RNA. Furthermore, the antigen used in the present disclosure can also be, for example, a lysate of cancer cells or tissues. These can be used alone or in combination of two or more. The cancer antigen may be, for example, derived from the subject to which the dendritic cell vaccine of the present disclosure is administered, or may be artificially synthesized.

[0029] The method for mixing dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen is not particularly limited, as long as the antigen is taken up by dendritic cells. For example, a method for mixing dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen in an aqueous medium may be used.

[0030] Examples of aqueous media include media commonly used in cell culture; dendritic cell culture medium; distilled water; and isotonic solutions such as physiological saline and aqueous glucose solutions.

[0031] In the step of mixing dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen, the concentration of dendritic cells in the mixture is not particularly limited and can be set appropriately. For example, 1.0 × 10 4 ~2.0×10 7 The cell density can be adjusted to about 1.0×10 5 ~5×10 6 It may be on the order of cells / ml.

[0032] In the step of mixing dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen, the concentration of the polymer compound having a group represented by general formula (1) in its side chain in the mixture can be, for example, about 0.1 μg / ml to 1 mg / ml, or about 5 μg / ml to 75 μg / ml.

[0033] In the step of mixing dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen, the concentration of the antigen in the mixture can be appropriately set depending on the type of antigen. For example, it can be about 0.5 μg / ml to 10 mg / ml, or it can be about 5 μg / ml to 100 μg / ml. For example, when a cell lysate is used as an antigen, the ratio of the number of dendritic cells to the number of cells used to prepare the cell lysate can be, for example, about 1:0.5 to 1:5.

[0034] The temperature at which dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen are mixed is not particularly limited as long as the dendritic cells can survive, and can be, for example, about 30 to 40°C, or about 34 to 38°C.

[0035] The time for mixing dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen is not particularly limited as long as the antigen is taken up by the dendritic cells, and may be, for example, about 15 minutes to 24 hours, or may be, for example, about 30 minutes to 3 hours.

[0036] The step of mixing dendritic cells, a polymer compound having a group represented by general formula (1) in its side chain, and an antigen may be performed by, for example, shaking, stirring, etc. Methods known in the art can be used for shaking and stirring, and any conditions can be adopted.

[0037] The method for producing a dendritic cell vaccine of the present disclosure may further comprise the step of washing the dendritic cells. The washing method may be any method known in the art, and any conditions may be employed.

[0038] The method for producing a dendritic cell vaccine of the present disclosure may further include a step of concentrating dendritic cells. The concentration method may be a method known in the art, and any conditions may be employed.

[0039] The dendritic cell vaccine of the present disclosure can be preferably used as a cancer vaccine, and therefore can be preferably used in the treatment of cancer.

[0040] The amount of dendritic cells contained in the dendritic cell vaccine of the present disclosure is not particularly limited and can be set as appropriate.

[0041] The dendritic cell vaccine of the present disclosure may or may not contain a polymer compound having a group represented by general formula (1) in its side chain.

[0042] In the dendritic cell vaccine of the present disclosure, the antigen is preferably incorporated into the dendritic cell. However, in the dendritic cell vaccine of the present disclosure, the antigen may be present outside the dendritic cell.

[0043] The dendritic cell vaccine of the present disclosure may further comprise other components. Examples of such other components include various pharmaceutically acceptable carriers (e.g., solvents, dispersants, isotonicity agents, chelating agents, stabilizers, pH adjusters, antiseptics, preservatives, antioxidants, solubilizers, thickeners, etc.). The dendritic cell vaccine of the present disclosure may also comprise an adjuvant. These may be used alone or in combination of two or more.

[0044] The formulation form of the dendritic cell vaccine of the present disclosure is not particularly limited, and examples include oral administration forms such as tablets, pills, capsules, powders, granules, liquids, syrups, and jellies; and parenteral administration forms such as injections, drip infusions, ointments, poultices, patches, nasal drops, inhalants, and suppositories. Of these, injections are preferred.

[0045] The dendritic cell vaccine of the present disclosure can be administered, for example, orally or parenterally (e.g., intravenously, intraarterially, intramuscularly, subcutaneously, intraperitoneally, rectally, transdermally, topically, etc.), with subcutaneous administration being preferred.

[0046] The dendritic cell vaccine of the present disclosure can be administered to, for example, mammals, including humans and non-human mammals such as rats, mice, rabbits, cows, pigs, dogs, cats, sheep, and monkeys.

[0047] The dose of the dendritic cell vaccine of the present disclosure to be administered (ingested) is not particularly limited, and is determined depending on the age, sex, severity of symptoms, administration method, etc. of the subject to be administered.

[0048] The present disclosure also encompasses a dendritic cell vaccine production adjuvant containing a polymer compound having a group represented by the following general formula (1) in its side chain. In this specification, the dendritic cell vaccine production adjuvant may be referred to as the "dendritic cell vaccine production adjuvant of the present disclosure." [ka]

[0049] The above description can be applied to the polymer compound having a group represented by general formula (1) on the side chain.

[0050] The content of the polymer compound having a group represented by general formula (1) in the side chain in the dendritic cell vaccine production adjuvant of the present disclosure is not particularly limited and can be set appropriately.

[0051] The dendritic cell vaccine production adjuvant of the present disclosure can further contain other components in addition to the polymer compound having a group represented by general formula (1) in its side chain. Examples of such other components include various pharmaceutically acceptable carriers (e.g., solvents, dispersants, isotonicity agents, chelating agents, stabilizers, pH adjusters, antiseptics, preservatives, antioxidants, solubilizers, thickeners, etc.). These can be used alone or in combination of two or more.

[0052] The form of the dendritic cell vaccine production adjuvant of the present disclosure is not particularly limited, and may be, for example, a liquid (e.g., a solution, a suspension, etc.), a paste, or a solid (e.g., a powder, etc.).

[0053] The dendritic cell vaccine production adjuvant of the present disclosure can promote antigen uptake by dendritic cells.

[0054] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." Furthermore, the present disclosure includes all arbitrary combinations of the constituent elements described in this specification.

[0055] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to specify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein. [Example]

[0056] The contents of the present disclosure will be specifically explained using the following experimental examples. However, the present disclosure is not limited to these in any way. In the following, unless otherwise specified, experiments were performed under atmospheric pressure and room temperature conditions. Furthermore, unless otherwise specified, "%" means "% by mass."

[0057] Experimental Example 1: Antigen uptake test by DC2.4 Cell lines and media used The C57BL / 6 mouse-derived dendritic cell line, Dendritic Cell 2.4 (DC2.4), was kindly provided by Kenneth L. Rock of the University of Assachusetts Medical School. DC2.4 were cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% fetal bovine serum (FBS) (Sigma Life Science), 1% non-essential amino acid solution (Nacalai Tesque), 1% sodium pyruvate solution (Nacalai Tesque), 1% penicilin streptomycin (Nacalai Tesque), and 0.004% 2-mercaptoethanol (FUJIFILM). However, for antigen transfection into DC2.4, RPMI medium containing only FBS but other reagents was used (FBS(-) RPMI medium).

[0058] Experimental procedure 1.0×10 5DC2.4 cells were seeded onto 24-well plates and cultured in RPMI medium for 1 day. The culture supernatant was removed and FBS-free RPMI medium was added. Ovalbumin-fluorescein conjugate (OVA) (Merck) was used as an antigen. OVA at 10 μg / ml (indicated as OVA in Fig. 1), a mixture of OVA at 10 μg / ml and Keyhole Limpet Hemocyanin (KLH) (Wako) at 50 μg / ml (indicated as OVA+KLH in Fig. 1), a mixture of OVA at 10 μg / ml and the membrane-permeable peptide-immobilized polymer HA-G4R8 at 50 μg / ml (indicated as OVA+HA in Fig. 1), and a mixture of OVA at 10 μg / ml and the membrane-permeable peptide-immobilized polymer VP-R8 at 50 μg / ml (indicated as OVA+VP in Fig. 1) were added in triplicate (n = 3 per group) to well plates cultured with DC2.4 cells. HA-G4R8 and VP-R8 were kindly provided by Professor Nobuyuki Sakuma of Setsunan University. Their structural formulas are shown below. The weight-average molecular weight of the hyaluronic acid in the main chain of the membrane-permeable peptide-immobilized polymer HA-G4R8 was 27 kDa. The weight-average molecular weight of the membrane-permeable peptide-immobilized polymer HA-G4R8 calculated from the molecular weight of the membrane-permeable peptide moiety and its immobilization rate was 114 kDa. The weight-average molecular weight of the backbone polymer (PNVA-co-AA: acrylic acid / N-vinylacetamide copolymer) in the membrane-permeable peptide-immobilized polymer VP-R8 was 350 kDa. The weight-average molecular weight of the membrane-permeable peptide-immobilized polymer VP-R8 calculated from the molecular weight of the membrane-permeable peptide, D-octaarginine, and its immobilization rate was 1100 kDa (calculated value).

[0059] [ka]

[0060] [ka]

[0061] The well plate was incubated at 37°C for 1 hour. The supernatant was removed and the plate was washed twice with Dulbecco's Phosphate Buffered Saline (D-PBS). Accutase™ (Nacalai Tesque) was added and the plate was incubated at 37°C for 5 minutes. The cell clumps were loosened by pipetting, transferred to a 15 ml tube, and centrifuged at 4°C, 2000 rpm, and 5 minutes at 4°C. After washing twice with D-PBS, the cells were suspended in 300 μl of D-PBS and the mean fluorescence intensity (MFI) was measured using Guava® easyCyte™ Flow Cytometers (Luminex Corporation). The results are shown in Figure 1.

[0062] As shown in FIG. 1, it was confirmed that antigen (OVA) uptake by DC2.4 was significantly increased when a mixture of OVA and the membrane-transportable polymer VP-R8 was added.

[0063] Experimental Example 2: DC2.4 vaccine in vivo experiment Animals and cell lines used Female C57BL / 6 mice used in the experiment were purchased from Charles River. DC2.4 were cultured in the same manner as in Experimental Example 1. EL4 cells, a malignant lymphoma cell line derived from C57BL / 6N mice, were used and cultured in RPMI medium containing 10% FBS.

[0064] Tumor cell lysate preparation EL4 cells cultured in a petri dish were washed twice with Dulbecco's Phosphate Buffered Saline (D-PBS). 0.25 w / v% trypsin-1 mmol / l EDTA 4Na solution (Wako) was added and incubated at 37°C for 5 minutes. The cells were detached from the petri dish, washed twice with D-PBS, and then 2 × 10 7The cells were suspended in D-PBS at 0.1% cells / mL. The cell suspension was immersed in liquid nitrogen for 3 minutes and then thawed at 42°C. This process was repeated five times. After centrifugation at 10,000 rpm at 4°C for 10 minutes, the supernatant was collected. The collected supernatant was filtered through a 0.22 μm Millex-GV Filter Unit (Merck), and the resulting solution was collected as the lysate.

[0065] Tumor inoculation into mice The supernatant of the cultured EL4 cells was removed, and the cells were washed once with D-PBS. Trypsin was added and the cells were incubated. The number of cells was counted, and the cell suspension was 1.4 × 10 5 The concentration was adjusted to cells / 70 μl. 70 μl was dispensed into 1.5 ml tubes, and the same amount of Corning® Matrigel® (Corning) was added. Mice were anesthetized with sevoflurane (Wako), and the cell solution was gently mixed thoroughly with a pipette. 1.4 × 10 cells were then injected into the right dorsal region. 5 The cells were administered subcutaneously at a concentration of 100 μl.

[0066] DC2.4 vaccine preparation and administration The supernatant of the cultured DC2.4 cells was removed, and 3 ml of FBS(-) RPMI medium was added. The DC2.4 cells were supplemented with lysate (shown as DC2.4+EL4Lysate in Fig. 2), a mixture of lysate and Keyhole Limpet Hemocyanin (KLH) (Wako) 50 μg / ml (shown as DC2.4+EL4Lysate+KLH in Fig. 2), or a mixture of lysate and VP-R8 12.5 μg / ml (shown as DC2.4+EL4Lysate+VP in Fig. 2). The lysate was prepared from DC2.4 (1.0 × 10 6Extraction was performed from three times the number of cells (1.0 × 10 cells / mouse). After addition, the mixture was incubated at 37°C for 1 hour with shaking. The supernatant was removed, and the mixture was washed twice with Dulbecco's Phosphate Buffered Saline (D-PBS). Accutase™ (Nacalai Tesque) was added, and the mixture was incubated at 37°C for 5 minutes. The cell clumps were loosened by pipetting to form single cells, transferred to a 15 ml tube, centrifuged at 4°C, 2000 rpm for 5 minutes, and washed twice with D-PBS. 1.0 × 10 cells / mouse were used. 6 The cells were suspended in D-PBS in 100 μl for administration to prepare a DC2.4 vaccine.

[0067] The day of tumor administration was designated day 0, and each DC2.4 vaccine was administered subcutaneously around the inguinal lymph nodes of mice on days 7, 10, and 13. In addition to DC2.4+EL4Lysate, DC2.4+EL4Lysate+VP, and DC2.4+EL4Lysate+KLH, PBS alone (shown as PBS in Figure 2) and DC2.4 cells cultured without the addition of lysate, KLH, or VP (shown as DC2.4 in Figure 2) were also administered subcutaneously around the inguinal lymph nodes of mice.

[0068] The tumor diameter was measured and the average tumor volume was calculated. The tumor volume was calculated as follows: (tumor volume) = (longest diameter of tumor) × (shortest diameter of tumor). 2 The calculation was performed using a ratio of 1 / 2. The results are shown in Figure 2.

[0069] As shown in Figure 2, when a suspension (DC2.4 vaccine) prepared by adding a mixture of DC2.4 cells lysate and VP-R8 to mice was administered, it was confirmed that the increase in tumor volume was suppressed.

[0070] Experimental Example 3: ICCS experiment using spleen cells extracted from treated mice Animals and cell lines used The same animals and cell lines as in Experimental Example 2 were used.

[0071] Tumor cell lysate preparation Lysates were prepared in the same manner as in Experimental Example 2.

[0072] DC2.4 vaccine preparation and administration DC2.4 vaccines (DC2.4+EL4Lysate, DC2.4+EL4Lysate+VP, DC2.4+EL4Lysate+KLH) suspended in D-PBS were prepared using the same method as in Experimental Example 2 and administered subcutaneously three times around the inguinal lymph nodes of mice (the first administration was designated day 0, and administrations were performed on days 0, 3, and 6). In addition to DC2.4+EL4Lysate, DC2.4+EL4Lysate+VP, and DC2.4+EL4Lysate+KLH, PBS alone (shown as PBS in Figure 3) and DC2.4 cells cultured without the addition of lysate, KLH, or VP (shown as DC2.4 in Figure 3) were also administered subcutaneously around the inguinal lymph nodes of mice.

[0073] Spleen collection Two weeks after the last administration (day 20), the mice were euthanized, and the spleens were collected and mashed using a cell scraper (Nihon Genetics) and sterile tweezers (As One). The spleen cells were filtered through a cell strainer (Corning). After centrifugation at 2000 rpm at 4°C for 5 minutes, the supernatant was removed. 1 ml of Red Blood Cell Lysis Buffer (Sigma Aldrich) was added and incubated at room temperature for 2 minutes. 5 ml of RPMI medium was added, mixed, and centrifuged at 2000 rpm at 4°C for 5 minutes. The supernatant was aspirated, washed twice with D-PBS, and suspended in 1 ml of RPMI medium. 100 μl of the cell suspension was added to a 96-well plate, and 2.0 × 10 cells of EL4 cells cultured in the wells were added. 5 The cells / sample were treated with 50 μg / ml of mitomycin C, and 100 μl of the treated cells was added for antigen stimulation.

[0074] ICCS experiment BD GolgiStop™ (BD Biosciences) was added to the antigen-stimulated splenocytes and cultured for 12 hours. The well plate was centrifuged (4°C, 2500 rpm, 5 min) and the supernatant was removed. The cells were washed once with 100 μl of staining buffer (1% FBS, 0.09% sodium azide in PBS) (4°C, 2500 rpm, 5 min) and the supernatant was removed. 1 μg of CD16 / 32 antibody (Bio Legend) was added in 100 μl of staining buffer and allowed to stand on ice for 15 minutes. The cells were washed once with 100 μl of staining buffer (4°C, 2500 rpm, 5 min) and the supernatant was removed. A sample of splenocytes not subjected to antibody reaction was prepared as a negative control (NC). 100 μl of FITC-anti-mouse CD4 (BD Biosciences), APC-anti-mouse CD8 (BD Biosciences), and PerCP-anti-mouse CD3 (BD ​​Biosciences) antibodies in staining buffer was added and incubated on ice for 30 minutes in the dark. The cells were washed once with 100 μl of staining buffer (4°C, 2500 rpm, 5 minutes), and the supernatant was removed. The cells were incubated on ice for 20 minutes in the dark with 100 μl of Fixation / Permeabilization solution (BD Biosciences). Then, the cells were washed twice with 1× BD Perm / Wash Buffer (BD Biosciences) (4°C, 3500 rpm, 5 minutes). The cells were incubated with PE-anti-mouse IFN-γ antibody (BD Biosciences) [0.5 μg in 100 μL Perm / Wash Buffer (final concentration 5 μg / ml)] on ice for 30 minutes in the dark. Then, they were washed twice with 1× BD Perm / Wash Buffer (4°C, 3500 rpm, 5 min). The cells were suspended in 300 μL staining buffer, and the mean fluorescence intensity (MFI) was measured using Guava® easyCyte™ Flow Cytometers (Luminex Corporation). The results are shown in Figure 3.

[0075] As shown in Figure 3, when DC2.4 vaccine, which was prepared by adding a mixture of DC2.4 cells and VP-R8 to the culture, was administered to mice, the number of IFN-γ-positive CD4 and CD8 cells tended to increase in response to stimulation with the antigen protein (EL4 cells), suggesting that the vaccine can induce antigen-specific cellular immunity.

Claims

1. A method for producing a dendritic cell vaccine, comprising mixing dendritic cells, a polymer compound having a group represented by the following general formula (1) in a side chain, and an antigen, wherein the trunk polymer of the polymer compound is a (meth)acrylic acid / N-vinylacetamide copolymer: 【Chemistry 1】 (In the formula, X １ represents residues obtained by removing the terminal amino group and the terminal carboxyl group from a membrane-permeable peptide, and the membrane-permeable peptide is a peptide consisting of an arginine oligomer having 7 to 10 repeating arginine residues; X ２ represents an amino group.)

2. The method for producing a dendritic cell vaccine according to claim 1 , wherein the antigen is a cancer antigen and the dendritic cell vaccine is a cancer vaccine.

3. A dendritic cell vaccine comprising: a dendritic cell; a polymer compound having a group represented by the following general formula (1) at its side chain; and an antigen, wherein the trunk polymer of the polymer compound is a (meth)acrylic acid / N-vinylacetamide copolymer: 【Chemistry 2】 (In the formula, X １ represents residues obtained by removing the terminal amino group and the terminal carboxyl group from a membrane-permeable peptide, and the membrane-permeable peptide is a peptide consisting of an arginine oligomer having 7 to 10 repeating arginine residues; X ２ represents an amino group.)

4. A dendritic cell vaccine production adjuvant comprising a polymer compound having a group represented by the following general formula (1) in a side chain, wherein the trunk polymer of the polymer compound is a (meth)acrylic acid / N-vinylacetamide copolymer: 【Transformation 3】 (In the formula, X １ represents residues obtained by removing the terminal amino group and the terminal carboxyl group from a membrane-permeable peptide, and the membrane-permeable peptide is a peptide consisting of an arginine oligomer having 7 to 10 repeating arginine residues; X ２ represents an amino group.)

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