Cancer vaccine

Abstract

To provide a technique that improves an antitumor effect of a tumor antigen epitope.SOLUTION: A cancer vaccine comprises a peptide comprising linkage of two or more amino acid sequences constituting a tumor antigen epitope, and a polymer compound comprising a group represented by the general formula (1) in a side chain (where X1 denotes a residue formed by excluding, from membrane permeable peptide, a terminal amino group and a terminal carboxyl group, and X2 denotes 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 cancer vaccines and the like.

Background Art

[0002] Conventionally, as cancer peptide vaccines, short peptide vaccines consisting of around 10 amino acids have been mainstream. However, since short peptides directly bind to MHC class I expressed on the surface of dendritic cells, T cells, and B cells, the problem has been that they cannot induce potent cytotoxic T lymphocytes (CTLs).

[0003] In order to induce potent CTLs, since it is necessary for tumor antigens to be taken up and processed inside dendritic cells, which are antigen-presenting cells, and for the epitopes, which are fragments thereof, to be expressed on the surface together with MHC Class I, in recent years, long peptide vaccines of around 30 amino acids combining multiple tumor antigen epitopes have attracted attention. However, in the case of long peptide vaccines, the problem has been how to efficiently incorporate antigen proteins into dendritic cells.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0006] The objective is to provide technology that enhances the antitumor effect of tumor antigen epitopes. [Means for solving the problem]

[0007] The inventors have discovered that antitumor immunity can be efficiently induced by using in combination a peptide in which multiple amino acid sequences constituting a tumor antigen epitope are linked with a polymer compound having a membrane-permeable peptide as a side chain, and have further refined this method.

[0008] This disclosure includes, for example, the following subjects: Section 1. A peptide consisting of two or more linked amino acid sequences constituting a tumor antigen epitope, and A cancer vaccine comprising a polymer compound having a group represented by the following general formula (1) in its side chain. [ka] (In the formula, X 1 This indicates the residue obtained by removing the terminal amino group and terminal carboxyl group from a membrane-permeable peptide, X 2 (This represents a hydroxyl group, an amino group, an alkoxyl group having 1 to 4 carbon atoms, or a benzyloxy group.) Section 2. A transmucosal cancer vaccine, as described in item 1. Section 3. A nasal cancer vaccine, as described in item 1 or 2. Section 4. The cancer vaccine according to any one of claims 1 to 3, wherein the number of amino acids constituting the peptide, which is formed by the linkage of two or more amino acid sequences constituting the tumor antigen epitope, is 15 to 100. Section 5. The amino acid sequence constituting the tumor antigen epitope is, VYDFAFRDL (Sequence ID 1), DKKQRFHNI (Sequence ID 2), YRDGNPYAV (Sequence ID 3), LCIVYRDGNPYAVCD (Sequence ID 4), KLPQLCTEL (Sequence ID 5), KLPQLCTEV (Sequence ID 6), KISEYRHYC (Sequence ID 7), ISEYRHYV (Sequence ID 8), QQYNKPLCDL(SEQ ID NO: 9), QLYNKPLCDV (Sequence ID 10), QLLRREVYDFAFRDL (Sequence ID 11), VYDFAFRDLC (SEQ ID NO: 12), RAHYNIVTF (SEQ ID NO: 13), LCVQSTHVD (Sequence ID 14), AEPDRAHYNIVTFCC (Sequence ID 15), YMLDLQPET (Sequence ID 16), YMLDLQPEV (Sequence ID 17), TLHEYMLDL (SEQ ID NO: 18), TLHEYMLDV (Sequence ID 19), RTLEDLLMGT (SEQ ID NO: 20), RTLEDLLMGV (SEQ ID NO: 21), and QAEPDRAHVYNIVTFCCKCD (Sequence ID 22) At least one selected from the group consisting of A cancer vaccine as described in any one of items 1 to 4. Section 6. The amino acid sequence constituting the tumor antigen epitope is, RMFPNAPYL (Sequence ID 23), CMTWNQMNL (Sequence ID 24), SENHTAPI (sequence number 25), KRYFKLSHLQMHSRKH (Sequence ID 26), SLGEQQYSV (Sequence ID 27), YMFPNAPYL (Sequence ID 28), CYTWNQMNL (Sequence ID 29), RWPSCQKKF (Sequence ID 30), RSDELVRHHNMHQRNMTKL (Sequence ID 31), ALLPAVPSL (SEQ ID NO: 32), WAPVLDFAPPGASAYGSL (Sequence ID 33), EQCLSAFTLHFSGQFTG (Sequence ID 34), FRGIQDVRRVSGVAPTLVR (Sequence ID 35), RVPGVAPTL (Sequence ID 36), PGCNKRYFKLSHLQMHSRKHTG (Sequence ID 37), SGQARMFPNAPYLPSCLES (SEQ ID NO: 38), and SGQAYMFPNAPYLPSCLES(Sequence ID 39) At least one selected from the group consisting of A cancer vaccine as described in any of items 1 to 4. Section 7. The cancer vaccine according to any one of claims 1 to 6, wherein the peptide, which is formed by linking two or more amino acid sequences constituting the tumor antigen epitope, is a peptide formed by linking two or more amino acid sequences constituting the tumor antigen epitope directly or via a linker. Section 8. The aforementioned membrane-permeable peptide A peptide having an arginine oligomer in which 2 to 30 arginine molecules are linked by peptide bonds, a peptide having the amino acid sequence GRKKRRQRRRPPQ, A peptide having the amino acid sequence TRQARRNRRRRWRERQR, A peptide having the amino acid sequence RRRRNRTRRNRRRVR, A peptide having the amino acid sequence TRRQRTRRARRNR, A peptide having the amino acid sequence KLTRAQRRAAARKNKRNTR, A peptide having the amino acid sequence KMTRAQRRAAARRNRWTAR, 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 cancer vaccine according to any one of claims 1 to 7, which is at least one membrane-permeable peptide selected from the group consisting of peptides having the amino acid sequence AGYLLGKINLKALAALAKKIL. Section 9. A peptide formed by linking two or more amino acid sequences that constitute the tumor antigen epitope is VYDFAFRDLRRDKKQRFHNIRRRAHYNIVTFRRLCVQSTHVD (Sequence ID 40), or A cancer vaccine as described in any one of items 1 to 8, which is a peptide consisting of the amino acid sequence RMFPNAPYLRRCMTWNQMNLRRSENHTAPIRRKRYFKLSHLQMHSRKH (Sequence ID 41). Section 10. The cancer vaccine according to any one of claims 1 to 9, wherein the stem polymer of a polymer compound having a group represented by the general formula (1) in its side chain is a vinyl hydrophilic polymer or a polysaccharide. Section 11. A polymer compound having a group represented by the general formula (1) in its side chain is, A cancer vaccine described in any one of items 1 to 10, which is a compound represented by the following general formula (2). [ka] (In the formula, a, x, and y represent integers greater than or equal to 1, and a:(x+y)=10:1~10. However, the units of a, x, and y are randomly combined. In the formula, X 1 and X 2 (This is the same as above.) Section 12. The cancer vaccine described in item 11, wherein x and y are integers satisfying x:y=0 to 40:10. Section 13. The cancer vaccine according to any one of items 1 to 12, wherein the polymer compound having the group represented by the general formula (1) in its side chain is a polymer compound having a weight-average molecular weight of 10 kDa to 30,000 kDa. [Effects of the Invention]

[0009] A cancer vaccine demonstrating excellent antitumor effects will be provided. [Brief explanation of the drawing]

[0010] [Figure 1] This shows the average tumor volume of mice. [Figure 2] This shows the average tumor volume of mice. [Figure 3] This shows the survival rate of mice. [Figure 4] A picture of a mouse tumor is shown. [Figure 5] This shows the weight changes of the mice. [Figure 6] The results of measuring the percentage of IFN-γ-producing CD8 T cells are shown. [Figure 7] The results of measuring the percentage of HPV E7 RAHYNIVTF49-57 specific CD8 T cells are shown. [Figure 8] The results of measuring cytotoxic activity against TC-1 cells are shown. [Figure 9] This shows the average tumor volume of mice. [Figure 10] This shows the survival rate of mice. [Figure 11] This shows the survival rate of mice. [Modes for carrying out the invention]

[0011] The embodiments included in this disclosure are described in further detail below. The cancer vaccines included in this disclosure include peptides consisting of two or more linked amino acid sequences constituting a tumor antigen epitope, and macromolecular compounds having a group represented by general formula (1), described later, in their side chains. In this specification, such cancer vaccines may be referred to as "cancer vaccines of this disclosure." In this specification, "cancer vaccine" means a vaccine administered to cancer patients and can be used for the treatment of cancer. In other words, in this specification, cancer vaccines can also be referred to as cancer treatment agents.

[0012] Tumor antigen epitopes are not particularly limited, as long as they can induce anti-tumor immunity. Examples include well-known tumor antigen epitopes.

[0013] The tumor antigen epitope may be, for example, a CD8 epitope (CD8-positive cytotoxic T cell recognition epitope) or a CD4 epitope (CD4-positive helper T cell recognition epitope).

[0014] The number of amino acids constituting a tumor antigen epitope can be, for example, around 8 to 20. When the tumor antigen epitope is presented on an MHC Class I molecule, the number of amino acids constituting the tumor antigen epitope may be, for example, around 8 to 10. Furthermore, when the tumor antigen epitope is presented on an MHC Class II molecule, the number of amino acids constituting the tumor antigen epitope may be, for example, around 15 to 20.

[0015] The tumor antigens to be targeted are not particularly limited. For example, they include human papillomavirus (HPV type 16) E6 protein, E7 protein; Wilms Tumor 1 (WT1) protein; MUC1; LMP2; EGFRvIII; HER-2 / neu; Idiotype; MAGE A3; p53 nonmutant; NY-ESO-1; PSMA; GD2; CEA; MelanA / MART1; neoantigen, etc.

[0016] When the tumor antigen to be targeted is the E6 protein of human papillomavirus (HPV type 16), the amino acid sequences constituting known tumor antigen epitopes include VYDFAFRDL 49-57 (CD8 epitope sequence: SEQ ID NO: 1), DKKQRFHNI 127-135 (CD8 epitope sequence: SEQ ID NO: 2), YRDGNPYAV 61-69 (CD8 epitope: SEQ ID NO: 3), LCIVYRDGNPYAVCD 57-71 (CD8 epitope: SEQ ID NO: 4), KLPQLCTEL 11-19 (CD8 epitope: SEQ ID NO: 5), KLPQLCTEV 11-19 (CD8 epitope: SEQ ID NO: 6), KISEYRHYC 72-80 (CD8 epitope: SEQ ID NO: 7), ISEYRHYV 72-80 (CD8 epitope: SEQ ID NO: 8), QQYNKPLCDL 90-99 (CD8 epitope: SEQ ID NO: 9), QLYNKPLCDV 90-99 (CD8 epitope: SEQ ID NO: 10), QLLRREVYDFAFRDL 43-57 (CD8 epitope: SEQ ID NO: 11), VYDFAFRDLC 49-58 (CD8 epitope: SEQ ID NO: 12) Examples include (Non-Patent Documents 1-3). In this specification, the numbers listed together with the amino acid sequence in the amino acid sequence showing the tumor antigen epitope of the E6 protein indicate the position in the amino acid sequence of the human E6 protein. In particular, the amino acid sequence that constitutes the tumor antigen (E6 protein) epitope is VYDFAFRDL (SEQ ID NO: 1), or DKKQRFHNI (Sequence ID 2) is preferred. If the target tumor antigen is the E7 protein of human papillomavirus (HPV type 16), the amino acid sequence constituting a known tumor antigen epitope is: RAHYNIVTF 49-57 (CD8 epitope sequence: SEQ ID NO: 13) LCVQSTHVD 67-75 (CD8 epitope sequence: SEQ ID NO: 14) AEPDRAHYNIVTFCC 45-59 (CD8 epitope: SEQ ID NO: 15) YMLDLQPET 11-19 (CD8 epitope: SEQ ID NO: 16) YMLDLQPEV 11-19 (CD8 epitope: SEQ ID NO: 17) TLHEYMLDL 7-15 (CD8 epitope: SEQ ID NO: 18) TLHEYMLDV 7-15 (CD8 epitope: SEQ ID NO: 19) RTLEDLLMGT 77-86 (CD8 epitope: SEQ ID NO: 20) RTLEDLLMGV 77-86 (CD8 epitope: SEQ ID NO: 21) QAEPDRAHVYNIVTFCCKCD 44-62 (CD8 epitope sequence: Sequence ID 22) Examples include (Non-Patent Documents 1-3). In this specification, the numbers listed together with the amino acid sequence in the amino acid sequence showing the tumor antigen epitope of the E7 protein indicate the position in the amino acid sequence of the human E7 protein. In particular, among the amino acid sequences that constitute the tumor antigen (E7 protein) epitope, RAHYNIVTF (SEQ ID NO: 13), or LCVQSTHVD (Sequence ID 14) is preferred. For example, a peptide consisting of two or more linked amino acid sequences that constitute a tumor antigen epitope is VYDFAFRDL (Sequence ID 1), DKKQRFHNI (Sequence ID 2), RAHYNIVTF (SEQ ID NO: 13), and It is preferable to include at least one selected from the group consisting of LCVQSTHVD (Sequence ID 14), more preferably at least two, even more preferably at least three, and particularly preferable to include all four.

[0017] If the target tumor antigen is the WT1 protein, the amino acid sequence constituting a known tumor antigen epitope is: RMFPNAPYL 126-134 (CD8 epitope: SEQ ID NO: 23) CMTWNQMNL 235-243 (CD8 epitope: SEQ ID NO: 24) SENHTAPI 273-280 (CD8 epitope: SEQ ID NO: 25) KRYFKLSHLQMHSRKH 332-337 (CD4 epitope: SEQ ID NO: 26) SLGEQQYSV 187-195 (CD8 epitope: SEQ ID NO: 27) YMFPNAPYL 126-134 (CD8 epitope: SEQ ID NO: 28) CYTWNQMNL 235-243 (CD8 epitope: SEQ ID NO: 29) RWPSCQKKF 417-425 (CD8 epitope: SEQ ID NO: 30) RSDELVRHHNMHQRNMTKL 427-445 (CD4 epitope: SEQ ID NO: 31) ALLPAVPSL 10-18 (CD8 epitope: SEQ ID NO: 32) WAPVLDFAPPGASAYGSL 35-52 (CD4 epitope: SEQ ID NO: 33) EQCLSAFTLHFSGQFTG 86-102 (CD4 epitope: SEQ ID NO: 34) FRGIQDVRRVSGVAPTLVR 294-312 (CD4 epitope: SEQ ID NO: 35) RVPGVAPTL 302-310 (CD8 epitope: SEQ ID NO: 36) PGCNKRYFKLSHLQMHSRKHTG 328-349 (CD4 epitope: SEQ ID NO: 37) SGQARMFPNAPYLPSCLES 122-140 (CD4 epitope: SEQ ID NO: 38) SGQAYMFPNAPYLPSCLES 122-140 (CD4 epitope: SEQ ID NO: 39) Examples include (Patent Document 1, Non-Patent Documents 4 and 5). In this specification, the numbers listed together with the amino acid sequence in the amino acid sequence representing the tumor antigen epitope of WT1 indicate the position in the amino acid sequence of human WT1. In particular, the amino acid sequence that constitutes the tumor antigen (WT1 protein) epitope is RMFPNAPYL (SEQ ID NO: 23), CMTWNQMNL (Sequence ID 24), SENHTAPI (sequence number 25), or KRYFKLSHLQMHSRKH (Sequence ID 26) is preferred. For example, a peptide consisting of two or more linked amino acid sequences that constitute a tumor antigen epitope is RMFPNAPYL (Sequence ID 23), CMTWNQMNL (Sequence ID 24), SENHTAPI (sequence number 25), and It is preferable to include at least one selected from the group consisting of KRYFKLSHLQMHSRKH (Sequence ID 26), more preferably at least two, even more preferably at least three, and particularly preferable to include all four.

[0018] The amino acid sequence constituting the tumor antigen epitope may be an amino acid sequence in which one or more amino acids are deleted, substituted, or added compared to the amino acid sequence constituting the known tumor antigen epitope described above, as long as it can induce antitumor immunity. The number of deleted, substituted, or added amino acids may be, for example, about 1 to 3, or about 1 to 2.

[0019] Techniques for introducing mutations such as deletions, substitutions, or additions of amino acids into specific amino acid sequences are publicly known in the art and can be performed using any method. For example, these can be done using restriction enzyme therapy, treatment with exonucleases or DNA ligases, site-directed mutagenesis, random mutagenesis, etc.

[0020] In a peptide formed by linking two or more amino acid sequences constituting a tumor antigen epitope, it is preferable that the number of amino acid sequences constituting the tumor antigen epitope to be linked is two or more. The lower limit of the number of linked tumor antigen epitopes may be, for example, three or more, or four or more. The upper limit of the number of amino acid sequences constituting the tumor antigen epitopes to be linked is not particularly limited and may be, for example, 10 or less, 8 or less, 6 or less, or 4 or less.

[0021] In a peptide formed by linking two or more amino acid sequences that constitute a tumor antigen epitope, the amino acid sequences constituting the tumor antigen epitope may be one type or two or more types. For example, in a peptide formed by linking two or more amino acid sequences that constitute a tumor antigen epitope, the amino acid sequences constituting each tumor antigen epitope may all be different.

[0022] In a peptide formed by linking two or more amino acid sequences constituting a tumor antigen epitope, the tumor antigen epitope may be a CD8 epitope, a CD4 epitope, or a combination thereof. For example, if a peptide formed by linking two or more amino acid sequences constituting a tumor antigen epitope contains both a CD8 epitope and a CD4 epitope, the ratio of CD8 epitopes to CD4 epitopes may be, for example, approximately 4:1 to 1:4, or approximately 3:1 to 1:3.

[0023] In a peptide formed by linking two or more amino acid sequences that constitute a tumor antigen epitope, the tumor antigen from which the tumor antigen epitope originates may be one type or two or more types.

[0024] In a peptide formed by linking two or more amino acid sequences constituting a tumor antigen epitope, the order in which the amino acid sequences constituting the tumor antigen epitope are linked is not particularly limited and can be set as appropriate.

[0025] In a peptide comprising two or more linked amino acid sequences constituting a tumor antigen epitope, the amino acid sequences constituting each tumor antigen epitope can be linked directly or via a linker.

[0026] Examples of linkers include linkers consisting of two or more amino acids. Examples of amino acids that constitute the linker include amino acids with basic side chains such as arginine, lysine, and histidine; and amino acids with non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine. These can be used individually or in combination of two or more. Amino acids with basic side chains are preferred as linker components, and arginine is more preferred. The number of amino acids that make up the linker may be, for example, 1 to 5, 1 to 3, or 1 to 2. Among these, arginine oligomers are preferred as linkers. Arginine oligomers with 2 to 5 arginine repeats are preferred, and arginine dimers (RR) are more preferred.

[0027] The number of amino acids constituting a peptide, which is formed by linking two or more amino acid sequences that make up a tumor antigen epitope, can be, for example, around 15 to 100. For example, it may also be around 30 to 90. A peptide formed by linking two or more amino acid sequences that constitute a tumor antigen epitope may, for example, be the tumor antigen protein itself.

[0028] Peptides consisting of two or more linked amino acid sequences that constitute a tumor antigen epitope include, specifically, VYDFAFRDLRRDKKQRFHNIRRRAHYNIVTFRRLCVQSTHVD (Sequence ID 40: HPV E6E7) Examples include RMFPNAPYLRRCMTWNQMNLRRSENHTAPIRRKRYFKLSHLQMHSRKH (Sequence ID 41:WT1).

[0029] The concentration of the peptide, which consists of two or more linked amino acid sequences constituting a tumor antigen epitope, contained in the cancer vaccine of this disclosure is not particularly limited and can be set as appropriate. For example, it can be approximately 0.05 to 50 μg / μl.

[0030] Peptides consisting of two or more linked amino acid sequences constituting a tumor antigen epitope can be produced using general chemical synthesis methods for peptides (e.g., liquid-phase and solid-phase methods) based on the known amino acid sequence information described above. Alternatively, peptides consisting of two or more linked amino acid sequences constituting a tumor antigen epitope can be produced using genetic engineering techniques with the polynucleotide encoding the peptide.

[0031] The polymer compounds used in this disclosure have a group represented by the following general formula (1) in their side chains.

[0032] [ka]

[0033] In general formula (1), X 1 This indicates the residues obtained by removing the terminal amino group and terminal carboxyl group from a membrane-permeable peptide.

[0034] Preferably, at least one of the amino acids constituting the membrane-permeable peptide residue is a basic amino acid. Furthermore, the basic amino acid may be either the L-form or the D-form.

[0035] Examples of basic amino acids include arginine, ornithine, lysine, hydroxylysine, and histidine, among which guanidino group-containing amino acids are preferred, and arginine is more preferred. The ratio of basic amino acids to the total amino acids constituting the membrane-permeable peptide residue is preferably 50% or more on a molar basis, and more preferably 70% or more. Among 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 the term "amino acid" is used, it means α-amino acid unless otherwise specified.

[0036] The number of amino acids constituting a membrane-permeable peptide residue can be, for example, 2 to 40. The upper or lower limit of this range may be, for example, 3, 4, 5, 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, 3 to 39.

[0037] Examples of membrane-permeable peptides include hydrophilic basic peptides such as peptides having arginine oligomers in which 2 to 30 arginine molecules are peptide-bonded, peptides having the amino acid sequence GRKKRRQRRRPPQ (e.g., HIV-1 Tat: SEQ ID NO: 42), peptides having the amino acid sequence TRQARRNRRRRWRERQR (e.g., HIV-1 Rev: SEQ ID NO: 43), peptides having the amino acid sequence RRRRNRTRRNRRRVR (e.g., FHV Coat: SEQ ID NO: 44), peptides having the amino acid sequence TRRQRTRRARRNR (e.g., HTLV-II Rex: SEQ ID NO: 45), peptides having the amino acid sequence KLTRAQRRAAARKNKRNTR (e.g., CCMV Gag: SEQ ID NO: 46); and peptides having the amino acid sequence KMTRAQRRAAARRNRWTAR (e.g., BMW Examples include amphiphilic basic peptides such as Gag (SEQ ID NO: 47), peptides having the amino acid sequence RQIKIWFQNRRMKWKK (e.g., penetratin: SEQ ID NO: 48), peptides having the amino acid sequence NAKTRRHERRRKLAIER (e.g., P22N: SEQ ID NO: 49), and peptides having the amino acid sequence DAATATRGRSAASRPTERPRAPARSASRPDDPVD (e.g., VP22: SEQ ID NO: 50); and hydrophobic basic peptides such as transportan (SEQ ID NO: 51) and TP-10 (SEQ ID NO: 52). Membrane-permeable peptides may also consist of the arginine oligomers described above or the amino acid sequences shown in SEQ ID NOs: 42-52. Among these, hydrophilic basic peptides are preferred as membrane-permeable peptides, peptides having arginine oligomers are more preferred, and peptides consisting of arginine oligomers are even more preferred. The number of arginine repeats in the arginine oligomer is preferably 2 to 20, more preferably 2 to 15, and preferably 4 to 10. For example, it may be 6 to 10.

[0038] In general formula (1), X 2 This represents a hydroxyl group, an amino group, an alkoxyl group having 1 to 4 carbon atoms, or a benzyloxy group. Examples of alkoxy groups having 1 to 4 carbon atoms include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, 1-methylpropoxy, and t-butoxy groups. 2 The preferred groups are hydroxyl groups, amino groups, t-butoxy groups, and benzyloxy groups, with hydroxyl groups and amino groups being more preferred, and amino groups being the most preferred. In a polymer compound having a group represented by general formula (1) in its side chain, if there are multiple groups represented by general formula (1), the X of each group represented by general formula (1) 1 and X 2 They may be the same or they may be different.

[0039] 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 the stem polymer.

[0040] The stem polymer is not particularly limited, but is preferably a hydrophilic polymer. Here, a hydrophilic polymer means a water-soluble polymer or a polymer that swells in water. In this specification, a water-soluble polymer means a polymer that dissolves uniformly in water at 25°C under normal pressure in an amount of 0.1% by mass or more.

[0041] Examples of hydrophilic polymers include 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, dextrin, and hyaluronic acid; water-soluble proteins or polypeptides such as albumin, casein, gelatin, polyglutamic acid, and polylysine; poly(meth)acrylic acid, poly(hydroxyethyl acrylate), poly(meth)acrylamide, and poly-N-vinylacetate. Examples include vinyl-based hydrophilic polymers such as amides, 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, alkyl vinyl ether / fumaric acid copolymer, etc.; and water-soluble polyurethanes. In this specification, (meth)acrylic acid means acrylic acid and / or methacrylic acid.

[0042] Since it facilitates the grafting of membrane-permeable peptide groups onto the stem polymer, the stem polymer is preferably a polymer having a carboxyl group, more preferably a hydrophilic polymer having a carboxyl group, even more preferably a copolymer of a monomer having a carboxyl group and a monomer not having a carboxyl group, and most preferably a (meth)acrylic acid / N-vinylacetamide copolymer.

[0043] For example, the group represented by general formula (1) may be bonded to the stem polymer via a linker. When the group represented by general formula (1) is bonded to the stem polymer via a linker, hyaluronic acid is preferred as the stem polymer. Examples of linkers include linkers consisting of two or more amino acids. The amino acids that make up the linker are not particularly limited, but examples include glycine, alanine, phenylalanine, and leucine. These can be used individually or in combination of two or more. Glycine and alanine are preferred as the amino acids that make up the linker, with glycine being more preferred. The number of amino acids that make up the linker may be, for example, around 2 to 4, or around 2 to 3. Among these, glycine oligomers are preferred as linkers. Specifically, glycine oligomers having 2 to 4 glycine repeats are preferred.

[0044] The ratio of monomer units having a carboxyl group to the total number of monomer units constituting the main polymer is preferably about 10-50%, and more preferably about 10-40%.

[0045] In a polymer compound having a group represented by general formula (1) in its side chain, the number of membrane-permeable peptide residues 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 constituting the stem polymer (monosaccharide units in the case of polysaccharides, and amino acid units in the case of water-soluble proteins or water-soluble polypeptides). For example, it may be about 0.05 to 0.5.

[0046] The weight-average molecular weight of the stem polymer is preferably, for example, 10 kDa to 10,000 kDa, more preferably 10 kDa to 5,000 kDa, and most preferably 10 kDa to 3,000 kDa. The weight-average molecular weight of the stem polymer may also be, for example, 100 kDa to 3,000 kDa, or 200 kDa to 3,000 kDa. In this specification, weight-average molecular weight refers to the weight-average molecular weight obtained when GPC analysis is performed using an aqueous solvent. If the stem polymer is a polysaccharide or a water-soluble protein, it refers to the weight-average molecular weight in terms of pullulan; if the stem polymer is a vinyl-based hydrophilic polymer, it refers to the weight-average molecular weight in terms of polyethylene glycol (PEG) or polyethylene oxide (PEO).

[0047] The weight-average molecular weight of a polymer compound having a group represented by general formula (1) in its side chain is preferably, for example, 10 kDa to 30,000 kDa, more preferably 10 kDa to 20,000 kDa, and most preferably 10 kDa to 10,000 kDa. Furthermore, if the polymer compound having a group represented by general formula (1) in its side chain is a compound represented by general formula (2), which will be described later, the weight-average molecular weight is preferably, for example, 10 kDa to 10,000 kDa, and more preferably 10 kDa to 1,000 kDa. If the polymer compound having a group represented by general formula (1) in its side chain is a compound represented by general formula (2), which will be described later, the weight-average molecular weight may be, for example, 500 kDa to 1,000 kDa.

[0048] Examples of polymer compounds having a group represented by general formula (1) in its side chain include the compound represented by the following general formula (2). [ka] In the formula, a, x, and y represent integers greater than or equal to 1, and a:(x+y)=10:1 to 10. However, the units of a, x, and y are randomly combined. In the formula, X 1 and X 2 The same applies as stated above. In this specification, the following structure may be referred to as unit a. [ka] Furthermore, in this specification, the following structures may be referred to as units of x. [ka] Furthermore, in this specification, the following structure may be referred to as a unit of y. [ka] In general formula (2), a:(x+y) is preferably 10:1 to 10, more preferably 10:2 to 8, and even more preferably 10:3 to 6.

[0049] In general formula (2), x and y are preferably integers satisfying x:y = 0 to 40:10. For example, x:y = 0 to 20:10 is preferred, and 0 to 10:10 is more preferred. For example, x:y = 1 to 10:10 is also acceptable.

[0050] In general formula (2), y / (a+x+y) 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. For example, it may be about 0.05 to 0.5.

[0051] A particularly favorable example is the general formula (2), where a:x:y = 70:10~20:10~20, etc.

[0052] 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, it 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 stem polymer. For example, it can be obtained by a peptide reaction between the carboxyl group of the stem polymer and the amino group of a membrane-permeable peptide. The reaction between the carboxyl group and the amino group can be carried out using known methods, such as esterifying the carboxyl group with N-hydroxysuccinimide and then reacting it with the amino group. More specifically, it can be produced by following the methods described in, for example, Japanese Patent Publication No. 5808082, Japanese Patent Publication No. 5281358, Japanese Patent Publication No. 6692051, Japanese Patent Publication No. 6880526, etc.

[0053] The concentration of the macromolecular compound having a group represented by general formula (1) in its side chain, contained in the cancer vaccine of this disclosure, is not particularly limited and can be set as appropriate. For example, it can be set to about 0.1 to 100 μg / μl.

[0054] The mass ratio of the peptide, which consists of two or more linked amino acid sequences constituting a tumor antigen epitope, and the macromolecular compound having a group represented by general formula (1) in its side chain, contained in the cancer vaccine of this disclosure is not particularly limited and can be set as appropriate. For example, it can be approximately 1:0.1 to 1:1000.

[0055] The cancer vaccine of this disclosure may further contain other components. Examples of such other components include various pharmaceutically acceptable carriers (e.g., solvents, dispersants, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity modifiers, etc.). These may be used individually or in combination of two or more. The cancer vaccine of this disclosure may or may not contain an adjuvant.

[0056] The formulation of the cancer vaccine disclosed herein is not particularly limited and may include, for example, oral preparations such as tablets, pills, capsules, powders, granules, liquids, syrups, jellies, and films; and parenteral preparations such as injections, intravenous infusions, ointments, poultices, patches, nasal sprays, inhalants, and suppositories. Among these, nasal sprays are preferred.

[0057] The method for producing the cancer vaccine described herein is not particularly limited and can be produced by methods known in the art. For example, it can be produced by mixing a peptide consisting of two or more linked amino acid sequences constituting a tumor antigen epitope, a polymer compound having a group represented by general formula (1) in its side chain, and other components as needed.

[0058] The cancer vaccine described herein can be administered by, for example, transmucosal administration, intravenous administration, transarterial administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, etc. Among these, transmucosal administration (e.g., transnasal, translungal, transoral (e.g., translingual), transgastrointestinal (e.g., transrectal), transvaginal, transocular, etc.) is preferred, transnasal, translungal, and transoral administration are more preferred, and transnasal administration is even more preferred. In other words, the cancer vaccine disclosed herein can be suitably used as a transmucosal cancer vaccine, and more suitably as a transnasal cancer vaccine. Since most conventional vaccines are administered by subcutaneous injection, transnasal cancer vaccines, in particular, can reduce patient discomfort and burden as a method of administration.

[0059] Examples of target animals for the cancer vaccine described herein include mammals. Examples of mammals include humans; rats, mice, rabbits, cattle, pigs, dogs, cats, sheep, monkeys, and other non-human mammals.

[0060] The dosage of the cancer vaccine disclosed herein is not particularly limited and will be determined based on the age, sex, severity of symptoms, and method of administration of the recipient.

[0061] The cancer vaccine disclosed herein exerts its antitumor effect by inducing cellular immune responses, such as the induction of tumor antigen-specific cytotoxic T cells. In other words, unlike conventional prophylactic vaccines that induce humoral immunity, the cancer vaccine disclosed herein can be suitably used in the treatment of cancer. Furthermore, the cancer vaccine disclosed herein can be suitably used to suppress the growth of cancer (tumors) or to shrink cancer (tumors). The cancers (tumors) targeted by the cancer vaccines disclosed herein are not particularly limited and are determined by the tumor antigen epitopes used. Examples include lung cancer, breast cancer, esophageal cancer, stomach cancer, colorectal cancer, anal cancer, pancreatic cancer, tongue cancer, pharyngeal cancer, thyroid cancer, cervical cancer, uterine cancer, ovarian cancer, childhood cancer, brain tumors, osteosarcoma, kidney cancer, urothelial carcinoma, and prostate cancer. The cancer vaccines described herein are not limited to patients with lung cancer, breast cancer, esophageal cancer, stomach cancer, colorectal cancer, anal cancer, pancreatic cancer, tongue cancer, pharyngeal cancer, thyroid cancer, cervical cancer, uterine cancer, ovarian cancer, childhood cancer, brain tumors, osteosarcoma, kidney cancer, urothelial carcinoma, prostate cancer, etc.

[0062] In this specification, the term "comprising" includes both "consisting essentially of" and "consisting of." Furthermore, this disclosure encompasses all combinations of the constituent elements described herein.

[0063] Furthermore, the various characteristics (properties, structure, function, etc.) described in each embodiment of this disclosure above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein. [Examples]

[0064] The contents of this disclosure will be specifically explained using the following experimental examples. However, this disclosure is not limited to these examples. Unless otherwise specified below, the experiments were conducted under atmospheric pressure and room temperature conditions. Unless otherwise specified, "%" means "mass%".

[0065] Study using HPV Preparation of purified long peptides Purified HPV E6E7 long peptide (amino acid sequence (SEQ ID NO: 40): VYDFAFRDLRRDKKQRFHNIRRRAHYNIVTFRRLCVQSTHVD) was synthesized using Eurofins Genomics. This long peptide contains the following H2-D proteins found in HPV type 16 E6 and E7 proteins. b and H2-K b Constraining CD8 epitope sequence [E6(VYDFAFRDL 49-57 (Sequence ID 1); DKKQRFHNI 127-135 (Sequence No. 2)) and E7(RAHYNIVTF) 49-57 (Sequence ID 13); LCVQSTHVD 67-75 The peptide sequence (SEQ ID NO: 14)) is linked by two arginine (R) residues. A 100 mg / ml stock solution of this long peptide was prepared using dimethyl sulfoxide (DMSO). The above HPV E7 RAHYNIVTF 49-57 Similarly, (SEQ ID NO: 13) was synthesized using Eurofins Genomics, and a 10 mg / ml stock solution was prepared using dimethyl sulfoxide (DMSO).

[0066] Membrane-permeable peptide-immobilized polymer VP-R8 The structural formula of VP-R8 is shown below. VP-R8 was manufactured by preparing the amount of monomer raw materials used, etc., according to the description in Japanese Patent Publication No. 5808082. The weight-average molecular weight of the core polymer (PNVA-co-AA: acrylic acid / N-vinylacetamide copolymer) of the membrane-permeable peptide immobilized polymer VP-R8 is 280 kDa. The weight-average molecular weight of the membrane-permeable peptide immobilized polymer VP-R8, calculated from the weight-average molecular weight of PNVA-co-AA, the molecular weight of D-octaarginine used as the membrane-permeable peptide, and its immobilization rate, was 925 kDa (calculated value).

[0067] [ka] Membrane-permeable peptide-immobilized polymer VP-R8 (a:x:y=70:15:15)

[0068] Animal studies using tumor-bearing mice 1 x 10 5 Each of the TC-1 cells was subcutaneously transplanted into 30 six-week-old female C57BL / 6 mice along with Matrigel (Corning) to induce tumor formation. TC-1 cells are a mouse lung cancer cell line that stably expresses HPV 16 E6 and E7 proteins through recombinant DNA. TC-1 cells were donated by Dr. TC Wu of Johns Hopkins University, USA. TC-1 cells were cultured in RPMI-1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, 100 U / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum. Seven days after tumor transplantation, the mice were randomly divided into the following six treatment groups (n=5). Treatment group 1-1: HPV E6E7 long peptide (10 μg) + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment groups 1-2: HPV E6E7 long peptide (100 μg) + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment groups 1-3: HPV E7 short peptide (RAHYNIVTF) 49-57 (10 μg) + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment groups 1-4: HPV E6E7 long peptide (10 μg) + physiological saline in 20 μL (intranasal administration) Treatment groups 1-5: Physiological saline + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment groups 1-6: HPV E6E7 long peptide (100 μg) + incomplete Freund's adjuvant (IFA) in 100 μL (intraperitoneal administration)

[0069] Treatment groups 1-1 and 1-2 received HPV E6E7 long peptide, while treatment group 1-3 received HPV E7 RAHYNIVTF. 49-57 60 μg, 600 μg, or 60 μg of the peptide stock solution were mixed with physiological saline to make 58 μL of each solution. 58 μL of VP-R8 solution (10 mg / mL) was added to each of these solutions to make a total of 116 μL. After thorough mixing, the solutions were allowed to stand at room temperature for at least 15 minutes to be used as the administration solution. For treatment groups 1-4, a stock solution of 60 μg of HPV E6E7 long peptide was mixed with physiological saline to make 58 μL. 58 μL of physiological saline was added to this solution to make a total of 116 μL, which was thoroughly mixed and allowed to stand at room temperature for at least 15 minutes to prepare the administration solution. For administration groups 1-5, 58 μL of physiological saline and 58 μL of VP-R8 solution (10 mg / mL) were added to a total volume of 116 μL. After thorough mixing, the solution was allowed to stand at room temperature for at least 15 minutes to prepare the administration solution. For treatment groups 1-6, 800 μg of HPV E6E7 long peptide was mixed with physiological saline to make 400 μL, which was then mixed with 400 μL of incomplete Freund's adjuvant (IFA, Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare the administration solution.

[0070] After classifying the mice on day 7 post-tumor transplantation, 20 μL of each administration solution was administered via micropipette into one nostril of the mice under anesthesia for administration groups 1-1, 1-2, 1-3, 1-4, and 1-5. For administration groups 1-6, 100 μL of the administration solution was administered intraperitoneally under anesthesia. Administration was performed on days 7, 14, 21, and 28 post-tumor transplantation. Tumor diameter was measured daily, and tumor volume was calculated using the following formula: [Tumor volume (mm²)]3 ) = major axis × (minor axis) 2 [×0.5]. Figure 1 shows the average tumor volume of treatment groups 1-1, 1-3, 1-4, and 1-5. Figure 2 shows the average tumor volume of treatment groups 1-2, 1-5, and 1-6, and Figure 3 shows the survival rate of the mice. Figure 4 shows representative mouse tumor images after administration.

[0071] As shown in Figures 1 and 4, intranasal administration of a mixture of HPV E6E7 long peptide and VP-R8 (administration group 1-1) significantly suppressed the increase in tumor volume compared to intranasal administration of each peptide individually (administration groups 1-4, 1-5). The tumor volume increase suppression effect was higher compared to intranasal administration of HPV E7 short peptide (administration group 1-3). As shown in Figure 2, intranasal administration of a mixture of HPV E6E7 long peptide and VP-R8 (administration groups 1-2) significantly suppressed the increase in tumor volume compared to intranasal administration of VP-R8 alone (administration groups 1-5). The tumor volume increase suppression effect was higher compared to intraperitoneal administration of a mixture of HPV E6E7 long peptide and incomplete Freund's adjuvant (administration groups 1-6). As shown in Figure 3, the survival rate of mice in treatment groups 1-2 was significantly improved compared to treatment groups 1-5 and 1-6. This indicates that administration of a mixture of HPV E6E7 long peptide and VP-R8 strongly exhibits an antitumor effect on E6E7 protein-expressing tumor cells and also improves their survival rate.

[0072] Immunology tests using mice Six-week-old female C57BL / 6 mice were classified into the following treatment groups (n=5). Treatment group 2-1: HPV E6E7 long peptide (10 μg) + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment group 2-2: HPV E7 short peptide (RAHYNIVTF) 49-57 (10 μg) + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment groups 2-3: HPV E6E7 long peptide (10 μg) + physiological saline in 20 μL (intranasal administration) Treatment groups 2-4: Physiological saline + VP-R8 (100 μg) in 20 μL (intranasal administration)

[0073] Treatment group 2-1 received HPV E6E7 long peptide, and treatment group 2-2 received HPV E7 short peptide (RAHYNIVTF). 49-57 Each of the following was mixed with physiological saline to make 58 μL of each solution. 58 μL of VP-R8 solution (10 mg / mL) was added to each of these solutions to make a total of 116 μL. After mixing well, the solution was allowed to stand at room temperature for at least 15 minutes to be used as the administration solution. For administration groups 2-3, 60 μg of HPV E6E7 long peptide was mixed with physiological saline to make 58 μL. 58 μL of physiological saline was added to this solution to make a total of 116 μL, which was thoroughly mixed and then allowed to stand at room temperature for at least 15 minutes to prepare the administration solution. For administration groups 2-4, 58 μL of physiological saline and 58 μL of VP-R8 solution (10 mg / mL) were added to a total volume of 116 μL. After thorough mixing, the solution was allowed to stand at room temperature for at least 15 minutes to be used as the administration solution.

[0074] To the mice classified as described above, 20 μL of each administration solution was administered via uninasal injection using a micropipette under anesthesia. Administration was performed on days 0, 7, and 14. As shown in Figure 5, no significant differences were observed in body weight changes. The mice were euthanized on day 15, and their spleens were collected. The isolated spleen cells were used in the following experiments.

[0075] Detection of HPV-specific T cells by intracellular cytokine staining 1 × 10⁶ mice isolated from treatment groups 2-1 to 2-4 6Individual spleen cells were stimulated with 5 μg / mL HPV E6E7 long peptide under 37°C and 5% CO2 conditions, after which the spleen cells were harvested. The culture time was 36 hours, and GolgiStop (BD Biosciences) was added to the culture medium from 24 to 36 hours after the start of culture. For TC-1 cells, those treated with 200 μg / mL mitomycin C (Nacalai Tesque) were used for stimulation culture. Spleen cells were cultured in RPMI-1640 medium supplemented with 2 mM glutamine, 10 mM HEPES, 100 U / mL penicillin, 100 μg / mL streptomycin, 1 mM non-essential amino acids, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, and 10% FBS. The collected spleen cells were centrifuged and reacted with 10 μg / mL anti-mouse CD16 / 32 antibody (BioLegend) at 4°C for 20 minutes. Staining buffer (phosphate-buffered saline containing 1% FBS and 0.09% sodium azide) was used as the antibody diluent. After the reaction, the cells were washed by centrifugation and stained with Staining buffer containing 2 μg / mL each of Fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4 antibody, PerCP-labeled anti-mouse CD3 antibody, and Allophycocyanin (APC)-labeled anti-mouse CD8 antibody (all from BD Biosciences) under light-shielded and ice-cold conditions for 30 minutes. After staining, the cells were washed and treated with the Fixation / Permeabilization solution included with the BD / Cytofix / Cytoperm Plus Fixation / Permeabilization Kit (BD Biosciences) on ice under light-shielded conditions for 20 minutes. The cells were centrifuged with 1× Perm / Wash Buffer (BD Biosciences) and then stained with PE-labeled anti-mouse interferon-γ (IFN-γ) antibody on ice for 30 minutes under light protection. The cells were then centrifuged again with 1× Perm / Wash Buffer and suspended in Staining Buffer. The cells were measured using a Guava easycyte flow cytometer (Luminex) and analyzed using Guava Incyte software (Luminex). The results are shown in Figure 6.

[0076] As shown in Figure 6, when spleen cells were stimulated with HPV E6E7 long peptide, the proportion of IFN-γ-producing CD8 T cells in treatment group 2-1 was significantly increased compared to treatment group 2-3 (p<0.05). This indicates that administration of a mixture of HPV E6E7 long peptide and VP-R8 induces an E6E7 peptide-specific cellular immune response.

[0077] HPV E7 RAHYNIVTF by MHC tetramer 49-57 Detection of specific CD8 T cells 1 × 10⁶ mice isolated from treatment groups 2-1 to 2-4 6Spleen cells were stimulated and cultured for 6 days at 37°C under 5% CO2 conditions using 5 μg / mL HPV E6E7 long peptide, and then harvested. 20 IU / mL interleukin-2 (IL-2) was added on days 2 and 4 of the culture. The cells were harvested and incubated on ice for 20 minutes with 10 μg / mL anti-mouse CD16 / 32 antibody (BioLegend). Staining buffer was used as the antibody diluent. After the reaction, the cells were centrifuged and washed, and then treated with 4 μg / mL T-Select H-2D b The cells were reacted with HPV16 E7 Tetramer-RAHYNIVTF-PE(MBL) under light-shielding conditions at room temperature for 30 minutes. After centrifugation, the cells were stained with PerCP-labeled anti-mouse CD3 antibody and allophycocyanin (APC)-labeled anti-mouse CD8 antibody under light-shielding conditions at 4°C for 30 minutes. After centrifugation, the cells were suspended in staining buffer, measured using a Guava easycyte flow cytometer, and then analyzed with Guava Incyte software. The results are shown in Figure 7.

[0078] As shown in Figure 7, when spleen cells were stimulated with HPV E6E7 long peptide, HPV E7 RAHYNIVTF was detected in treatment group 2-1. 49-57 The proportion of specific CD8 T cells was significantly increased compared to other treatment groups (p<0.05). This suggests that administration of a mixture of HPV E6E7 long peptide and VP-R8 is effective in promoting HPV E7 RAHYNIVTF 49-57 It was shown to induce specific cytotoxic T cells.

[0079] Detection of HPV E6E7-specific cytotoxic activity in spleen cells 2 × 10⁶ mice isolated from treatment groups 2-1 to 2-4 7 2 × 10⁶ mouse spleen cells 6 Mitomycin C-treated TC-1 cells were mixed with cells / well and cultured at 37°C under 5% CO2 conditions for 6 days. 20 IU / mL IL-2 was added on days 2 and 4 of culture. Splenocytes were harvested and plated in a 96-well plate with 5 × 10⁶ cells. 3After mixing TC-1 cells in a 40:1 ratio, the cells were cultured at 37°C under 5% CO2 conditions for 8 hours. The culture supernatant was collected, and the lactate dehydrogenase activity in the culture supernatant was measured using the Cytotox 96 Non-radioactive Cytotoxicity Assay Kit (Promega). The measurement method followed the instructions in the kit. Cytotoxic activity was calculated from the absorbance values. The results are shown in Figure 8.

[0080] As shown in Figure 8, when spleen cells were stimulated with TC-1 cells, the cytotoxic activity against TC-1 cells was significantly increased in the treatment group 2-1 compared to the treatment group 2-1 (p<0.05). This indicates that administration of a mixture of HPV E6E7 long peptide and VP-R8 induces a cellular immune response with HPV E6E7-specific cytotoxic activity.

[0081] Study using WT1 Preparation of purified long peptides Purified Wilms' tumor 1 (WT1) long peptide (amino acid sequence (SEQ ID NO: 41): RMFPNAPYLRRCMTWNQMNLRRSENHTAPIRRKRYFKLSHLQMHSRKH) was synthesized using Eurofins Genomics. This long peptide is derived from the following epitope sequence in the WT1 protein (RMFPNAPYL 126-134 (Sequence ID 23): H-2D b Constraining CD8 epitope, CMTWNQMNL 235-243 (Sequence ID 24): HLA-A * 24:02 Restrictive CD8 epitope, SENHTAPI 273-280 (Sequence ID 25): H-2Kk-restricted CD8 epitope, KRYFKLSHLQMHSRKH 332-337 (Sequence ID 26): HLA-DRB1 * The peptide sequence consists of a 0405-restrictive CD4 epitope linked by two arginine (R) residues. A 100 mg / ml stock solution of this long peptide was prepared using dimethyl sulfoxide (DMSO). RMFPNAPYL (SEQ ID NO: 23) was also synthesized by Eurofins Genomics, and a 100 mg / ml stock solution was prepared using dimethyl sulfoxide (DMSO).

[0082] Animal studies using tumor-bearing mice 1 x 10 5 Forty C1498-WT1 cells were subcutaneously transplanted with Matrigel into 40 six-week-old female C57BL / 6 mice to induce tumor formation. C1498-WT1 cells are a mouse leukemia cell line that stably expresses the mouse WT1 protein through recombinant DNA. The C1498-WT1 cells were donated by Dr. Yoshiko Hashii of Osaka University. For culturing the C1498-WT1 cells, RPMI-1640 medium supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μM 2-mercaptoethanol, 0.5 mg / mL G418, and 10% fetal bovine serum was used. Seven days after tumor transplantation, the mice were randomly classified into the following eight treatment groups (n=5). Treatment group 3-1: WT1 long peptide (100 μg) + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment group 3-2: WT1 long peptide (100 μg) + physiological saline in 20 μL (intranasal administration) Treatment group 3-3: Physiological saline + VP-R8 (100 μg) in 20 μL (intranasal administration) Treatment groups 3-4: WT1 long peptide (100 μg) + incomplete Freund's adjuvant (IFA) in 100 μL (intraperitoneal administration) Treatment groups 3-5: RMFPNAPYL 126-134 + Incomplete Freund's adjuvant (IFA) in 100 μL (administered intraperitoneally)

[0083] For treatment group 3-1, 600 μg of WT1 long peptide was mixed with physiological saline to make 58 μL of each solution. To each of these solutions, 58 μL of VP-R8 solution (10 mg / mL) was added to make a total of 116 μL. After thorough mixing, the solution was allowed to stand at room temperature for at least 15 minutes to be used as the administration solution. For treatment group 3-2, 600 μg of WT1 long peptide was mixed with physiological saline to make 58 μL. 58 μL of physiological saline was added to this solution to make a total of 116 μL, which was thoroughly mixed and then allowed to stand at room temperature for at least 15 minutes to prepare the administration solution. For administration group 3-3, 58 μL of physiological saline and 58 μL of VP-R8 solution (10 mg / mL) were added to a total volume of 116 μL. After thorough mixing, the solution was allowed to stand at room temperature for at least 15 minutes to be used as the administration solution. For treatment groups 3-4, 800 μg of WT1 long peptide was mixed with physiological saline to make 400 μL, which was then mixed with 400 μL of incomplete Freund's adjuvant to prepare the administration solution. For treatment groups 3-5, 800 μg of RMFPNAPYL was administered. 126-134 The peptide was mixed with physiological saline to make 400 μL, which was then mixed with 400 μL of incomplete Freund's adjuvant to prepare the administration solution.

[0084] After classifying the mice on day 7 post-tumor transplantation, 20 μL of each administration solution was administered to uninasal cavities of mice under anesthesia using a micropipette for administration groups 3-1 to 3. For administration groups 3-4 and 3-5, 100 μL of the administration solution was administered intraperitoneally under anesthesia. Administration was performed on days 7, 14, 21, and 28 post-tumor transplantation. Tumor diameter was measured daily, and tumor volume was calculated using the following formula: [Tumor volume (mm²)] 3 ) = major axis × (minor axis) 2 [×0.5]. The average tumor volume for treatment groups 3-1 to 3-4 is shown in Figure 9, and the survival rate of the mice is shown in Figure 10. The survival rates of mice in treatment groups 3-1 and 3-5 are also shown in Figure 11.

[0085] As shown in Figures 9 and 10, intranasal administration of a mixture of WT1 long peptide and VP-R8 (administration group 3-1) significantly suppressed the increase in tumor volume and significantly improved survival rates compared to intraperitoneal administration of a mixture of WT1 long peptide and incomplete Freund's adjuvant (administration group 3-4). As shown in Figure 11, intranasal administration of a mixture of WT1 long peptide and VP-R8 (administration group 3-1) was found to be more effective in improving survival rates compared to intraperitoneal administration of a mixture of WT1 short peptide and incomplete Freund's adjuvant (administration group 3-5). This indicates that administration of a mixture of WT1 long peptide and VP-R8 induces an antitumor effect against WT1 protein-expressing tumor cells.

Claims

1. A cancer vaccine comprising a peptide consisting of two or more linked amino acid sequences constituting a tumor antigen epitope, and a high-molecular-weight compound having a group represented by the following general formula (1) in its side chain, 【Chemistry 1】 (In the formula, X １ This indicates the residue obtained by removing the terminal amino group and terminal carboxyl group from a membrane-permeable peptide, X ２ This indicates an amino group. The membrane-permeable peptide is a peptide having an arginine oligomer in which 2 to 30 arginine molecules are linked by peptide bonds. The stem polymer of the polymer compound having the group represented by the general formula (1) in its side chain is a (meth)acrylic acid / N-vinylacetamide copolymer. A cancer vaccine in which the amino acid sequences constituting the tumor antigen epitope are at least two selected from the group consisting of VYDFAFFRDL (SEQ ID NO: 1), DKKQRFHNI (SEQ ID NO: 2), RAHYNIVTF (SEQ ID NO: 13), and LCVQSTHVD (SEQ ID NO: 14).

2. A cancer vaccine comprising a peptide formed by linking two or more amino acid sequences constituting a tumor antigen epitope, and a polymer compound having a group represented by the following general formula (1) in its side chain, 【Chemistry 1】 (In the formula, X1 represents a residue obtained by removing the terminal amino group and terminal carboxyl group from a membrane-permeable peptide, and X2 represents an amino group.) The membrane-permeable peptide is a peptide having an arginine oligomer in which 2 to 30 arginine molecules are linked by peptide bonds. The stem polymer of the polymer compound having the group represented by the general formula (1) in its side chain is a (meth)acrylic acid / N-vinylacetamide copolymer. A cancer vaccine in which the amino acid sequences constituting the tumor antigen epitope are at least two selected from the group consisting of RMFPNAPYL (SEQ ID NO: 23), CMTWNQMNL (SEQ ID NO: 24), SENHTAPI (SEQ ID NO: 25), and KRYFKLSHLQMHSRRKH (SEQ ID NO: 26).

3. The cancer vaccine according to claim 1 or 2, which is a transmucosal cancer vaccine.

4. A cancer vaccine according to any one of claims 1 to 3, which is a nasal cancer vaccine.

5. The cancer vaccine according to any one of claims 1 to 4, wherein the number of amino acids constituting the peptide, which is formed by linking two or more amino acid sequences constituting the tumor antigen epitope, is 15 to 100.

6. The cancer vaccine according to any one of claims 1 and 3 to 5, wherein the peptide comprising two or more linked amino acid sequences constituting the tumor antigen epitope comprises VYDFAFFRDL (SEQ ID NO: 1), DKKQRFHNI (SEQ ID NO: 2), RAHYNIVTF (SEQ ID NO: 13), and LCVQSTHVD (SEQ ID NO: 14).

7. The cancer vaccine according to any one of claims 2 to 5, wherein the peptide, which consists of two or more linked amino acid sequences constituting the tumor antigen epitope, comprises RMFPNAPYL (SEQ ID NO: 23), CMTWNQMNL (SEQ ID NO: 24), SENHTAPI (SEQ ID NO: 25), and KRYFKLSHLQMHSRRKH (SEQ ID NO: 26).

8. The cancer vaccine according to any one of claims 1 to 7, wherein the peptide, which is formed by linking two or more amino acid sequences constituting the tumor antigen epitope, is a peptide formed by linking two or more amino acid sequences constituting the tumor antigen epitope directly or via a linker.

9. The peptide, which consists of two or more linked amino acid sequences constituting the tumor antigen epitope, is VYDFAFFRDLRRRDKKQRFHNIRRRAHYNIVTFRRLCVQSTHVD (SEQ ID NO: 40), or A cancer vaccine according to any one of claims 1 to 8, wherein the peptide consists of the amino acid sequence RMFPNAPYLRRCMTWNQMNLRRSENHTAPIIRRKRYFKLSHLQMHSRRKH (Sequence ID 41).

10. A polymer compound having a group represented by the general formula (1) in its side chain is A cancer vaccine according to any one of claims 1 to 9, wherein the compound is represented by the following general formula (2). 【Chemistry 2】 (In the formula, a, x, and y represent integers greater than or equal to 1, and a:(x+y)=10:1 to 10. However, the units of a, x, and y are randomly combined. In the formula, X １ and X ２ (This is the same as above.)

11. The cancer vaccine according to claim 10, wherein x and y are integers satisfying x:y = 0 to 40:

10.

12. The cancer vaccine according to any one of claims 1 to 11, wherein the polymer compound having the group represented by the general formula (1) in its side chain is a polymer compound having a weight-average molecular weight of 10 kDa to 30,000 kDa.

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