KOC1-derived peptides and vaccines containing the same

Abstract

The present invention provides KOC1-derived epitope peptides having the ability to induce cytotoxic T cells. The present invention also provides polynucleotides encoding the peptides, antigen-presenting cells presenting the peptides, and cytotoxic T cells targeting the peptides, as well as methods for inducing antigen-presenting cells or CTLs. The present invention also provides compositions and pharmaceutical compositions containing them as active ingredients. In addition, the present invention provides methods for treating and / or preventing cancer, and / or preventing its postoperative recurrence using the peptides, polynucleotides, antigen-presenting cells, cytotoxic T cells or pharmaceutical compositions of the present invention. A method for inducing an immune response against cancer is also provided.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of July 31, 2015, application number 201580052377.4, and invention name “KOC1-derived peptides and vaccines containing the same”. Technical Field

[0002] The present invention relates to the field of biological sciences, and more particularly to the field of cancer treatment. Specifically, the present invention relates to novel peptides that are effective as cancer vaccines, methods for treating and / or preventing tumors using the peptides, and pharmaceutical compositions containing the peptides.

[0003] This application claims the benefit of Japanese patent applications filed on August 4, 2014 (Japanese Patent Application Nos. 2014-158919, 2014-158920, and 2014-158921), which are hereby incorporated by reference herein in their entirety. Background of the Invention

[0004] It has been shown that cytotoxic T cells (CTLs) can recognize epitope peptides derived from tumor-associated antigens (TAAs) found on major histocompatibility complex (MHC) class I molecules and then kill tumor cells. Since the discovery of the melanoma antigen (MAGE) family, many other TAAs have been discovered by immunological methods (NPL1: Boon T, Int J Cancer 1993 May 8, 54 (2): 177-80; NPL2: Boon T & van der Bruggen P, J Exp Med 1996 Mar 1, 183 (3): 725-9). Some of these TAAs are currently being clinically developed as immunotherapy targets.

[0005] Among several of these TAAs, epitope peptides that can be recognized by CTLs were identified, and their application in immunotherapy for various types of cancer is expected (NPL3: Harris CC, J Natl Cancer Inst 1996 Oct 16, 88(20):1442-55; NPL4: Butterfield LH et al., Cancer Res 1999 Jul 1, 59(13):3134-42; NPL5: Vissers JL et al., Cancer Res 1999 Nov 1, 59(21):5554-9; NPL6: van der Burg SH et al., J Immunol 1996 May 1, 156(9):3308-14; NPL7: Tanaka F et al., Cancer Res 1997 Oct 15, 57(20):4465-8; NPL8: Fujie T et al., Int J Cancer 1999 Jan 18, 80(2): 169-72; NPL9: Kikuchi M et al., Int J Cancer 1999 May 5, 81(3): 439-66; NPL10: Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94. To date, several clinical trials using these TAA-derived CTL epitope peptides have been reported. Unfortunately, many of these clinical trials showed low objective response rates (NPL11: Belli F et al., J Clin Oncol 2002 Oct 15, 20(20): 4169-80; NPL12: Coulie PG et al., Immunol Rev 2002 Oct, 188: 33-42; NPL13: Rosenberg SA et al., Nat Med 2004 Sep, 10(9): 909-15). Therefore, there is still a need to identify new CTL epitopes that can be used for cancer immunotherapy.

[0006] KOC1 (insulin-like growth factor II mRNA binding protein 3, also described as IGF2BP3 or IMP-3; reference sequence: GeneBank accession number NM_006547.2 (SEQ ID NO: 109)) was identified as a gene upregulated in lung cancer by gene expression profiling using a whole-genome cDNA microarray containing 23,040 genes (NPL14: Kikuchi T et al., Oncogene. 2003 Apr 10; 22(14): 2192-205; PTL1: WO2004 / 031413). KOC1 expression was specifically upregulated in tumor cells of more than 90% of lung cancer patients, but it was not expressed in other normal vital organs except testis and placenta. In addition, it was shown that as a result of downregulating KOC1 expression by RNA interference, cell proliferation in cancer cell lines expressing KOC1 was inhibited.

[0007] Recently, KOC1-derived HLA-A24-restricted CTL epitope peptides (PTL2: WO2006 / 090810; NPL15: Suda T et al., Cancer Sci. 2007 Nov; 98(11): 1803-8) and HLA-A2-restricted CTL epitope peptides (PTL3: WO2011 / 067920; NPL16: Tomita Y et al., Cancer Sci. 2011 Jan; 102(1): 71-8) have been identified. These peptides are effective in cancer patients with HLA-A24 or HLA-A2 types, but are not expected to be effective in cancer patients who do not have these HLA types.

[0008] [Citation List]

[0009] [Patent Document]

[0010] [PTL 1] WO2004 / 031413

[0011] [PTL2] WO2006 / 090810

[0012] [PTL3] WO2011 / 067920

[0013] [Non-patent literature]

[0014] [NPL 1]Boon T,Int J Cancer 1993May 8,54(2):177-80

[0015] [NPL 2]Boon T&van der Bruggen P,J Exp Med 1996Mar 1,183(3):

[0016] 725-9

[0017] [NPL 3]Harris CC,J Natl Cancer Inst 1996Oct 16,88(20):1442-55

[0018] [NPL 4] Butterfield LH et al., Cancer Res 1999 Jul 1, 59(13): 3134-42

[0019] [NPL 5] Vissers JL et al., Cancer Res 1999 Nov 1, 59(21): 5554-9

[0020] [NPL 6] van der Burg SH et al., J Immunol 1996 May 1, 156(9): 3308-14

[0021] [NPL 7] Tanaka F et al., Cancer Res 1997 Oct 15, 57(20): 4465-8

[0022] [NPL 8] Fujie T et al., Int J Cancer 1999 Jan 18, 80(2): 169-72

[0023] [NPL 9] Kikuchi M et al., Int J Cancer 1999May 5,81(3):459-66

[0024] [NPL 10] Oiso M et al., Int J Cancer 1999 May 5, 81(3): 387-94

[0025] [NPL 11] Belli F et al., J Clin Oncol 2002 Oct 15, 20(20): 4169-80

[0026] [NPL 12] Coulie PG et al., Immunol Rev 2002 Oct, 188: 33-42

[0027] [NPL 13] Rosenberg SA et al., Nat Med 2004 Sep, 10(9):909-15

[0028] [NPL 14] Kikuchi T et al., Oncogene. 2003 Apr 10; 22(14): 2192-205

[0029] [NPL 15] Suda T et al., Cancer Sci. 2007 Nov; 98(11): 1803-8

[0030] [NPL 16] Tomita Y et al., Cancer Sci. 2011 Jan; 102(1): 71-8 SUMMARY OF THE INVENTION

[0031] The present invention relates to peptides that can induce CTLs specific for cells expressing KOC1. When these peptides are presented on antigen-presenting cells (APCs) via human leukocyte antigens (HLA), CTLs that exhibit specific cytotoxic activity against cells expressing KOC1 are induced. KOC1-derived peptides with CTL inducibility (CTL inducibility) that have been identified to date are HLA-A2-restricted peptides or HLA-A24-restricted peptides, which cannot induce CTLs against cells that do not express these HLAs. Therefore, conventional peptides are not suitable for immunotherapy in subjects who do not have these HLAs. HLA-A11 and HLA-A33 are common alleles in Asians (Sette A, Sidney J., Immunogenetics 1999, 50:201-12), while HLA-A03 and HLA-A01 are common alleles in Caucasians (Cao et al., Hum Immunol 2001; 62(9):1009-30). It is desirable to administer HLA-A11-restricted peptides to HLA-A11-positive subjects, HLA-A33-restricted peptides to HLA-A33-positive subjects, HLA-A03-restricted peptides to HLA-A03-positive subjects, and HLA-A01-restricted peptides to HLA-A01-positive subjects. Therefore, the present invention relates to KOC1-derived peptides that have the ability to induce CTLs restricted to HLA-A11, HLA-A33, HLA-A03, or HLA-A01. Based on the results disclosed herein, it has been demonstrated that the peptides of the present invention are epitope peptides capable of inducing a potent and specific immune response against cells expressing KOC1 and HLA-A11, HLA-A33, HLA-A03, or HLA-A01.

[0032] Therefore, one of the objectives of the present invention is to provide KOC1-derived peptides capable of inducing CTLs in an HLA-A11-, HLA-A33-, HLA-A03-, or HLA-A01-restricted manner. These peptides can be used to induce CTLs in vitro, ex vivo, or in vivo, or can be administered to a subject for the purpose of inducing an immune response against KOC1-expressing cancer cells. Preferred peptides are peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41; more preferred peptides are nonapeptides or decapeptides; and even more preferred peptides are peptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41.

[0033] The peptides of the present invention include peptides in which one, two or more amino acids are substituted, deleted, inserted and / or added, as long as the resulting modified peptide retains the CTL inducibility of the original peptide.

[0034] The present invention also provides isolated polynucleotides encoding any of the peptides of the present invention. Similar to the peptides of the present invention, these polynucleotides can be used to induce APCs with CTL inducibility and can be administered to a subject to induce an immune response against KOC1-expressing cancer cells.

[0035] The present invention also provides a composition comprising one or more types of peptides of the present invention, one or more types of polynucleotides encoding one or more types of peptides of the present invention, APCs of the present invention, exosomes presenting the peptides of the present invention, and / or CTLs of the present invention. The composition of the present invention is preferably a pharmaceutical composition. The pharmaceutical composition of the present invention can be used to treat and / or prevent cancer, as well as to prevent its postoperative recurrence. They can also be used to induce an immune response against cancer. When administered to a subject, the peptides of the present invention are presented on the surface of the APCs, resulting in the induction of CTLs targeting the peptides. Therefore, another object of the present invention is to provide a composition for inducing CTLs, wherein the composition comprises one or more types of peptides of the present invention, one or more types of polynucleotides encoding one or more types of peptides of the present invention, APCs of the present invention, and / or exosomes presenting the peptides of the present invention.

[0036] Another object of the present invention is to provide a method for inducing APCs having CTL inducibility, wherein the method comprises the step of contacting one or more types of peptides of the present invention with APCs, or the step of introducing a polynucleotide encoding any one of the peptides of the present invention into APCs.

[0037] The present invention also provides a method for inducing CTLs, comprising the steps of co-culturing CD8-positive T cells with APCs presenting a complex of an HLA antigen and a peptide of the present invention on their surface, co-culturing CD8-positive T cells with exosomes presenting a complex of an HLA antigen and a peptide of the present invention on their surface, or introducing a vector into CD8-positive T cells, the vector comprising a polynucleotide encoding each subunit of a T cell receptor (TCR), the TCR being capable of binding to a peptide of the present invention presented by an HLA antigen on the cell surface. Preferred HLA antigens in the present invention are HLA-A11, HLA-A33, HLA-A03, or HLA-A01.

[0038] Another object of the present invention is to provide isolated APCs that present a complex of an HLA antigen and the peptide of the present invention on their surface. The present invention also provides isolated CTLs that target the peptide of the present invention. These APCs and CTLs can be used for immunotherapy against cancers that express KOC1. In the present invention, the cancers subject to immunotherapy are, for example, cancers present in patients who are homozygous or heterozygous for HLA-A11, HLA-A33, HLA-A03, or HLA-A01. That is, the present invention provides immunotherapy for cancers that express KOC1 and at least one HLA antigen selected from HLA-A11, HLA-A33, HLA-A03, and HLA-A01.

[0039] Another object of the present invention is to provide a method for inducing an immune response against cancer in a subject, wherein the method comprises administering to the subject a peptide of the present invention, a polynucleotide encoding the peptide, an APC of the present invention, an exosome presenting the peptide of the present invention, and / or a CTL of the present invention. Another object of the present invention is to provide a method for treating and / or preventing cancer and preventing its postoperative recurrence in a subject, wherein the method comprises administering to the subject a peptide of the present invention, a polynucleotide encoding the peptide, an APC of the present invention, an exosome presenting the peptide of the present invention, and / or a CTL of the present invention.

[0040] In addition to the above, other objects and features of the present invention will become more apparent when the following detailed description is read in conjunction with the accompanying drawings and examples. However, it should be understood that the foregoing overview of the present invention and the following detailed description are exemplary embodiments and are not restrictive of the present invention or other alternative embodiments of the present invention. In particular, although the present invention is described herein with reference to a plurality of specific embodiments, it should be understood that this description is an illustration of the present invention and does not constitute a limitation of the present invention. Various modifications and applications can be conceived by those skilled in the art without departing from the spirit and scope of the present invention as described in the appended claims. Similarly, other objects, features, benefits and advantages of the present invention will become apparent from this overview and certain embodiments described below, and will be apparent to those skilled in the art. From the above, in conjunction with the accompanying examples, data, drawings and all reasonable inferences drawn therefrom, such objects, features, benefits and advantages will be apparent, either alone or considered together with the references incorporated herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Composed of photos (a) to (e), it shows the results of interferon (IFN)-gamma enzyme-linked immunosorbent assay (ELISPOT) assays in CTLs induced using peptides derived from KOC1. Compared to the control, CTLs in well #6 with KOC1-A11-9-415 (SEQ ID NO: 5) (a), well #4 with KOC1-A11-10-414 (SEQ ID NO: 28) (b); well #5 with KOC1-A11-10-204 (SEQ ID NO: 30) (c), and well #8 with KOC1-A11-10-14 (SEQ ID NO: 32) (d) showed potent IFN-gamma production. In these photos, the squares above the wells indicate that cells from the corresponding wells were propagated for the establishment of CTL lines. In contrast, KOC1-A11-9-258 (SEQ ID NO: 1) (e) is shown as an example of typical negative data in which no specific IFN-gamma is produced. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide.

[0042] Figure 2Consists of a series of line graphs (a) to (c), which show the results of IFN-gamma enzyme-linked immunosorbent assay (ELISA), demonstrating the production of IFN-gamma in CTL lines stimulated with KOC1-A11-9-415 (SEQ ID NO: 5) (a), KOC1-A11-10-204 (SEQ ID NO: 30) (b), or KOC1-A11-10-14 (SEQ ID NO: 32) (c). These results demonstrate that CTL lines established by stimulation with each peptide exhibit potent IFN-gamma production compared to controls. In the figures, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide. The R / S ratio represents the ratio of the number of CTL clones (responding cells) to the number of target cells (stimulator cells).

[0043] Figure 3 Consisting of line graphs (a) and (b), IFN-gamma production in CTL clones established by limiting dilution of CTL lines stimulated with KOC1-A11-9-415 (SEQ ID NO: 5) or KOC1-A11-10-14 (SEQ ID NO: 32) is shown. These results demonstrate that CTL clones established by stimulation with each peptide exhibited potent IFN-gamma production compared to the control. In the graph, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide. The R / S ratio represents the ratio of the number of cells of the CTL line (responder cells) to the number of cells of the target cells (stimulator cells).

[0044] Figure 4 The line graph shows specific CTL activity against target cells expressing both KOC1 and HLA-A*1101. COS7 cells transfected with either HLA-A*1101 or the full-length KOC1 gene were prepared as controls. CTL clones established using KOC1-A11-10-14 (SEQ ID NO: 32) exhibited specific CTL activity against COS7 cells transfected with both KOC1 and HLA-A*1101 (black diamonds). On the other hand, no significant specific CTL activity was observed against target cells transfected with either HLA-A*1101 (white triangles) or KOC1 (white circles).

[0045] Figure 5Comprising photos (a) to (l), there are shown the results of IFN-gamma ELISPOT assay in CTLs induced using peptides derived from KOC1. Compared to the control, the expression of KOC1-A33-9-543 (SEQ ID NO: 61) in well #1 (a), KOC1-A33-9-282 (SEQ ID NO: 62) in well #2 (b), KOC1-A33-9-317 (SEQ ID NO: 63) in well #3 (c), KOC1-A33-9-485 (SEQ ID NO: 64) in well #8 (d), KOC1-A33-9-286 (SEQ ID NO: 67) in well #3 (e), KOC1-A33-9-34 (SEQ ID NO: 74) in well #8 (f), KOC1-A33-10-542 (SEQ ID NO: 77) in well #4 (g), KOC1-A33-10-281 (SEQ ID NO: 78) in well #5 (h), KOC1-A33-10-317 (SEQ ID NO: 79) in well #6 Strong IFN-gamma production was demonstrated in CTLs in well #3 with KOC1-A33-10-543 (SEQ ID NO: 79) (h), well #6 with KOC1-A33-10-424 (SEQ ID NO: 80) (j), and well #5 with KOC1-A33-10-285 (SEQ ID NO: 85) (k). In these photographs, the squares above the wells indicate that cells from the corresponding wells were propagated for establishing CTL lines. In contrast, KOC1-A33-9-517 (SEQ ID NO: 4) (l) is shown as an example of typical negative data in which no specific IFN-gamma was produced. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that had not been pulsed with any peptide.

[0046] Figure 6Consisting of a series of line graphs (a) to (f), the results of IFN-gamma ELISA are shown, demonstrating IFN-gamma production in CTL lines stimulated with KOC1-A33-9-543 (SEQ ID NO: 61) (a), KOC1-A33-9-282 (SEQ ID NO: 62) (b), KOC1-A33-9-485 (SEQ ID NO: 64) (c), KOC1-A33-9-286 (SEQ ID NO: 67) (d), KOC1-A33-10-542 (SEQ ID NO: 77) (e), or KOC1-A33-10-281 (SEQ ID NO: 52) (f). These results demonstrate that CTL lines established by stimulation with each peptide exhibit potent IFN-gamma production compared to the control. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells not pulsed with any peptide. The R / S ratio indicates the ratio of the number of CTL lines (responder cells) to the number of target cells (stimulator cells).

[0047] Figure 7 Consists of a series of line graphs (a) to (f), showing IFN-gamma production in CTL clones established by limiting dilution from CTL lines stimulated with: KOC1-A33-9-543 (SEQ ID NO: 61) (a), KOC1-A33-9-282 (SEQ ID NO: 62) (b), KOC1-A33-9-485 (SEQ ID NO: 64) (c), KOC1-A33-9-286 (SEQ ID NO: 67) (d), KOC1-A33-10-542 (SEQ ID NO: 77) (e), or KOC1-A33-10-281 (SEQ ID NO: 52) (f). These results demonstrate that CTL clones established by stimulation with each peptide exhibit potent IFN-gamma production compared to controls. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells not pulsed with any peptide. The R / S ratio indicates the ratio of the number of CTL lines (responder cells) to the number of target cells (stimulator cells).

[0048] Figure 8The figure consists of a series of line graphs (a) to (c) showing specific CTL activity against target cells expressing both KOC1 and HLA-A*3303. COS7 cells transfected with either HLA-A*3303 or the full-length KOC1 gene were prepared as controls. CTL clones established using KOC1-A33-9-485 (SEQ ID NO: 64) (a), KOC1-A33-9-286 (SEQ ID NO: 67) (b), and KOC1-A33-10-542 (SEQ ID NO: 77) (c) exhibited specific CTL activity against COS7 cells transfected with both KOC1 and HLA-A*3303 (black diamonds). On the other hand, no significant specific CTL activity was demonstrated against target cells transfected with either HLA-A*3303 (white triangles) or KOC1 (white circles).

[0049] Figure 9 Comprising photos (a) to (d), the results of IFN-gamma ELISPOT assays in CTLs induced with peptides derived from KOC1 are shown. Compared to the control, CTLs in well #5 with KOC1-A03-10-120 (SEQ ID NO: 27) (a), well #3 with KOC1-A03-10-204 (SEQ ID NO: 30) (b), and well #5 with KOC1-A03-10-281 (SEQ ID NO: 52) (c) showed strong IFN-gamma production. In these photos, the squares above the wells indicate that cells from the corresponding wells were propagated for establishing CTL lines. In contrast, KOC1-A03-10-414 (SEQ ID NO: 28) (d) is shown as an example of typical negative data in which no specific IFN-gamma is produced. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide.

[0050] Figure 10 is a series of line graphs showing the results of IFN-gamma ELISA, demonstrating IFN-gamma production in CTL lines stimulated with KOC1-A03-10-120 (SEQ ID NO: 27). These results demonstrate that CTL lines established by stimulation with each peptide exhibit robust IFN-gamma production compared to controls. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide. The R / S ratio represents the ratio of the number of CTL lines (responding cells) to the number of target cells (stimulating cells).

[0051] Figure 11 It is a line graph showing the production of IFN-gamma in CTL clones established by limiting dilution from a CTL line stimulated with KOC1-A03-10-120 (SEQ ID NO: 27). These results confirm that the CTL lines established by stimulation with each peptide exhibited strong IFN-gamma production compared to the control. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide. The R / S ratio represents the ratio of the number of CTL lines (responding cells) to the number of target cells (stimulating cells).

[0052] Figure 12 The figure is a line graph showing specific CTL activity against target cells expressing both KOC1 and HLA-A*0301. COS7 cells transfected with either HLA-A*0301 or the full-length KOC1 gene were prepared as controls. CTL clones established using KOC1-A03-10-120 (SEQ ID NO: 27) exhibited specific CTL activity against COS7 cells transfected with both KOC1 and HLA-A*0301 (black diamonds). On the other hand, no significant specific CTL activity was observed against target cells transfected with either HLA-A*3301 (white triangles) or KOC1 (white circles).

[0053] Figure 13Comprising photos (a) to (h), there are shown the results of IFN-gamma ELISPOT assay in CTLs induced using peptides derived from KOC1. Compared to the control, CTLs in well #2 with KOC1-A01-9-96 (SEQ ID NO: 86) (a), well #4 with KOC1-A01-9-118 (SEQ ID NO: 87) (b), well #3 with KOC1-A01-9-404 (SEQ ID NO: 90), well #4 with KOC1-A01-9-402 (SEQ ID NO: 92) (d), well #7 with KOC1-A01-10-312 (SEQ ID NO: 46) (e), well #1 with KOC1-A01-10-402 (SEQ ID NO: 95) (f), and well #1 with KOC1-A01-10-535 (SEQ ID NO: 41) (g) showed potent IFN-gamma production. In these photos, the squares above the wells show that cells from the corresponding wells were propagated to establish CTL lines. In contrast, KOC1-A01-9-536 (SEQ ID NO: 13) (h) is shown as an example of typical negative data in which no specific IFN-gamma was produced. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide.

[0054] Figure 14 Consists of a series of line graphs (a) and (b), showing the results of IFN-gamma ELISA, demonstrating IFN-gamma production in CTL lines stimulated with KOC1-A01-9-96 (SEQ ID NO: 86) (a) or KOC1-A01-9-118 (SEQ ID NO: 87) (b). These results demonstrate that CTL lines established by stimulation with each peptide exhibit potent IFN-gamma production compared to controls. In the figure, "+" indicates IFN-gamma production against target cells pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide. The R / S ratio represents the ratio of the number of cells of the CTL line (responder cells) to the number of cells of the target cells (stimulator cells).

[0055] Figure 15Is a line graph showing IFN-gamma production in CTL clones established by limiting dilution from a CTL line stimulated with KOC1-A01-9-96 (SEQ ID NO: 86). These results demonstrate that CTL clones established by stimulation with each peptide exhibit robust IFN-gamma production compared to controls. In the figure, "+" indicates IFN-gamma production against target cells that were pulsed with the appropriate peptide; and "-" indicates IFN-gamma production against target cells that have not been pulsed with any peptide. The R / S ratio represents the ratio of the number of CTL lines (responding cells) to the number of target cells (stimulating cells).

[0056] Figure 16 The figure is a line graph showing specific CTL activity against target cells expressing both KOC1 and HLA-A*0101. COS7 cells transfected with either HLA-A*0101 or the full-length KOC1 gene were prepared as controls. A CTL clone established using KOC1-A01-9-96 (SEQ ID NO: 86) showed specific CTL activity against COS7 cells transfected with both KOC1 and HLA-A*0101 (black diamonds). On the other hand, no significant specific CTL activity was shown against target cells transfected with either HLA-A*0101 (white triangles) or KOC1 (white circles). Detailed Description of the Invention

[0057] Description of Specific Embodiments

[0058] Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing embodiments of the present invention, preferred methods, apparatus, and materials are now described. However, before describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these can be varied based on routine experimentation and optimization. It should also be understood that the terms used in this specification are used only for the purpose of describing specific versions or embodiments and are not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0059] I. Definition

[0060] As used herein, the terms "a", "an", "the", and "said" mean "at least one", unless expressly stated otherwise.

[0061] The terms "isolated" and "purified," when used in connection with a substance (e.g., a peptide, antibody, polynucleotide, etc.), refer to a substance that is substantially free of at least one substance that may be present in its natural source. Thus, an isolated or purified peptide is substantially free of other cellular material, such as carbohydrates, lipids, or other contaminating proteins from the cell or tissue from which the peptide originates. When the peptide is chemically synthesized, an isolated or purified peptide is substantially free of precursor substances or other chemical substances. The phrase "substantially free of cellular material" includes peptide preparations in which the peptide is separated from cellular components of the cells from which the peptide was isolated or recombinantly produced. Thus, a peptide that is substantially free of cellular material includes peptide preparations that contain less than about 30%, 20%, 10%, 5%, 3%, 2%, or 1% (by dry weight) of other cellular material. When the peptide is recombinantly produced, an isolated or purified peptide that is substantially free of culture medium includes a peptide preparation that contains less than about 20%, 10%, or 5%, 3%, 2%, or 1% (by dry weight) of culture medium by volume of the peptide preparation. When the peptide is chemically synthesized, an isolated or purified peptide is substantially free of precursors or other chemicals and comprises a peptide preparation containing less than about 30%, 20%, 10%, 5%, 3%, 2% or 1% (by dry weight) of precursors or other chemicals by volume of the peptide preparation. A peptide preparation can be confirmed to be isolated or purified, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining or such gels. In preferred embodiments, the peptides and polynucleotides of the present invention are isolated or purified.

[0062] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In addition to naturally occurring amino acid polymers, these terms also apply to non-naturally occurring amino acid polymers that comprise one or more non-naturally occurring amino acid residues. Non-naturally occurring amino acids include amino acid analogs, amino acid mimetics, and the like.

[0063] As used herein, the term "amino acid" refers to naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that perform similar functions to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code in cells, as well as those that are post-translationally modified (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine, etc.). The phrase "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid (alpha carbon bound to hydrogen, carboxyl group, amino group, and R group) but has a modified R group or a modified backbone (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc.). The phrase "amino acid mimetics" refers to a compound that has a different structure from a typical amino acid but has a similar function. Amino acids can be L-amino acids or D-amino acids, and the peptides of the present invention are preferably L-amino acid polymers.

[0064] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymer of nucleotides.

[0065] The term "composition" as used in this specification is intended to include a product comprising a specified ingredient in a specified amount, as well as any product resulting directly or indirectly from a combination of specified ingredients in a specified amount. When the composition is a pharmaceutical composition, the term "composition" is intended to include a product of active ingredients and inert ingredients, as well as any product resulting directly or indirectly from the combination, complexation or aggregation of any two or more ingredients, from the dissociation of one or more ingredients, or from other types of reactions or interactions of one or more ingredients. Thus, the pharmaceutical compositions of the present invention include any composition prepared by mixing the compounds or cells of the present invention with a pharmaceutically or physiologically acceptable carrier. Without limitation, the terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used in this specification include liquid or solid fillers, diluents, excipients, solvents and encapsulating materials; and refer to a pharmaceutically or physiologically acceptable material, composition, substance or medium.

[0066] Unless otherwise indicated, the term "cancer" refers to cancers that overexpress the KOC1 gene; examples thereof include, but are not limited to, bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head and neck cancer, soft tissue tumors, testicular cancer, etc. In an exemplary embodiment, the "cancer" is a cancer that expresses KOC1 and HLA-A11, HLA-A33, HLA-A03, and / or HLA-A01.

[0067] Unless otherwise indicated, the terms "cytotoxic T lymphocytes" and "cytotoxic T cells" and "CTLs" are used interchangeably herein. Unless otherwise indicated, they refer to a subset of T lymphocytes that can recognize non-self cells (e.g., tumor / cancer cells, virus-infected cells) and induce the death of such cells.

[0068] Unless otherwise specified, the term "HLA-A11" refers to the HLA-A11 type including subtypes such as HLA-A*1101, HLA-A*1102, HLA-A*1103, and HLA-A*1104.

[0069] Unless otherwise indicated, the term "HLA-A33" refers to HLA-A33 types including subtypes such as HLA-A*3303, HLA-A*3301, and HLA-A*3304.

[0070] Unless otherwise specified, the term "HLA-A03" refers to HLA-A03 types including subtypes such as HLA-A*0301, HLA-A*0302, and HLA-A*0305.

[0071] Unless otherwise specified, the term "HLA-A01" refers to HLA-A01 types including subtypes such as HLA-A*0101, HLA-A*0102, HLA-A*0103, and HLA-A*0104.

[0072] In the context of a subject or patient, the phrase "the HLA antigen of the subject (or patient) is HLA-A11" means that the subject or patient has a homozygous or heterozygous HLA-A11 antigen gene as an MHC (major histocompatibility complex) class I molecule, and that the HLA-A11 antigen is expressed as an HLA antigen in cells of the subject or patient. Similarly, the phrases “the HLA antigen of the subject (or patient) is HLA-A33,” “the HLA antigen of the subject (or patient) is HLA-A03,” and “the HLA antigen of the subject (or patient) is HLA-A01,” as used herein, respectively, mean that the subject or patient has a homozygous or heterozygous HLA-A33 antigen gene as an MHC (major histocompatibility complex) class I molecule, and the HLA-A33 antigen is expressed as an HLA antigen in the cells of the subject or patient; that the subject or patient has a homozygous or heterozygous HLA-A03 antigen gene as an MHC (major histocompatibility complex) class I molecule, and the HLA-A03 antigen is expressed as an HLA antigen in the cells of the subject or patient; and that the subject or patient has a homozygous or heterozygous HLA-A01 antigen gene as an MHC (histocompatibility complex) class I molecule, and the HLA-A01 antigen is expressed as an HLA antigen in the cells of the subject or patient.

[0073] As long as the methods and compositions of the present invention are useful in the context of "treating" cancer, the treatment is considered "effective" when it achieves a clinical advantage, for example, by reducing the size, spread, or metastatic capacity of the cancer in the subject, delaying cancer progression, alleviating clinical symptoms of the cancer, prolonging survival, or inhibiting postoperative recurrence. When the treatment is administered prophylactically, "effective" means that the treatment delays or prevents the formation of cancer, or prevents or alleviates clinical symptoms of cancer. Effectiveness is determined relative to any known methods for diagnosing or treating a particular tumor type.

[0074] As long as the methods and compositions of the present invention are used in the context of cancer "prevention", the term "prevention" herein includes any work that reduces the burden of mortality or morbidity associated with cancer. Prevention can be carried out at the "primary, secondary and tertiary prevention levels". Although primary prevention avoids the development of the disease, prevention at the secondary and tertiary levels includes preventing the progression of the disease and the appearance of symptoms, as well as aiming to reduce the adverse effects of existing diseases by restoring function and reducing disease-related complications. Alternatively, prevention can include alleviating the severity of a particular condition, such as a broad range of preventive treatments aimed at reducing tumor growth and metastasis.

[0075] In the context of the present invention, the treatment and / or prevention (prevention) of cancer and / or the prevention (prevention) of its postoperative recurrence includes any event, such as inhibition of cancer cell proliferation, tumor regression or regression, induction of remission and inhibition of cancer development, tumor regression, as well as reduction or inhibition of metastasis, inhibition of postoperative recurrence of cancer and prolonged survival. Effective treatment and / or prevention (prevention) of cancer reduces mortality, improves the prognosis of individuals with cancer, reduces blood levels of tumor markers, and alleviates detectable symptoms associated with cancer. For example, alleviation or improvement of symptoms constitutes effective treatment and / or prevention (prevention) and includes patients in whom symptoms are stabilized or alleviated by 10%, 20%, 30% or more.

[0076] In the context of the present invention, the term "antibody" refers to an immunoglobulin and a fragment thereof that specifically reacts with a specified protein or its peptide. Antibodies can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused with other proteins or radiolabels, and antibody fragments. In addition, "antibody" herein is used in the broadest sense, and specifically encompasses complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies, multispecific antibodies (such as bispecific antibodies) and antibody fragments formed by two or more complete antibodies, as long as they exhibit the desired biological activity. "Antibody" can be all classes of antibodies (such as IgA, IgD, IgE, IgG and IgM).

[0077] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0078] II. Peptides

[0079] HLA-A11 and HLA-A33 are common alleles in Asians (Sette A, Sidney J., Immunogenetics 1999, 50: 201-12), and HLA-A03 and HLA-A01 are common alleles in Caucasians (Cao et al., Hum Immunol 2001; 62(9): 1009-30). Therefore, for large populations of Asians or Caucasians, an effective method for treating cancers expressing KOC1 can be provided by providing KOC1-derived CTL-inducing peptides that are restricted to HLA-A11, HLA-A33, HLA-A03, or HLA-A01. Therefore, the present invention provides KOC1-derived peptides that can induce CTLs in an HLA-A11-, HLA-A33-, HLA-A03-, or HLA-A01-restricted manner.

[0080] The peptides of the present invention are KOC1-derived peptides that can induce CTLs in an HLA-A11-, HLA-A33-, HLA-A03-, or HLA-A01-restricted manner. Peptides capable of inducing CTLs in an HLA-A11-restricted manner include peptides having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32. Similarly, peptides capable of inducing CTLs in an HLA-A33-restricted manner include peptides having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85. Similarly, peptides capable of inducing CTLs in an HLA-A03-restricted manner include peptides having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52. Similarly, peptides capable of inducing CTLs in an HLA-A01-restricted manner include peptides having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41.

[0081] CTLs having cytotoxic activity specific to these peptides can be established by stimulating T cells in vitro with dendritic cells (DCs) pulsed with these peptides. The established CTLs exhibit specific cytotoxic activity against target cells pulsed with each peptide.

[0082] The KOC1 gene is overexpressed in cancer cells, such as bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, testicular cancer, etc., but is not expressed in most normal organs. Therefore, it is an excellent target for immunotherapy. Therefore, the peptide of the present invention can be appropriately used for cancer immunotherapy. Preferred peptides are nonapeptides (peptides consisting of 9 amino acid residues) or decapeptides (peptides consisting of 10 amino acid residues), and more preferably are peptides selected from the group consisting of the following amino acid sequences: SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41. For example, a peptide having the amino acid sequence of SEQ ID NO:32 is suitable for inducing CTLs that exhibit specific cytotoxic activity against cells expressing HLA-A11 and KOC1, and can be used for cancer immunotherapy in HLA-A11-positive patients. Furthermore, for example, a peptide having an amino acid sequence selected from SEQ ID NOs:64, 67, and 77 is suitable for inducing CTLs that exhibit specific cytotoxic activity against cells expressing HLA-A33 and KOC1, and can be used for cancer immunotherapy in HLA-A33-positive patients. Furthermore, for example, a peptide having the amino acid sequence of SEQ ID NO:27 is suitable for inducing CTLs that exhibit specific cytotoxic activity against cells expressing HLA-A03 and KOC1, and can be used for cancer immunotherapy in HLA-A03-positive patients. Furthermore, for example, a peptide having the amino acid sequence of SEQ ID NO:86 is suitable for inducing CTLs that exhibit specific cytotoxic activity against cells expressing HLA-A01 and KOC1, and can be used for cancer immunotherapy in HLA-A01-positive patients. In a more preferred embodiment, the peptide of the present invention is a peptide selected from the group consisting of the following amino acid sequences: SEQ ID NOs: 32, 64, 67, 77, 27 and 86.

[0083] For the peptides of the present invention, additional amino acid residues may be added to the amino acid sequence of the peptides of the present invention, as long as the resulting peptide retains the CTL inducibility of the original peptide. The additional amino acid residues may be composed of any type of amino acid, as long as they do not impair the CTL inducibility of the original peptide. Thus, the peptides of the present invention include peptides with CTL inducibility comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41. Such peptides, for example, have fewer than about 40 amino acids, in many cases fewer than about 20 amino acids, and typically fewer than about 15 amino acids. Thus, if the original peptide is a nonapeptide, the peptides of the present invention include peptides that are 10 amino acids long or 11-40 amino acids long, generated by adding additional amino acids to the peptide. In addition, if the original peptide is a decapeptide, the peptides of the present invention include peptides of 11-40 amino acids in length, which are produced by appending additional amino acids to the peptide. Such peptides can be, for example, peptides of 11-20 amino acids in length or peptides of 11-15 amino acids in length. Preferred examples of additional amino acid residues are amino acid residues adjacent to the amino acid sequence of the peptide of the present invention in the full-length amino acid sequence of KOC1 (e.g., SEQ ID NO: 110). Therefore, the peptides of the present invention include peptides containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41, wherein the peptides are peptide fragments of KOC1 and have CTL inducing activity.

[0084] Generally speaking, modification of one, two or more amino acids in certain peptides does not affect the function of the peptide, or in some cases may even enhance the desired function of the original protein. In fact, it is known that modified peptides (i.e., peptides consisting of an amino acid sequence in which one, two or several amino acid residues are modified (i.e., substituted, added, deleted and / or inserted) compared to the original reference sequence) retain the biological activity of the original peptide (Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Therefore, in one embodiment, the peptide of the present invention may be a peptide comprising the following amino acid sequence, wherein one, two or several amino acid residues of the amino acid sequence selected from SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41 are substituted, deleted, inserted and / or added, and has CTL inducing activity.

[0085] Those skilled in the art will recognize that individual substitutions of amino acid sequences (which change a single amino acid or a small percentage of amino acids) tend to result in the retention of the properties of the original amino acid side chains. This is generally referred to as "conservative substitutions" or "conservative modifications"; and modification of a protein by "conservative substitutions" or "conservative modifications" can result in a modified protein having a function similar to the original protein. Conservative substitution tables providing functionally similar amino acids are well known in the art. Examples of functionally similar amino acid side chain features include, for example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following common functional groups or features: aliphatic side chains (G, A, V, L, I, P); hydroxyl-containing side chains (S, T, Y); sulfur-containing side chains (C, M); carboxylic acid and amide-containing side chains (D, N, E, Q); basic side chains (R, K, H); and aromatic side chains (H, F, Y, W). In addition, the following eight groups each contain amino acids that are recognized in the art as conservative substitutions for each other:

[0086] 1) Alanine (A), Glycine (G);

[0087] 2) Aspartic acid (D), glutamic acid (E);

[0088] 3) Asparagine (N), glutamine (Q);

[0089] 4) Arginine (R), Lysine (K);

[0090] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0091] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0092] 7) Serine (S), Threonine (T); and

[0093] 8) Cysteine ​​(C), Methionine (M) (see, for example, Creighton, Proteins 1984).

[0094] Such conservatively modified peptides are also included in the peptides of the present invention. However, the peptides of the present invention are not limited thereto and may include non-conservative modifications as long as the peptide retains the CTL inducibility of the original peptide. In addition, the modified peptides do not exclude CTL-inducible peptides derived from polymorphic variants, interspecies homologs, and alleles of KOC1.

[0095] As long as the peptide retains the CTL inducibility of the original peptide, a small number (e.g., 1, 2, or several) or a small percentage of amino acids may be modified (i.e., substituted, deleted, inserted, and / or added). Here, the term "several" means 5 or fewer amino acids, such as 4 or 3 or fewer. The percentage of modified amino acids is preferably 20% or less, more preferably 15% or less, even more preferably 10% or less, for example 1 to 5%.

[0096] When used in the context of cancer immunotherapy, the peptides of the present invention are presented on the surface of cells or exosomes, preferably as a complex with HLA antigens. Therefore, it is preferred that the peptides of the present invention have a high binding affinity to HLA antigens. For this purpose, the peptides can be modified by substitution, deletion, insertion and / or addition of amino acid residues to produce modified peptides with improved binding affinity. Since the sequence regularity of peptides displayed by binding to HLA antigens is known (Falk, et al., Immunogenetics 1994 40 232-41; Chujoh, et al., Tissue Antigens 1998:52:501-9; Takiguchi, et al., Tissue Antigens 2000:55:296-302.), modifications based on such regularities can be introduced into the peptides of the present invention.

[0097] For example, in peptides with binding affinity to HLA class I molecules, the second amino acid from the N-terminus and the C-terminal amino acid are generally involved in binding to HLA class I anchor residues (Rammensee HG, et al., Immunogenetics. 1995; 41 (4): 178-228.). For example, for HLA-A11, threonine, valine, isoleucine, leucine, phenylalanine and tyrosine are the second amino acid at the N-terminus, and lysine and arginine are considered to be anchor residues with high binding affinity to HLA-A11 as the C-terminal amino acids (Falk, et al., Immunogenetics 1994, 40: 232-41; Chujoh, et al., Tissue Antigens 1998: 52: 501-9). Furthermore, in HLA-A11, there are auxiliary anchor residues at positions 3 and 7 of the N-terminus. Furthermore, it is known that leucine, phenylalanine, tyrosine, isoleucine, and alanine are preferred as the third amino acid from the N-terminus, and leucine, isoleucine, tyrosine, valine, and phenylalanine are preferred as the seventh amino acid from the N-terminus (Falk et al., Immunogenetics 1994, 40:232-41; Chujoh et al., Tissue Antigens 1998:52:501-9). Therefore, to maintain or enhance HLA-A11 binding affinity, it may be desirable to substitute the second amino acid from the N-terminus with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine, and / or substitute the C-terminal amino acid with lysine or arginine. In addition, it may be necessary to substitute the third amino acid from the N-terminus with leucine, phenylalanine, tyrosine, isoleucine, or alanine, and / or substitute the seventh amino acid from the N-terminus with leucine, isoleucine, tyrosine, valine, or phenylalanine. Thus, peptides comprising an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32 having CTL inducibility are included in the peptides of the present invention: wherein the second amino acid selected from the group consisting of SEQ ID NOs: 5, 28, 30, and 32 is substituted with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine; the third amino acid from the N-terminus is substituted with leucine, phenylalanine, tyrosine, isoleucine, or alanine; the seventh amino acid from the N-terminus is substituted with leucine, isoleucine, tyrosine, valine, or phenylalanine; and / or the C-terminal amino acid is substituted with lysine or arginine.In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence, wherein in the amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32, the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine; the third amino acid from the N-terminus is substituted with leucine, phenylalanine, tyrosine, isoleucine, or alanine; the seventh amino acid from the N-terminus is substituted with leucine, isoleucine, tyrosine, valine, or phenylalanine; and / or the C-terminal amino acid is substituted with lysine or arginine. Specifically, the peptide of the present invention includes a peptide having CTL inducibility comprising an amino acid sequence, wherein one or more substitutions selected from the following (a) to (d) are introduced into the amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32:

[0098] (a) the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine;

[0099] (b) the third amino acid from the N-terminus is substituted with leucine, phenylalanine, tyrosine, isoleucine or alanine;

[0100] (c) the seventh amino acid from the N-terminus is substituted with leucine, isoleucine, tyrosine, valine or phenylalanine; and

[0101] (d) The C-terminal amino acid is substituted with lysine or arginine.

[0102] In a preferred embodiment, the peptide of the present invention may be a peptide consisting of amino acids having CTL inducibility, wherein one or more substitutions selected from (a) to (d) above are introduced into an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32. In the present invention, the preferred number of substitutions is 1, 2, 3, or 4 substitutions selected from (a) to (d) above.

[0103] The peptide of the present invention may be a peptide having CTL inducibility comprising an amino acid sequence in which, in an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32, the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine, and / or the C-terminal amino acid is substituted with lysine or arginine. Preferably, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence in which, in an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32, the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine, and / or the C-terminal amino acid is substituted with lysine or arginine. That is, the peptide of the present invention may be a peptide having CTL inducibility comprising an amino acid sequence in which one or more substitutions selected from the following (a) and (b) are introduced into an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32:

[0104] (a) the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine; and

[0105] (b) The C-terminal amino acid is substituted with lysine or arginine.

[0106] In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence, wherein one or more substitutions selected from (a) to (b) above are introduced into an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32. In a more preferred embodiment, the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, or leucine.

[0107] In HLA-A33, phenylalanine, tyrosine, alanine, isoleucine, leucine, and valine, which are the second amino acids from the N-terminus, and lysine and arginine, which are the C-terminal amino acids, are known to serve as anchor residues with high binding affinity for HLA-A33 (Falk et al., Immunogenetics 1994, 40:232-41; Takiguchi et al., Tissue Antigens 2000, 55:296-302). Furthermore, in HLA-A33, the first amino acid residue from the N-terminus is also known to serve as an anchor residue, and aspartic acid and glutamic acid are preferred as the first amino acid from the N-terminus (Falk et al., Immunogenetics 1994, 40:232-41; Takiguchi et al., Tissue Antigens 2000: 55:296-302). Therefore, in order to maintain or enhance HLA-A33 binding affinity, it may be desirable to substitute the first amino acid from the N-terminus with aspartic acid or glutamic acid, the second amino acid from the N-terminus with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine, and / or the C-terminal amino acid with lysine or arginine. Thus, peptides comprising the following amino acid sequences with CTL inducibility are included in the peptides of the present invention: the peptides having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, wherein the first amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine, and / or the C-terminal amino acid is substituted with lysine or arginine. In a preferred embodiment, the peptide of the present invention may be a peptide composed of amino acids having CTL inducibility, wherein in an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, the first amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine, and / or the C-terminal amino acid is substituted with lysine or arginine. Specifically, the peptide of the present invention includes a peptide comprising an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from the following (a) to (c) are introduced into an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85:

[0108] (a) The first amino acid from the N-terminus is substituted with aspartic acid or glutamic acid;

[0109] (b) the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine; and

[0110] (c) The C-terminal amino acid is substituted with arginine or lysine.

[0111] In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence, wherein one or more substitutions selected from (a) to (c) above are introduced into an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85. In the present invention, the preferred number of substitutions is 1, 2 or 3 substitutions selected from (a) to (c) above.

[0112] Furthermore, the peptide of the present invention may be a peptide having CTL inducibility comprising an amino acid sequence wherein, in an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine, and / or the C-terminal amino acid is substituted with arginine or lysine. Preferably, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence wherein, in an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine, and / or the C-terminal amino acid is substituted with arginine or lysine. That is, the peptide of the present invention may be a peptide having CTL inducibility comprising an amino acid sequence wherein one or more substitutions selected from the following (a) and (b) are introduced into an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85:

[0113] (a) the second amino acid from the N-terminus is substituted with phenylalanine, tyrosine, alanine, isoleucine, leucine or valine; and

[0114] (b) The C-terminal amino acid is substituted with arginine or lysine.

[0115] In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence, wherein one or more substitutions selected from the above (a) and (b) are introduced into an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85. In a more preferred embodiment, the second amino acid from the N-terminus is substituted with phenylalanine or tyrosine.

[0116] In HLA-A03, alanine, isoleucine, serine, and threonine are the second amino acid from the N-terminus, and arginine, lysine, tyrosine, and phenylalanine are the C-terminal amino acids, which are known to be anchor residues with high binding affinity for HLA-A03 (Kubo RT. et al., J Immunol. 1994 Apr 15; 152(8): 3913-24; Sidney J et al., Hum Immunol. 1996 Feb; 45(2): 79-93; Gambacorti-Passerini C et al., Clin Cancer Res. 1997 May; 3(5): 675-83). Therefore, in order to maintain or enhance HLA-A03 binding affinity, it may be preferable to substitute the second amino acid from the N-terminus with leucine, methionine, valine, alanine, isoleucine, serine, or threonine, and / or substitute the C-terminal amino acid with arginine, lysine, tyrosine, or phenylalanine. Therefore, peptides having CTL inducibility comprising an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52, wherein the second amino acid from the N-terminus is substituted with leucine, methionine, valine, alanine, isoleucine, serine, or threonine, and / or the C-terminal amino acid is substituted with arginine, lysine, tyrosine, or phenylalanine, are included in the peptides of the present invention. In a preferred embodiment, the peptides of the present invention may be peptides having CTL inducibility consisting of amino acids wherein the second amino acid from the N-terminus is substituted with leucine, methionine, valine, alanine, isoleucine, serine, or threonine, and / or the C-terminal amino acid is substituted with arginine, lysine, tyrosine, or phenylalanine. That is, the peptides of the present invention include peptides having CTL inducibility comprising an amino acid sequence wherein one or more substitutions selected from the following (a) and (b) are introduced into the amino acid sequence selected from SEQ ID NOs: 27, 30, and 52:

[0117] (a) the second amino acid from the N-terminus is substituted with leucine, methionine, valine, alanine, isoleucine, serine or threonine; and

[0118] (b) The C-terminal amino acid is substituted with arginine, lysine, tyrosine, or phenylalanine.

[0119] In a preferred embodiment, the peptide of the present invention can be a peptide having CTL inducibility consisting of an amino acid sequence in which one or more substitutions selected from (a) and (b) above are introduced into an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52. In a more preferred embodiment, the second amino acid from the N-terminus is substituted with leucine, methionine, or valine. In the present invention, the preferred number of substitutions is 1 or 2 substitutions selected from (a) and (b) above.

[0120] In HLA-A01, aspartic acid and glutamic acid, the third amino acid from the N-terminus, and tyrosine, the C-terminal amino acid, are known to be anchor residues with high binding affinity for HLA-A01. Furthermore, an auxiliary anchor residue is known at position 2 from the N-terminus of HLA-A01, and threonine and serine are known to be preferred as the second amino acid from the N-terminus (Kubo, RT Journal of Immunology 1994, 152:3913; Gambacorti-Passerini, C. Clinical Cancer Research 1997, 3:675-83; Falk, K. Immunogenetics 1994, 40:238-41). Therefore, to maintain or enhance HLA-A01 binding affinity, it may be necessary to replace the third amino acid from the N-terminus with aspartic acid or glutamic acid and / or the C-terminal amino acid with tyrosine. Another possibility is to replace the second amino acid from the N-terminus with threonine or serine. Therefore, peptides having CTL inducibility comprising an amino acid sequence wherein, in an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, the second amino acid from the N-terminus is substituted with threonine or serine, the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and / or the C-terminal amino acid is substituted with tyrosine are included in the peptides of the present invention. In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence wherein, in an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, the second amino acid from the N-terminus is substituted with threonine or serine, the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and / or the C-terminal amino acid is substituted with tyrosine. That is, the peptides of the present invention include peptides having CTL inducibility comprising an amino acid sequence, wherein one or more substitutions selected from the following (a) to (c) are introduced into an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41:

[0121] (a) The second amino acid from the N-terminus is substituted with threonine or serine;

[0122] (b) the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid; and

[0123] (c) The C-terminal amino acid is substituted with tyrosine.

[0124] In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence, wherein one or more substitutions selected from (a) to (c) above are introduced into an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41. In the present invention, the preferred number of substitutions is 1, 2 or 3 substitutions selected from (a) to (c) above.

[0125] Furthermore, the peptides of the present invention may be peptides having CTL inducibility comprising an amino acid sequence in which, in an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and / or the C-terminal amino acid is substituted with tyrosine. Preferably, the peptides of the present invention may be peptides having CTL inducibility consisting of an amino acid sequence in which, in SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid, and / or the C-terminal amino acid is substituted with tyrosine. Specifically, the peptides of the present invention include peptides having CTL inducibility comprising an amino acid sequence in which one or more substitutions selected from the following (a) and (b) are introduced into an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41:

[0126] (a) the third amino acid from the N-terminus is substituted with aspartic acid or glutamic acid; and

[0127] (b) The C-terminal amino acid was substituted with tyrosine.

[0128] In a preferred embodiment, the peptide of the present invention may be a peptide having CTL inducibility consisting of an amino acid sequence, wherein one or more substitutions selected from the above (a) and (b) are introduced into an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41.

[0129] Not only can amino acid substitutions be introduced at anchor sites, but substitutions can also be introduced at potential T cell receptor (TCR) recognition sites of the peptide. Several studies have demonstrated that peptides with amino acid substitutions can function equivalent to or better than the original ones, such as CAP1, p53 (264-272) 、Her-2 / neu (369-377) or gp100 (209-217)(Zaremba et al. Cancer Res. 57, 4570-4577, 1997, TK Hoffmann et al. J Immunol. (2002); 168(3): 1338-47., SODionne et al. Cancer Immunol immunother. (2003) 52: 199-206 and SODionne et al. Cancer Immunology, Immunotherapy (2004) 53, 307-314).

[0130] The present invention also contemplates the addition of one, two, or several amino acids to the N-terminus and / or C-terminus of the peptides of the present invention (e.g., peptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41). Such modified peptides that retain CTL inducibility are also encompassed by the present invention. For example, when a peptide (in which one, two, or several amino acids are added to the N-terminus and / or C-terminus of a peptide consisting of a sequence selected from the group consisting of SEQ ID NOs: 32, 64, 67, 77, 27, and 86) is contacted with APCs, it is incorporated into the APCs and processed into a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 64, 67, 77, 27, and 86. It can then induce CTLs by presenting on the cell surface of APCs via an antigen presentation pathway.More specifically, the peptide of the present invention may be a peptide in which one, two or several amino acids are added to either or both of the N-terminus and the C-terminus.

[0131] However, when the amino acid sequence of a peptide is partially identical to that of an endogenous or exogenous protein with a different function, side effects such as autoimmune diseases and / or allergic symptoms directed against a specific substance may be induced. Therefore, it is preferable to perform a homology search using available databases to avoid situations where the amino acid sequence of the peptide matches that of another protein. If a homology search reveals that there are no peptides that differ from the target peptide by only one or two amino acids, the target peptide can be modified to increase its binding affinity for HLA antigens and / or enhance its CTL inducibility without any risk of such side effects.

[0132] Peptides of the present invention in which one, two, or several amino acids have been modified are predicted to retain the CTL inducibility of the original peptide; however, it is preferred to verify the CTL inducibility of the modified peptide. Here, a "peptide having CTL inducibility" refers to a peptide that induces CTLs upon stimulation of APCs with the peptide. "CTL induction" includes induction of CTL differentiation, induction of CTL activation, induction of CTL proliferation, induction of CTL cytotoxic activity, induction of CTL-mediated target cell lysis, and induction of increased IFN-gamma production by CTLs.

[0133] The ability to induce CTLs can be confirmed by inducing and stimulating APCs (e.g., B lymphocytes, macrophages, and dendritic cells) that retain HLA antigens with peptides and mixing them with CD8-positive cells; then measuring IFN-gamma released by CTLs against target cells. For APCs, dendritic cells derived from human peripheral blood mononuclear cells can be preferably used. As a reaction system, transgenic animals that have been made to express HLA antigens can be used. Alternatively, for example, 51 Cr or other radioactive materials are used to label target cells, and the cytotoxic activity of peptide-induced CTLs can be calculated from the radioactivity released from the target cells. Alternatively, in the presence of peptide-stimulated APCs, CTL inducibility can be assessed by measuring IFN-gamma produced and released by CTLs and visualizing the inhibition zone on the culture medium using an anti-IFN-gamma monoclonal antibody.

[0134] In addition to the modifications described above, the peptides of the present invention can also be linked to other peptides, as long as the resulting linked peptide retains CTL inducibility. Examples of suitable peptides for linking to the peptides of the present invention include TAA-derived CTL-inducing peptides. In addition, the peptides of the present invention can also be linked to each other. Suitable linkers for linking peptides are known in the art, and for example, linkers such as AAY (PM Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (SEQ ID NO:111) (RPM Sutmuller et al., J Immunol. 2000, 165:7308-15), or K (S. Ota et al., Can Res. 62, 1471-6, KS Kawamura et al., J Immunol. 2002, 168:5709-15) can be used. The peptides can be linked in various arrangements (e.g., chain, repeat, etc.), and three or more peptides can also be linked.

[0135] The peptides of the present invention may also be linked to other substances, as long as the resulting linked peptide retains CTL inducibility. Examples of suitable substances to which the peptides of the present invention may be linked include, for example, peptides, lipids, sugars or sugar chains, acetyl groups, and natural or synthetic polymers. The peptides of the present invention may be modified by glycosylation, side chain oxidation, or phosphorylation, etc., as long as their CTL inducibility is not impaired. Such modifications may also be performed to impart additional functionality (e.g., targeting and delivery) or to stabilize the peptide.

[0136] For example, to improve the in vivo stability of peptides, it is known in the art to introduce D-amino acids, amino acid mimetics, or unnatural amino acids; this concept is also applicable to the peptides of the present invention. The stability of peptides can be determined in a variety of ways. For example, stability can be tested using peptidases and various biological media such as human plasma and serum (see, for example, Verhoef et al., Eur J Drug Metab Pharmacokin 1986, 11: 291-302).

[0137] Furthermore, as described above, among the modified peptides comprising the substitution, deletion, insertion, and / or addition of one, two, or several amino acid residues, those having the same or higher activity as the original peptide can be screened or selected. Therefore, the present invention also provides a method for screening or selecting modified peptides having the same or higher activity as the original peptide. Specifically, the present invention provides a method for screening for peptides having CTL inducibility, wherein the method comprises the following steps:

[0138] (a) generating a candidate sequence consisting of an amino acid sequence modified by substituting, deleting, inserting and / or adding one, two or several amino acid residues to an original amino acid sequence, wherein the original amino acid sequence is selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41;

[0139] (b) selecting, from the candidate sequences generated in (a), candidate sequences that have no significant homology (or sequence homology) to any peptide of a known human gene product other than KOC1;

[0140] (c) contacting the peptide consisting of the candidate sequence selected in (b) with APCs;

[0141] (d) contacting the APCs of (c) with CD8-positive T cells; and

[0142] (e) Selecting a peptide having CTL inducibility equal to or higher than that of a peptide consisting of the original amino acid sequence.

[0143] Herein, the peptides of the present invention are also described as "KOC1 peptides" or "KOC1 polypeptides."

[0144] III. Preparation of the Peptides of the Invention

[0145] The peptides of the present invention can be prepared using known techniques. For example, the peptides of the present invention can be prepared using recombinant DNA techniques or chemical synthesis. The peptides of the present invention can be synthesized independently or as longer polypeptides comprising two or more peptides. The peptides of the present invention can be produced in host cells using recombinant DNA techniques or isolated from host cells or synthetic reaction products after chemical synthesis. That is, the peptides of the present invention can be purified or isolated so that they are substantially free of other host cell proteins and fragments thereof, or any other chemical substances.

[0146] The peptides of the present invention may contain modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the original peptide. Other exemplary modifications include incorporation of one or more D-amino acids or other available amino acid mimetics, such as to increase the serum half-life of the peptide.

[0147] The peptide of the present invention can be obtained by chemical synthesis based on the selected amino acid sequence. Examples of conventional peptide synthesis methods that can be suitable for synthesis include the methods described in the following documents:

[0148] (i) Peptide Synthesis, Interscience, New York, 1966;

[0149] (ii)The Proteins,Vol.2,Academic Press,New York,1976;

[0150] (iii) “Peptide Synthesis” (Japanese), Maruzen Co., 1975;

[0151] (iv) "Basics and Experiment of Peptide Synthesis" (in Japanese), MaruzenCo., 1985;

[0152] (v) "Development of Pharmaceuticals" (Japanese), Continued Vol.14 (peptide synthesis), Hirokawa, 1991;

[0153] (vi)WO99 / 67288; and

[0154] (vii) Barany G. & Merrifield RB, Peptides Vol. 2, Solid Phase Peptide Synthesis, Academic Press, New York, 1980, 100-118.

[0155] Alternatively, any known genetic engineering peptide production method can be applied to obtain the peptides of the present invention (e.g., Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62). For example, first, a suitable vector containing a polynucleotide encoding the target peptide in an expressible form (e.g., downstream of a regulatory sequence equivalent to a promoter sequence) is prepared and transformed into a suitable host cell. The present invention also provides such vectors and host cells. The host cells are then cultured to produce the peptide of interest. The peptides of the present invention can also be produced in vitro using an in vitro translation system.

[0156] IV. Polynucleotides

[0157] The present invention also provides polynucleotides encoding any of the peptides of the present invention. These include polynucleotides derived from the naturally occurring KOC1 gene (e.g., GenBank Accession No. NM_006547 (SEQ ID NO: 109)) and polynucleotides having conservatively modified nucleotide sequences thereof. As used herein, the phrase "conservatively modified nucleotide sequence" refers to sequences that encode identical or substantially identical amino acid sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position specified by a codon for alanine, the codon can be changed to any corresponding codon without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. Each nucleic acid sequence encoding a peptide herein also describes every possible silent variation of that nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is normally the only codon for methionine, and TGG, which is normally the only codon for tryptophan) can be modified to produce functionally identical molecules. Accordingly, each silent variation of a nucleic acid that encodes a peptide is implicitly described in each disclosed sequence.

[0158] The polynucleotide of the present invention can be composed of DNA, RNA, and derivatives thereof. DNA is appropriately composed of bases such as A, T, C, and G, and T is replaced by U in RNA.

[0159] The polynucleotides of the present invention can encode a plurality of peptides of the present invention, with or without intervening amino acid sequences. For example, the intervening amino acid sequence can provide a cleavage site (e.g., an enzyme recognition sequence) for the polynucleotide or the translated peptide. In addition, the polynucleotide can include any additional sequences of the coding sequence encoding the peptide of the present invention. For example, the polynucleotide can be a recombinant polynucleotide comprising the regulatory sequences required for expressing the peptide, or can be an expression vector (e.g., a plasmid) having a marker gene or the like. In general, such recombinant polynucleotides can be prepared by manipulating the polynucleotides using conventional recombinant techniques, for example, by using a polymerase and an endonuclease.

[0160] Both recombinant and chemical synthesis techniques can be used to generate the polynucleotides of the present invention. For example, the polynucleotides of the present invention can be generated by inserting a suitable vector that can be expressed after transfection into competent cells. Alternatively, polynucleotides can be amplified using PCR technology or expressed in a suitable host (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1989). Alternatively, solid phase techniques can be used to synthesize polynucleotides, as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48: 2223-311; Matthes et al., EMBO J 1984, 3: 801-5.

[0161] V. Exosomes

[0162] The present invention further provides intracellular vesicles called exosomes, which present complexes formed between the peptides of the present invention and HLA antigens on their surfaces. Exosomes can be prepared, for example, by the methods described in detail in JPH11-510507 and WO99 / 03499, and can be prepared using APCs obtained from patients who are the subjects of treatment and / or prevention. The exosomes of the present invention can be used as vaccines for inoculation in a manner similar to that of the peptides of the present invention.

[0163] The type of HLA antigen contained in the complex must match the type of subject in need of treatment and / or prevention (prevention). For example, in Asian populations, HLA-A11 (e.g., HLA-A*1101) and HLA-A33 (e.g., HLA-A*3303) are alleles that are widely and commonly found in Asian populations, and these HLA antigen types are considered suitable for treatment in Asian patients. In addition, HLA-A03 (e.g., HLA-A*0301) and HLA-A01 (e.g., HLA-A*0101) are alleles that are widely and commonly found in Caucasian populations, and these HLA antigen types are considered suitable for treatment in Caucasian patients. Typically, in clinical practice, by pre-study of the HLA antigen type of the patient in need of treatment, suitable peptides can be selected that have a high level of binding affinity for a specific HLA antigen or CTL induction ability through antigen presentation mediated by a specific HLA antigen.

[0164] The exosomes of the present invention present on their surfaces a complex of the peptide of the present invention with HLA-A11, HLA-A33, HLA-A03, or HLA-A01. When the HLA complexed with the peptide of the present invention is HLA-A11, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32, or a modified peptide thereof. Furthermore, when the HLA complexed with the peptide of the present invention is HLA-A33, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, or a modified peptide thereof. Furthermore, when the HLA that forms a complex with the peptide of the present invention is HLA-A03, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52, or a modified peptide thereof. Furthermore, when the HLA that forms a complex with the peptide of the present invention is HLA-A01, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, or a modified peptide thereof.

[0165] VI. Antigen Presenting Cells (APCs)

[0166] The present invention also provides APCs that present complexes formed between HLA antigens and peptides of the present invention on their surfaces. Alternatively, the present invention provides APCs that have complexes formed between HLA antigens and peptides of the present invention on their surfaces. The APCs of the present invention may be isolated APCs. When used in the context of cells (APCs, CTLs, etc.), the term "isolated" means that the cells are separated from another type of cell. The APCs of the present invention may be APCs induced from APCs derived from patients to be treated and / or prevented (prevented), and may be administered as vaccines themselves or in combination with other drugs (including peptides of the present invention, exosomes or CTLs).

[0167] The APCs of the present invention are not limited to a specific type of cell, and include cells that inhibit the presentation of protein antigens on their cell surfaces for recognition by lymphocytes (e.g., dendritic cells (DC), Langerhans cells, macrophages, B cells, and activated T cells). Since DC is a representative APC with the strongest CTL induction activity among APCs, DC can be preferably used as the APCs of the present invention. In the present invention, preferred DC is isolated DC derived from humans. In addition, the APCs of the present invention do not necessarily have to be homogeneous, and they can be a mixture of multiple types of cells with antigen presenting function, and can be a mixture of APCs, wherein each APC presents a different type of peptide of the present invention.

[0168] For example, the APCs of the present invention can be obtained by isolating DCs from peripheral blood mononuclear cells and then stimulating them in vitro with the peptides of the present invention. When the peptides of the present invention are administered to a subject, APCs that present the peptides of the present invention are induced in the subject's body. Therefore, after administering the peptides of the present invention to a subject, the APCs of the present invention can be obtained by collecting APCs from the subject. Alternatively, the APCs of the present invention can be obtained by contacting APCs collected from a subject with the peptides of the present invention.

[0169] To induce an immune response against KOC1-expressing cancer cells in a subject, the APCs of the present invention can be administered to the subject alone or in combination with other drugs (including the peptides, exosomes, or CTLs of the present invention). For example, ex vivo administration can include the following steps:

[0170] (a) collecting APCs from a first subject;

[0171] (b) contacting the APCs of step (a) with the peptide of the present invention; and

[0172] (c) administering the APCs of step (b) to a second subject.

[0173] The first subject and the second subject may be the same individual or different individuals. When the first subject and the second subject are different individuals, preferably the first subject and the second subject have the same HLA type. The APCs obtained in step (b) above can be used as a vaccine for cancer treatment and / or prevention.

[0174] The APCs of the present invention obtained by the method described above have CTL inducibility. The term "CTL inducibility" in the context of APCs refers to the ability of an APC to induce CTLs when in contact with CD8-positive cells. In addition, "CTL inducibility" includes the ability of an APC to induce CTL activation, the ability of an APC to induce CTL proliferation, the ability of an APC to promote CTL-mediated lysis of target cells, and the ability of an APC to enhance CTL-mediated IFN-gamma production. The CTLs induced by the APCs of the present invention are specific for KOC1 and exhibit specific cytotoxic activity against cells expressing KOC1.

[0175] In addition to the above methods, the APCs of the present invention can be prepared by introducing a polynucleotide encoding the peptide of the present invention into APCs in vitro. The polynucleotide to be introduced can be in the form of DNA or RNA. The method of introduction is not particularly limited, and examples thereof include various methods conventionally practiced in the art, such as lipofection, electroporation, and the calcium phosphate method. More specifically, the methods described in Cancer Res 1996, 56:5672-7; J Immunol 1998, 161:5607-13; J Exp Med 1996, 184:465-72, and JP 2000-509281 can be used. By introducing a polynucleotide encoding the peptide of the present invention into APCs, the polynucleotide is transcribed and translated in the cells, and the resulting peptide is then processed by MHC class I and presented on the cell surface of the APC via a presentation pathway.

[0176] In a preferred embodiment, the APCs of the present invention present a complex formed between the peptide of the present invention and HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301), or HLA-A01 (more preferably HLA-A*0101) on their cell surfaces. When the HLA complexed with the peptide of the present invention is HLA-A11, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32. When the HLA that forms a complex with the peptide of the present invention is HLA-A33, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85. When the HLA that forms a complex with the peptide of the present invention is HLA-A03, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30 and 52, or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 27, 30 and 52. When the HLA that forms a complex with the peptide of the present invention is HLA-A01, the peptide of the present invention is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41.

[0177] The APCs of the present invention are preferably APCs induced by a method comprising the following steps (a) or (b):

[0178] (a) contacting APCs expressing at least one HLA selected from the group consisting of HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) and HLA-A01 (more preferably HLA-A*0101) with the peptide of the present invention; or

[0179] (b) introducing a polynucleotide encoding the peptide of the present invention into APCs expressing at least one HLA selected from the group consisting of HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) and HLA-A01 (more preferably HLA-A*0101).

[0180] The peptide of the present invention that contacts the APC expressing HLA-A11 is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30 and 32 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 5, 28, 30 and 32.

[0181] The peptide of the present invention that comes into contact with APCs expressing HLA-A33 is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85.

[0182] The peptide of the present invention that contacts the HLA-A03-expressing APC is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30 and 52 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 27, 30 and 52.

[0183] The peptide of the present invention that comes into contact with APCs expressing HLA-A01 is preferably a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41.

[0184] The present invention provides the use of the peptides of the present invention for preparing a pharmaceutical composition for inducing APCs with CTL inducibility. Furthermore, the present invention provides a method or process for preparing a pharmaceutical composition for inducing APCs with CTL inducibility. Furthermore, the present invention provides the peptides of the present invention for inducing APCs with CTL inducibility.

[0185] VII. Cytotoxic T Lymphocytes (CTL)

[0186] The CTLs induced by the peptides of the present invention can be used as vaccines in a manner similar to the peptides of the present invention for enhancing the immune response of cells that target and express KOC1 in vivo. Therefore, the present invention provides CTLs that are induced or activated by the peptides of the present invention. The CTLs of the present invention are CTLs that target the peptides of the present invention and are capable of binding to a complex of the peptides of the present invention and HLA antigens. The binding of CTL to the complex is mediated by the T cell receptor (TCR) present on the cell surface of the CTL. The CTLs of the present invention can be isolated CTLs. Preferred CTLs are isolated human CTLs. The CTLs of the present invention do not need to be homogeneous and can be a mixture of CTLs, each of which targets a different type of peptide of the present invention.

[0187] The CTLs of the present invention can be obtained by (1) administering the peptide of the present invention to a subject, (2) stimulating APCs and CD8-positive T cells or peripheral blood mononuclear cells (PBMCs) derived from the subject with the peptide of the present invention in vitro, (3) contacting CD8-positive T cells or PBMCs with APCs or exosomes in vitro, said APCs or exosomes presenting a complex of an HLA antigen and the peptide of the present invention on their surface, or (4) introducing a vector containing a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to the peptide of the present invention presented by an HLA antigen on the cell surface into CD8-positive T cells. Exosomes and APCs used in the method of (2) or (3) above can be prepared by the methods described in the sections "V. Exosomes" and "VI. Antigen-presenting cells (APCs)", respectively, and the details of the method of (4) above will be described in the section "VIII. T cell receptor (TCR)".

[0188] The CTLs of the present invention can be administered alone to patients undergoing treatment and / or prevention (prevention), or in combination with other drugs (including peptides of the present invention, APCs or exosomes) for the purpose of regulating the effect. Further, the CTLs of the present invention can be CTLs induced from CD8 positive T cells, which are derived from patients undergoing administration of CTLs. The CTLs of the present invention specifically act on target cells presenting the peptides of the present invention, for example, the same peptides used to induce the CTLs of the present invention. The target cells can be cells endogenously expressing KOC1, such as cancer cells, or cells transfected with the KOC1 gene. Cells presenting the peptides of the present invention on the cell surface due to the stimulation of the peptide can also become attack targets of the CTLs of the present invention. The cells targeted by the CTL of the present invention are preferably cells positive for at least one of HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301), and HLA-A01 (more preferably HLA-A*0101).

[0189] In a preferred embodiment, the CTLs of the present invention specifically target cells expressing KOC1 and at least one HLA selected from the group consisting of HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301), and HLA-A01 (more preferably HLA-A*0101). In the present invention, the cells targeted by the CTLs may be cells homozygous or heterozygous for any of the HLA-A11, HLA-A33, HLA-A03, and HLA-A01 alleles.

[0190] Herein, CTL "targeting" cells refers to the recognition of cells that present a complex of HLA and the peptide of the present invention on their cell surface by CTLs, and the expression of cytotoxic activity against these cells. Further, "specific targeting" refers to the expression of CTLs that exhibit cytotoxic activity against these cells, but do not show destructive activity against other cells. The expression "recognizing cells" used in the context of CTLs refers to binding to a complex of HLA and the peptide of the present invention presented on the cell surface via its TCR, and exhibiting specific cytotoxic activity against the cell. Therefore, the CTLs of the present invention are preferably CTLs that can bind to a complex presented on the cell surface via TCR, the complex being formed between the peptide of the present invention and HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) or HLA-A01 (more preferably HLA-A*0101).

[0191] Furthermore, the CTLs of the present invention are preferably CTLs induced by a method comprising the steps described in the following (a) or (b):

[0192] (a) contacting CD8-positive T cells in vitro with APCs or exosomes presenting on their surface a complex of the peptide of the present invention and HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) or HLA-A01 (more preferably HLA-A*0101); or

[0193] (b) introducing a polynucleotide comprising a subunit encoding each TCR capable of binding to the peptide of the present invention presented on the cell surface through HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), HLA-A03 (more preferably HLA-A*0301) or HLA-A01 (more preferably HLA-A*0101) into CD8-positive T cells.

[0194] VIII. T cell receptor (TCR)

[0195] The present invention also provides compositions and methods for using the compositions, the compositions comprising polynucleotides encoding each TCR subunit, the TCR being capable of binding to the peptides of the present invention presented on the cell surface by HLA antigens. The polynucleotides confer specificity to CD8 positive T cells for cancer cells expressing KOC1, which is achieved by expressing TCRs capable of binding to the peptides of the present invention presented on the cell surface by HLA antigens. By using methods known in the art, polynucleotides encoding the alpha and beta chains of TCR subunits in CTLs induced by the peptides of the present invention can be identified (WO2007 / 032255 and Morgan et al., J Immunol, 171, 3288 (2003)). For example, preferably, PCR methods are used for TCR analysis. Without being limited thereto, the PCR primers used for analysis can be, for example, a primer set for amplification by combining the following 5' side primers and 3' side primers:

[0196] 5' side primer:

[0197] 5'-R primer (5'-gtctaccaggcattcgcttcat-3') (SEQ ID NO: 105)

[0198] 3' primer

[0199] TCR-alpha-chain C-region-specificity

[0200] 3-TRa-C primer (5'-tcagctggaccacagccgcagcgt-3') (SEQ ID NO: 106)

[0201] TCR-beta-chain C1-region-specificity

[0202] 3-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3') (SEQ ID NO: 107) or

[0203] TCR-beta-chain C2-region-specificity

[0204] 3-TR-beta-C2 primer (5'-ctagcctctggaatcctttctctt-3') (SEQ ID NO: 108)

[0205] TCRs formed by introducing the identified polynucleotides into CD8-positive T cells are able to bind to target cells presenting the peptides of the present invention with high affinity and mediate efficient killing of target cells presenting the peptides of the present invention in vivo and in vitro.

[0206] The polynucleotide encoding each TCR subunit can be incorporated into a suitable vector, such as a retroviral vector. These vectors are well known in the art. The polynucleotide or a vector comprising them can be introduced into CD8 positive T cells, such as CD8 positive T cells derived from a patient. The present invention provides a ready-to-use composition that allows rapid modification of a patient's own T cells (or T cells derived from other subjects) to quickly and easily generate modified T cells with excellent cancer cell killing properties.

[0207] Herein, specific TCR is when TCR is presented on the surface of CD8 positive T cells, can be presented on the target cell surface by specific recognition peptide of the present invention and the complex of HLA antigen, give TCR for target cell specific cytotoxic activity.The specific recognition of above-mentioned complex can be confirmed by any known method, and its preferred example includes HLA multimer staining analysis using HLA molecules and peptide of the present invention, and ELISPOT determination method.By implementing ELISPOT determination method, can confirm by the identification of the specific TCR mediation of target cell of T cell and intracellular signal transduction that are imported with above-mentioned polynucleotide.When above-mentioned TCR is presented on CD8 positive T cell surface, can also confirm by known method whether TCR can give target cell specific cytotoxic activity for CD8 positive T cells.Preferred method includes for example measuring the cytotoxic activity for HLA positive target cells by chromium release assay etc.

[0208] In the context of HLA-A11, the present invention provides CTLs prepared by transforming CD8-positive T cells with a polynucleotide encoding each TCR subunit that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 5, 28, 30, and 32.

[0209] In the context of HLA-A33, the present invention provides CTLs prepared by transforming CD8-positive T cells with a polynucleotide encoding each TCR subunit that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85.

[0210] In the context of HLA-A03, the present invention provides CTLs prepared by transforming CD8-positive T cells with a polynucleotide encoding each TCR subunit that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 27, 30, and 52.

[0211] In the context of HLA-A01, the present invention provides CTLs prepared by transforming CD8-positive T cells with a polynucleotide encoding each TCR subunit that binds to a peptide having an amino acid sequence selected from, for example, SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41.

[0212] The transformed CTLs are capable of homing in vivo and can be expanded using known in vitro culture methods (e.g., Kawakami et al., J Immunol., 142, 3452-3461 (1989)). The CTLs of the present invention can be used to form immunogenic compositions, which are useful for treating or preventing diseases in patients in need of treatment and / or prevention (for reference WO2006 / 031221, the contents of which are incorporated herein by reference).

[0213] IX. Pharmaceutical Compositions

[0214] The present invention also provides a composition or pharmaceutical composition comprising at least one active ingredient selected from the group consisting of:

[0215] (a) a peptide of the present invention;

[0216] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0217] (c) APC of the present invention;

[0218] (d) exosomes of the present invention; and

[0219] (e) CTL of the present invention.

[0220] In addition to the above-mentioned active ingredients, the pharmaceutical composition of the present invention may contain a carrier, excipient or such commonly used in medicines as needed without particular limitation. Examples of carriers that can be used in the pharmaceutical composition of the present invention include sterile water, physiological saline, phosphate buffer, culture medium, etc. Therefore, the present invention also provides a pharmaceutical composition comprising at least one active ingredient selected from the following (a) to (e) and a pharmaceutically acceptable carrier:

[0221] (a) a peptide of the present invention;

[0222] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0223] (c) APC of the present invention;

[0224] (d) exosomes of the present invention; and

[0225] (e) CTL of the present invention.

[0226] In addition, the pharmaceutical composition of the present invention may contain stabilizers, suspending agents, preservatives, surfactants, solubilizers, pH regulators, aggregation inhibitors, etc. as needed.

[0227] Compared to normal tissues, KOC1 expression is significantly upregulated in cancer cells. Therefore, the peptides of the present invention or polynucleotides encoding the peptides can be used to treat and / or prevent cancer, and / or prevent its postoperative recurrence. Therefore, the present invention provides pharmaceutical compositions for treating and / or preventing cancer, and / or preventing its postoperative recurrence, comprising one or more types of peptides or polynucleotides of the present invention as active ingredients. Alternatively, the peptides of the present invention can be prepared for presentation on the surface of exosomes or APCs for use as pharmaceutical compositions. In addition, CTLs of the present invention targeting any of the peptides of the present invention can also be used as active ingredients of the pharmaceutical compositions of the present invention. The pharmaceutical compositions of the present invention may comprise a therapeutically effective amount or a pharmaceutically effective amount of the above-mentioned active ingredients.

[0228] The pharmaceutical compositions of the present invention can also be used as vaccines. In the context of the present invention, the term "vaccine" (also referred to as "immunogenic composition") refers to a composition that, when inoculated into an animal, has the function of inducing an immune response that results in an anti-tumor effect. Therefore, the pharmaceutical compositions of the present invention can be used to induce an immune response that results in an anti-tumor effect. The immune response induced by the peptides, polynucleotides, APCs, CTLs, and pharmaceutical compositions of the present invention is not particularly limited, as long as it is an immune response that results in an anti-tumor effect, and examples include inducing cancer cell-specific CTLs and inducing cancer cell-specific cytotoxic activity.

[0229] The pharmaceutical compositions of the present invention can be used to treat and / or prevent cancer in human subjects or patients, and / or prevent postoperative recurrence thereof. The pharmaceutical compositions of the present invention can preferably be used in subjects who are positive for at least one HLA selected from HLA-A11, HLA-A33, HLA-A03, and HLA-A01. Furthermore, the pharmaceutical compositions of the present invention can preferably be used to treat and / or prevent cancer expressing KOC1 and at least one HLA selected from HLA-A11, HLA-A33, HLA-A03, and HLA-A01, and / or prevent postoperative recurrence thereof.

[0230] In another embodiment, the present invention provides the use of an active ingredient selected from the following in the preparation of a pharmaceutical composition for treating or preventing cancer:

[0231] (a) a peptide of the present invention;

[0232] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0233] (c) APCs presenting the peptide of the present invention on their surface;

[0234] (d) exosomes presenting the peptide of the present invention on their surface; and

[0235] (e) CTL of the present invention.

[0236] Alternatively, the present invention further provides an active ingredient selected from the following for treating or preventing cancer:

[0237] (a) a peptide of the present invention;

[0238] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0239] (c) APCs presenting the peptide of the present invention on their surface;

[0240] (d) exosomes presenting the peptide of the present invention on their surface; and

[0241] (e) CTL of the present invention.

[0242] Alternatively, the present invention also provides a method or process for preparing a pharmaceutical composition for treating or preventing cancer, wherein the method or process comprises the step of formulating at least one active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier:

[0243] (a) a peptide of the present invention;

[0244] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0245] (c) APCs presenting the peptide of the present invention on their surface;

[0246] (d) exosomes presenting the peptide of the present invention on their surface; and

[0247] (e) CTL of the present invention.

[0248] In another embodiment, the present invention also provides a method or process for preparing a pharmaceutical composition for treating or preventing cancer, wherein the method or process comprises the step of mixing an active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier:

[0249] (a) a peptide of the present invention;

[0250] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0251] (c) APCs presenting the peptide of the present invention on their surface;

[0252] (d) exosomes presenting the peptide of the present invention on their surface; and

[0253] (e) CTL of the present invention.

[0254] In another embodiment, the present invention further provides a method for treating or preventing cancer, comprising the step of administering to a subject at least one active ingredient selected from the following:

[0255] (a) a peptide of the present invention;

[0256] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0257] (c) APCs presenting the peptide of the present invention on their surface;

[0258] (d) exosomes presenting the peptide of the present invention on their surface; and

[0259] (e) CTL of the present invention.

[0260] In the present invention, peptides having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32 have been identified as HLA-A11-restricted epitope peptides that can induce potent and specific immune responses. Therefore, pharmaceutical compositions of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32 are particularly suitable for administration to subjects possessing HLA-A11 (e.g., HLA-A*1101) as an HLA antigen. This also applies to pharmaceutical compositions comprising: polynucleotides encoding any of these peptides (i.e., polynucleotides of the present invention), APCs or exosomes that present these peptides (i.e., APCs or exosomes of the present invention), or CTLs that target these peptides (i.e., CTLs of the present invention). In other words, pharmaceutical compositions comprising an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32 are suitable for administration to subjects possessing HLA-A11 (i.e., HLA-A11-positive subjects). In a more preferred embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NO:32.

[0261] Similarly, in the present invention, peptides having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85 have been identified as HLA-A33-restricted epitope peptides that can induce potent and specific immune responses. Therefore, the pharmaceutical compositions of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85 are particularly suitable for administration to subjects having HLA-A33 (e.g., HLA-A*3303) as an HLA antigen. The same applies to pharmaceutical compositions comprising polynucleotides encoding any of these peptides (i.e., polynucleotides of the present invention), APCs or exosomes presenting these peptides (i.e., APCs or exosomes of the present invention), or CTLs targeting these peptides (i.e., CTLs of the present invention). That is, pharmaceutical compositions comprising an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85 are suitable for administration to subjects having HLA-A33 (i.e., HLA-A33-positive subjects). In a more preferred embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising at least one peptide having an amino acid sequence of SEQ ID NOs: 64, 67, and 77.

[0262] Similarly, in the present invention, peptides having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52 have been identified as HLA-A03-restricted epitope peptides that can induce potent and specific immune responses. Therefore, pharmaceutical compositions of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52 are particularly suitable for administration to subjects possessing HLA-A03 (e.g., HLA-A*0301) as an HLA antigen. This also applies to pharmaceutical compositions comprising: a polynucleotide encoding any of these peptides (i.e., a polynucleotide of the present invention); an APC or exosome that presents these peptides (i.e., an APC or exosome of the present invention); or a CTL that targets these peptides (i.e., a CTL of the present invention). In other words, pharmaceutical compositions comprising an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52 are suitable for administration to subjects possessing HLA-A03 (i.e., HLA-A03-positive subjects). In a more preferred embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a peptide having the amino acid sequence of SEQ ID NO:27.

[0263] Similarly, in the present invention, peptides having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41 have been identified as HLA-A01-restricted epitope peptides that can induce potent and specific immune responses. Therefore, the pharmaceutical compositions of the present invention comprising at least one peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41 are particularly suitable for administration to subjects having HLA-A01 (e.g., HLA-A*0101) as an HLA antigen. The same applies to pharmaceutical compositions comprising: a polynucleotide encoding any of these peptides (i.e., a polynucleotide of the present invention); an APC or exosome presenting these peptides (i.e., an APC or exosome of the present invention); or a CTL targeting these peptides (i.e., a CTL of the present invention). That is, a pharmaceutical composition comprising an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41 is suitable for administration to subjects having HLA-A01 (i.e., HLA-A01-positive subjects). In a more preferred embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising a peptide having an amino acid sequence of SEQ ID NO: 86.

[0264] Cancers to be treated and / or prevented by the pharmaceutical composition of the present invention are not particularly limited as long as they are cancers expressing KOC1, and include bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, testicular cancer, etc. The pharmaceutical composition of the present invention is preferably used in a subject homozygous or heterozygous for an HLA allele selected from HLA-A11, HLA-A33, HLA-A03, and HLA-A01.

[0265] In addition to the above-mentioned active ingredients, the pharmaceutical composition of the present invention may contain other peptides having the ability to induce CTLs against cancer cells (e.g., other TAA-derived CTL-inducing peptides), other polynucleotides encoding other peptides, other cells presenting other peptides, etc.

[0266] The pharmaceutical composition of the present invention may also optionally include other therapeutic substances as active ingredients, as long as they do not inhibit the anti-tumor effect of the above-mentioned active ingredients (e.g., peptides of the present invention). For example, the pharmaceutical composition of the present invention may optionally include anti-inflammatory compositions, analgesics, chemotherapeutic agents, etc. In addition to including other therapeutic substances in the pharmaceutical composition of the present invention itself, the pharmaceutical composition of the present invention may also be administered sequentially or simultaneously with one or more other pharmaceutical compositions. The dosage of the pharmaceutical composition of the present invention and other pharmaceutical compositions depends, for example, on the type of pharmaceutical composition used and the disease being treated, as well as on the scheduling and route of administration.

[0267] It should be understood that the pharmaceutical compositions of this invention may contain, in addition to the ingredients specifically mentioned herein, other ingredients conventional in the art having regard to the type of formulation.

[0268] The present invention also provides an article or kit comprising the pharmaceutical composition of the present invention. The article or kit of the present invention may include a container for holding the pharmaceutical composition of the present invention. Examples of suitable containers include, but are not limited to, bottles, vials, or test tubes. The container may be made of a variety of materials, such as glass or plastic. A label may be attached to the container and may describe the disease or condition for which the pharmaceutical composition of the present invention is intended. The label may also provide instructions for administration, etc.

[0269] In addition to the container containing the pharmaceutical composition of the present invention, the article of manufacture or kit of the present invention may optionally include a second container containing a pharmaceutically acceptable diluent. The article of manufacture or kit of the present invention may also include other materials required from a commercial standpoint and a user's perspective, such as other buffers, diluents, filters, injection needles, syringes, and package inserts with instructions for use.

[0270] If desired, the pharmaceutical composition of the present invention may be provided in a pack or dispensing device containing one or more unit dosage forms of the active ingredient. For example, the pack may comprise, for example, metal or plastic foil, such as a blister pack. The pack or dispensing device may be accompanied by instructions for administration.

[0271] (1) Pharmaceutical compositions containing peptides as active ingredients

[0272] The pharmaceutical composition comprising the peptide of the present invention can be formulated as needed by conventional preparation methods. In addition to the peptide of the present invention, the pharmaceutical composition of the present invention may contain carriers, excipients, etc. commonly used in medicines, without particular limitation. Examples of carriers that can be used in the pharmaceutical composition of the present invention include sterile water (e.g., water for injection), physiological saline, phosphate buffer, phosphate-buffered saline, Tris-buffered saline, 0.3% glycine, culture medium, and the like. In addition, the pharmaceutical composition of the present invention may contain stabilizers, suspending agents, preservatives, surfactants, solubilizers, pH regulators, aggregation inhibitors, and the like as needed. The pharmaceutical composition of the present invention can induce specific immunity against cancer cells expressing KOC1, and therefore can be used for the purpose of cancer treatment or prevention (prevention).

[0273] For example, the pharmaceutical composition of the present invention can be prepared by dissolving in a pharmaceutically or physiologically acceptable water-soluble carrier, such as sterile water (e.g., water for injection), physiological saline, phosphate buffer, phosphate-buffered saline, and Tris-buffered saline, and adding stabilizers, suspending agents, preservatives, surfactants, solubilizers, pH regulators, aggregation inhibitors, etc. as needed, and then sterilizing the peptide solution. The method for sterilizing the peptide solution is not particularly limited, and is preferably performed by filtration sterilization. Filter sterilization can be performed using, for example, a filter sterilizing filter with a pore size of 0.22 μm or less. The filter-sterilized peptide solution can be administered to a subject, for example, as an injection, but is not limited thereto. The pharmaceutical composition of the present invention can be prepared as a freeze-dried preparation by freeze-drying the above-mentioned peptide solution. The freeze-dried preparation can be prepared by filling the peptide solution prepared as described above into an appropriate container, such as an ampoule, a vial, or a plastic container, followed by freeze drying and encapsulating it in the container with a washed and sterilized rubber stopper, etc., after pressure recovery. The freeze-dried preparation can be administered to a subject after it is redissolved in a pharmaceutically or physiologically acceptable water-soluble carrier, such as sterile water (e.g., water for injection), physiological saline, phosphate buffer, phosphate-buffered saline, and Tris-buffered saline. Preferred examples of the pharmaceutical composition of the present invention include injections of such filter-sterilized peptide solutions and freeze-dried preparations produced by lyophilizing the peptide solutions. The present invention also includes a kit containing such freeze-dried preparations and a reconstitution solution. The present invention also includes a kit containing a container for the freeze-dried preparation (which is a pharmaceutical composition of the present invention) and a container for the reconstitution solution.

[0274] The pharmaceutical composition of the present invention may comprise a combination of two or more types of peptides of the present invention. The combination of peptides may take the form of a cocktail of mixed peptides, or may be conjugated to each other using standard techniques. For example, the peptides may be chemically linked or expressed as a single fusion polypeptide. By administering the peptides of the present invention, the peptides are displayed at high density on APCs using HLA antigens, and then CTLs that specifically react with the complex formed between the displayed peptides and HLA antigens are induced. Alternatively, APCs (e.g., DCs) may be isolated from a subject and then stimulated with the peptides of the present invention to obtain APCs that display any of the peptides of the present invention on their cell surfaces. These APCs are re-administered to the subject to induce CTLs in the subject, and as a result, the aggressiveness against cancer cells expressing KOC1 can be increased.

[0275] The pharmaceutical composition of the present invention may also include adjuvants known for effectively establishing cellular immunity. An adjuvant refers to a compound that enhances the immune response to an antigen with immunological activity when administered together with an antigen (or continuously). Known adjuvants described in the literature can be used, such as Clin Microbiol Rev 1994, 7: 277-89. Examples of suitable adjuvants include aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum oxyhydroxide, etc.), alum, cholera toxin, salmonella toxin, IFA (incomplete Freund's adjuvant), CFA (complete Freund's adjuvant), ISCOMatrix, GM-CSF and other immunostimulatory cytokines, oligodeoxynucleotides containing CpG motifs (CpG7909, etc.), oil-in-water emulsions, saponins or their derivatives (QS21, etc.), lipopolysaccharides such as lipid A or its derivatives (MPL, RC5 29, GLA, E6020, etc.), lipopeptides, lactoferrin, flagellin, double-stranded RNA or its derivatives (poliIC, etc.), bacterial DNA, imidazoquinoline (Imiquimod, R848, etc.), C-type lectin ligands (trehalose-6,6'-dibehenic acid / ester (TDB), etc.), CD1d ligands (alpha-galactosylceramide, etc.), squalene emulsions (MF59, AS03, AF03, etc.), PLGA, etc., but not limited thereto. In a kit comprising the pharmaceutical composition of the present invention, the adjuvant may be contained in another container separate from the pharmaceutical composition comprising the peptide of the present invention. In this case, the adjuvant and the pharmaceutical composition can be administered to the subject continuously or mixed together immediately before administration to the subject. The present invention also provides such a kit comprising a pharmaceutical composition containing the peptide of the present invention and an adjuvant. When the pharmaceutical composition of the present invention is a freeze-dried preparation, the kit may further comprise a reconstitution solution. In addition, the present invention provides a kit comprising a container for containing the pharmaceutical composition of the present invention and a container for storing the adjuvant. The kit may further comprise a container for storing the reconstituted solution as needed.

[0276] When an oil adjuvant is used as an adjuvant, the pharmaceutical composition of the present invention can be prepared as an emulsion. For example, an emulsion can be prepared by mixing and stirring the peptide solution and the oil adjuvant prepared as described above. The peptide solution can be a solution that is redissolved after freeze drying. The emulsion can be a W / O emulsion and an O / W emulsion, and the W / O emulsion is preferably used to obtain a high immune response enhancement effect. IFA can be preferably used as an oil adjuvant, but is not limited to this. The preparation of the emulsion can be carried out immediately before administering the subject, and in this case, the pharmaceutical composition of the present invention can be provided as a kit comprising the peptide solution of the present invention and the oily adjuvant. When the pharmaceutical composition of the present invention is a freeze-dried preparation, the kit can further include a redissolved solution.

[0277] Furthermore, the pharmaceutical composition of the present invention may be a liposome preparation in which the peptide of the present invention is encapsulated, a particle formulation in which the peptide is bound to beads having a diameter of several microns, or a formulation in which lipids are bound to the peptide.

[0278] In another embodiment of the present invention, the peptide of the present invention can also be administered in the form of a pharmaceutically acceptable salt. Examples of preferred salts include salts with alkali metals (lithium, potassium, sodium, etc.), salts with alkaline earth metals (calcium, magnesium, etc.), salts with other metals (copper, iron, zinc, manganese, etc.), salts with organic bases, salts with amines, salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, etc.), and salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.). As used herein, the phrase "pharmaceutically acceptable salt" refers to salts that retain the biological, physiological, pharmacological, and pharmaceutical effectiveness and properties of the compound. Therefore, pharmaceutical compositions comprising pharmaceutically acceptable salts of the peptide of the present invention are also included in the present invention. In addition, in addition to the free peptide, the "peptide of the present invention" also includes its pharmaceutically acceptable salt.

[0279] In some embodiments, pharmaceutical composition of the present invention can further include the composition of initiating (prime) CTLs. It has been confirmed that lipid is a component that can induce CTL for viral antigens in vivo. For example, palmitic acid residues can be connected to the ε- and α-amino groups of lysine residues and then be connected to peptides of the present invention. The lipidated peptide can then be directly used in micelles or particles, incorporated into liposomes, or emulsified in adjuvants. As other examples of lipid-induced CTL responses, escherichia coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinylseryl-serine (P3CSS), when covalently linked with suitable peptides, can be used to induce CTL (see, for example, Deres et al., Nature 1989, 342:561-4).

[0280] Examples of suitable methods for administering the peptides or pharmaceutical compositions of the present invention include oral, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal and intravenous injections, as well as systemic administration or local administration near the target site, but are not limited thereto. Preferred methods of administration include subcutaneous injection near lymph nodes such as the axilla or groin. Administration can be performed by a single administration or reinforced by multiple administrations. The peptides of the present invention can be administered to a subject in a therapeutically or pharmaceutically effective amount to treat cancer, or in a therapeutically or pharmaceutically effective amount to induce immunity (more specifically CTLs) against cancer cells expressing KOC1. The dosage of the peptides of the present invention can be appropriately adjusted based on the disease to be treated, the patient's age, weight, method of administration, and the like. For each peptide of the present invention, the dosage is typically 0.001 mg-1000 mg, such as 0.01 mg-100 mg, such as 0.1 mg-30 mg, such as 0.1 mg-10 mg, such as 0.5 mg-5 mg. The dosing interval can be once every few days to several months, and for example, administration can be performed at intervals of once a week. Those skilled in the art can appropriately select an appropriate dose (dosage).

[0281] In a preferred embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of the peptide of the present invention and a pharmaceutically or physiologically acceptable carrier. In another embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of the peptide of the present invention, a pharmaceutically or physiologically acceptable carrier and an adjuvant. The pharmaceutical composition of the present invention may comprise 0.001mg-1000mg, preferably 0.01mg-100mg, more preferably 0.1mg-30mg, even more preferably 0.1mg-10mg, for example 0.5mg-5mg of the peptide of the present invention. When the pharmaceutical composition of the present invention is an injection, it may comprise the following concentration of the peptide of the present invention: 0.001mg / ml-1000mg / ml, preferably 0.01mg / ml-100mg / ml, more preferably 0.1mg / ml-30mg / ml, even more preferably 0.1mg / ml-10mg / ml, for example 0.5mg / ml-5mg / ml. In this case, for example, 0.1 to 5ml, preferably 0.5ml to 2ml of the pharmaceutical composition of the present invention may be administered to a subject by injection.

[0282] In addition, the present invention provides a method for treating and / or preventing cancer and / or preventing its postoperative recurrence, which comprises administering a therapeutically effective amount of a peptide of the present invention or a pharmaceutical composition of the present invention to a subject. As described above, the peptide of the present invention can be administered to a subject in a single dose generally in the following amounts: 0.001 mg-1000 mg, for example, 0.01 mg-100 mg, for example, 0.1 mg-30 mg, for example, 0.1 mg-10 mg, or for example, 0.5 mg-5 mg. In a preferred embodiment, the peptide of the present invention is administered to a subject together with an adjuvant. In addition, the dosing interval can be once every few days to several months, preferably once every few days to once a month, for example once a week or once every two weeks.

[0283] (2) Pharmaceutical compositions containing polynucleotides as active ingredients

[0284] The pharmaceutical composition of the present invention may also contain polynucleotides encoding the peptide disclosed in the present invention in an expressible form. In this article, the phrase "in an expressible form" means that the polynucleotide will be expressed as a peptide of the present invention when introduced into a cell. In an exemplary embodiment, the sequence of the polynucleotide of the present invention includes regulatory elements necessary for the expression of the peptide of the present invention. The polynucleotide of the present invention may have the sequence required for stable insertion into the genome of the target cell (for a description of homologous recombination cassette vectors, see, for example, Thomas KR & Capecchi MR, Cell 1987, 51: 503-12). See, for example, Wolff et al., Science 1990, 247: 1465-8; U.S. Patent Nos. 5,580,859, 5,589,466, 5,804,566, 5,739,118, 5,736,524, 5,679,647; and WO98 / 04720. Examples of DNA-based delivery technologies include "naked DNA," facilitated (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).

[0285] The peptides of the present invention can also be expressed using viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, such as cowpox or fowlpox. For example, cowpox virus can be used as a vector for expressing the peptides of the present invention. After being introduced into the host, the recombinant cowpox virus expresses the immunogenic peptide and thereby triggers an immune response. Vaccinia vectors and methods that can be used for vaccination protocols are described in, for example, U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. Various other vectors that can be used for therapeutic administration or vaccination are obvious, such as adenovirus and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, etc. See, eg, Shata et al., Mol Med Today 2000, 6:66-71; Shedlock et al., J Leukoc Biol 2000, 68:793-806; Hipp et al., In Vivo 2000, 14:571-85.

[0286] Delivery of the polynucleotides of the invention to a patient can be direct, in which case the patient is directly exposed to a vector carrying the polynucleotide of the invention, or indirect, in which case cells are first transformed in vitro with a vector carrying the polynucleotide of the invention and then transplanted into the patient. These two approaches are referred to as in vivo and ex vivo gene therapy, respectively.

[0287] For a general review of gene therapy methods, see Goldspiel et al., Clinical Pharmacy 1993, 12:488-505; Wu and Wu, Biotherapy 1991, 3:87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33:573-96; Mulligan, Science 1993, 260:926-32; Morgan & Anderson, Ann Rev Biochem 1993, 62:191-217; Trends in Biotechnology 1993, 11(5):155-215. Well-known methods in the field of recombinant DNA technology that can be applied to the present invention are described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1993; and Krieger, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY, 1990.

[0288] Similar to the administration of peptides, the administration of polynucleotides can be carried out by oral, intradermal, subcutaneous, intravenous, intramuscular, intraosseous, or intraperitoneal injection, etc. Administration can be systemic administration or local administration near the target site. Administration can be implemented by a single administration, or strengthened by multiple administrations. The polynucleotides of the present invention can be administered to a subject in a therapeutically or pharmaceutically effective amount for inducing immunity (more specifically, CTL) against cancer cells expressing KOC1, or in a therapeutically or pharmaceutically effective amount for treating cancer. The dosage of the polynucleotide in a suitable carrier or the dosage of the polynucleotide in a cell transformed with the polynucleotide encoding the peptide of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age, weight, method of administration, etc., and is typically 0.001 mg-1000 mg, for example, 0.01 mg-100 mg, for example, 0.1 mg-30 mg, for example, 0.1 mg-10 mg, or for example 0.5 mg-5 mg. The dosing interval can be once every few days to several months, for example, administration can be carried out at intervals of once a week. Those skilled in the art can appropriately select an appropriate dose (dosage).

[0289] X. Methods Using Peptides, Exosomes, APCs, and CTLs

[0290] The peptides and polynucleotides of the present invention can be used to induce APCs and CTLs. Exosomes and APCs of the present invention can also be used to induce CTLs. Peptides, polynucleotides, exosomes, and APCs can be used in combination with any other compound, as long as their CTL-inducing ability is not inhibited. Therefore, pharmaceutical compositions containing any of the peptides, polynucleotides, APCs, and exosomes of the present invention can be used to induce the CTLs of the present invention. Furthermore, pharmaceutical compositions containing the peptides or polynucleotides of the present invention can be used to induce the APCs of the present invention.

[0291] (1) Methods for inducing APCs

[0292] The present invention provides a method for inducing APCs having CTL inducibility using the peptides or polynucleotides of the present invention.

[0293] The method of the present invention includes the steps of contacting APC with the peptide of the present invention in vitro, ex vivo or in vivo. For example, the method of contacting APC with the peptide in vitro may include the following steps:

[0294] (a) collecting APCs from a subject; and

[0295] (b) contacting the APCs of step (a) with the peptide of the present invention.

[0296] The above-mentioned APCs are not limited to specific types of cells, and cells known to present protein antigens on their cell surfaces for recognition by lymphocytes, such as DCs, Langerhans cells, macrophages, B cells, and activated T cells, can be used. DCs have the strongest CTL induction ability among APCs, so DCs are preferably used. Any peptide of the present invention can be used alone or in combination with other peptides of the present invention. In addition, the peptides of the present invention can be used in combination with other CTL-inducing peptides (e.g., other TAA-derived CTL-inducing peptides).

[0297] At the same time, when the peptides of the present invention are administered to a subject, APCs come into contact with the peptides in vivo, resulting in the induction of APCs with high CTL inducibility in the subject's body. Therefore, the methods of the present invention may include the step of administering the peptides of the present invention to a subject. Similarly, when an expressible polynucleotide of the present invention is administered to a subject, the peptides of the present invention are expressed in vivo, and the expressed peptides come into contact with APCs in vivo, resulting in the induction of APCs with high CTL inducibility in the subject's body. Therefore, the present invention may also include the step of administering the polynucleotides of the present invention to a subject.

[0298] In order to induce APCs with CTL inducibility, the present invention also includes the step of introducing the polynucleotide of the present invention into APCs. For example, the method may include the following steps:

[0299] (a) collecting APCs from a subject; and

[0300] (b) introducing a polynucleotide encoding the peptide of the present invention into the APCs of step (a).

[0301] Step (b) can be performed as described above in the section "VI. Antigen presenting cells (APCs)".

[0302] Therefore, in one embodiment, the present invention provides a method for inducing APCs with CTL inducibility, comprising the following steps (a) or (b):

[0303] (a) contacting APCs with the peptide of the present invention; and

[0304] (b) Introducing a polynucleotide encoding the peptide of the present invention into APCs.

[0305] In addition, the present invention provides a method for preparing APCs having CTL inducibility, comprising the following steps (a) or (b):

[0306] (a) contacting APCs with the peptide of the present invention; or

[0307] (b) Introducing a polynucleotide encoding the peptide of the present invention into APCs.

[0308] The above methods can be implemented in vitro, in vitro or in vivo, and are preferably implemented in vitro or in vitro. The APCs used in the above methods can be derived from the subject to which the induced APCs are planned to be administered, or they can be derived from different subjects. When APCs from a subject (donor) different from the subject to be administered are used, the administering subject and the donor must have the same HLA type. In the method of the present invention, when a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30 and 32 or a modified peptide thereof is used as the peptide of the present invention, the HLA type in the administering subject and the donor is preferably HLA-A11 (more preferably HLA-A*1101). Alternatively, the APCs used in the above methods are preferably APCs expressing HLA-A11 (more preferably HLA-A*1101). Similarly, when a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, or a modified peptide thereof, is used as a peptide of the present invention, the HLA type in the subject or donor is preferably HLA-A33 (more preferably HLA-A*3303). Alternatively, the APCs used in the above method are preferably APCs expressing HLA-A33 (more preferably HLA-A*3303). Similarly, when a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52, or a modified peptide thereof, is used as a peptide of the present invention, the HLA type in the subject or donor is preferably HLA-A03 (more preferably HLA-A*0301). Alternatively, the APCs used in the above method are preferably APCs expressing HLA-A03 (more preferably HLA-A*0301). Similarly, when a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, or a modified peptide thereof, is used as the peptide of the present invention, the HLA type in the administration subject and the donor is preferably HLA-A01 (more preferably HLA-A*0101). Alternatively, the APCs used in the above method are preferably APCs expressing HLA-A01 (more preferably HLA-A*0101). After separating PBMCs from blood collected from a donor by gravity centrifugation or the like, APCs can be prepared from the PBMCs using known methods.

[0309] In another embodiment, the present invention also provides a pharmaceutical composition comprising the peptide of the present invention or a polynucleotide encoding the peptide for inducing APCs with CTL inducibility.

[0310] Alternatively, the present invention also provides use of the peptide of the present invention or a polynucleotide encoding the peptide in the preparation of a pharmaceutical composition for inducing APCs with CTL inducibility.

[0311] Alternatively, the present invention also provides the peptide of the present invention or a polynucleotide encoding the peptide, for use in inducing APCs having CTL inducibility.

[0312] Alternatively, the present invention further provides a method or process for preparing a pharmaceutical composition for inducing APCs, wherein the method or process comprises the step of formulating the peptide of the present invention or the polynucleotide of the present invention with a pharmaceutically or physiologically acceptable carrier.

[0313] In another embodiment, the present invention further provides a method or process for preparing a pharmaceutical composition for inducing APCs with CTL inducibility, wherein the method or process comprises the step of mixing the peptide of the present invention or the polynucleotide of the present invention with a pharmaceutically or physiologically acceptable carrier.

[0314] The APCs induced by the method of the present invention can induce CTLs specific for KOC1 (ie, the CTLs of the present invention).

[0315] (2) Methods for inducing CTL

[0316] The present invention also provides methods for inducing CTLs using the peptides, polynucleotides, exosomes, or APCs of the present invention. The present invention also provides methods for inducing CTLs using one or more polynucleotides encoding polypeptides capable of forming a T cell receptor (TCR) (i.e., TCR subunits), wherein the TCR is capable of recognizing a complex of the peptides of the present invention and an HLA antigen. Preferably, the method for inducing CTLs comprises at least one step selected from the following:

[0317] (a) contacting CD8-positive T cells with antigen-presenting cells presenting a complex of an HLA antigen and the peptide of the present invention on its surface;

[0318] (b) contacting CD8-positive T cells with exosomes presenting a complex of an HLA antigen and the peptide of the present invention on its surface; and

[0319] (c) introducing one or more polynucleotides encoding polypeptides capable of forming a TCR into CD8-positive T cells, wherein the TCR is capable of recognizing a complex of the peptide of the present invention and an HLA antigen.

[0320] When the peptides, polynucleotides, APCs, or exosomes of the present invention are administered to a subject, CTLs are induced in the subject's body, and the intensity of the immune response targeting KOC1-expressing cancer cells is enhanced. Therefore, the methods of the present invention may include the step of administering the peptides, polynucleotides, APCs, or exosomes of the present invention to a subject.

[0321] Alternatively, they can be used in vitro or ex vivo to induce CTLs. For example, the method of the present invention can include the following steps:

[0322] (a) collecting APCs from subjects;

[0323] (b) contacting the APCs of step (a) with the peptide of the present invention; and

[0324] (c) Co-culturing the APCs from step (b) with CD8-positive T cells.

[0325] The induced CTLs can then be returned to the subject.

[0326] APCs to be co-cultured with CD8-positive T cells in step (c) above can also be prepared by introducing a polynucleotide encoding the peptide of the present invention into APCs, as described above in the section "VI. Antigen-presenting cells (APCs)". However, the APCs used in the method of the present invention are not limited thereto, and any APCs that present a complex of an HLA antigen and the peptide of the present invention on its surface can be used.

[0327] In addition to such APCs, exosomes presenting a complex of an HLA antigen and a peptide of the present invention on their surface can also be used in the methods of the present invention. Specifically, the methods of the present invention can include a step of co-culturing exosomes presenting a complex of an HLA antigen and a peptide of the present invention on their surface. Such exosomes can be prepared by the methods described above in section "V. Exosomes."

[0328] In addition, CTLs can be induced by introducing a vector comprising a polynucleotide encoding each subunit of a TCR into CD8-positive T cells, wherein the TCR is capable of binding to the peptide of the present invention presented by the HLA antigen on the cell surface. Such conversion can be performed as described in the "VIII. T cell receptor (TCR)" section above.

[0329] Therefore, in one embodiment, the present invention provides a method for inducing CTLs, comprising the steps selected from the group consisting of:

[0330] (a) co-culturing CD8-positive T cells with APCs, wherein the APCs present a complex of an HLA antigen and the peptide of the present invention on their surface;

[0331] (b) co-culturing CD8-positive T cells with exosomes presenting a complex of an HLA antigen and the peptide of the present invention on its surface; and

[0332] (c) introducing a vector containing a polynucleotide encoding each subunit of TCR capable of binding to the peptide of the present invention presented via HLA antigen on the cell surface into CD8-positive T cells.

[0333] The above methods can be implemented in vitro, in vitro or in vivo, and are preferably implemented in vitro or in vitro. The APCs or exosomes and CD8-positive T cells used in the above methods can be derived from the subject to which the induced CTL is to be administered, or they can be derived from different subjects. When using APCs or exosomes and CD8-positive T cells from a subject (donor) different from the subject to be administered, the subject to be administered and the donor must have the same HLA type. For example, when a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30 and 32 or a modified peptide thereof is used as the peptide of the present invention, the HLA type in the subject to be administered and the donor is preferably HLA-A11 (more preferably HLA-A*1101). Alternatively, the APCs or exosomes used in the above methods are preferably APCs or exosomes that present a complex of HLA-A11 (more preferably HLA-A*1101) and the peptide of the present invention (a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30 and 32 or a modified peptide thereof) on its surface. In this case, the induced CTLs exhibit cytotoxic activity against cells presenting a complex of HLA-A11 and the peptide of the present invention (e.g., HLA-A11-positive cells expressing KOC1). Alternatively, when a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, or a modified peptide thereof, is used as the peptide of the present invention, the HLA of the subject and the donor are preferably both HLA-A33 (more preferably HLA-A*3303). Alternatively, the APCs or exosomes used in the above method are preferably APCs or exosomes that present a complex of HLA-A33 (more preferably HLA-A*3303) and the peptide of the present invention (a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, or a modified peptide thereof) on their surface. In this case, the induced CTLs exhibit cytotoxic activity against cells presenting a complex of HLA-A33 and the peptide of the present invention (e.g., HLA-A33-positive cells expressing KOC1). Alternatively, when a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52, or a modified peptide thereof, is used as the peptide of the present invention, the HLA of the subject and the donor are preferably both HLA-A03 (more preferably HLA-A*0301). Alternatively, the APCs or exosomes used in the above method are preferably those that present a complex of HLA-A03 (more preferably HLA-A*0301) and the peptide of the present invention (a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52, or a modified peptide thereof) on their surface.In this case, the induced CTLs exhibit cytotoxic activity against cells presenting a complex of HLA-A03 and the peptide of the present invention (e.g., HLA-A03-positive cells expressing KOC1). Alternatively, when a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, or a modified peptide thereof, is used as the peptide of the present invention, the HLA of the subject and the donor are preferably both HLA-A01 (more preferably HLA-A*0101). Alternatively, the APCs or exosomes used in the above method are preferably APCs or exosomes that present a complex of HLA-A01 (more preferably HLA-A*0101) and the peptide of the present invention (a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, or a modified peptide thereof) on their surface. In this case, the induced CTLs exhibit cytotoxic activity against cells presenting a complex of HLA-A01 and the peptide of the present invention (eg, HLA-A01-positive cells expressing KOC1).

[0334] In another embodiment, the present invention also provides a composition or pharmaceutical composition for inducing CTL, comprising at least one active ingredient selected from the following:

[0335] (a) a peptide of the present invention;

[0336] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0337] (c) APCs presenting the peptide of the present invention on their surface; and

[0338] (d) Exosomes presenting the peptide of the present invention on their surface.

[0339] In another embodiment, the present invention also provides use of an active ingredient selected from the following in the preparation of a composition or pharmaceutical composition for inducing CTL:

[0340] (a) a peptide of the present invention;

[0341] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0342] (c) APCs presenting the peptide of the present invention on their surface; and

[0343] (d) Exosomes presenting the peptide of the present invention on their surface.

[0344] Alternatively, the present invention further provides an active ingredient selected from the following for inducing CTLs:

[0345] (a) a peptide of the present invention;

[0346] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0347] (c) APCs presenting the peptide of the present invention on their surface; and

[0348] (d) Exosomes presenting the peptide of the present invention on their surface.

[0349] Alternatively, the present invention further provides a method or process for preparing a composition or pharmaceutical composition for inducing CTLs, which comprises formulating an active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier:

[0350] (a) a peptide of the present invention;

[0351] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0352] (c) APCs presenting the peptide of the present invention on their surface; and

[0353] (d) Exosomes presenting the peptide of the present invention on their surface.

[0354] In another embodiment, the present invention further provides a method or process for preparing a composition or pharmaceutical composition for inducing CTLs, which comprises the step of mixing an active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier:

[0355] (a) a peptide of the present invention;

[0356] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0357] (c) APCs presenting the peptide of the present invention on their surface; and

[0358] (d) Exosomes presenting the peptide of the present invention on their surface.

[0359] XI. Methods of Inducing an Immune Response

[0360] The present invention also provides methods for inducing an immune response against cancers expressing KOC1. Applicable cancers include, but are not limited to, bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, testicular cancer, and the like. Preferably, the cancer expresses at least one HLA selected from HLA-A11, HLA-A33, HLA-A03, and HLA-A01.

[0361] The present invention also provides methods for inducing an immune response against cancer cells expressing KOC1. KOC1 is believed to be overexpressed in the various types of cancer described above. Therefore, when an immune response is induced against cancer cells expressing KOC1, the proliferation of the cancer cells is inhibited. Therefore, the present invention also provides methods for inhibiting the proliferation of cancer cells expressing KOC1. The methods of the present invention are particularly suitable for inhibiting the proliferation of cancer cells expressing KOC1 and at least one HLA selected from HLA-A11, HLA-A33, HLA-A03, and HLA-A01.

[0362] The methods of the present invention may include the step of administering a composition comprising any of the peptides of the present invention or a polynucleotide encoding the peptide. The methods of the present invention also encompass administering APCs or exosomes that present any of the peptides of the present invention. For details, see the "IX. Pharmaceutical Compositions" section, particularly the section describing the use of the pharmaceutical compositions of the present invention as vaccines. In addition, exosomes and APCs that can be used in the methods of inducing an immune response of the present invention are described in detail above in "V. Exosomes," "VI. Antigen Presenting Cells (APCs)," and "X. Methods Using Peptides, Exosomes, APCs, and CTLs" (1) and (2).

[0363] In another embodiment, the present invention provides a pharmaceutical composition or vaccine for inducing an immune response against a cancer expressing KOC1, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from the group consisting of:

[0364] (a) a peptide of the present invention;

[0365] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0366] (c) APCs presenting the peptide of the present invention on their surface;

[0367] (d) exosomes presenting the peptide of the present invention on their surface; and

[0368] (e) CTL of the present invention.

[0369] Alternatively, the present invention further provides a pharmaceutical composition or vaccine for inducing an immune response against cancer cells expressing KOC1, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from the following:

[0370] (a) a peptide of the present invention;

[0371] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0372] (c) APCs presenting the peptide of the present invention on their surface;

[0373] (d) exosomes presenting the peptide of the present invention on their surface; and

[0374] (e) CTL of the present invention.

[0375] Alternatively, the present invention further provides a pharmaceutical composition or vaccine for inhibiting the proliferation of cancer cells expressing KOC1, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from the following:

[0376] (a) a peptide of the present invention;

[0377] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0378] (c) APCs presenting the peptide of the present invention on their surface;

[0379] (d) exosomes presenting the peptide of the present invention on their surface; and

[0380] (e) CTL of the present invention.

[0381] In another embodiment, the present invention provides use of an active ingredient selected from the following in the preparation of a pharmaceutical composition or vaccine for inducing an immune response against a cancer expressing KOC1:

[0382] (a) a peptide of the present invention;

[0383] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0384] (c) APCs presenting the peptide of the present invention on their surface;

[0385] (d) exosomes presenting the peptide of the present invention on their surface; and

[0386] (e) CTL of the present invention.

[0387] Alternatively, the present invention also provides use of an active ingredient selected from the following in the preparation of a pharmaceutical composition or vaccine for inducing an immune response against cancer cells expressing KOC1:

[0388] (a) a peptide of the present invention;

[0389] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0390] (c) APCs presenting the peptide of the present invention on their surface;

[0391] (d) exosomes presenting the peptide of the present invention on their surface; and

[0392] (e) CTL of the present invention.

[0393] Alternatively, the present invention further provides use of an active ingredient selected from the following in the preparation of a pharmaceutical composition or vaccine for inhibiting the proliferation of cancer cells expressing KOC1:

[0394] (a) a peptide of the present invention;

[0395] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0396] (c) APCs presenting the peptide of the present invention on their surface;

[0397] (d) exosomes presenting the peptide of the present invention on their surface; and

[0398] (e) CTL of the present invention.

[0399] The present invention also provides a method or process for preparing a pharmaceutical composition for inducing an immune response against cancer expressing KOC1, wherein the method may include the step of mixing or formulating the peptide or polynucleotide of the present invention with a pharmaceutically acceptable carrier.

[0400] Alternatively, the present invention provides a method for inhibiting the proliferation of cancer cells expressing KOC1 or a method for inducing an immune response against cancer expressing KOC1, comprising the step of administering to a subject a vaccine or pharmaceutical composition comprising an active ingredient selected from the group consisting of:

[0401] (a) a peptide of the present invention;

[0402] (b) a polynucleotide encoding the peptide of the present invention in an expressible form;

[0403] (c) APCs presenting the peptide of the present invention on their surface;

[0404] (d) exosomes presenting the peptide of the present invention on their surface; and

[0405] (e) CTL of the present invention.

[0406] In the context of the present invention, KOC1-expressing cancers can be treated by administering the peptides, polynucleotides, APCs, exosomes, and / or CTLs of the present invention. Alternatively, an immune response against KOC1-expressing cancers can be induced by administering the peptides, polynucleotides, APCs, exosomes, and / or CTLs of the present invention. Examples of such cancers include, but are not limited to, bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, testicular cancer, and the like. Furthermore, an immune response against KOC1-expressing cancers can be induced by administering the peptides, polynucleotides, APCs, exosomes, and / or CTLs of the present invention. Therefore, prior to administering a vaccine or pharmaceutical composition comprising the above-described active ingredients, it is preferably determined whether KOC1 expression levels in the diseased area of ​​the subject to be treated are increased.

[0407] Thus, in one embodiment, the present invention provides a method of treating a cancer expressing KOC1 in a patient in need of cancer treatment, wherein the method comprises the steps of:

[0408] (i) measuring the level of KOC1 expression in a biological sample collected from a diseased site of a subject suffering from cancer;

[0409] (ii) identifying a subject having a cancer expressing KOC1 based on the KOC1 expression level measured in (i); and

[0410] (iii) administering at least one component selected from the group consisting of (a) to (e) above to a cancer patient expressing KOC1.

[0411] Alternatively, the present invention further provides vaccines and pharmaceutical compositions comprising at least one active ingredient selected from the group consisting of (a) to (e) above, for administration to a patient with a cancer expressing KOC1. The present invention also provides a method for identifying or selecting a subject to be treated with at least one active ingredient selected from (a) to (e) above, wherein the method comprises the following steps:

[0412] (i) measuring the level of KOC1 expression in a biological sample collected from a diseased site of a subject suffering from cancer;

[0413] (ii) identifying a subject having a cancer expressing KOC1 based on the KOC1 expression level measured in (i); and

[0414] (iii) identifying or selecting the subject identified in (ii) as a subject that can be treated with at least one active ingredient selected from the group consisting of (a) to (e).

[0415] The biological sample collected from the subject for measuring KOC1 expression levels in the above-mentioned method is not particularly limited, and for example, a tissue sample containing cancer cells collected by biopsy or the like can be preferably used. KOC1 expression levels in biological samples can be measured by known methods, such as methods for detecting transcription products of the KOC1 gene using probes or PCR methods (e.g., cDNA microarrays, Northern blotting, RT-PCR, etc.), methods for detecting translation products of the KOC1 gene using antibodies or the like (e.g., Western blotting, immunostaining, etc.), and the like. Furthermore, the biological sample can be a blood sample, in which case the blood level of antibodies against KOC1 is measured, and the KOC1 expression level at the diseased site can be assessed based on the blood level. Blood levels of antibodies against KOC1 or a fragment thereof can be measured using known methods, such as enzyme immunoassays (EIAs), enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), etc., using KOC1 proteins or peptides of the present invention as antigens.

[0416] Typically, KOC1 transcription and translation products are rarely detected in tissues and cells that do not express KOC1. Therefore, when KOC1 transcription or translation products are detected in cancer cells or tissue samples containing cancer cells collected from a subject, it can be determined that the subject's cancer expresses KOC1. In blood samples from subjects with cancer that does not express KOC1, antibodies against KOC1 or its fragments are rarely detected. Therefore, when antibodies against KOC1 or its fragments are detected in a blood sample collected from a subject, it can be determined that the subject's cancer expresses KOC1. Whether a subject's cancer expresses KOC1 can also be determined by comparing the measurement results of the same type of biological material collected from a non-cancer site of the subject or by comparing the same type of biological material collected from a subject without cancer (normal control sample). That is, when the level in the test subject's biological sample is elevated when compared to the measured target level (normal control level) in the normal control sample, the subject's cancer is assessed to express KOC1. For example, when the amount of the target detected increases by at least 10% or more compared to the normal control level, the subject's cancer can be assessed to express KOC1. It is desirable that the amount of the target substance detected is preferably 25% or higher, more preferably 50% or higher than the normal control level. In addition, the amount of the transcription product or translation product of KOC1 detected can be evaluated by normalization relative to the amount of a known housekeeping gene (e.g., β-actin, glyceraldehyde-3-phosphate dehydrogenase, or ribosomal protein P1) detected.

[0417] In a preferred embodiment, the HLA type of the subject is preferably confirmed before administering at least one active ingredient selected from the group consisting of (a) to (e) above. For example, for subjects to be administered with an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 5, 28, 30, and 32, it is preferred to select an HLA-A11-positive subject. For subjects to be administered with an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, and 85, it is preferred to select an HLA-A33-positive subject. For subjects to be administered with an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 30, and 52, it is preferred to select an HLA-A03-positive subject. For subjects to be administered with an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs: 86, 87, 90, 92, 46, 95, and 41, it is preferred to select an HLA-A01-positive subject.

[0418] The present invention also provides a complex of the peptide of the present invention and HLA. The complex of the present invention can be a monomer or a multimer. When the complex of the present invention is a multimer, the number of polymerizations is not particularly limited, and the multimer can be any number of polymerizations. Examples include, but are not limited to, tetramers, pentamers, hexamers, etc. Multimers of the present invention also include dextramers (WO2002 / 072631) and streptamers (Knabel M et al., Nat Med. 2002 Jun; 8(6): 631-7). Complexes of the peptide of the present invention and HLA can be prepared according to known methods (e.g., Altman JD et al., Science. 1996, 274(5284): 94-6; WO2002 / 072631; WO2009 / 003492; Knabel M, Nat Med. 2002 Jun; 8(6): 631-7, etc.). For example, the complex of the present invention can be used to quantify CTLs specific for the peptide of the present invention. For example, a blood sample is collected from a subject to whom a pharmaceutical composition of the present invention is administered, and after isolating PBMCs, CD4-negative cells are prepared and contacted with the fluorescent dye-conjugated complex of the present invention. The percentage of CTLs specific for the peptide of the present invention can then be measured by flow cytometry analysis. For example, the immune response induction effect of the pharmaceutical composition of the present invention can be monitored by measuring CTLs specific for the peptide of the present invention before, during, and / or after administration of the pharmaceutical composition of the present invention.

[0419] XII. Antibodies

[0420] The present invention further provides antibodies that bind to the peptides of the present invention. Preferred antibodies specifically bind to the peptides of the present invention and do not bind (or weakly bind) to peptides other than the present invention. In another embodiment, such antibodies may include antibodies that recognize the peptide in the context of an HLA molecule, i.e., antibodies that bind to a peptide-MHC complex. The binding specificity of the antibody can be confirmed by inhibition assays. That is, if the binding between the antibody to be analyzed and the full-length KOC1 polypeptide is inhibited in the presence of the peptide of the present invention, the antibody demonstrates specific binding to the peptide of the present invention. Antibodies against the peptides of the present invention can be used for disease diagnosis and prognosis assays, as well as for selecting subjects to administer the pharmaceutical composition of the present invention and monitoring the pharmaceutical composition of the present invention.

[0421] The present invention also provides various immunoassays for detecting and / or quantifying the peptides of the present invention or fragments thereof. Such immunoassays include, but are not limited to, radioimmunoassays, immunochromatographic assays, enzyme-linked immunosorbent assays (ELISAs), enzyme-linked immunofluorescence assays (ELIFAs), and the like, and are performed within the scope of various immunoassay formats well known in the art.

[0422] The antibodies of the present invention can be used in immunological imaging methods that can detect cancers expressing KOC1, and examples thereof include, but are not limited to, radioscintigraphic imaging using the labeled antibodies of the present invention. Such assays are clinically used to detect, monitor, and prognose cancers that express KOC1; and examples of such cancers include, but are not limited to, bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, testicular cancer, and the like.

[0423] The antibodies of the present invention can be used in any arbitrary form, such as monoclonal or polyclonal antibodies, and can also include antisera obtained by immunizing animals (such as rabbits) with the peptides of the present invention, all kinds of polyclonal and monoclonal antibodies, human antibodies, and chimeric antibodies and humanized antibodies produced by genetic recombination.

[0424] The peptide of the present invention or a fragment thereof used as an antigen for obtaining antibodies can be obtained by chemical synthesis or genetic engineering technology based on the amino acid sequence disclosed herein.

[0425] The peptide used as an immunizing antigen can be a peptide of the present invention or a fragment of the peptide of the present invention. In addition, the peptide can be bound to a carrier or conjugated to a carrier to increase immunogenicity. Keyhole limpet hemocyanin (KLH) is well known as a carrier. Methods for conjugating KLH to peptides are also well known in the art.

[0426] Any mammal can be immunized with the above antigens, and when producing monoclonal antibodies, compatibility with the parental cells used for cell fusion is preferably considered. Generally, animals of the order Rodentia, Lagomorpha, or Primates can be used. Rodents include, for example, mice, rats, and hamsters. Lagomorpha include, for example, rabbits. Primates include, for example, sphenodontids (old world monkeys) such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys, baboons, and chimpanzees.

[0427] Methods for immunizing animals with antigens are known in the art. Intraperitoneal and subcutaneous injections of antigens are standard methods for immunizing mammals. More specifically, the antigen can be diluted and suspended in an appropriate amount of phosphate buffered saline (PBS), normal saline, etc. If necessary, the antigen suspension can be mixed with an appropriate amount of standard adjuvant such as Freund's complete adjuvant, made into an emulsion, and then applied to a mammal. Then, it is preferred to use an antigen mixed with an appropriate amount of Freund's incomplete adjuvant several times every 4 to 21 days. Suitable carriers can be used for immunization. After immunization as described above, the increase in the amount of the desired antibody in the serum can be tested by standard methods.

[0428] Polyclonal antibodies against the peptides of the present invention can be prepared by collecting blood from a mammal (in which an increase in the desired antibody serum level has been confirmed after immunization) and separating serum from the blood by any conventional method. The polyclonal antibodies can be serum containing the polyclonal antibodies, or fractions containing the polyclonal antibodies that can be separated from the serum. Immunoglobulin G or M can be prepared from the fraction that recognizes only the peptides of the present invention using, for example, an affinity column coupled with an affinity column of the peptides of the present invention, and the fractions can be further purified using a protein A or protein G column.

[0429] To prepare monoclonal antibodies, after confirming an increase in serum levels of the desired antibody after immunization, immune cells are collected from a mammal and cell fusion is performed. The immune cells used for cell fusion can preferably be obtained from the spleen. As other parent cells to be fused with the above-mentioned immune cells, for example, mammalian myeloma cells can be used, preferably myeloma cells that have acquired drug selectivity properties for the fused cells.

[0430] The above-mentioned immune cells can be fused with myeloma cells according to a known method, for example, the method of Milstein et al. (Galfre and Milstein, Methods Enzymol 73:3-46 (1981)).

[0431] For the hybridoma obtained by cell fusion, it can be cultivated in standard selection culture medium such as HAT culture medium (culture medium containing hypoxanthine, aminopterin and thymidine) to select. In HAT culture medium, continue cell culture enough time (for example, a few days to a few weeks), to allow all other cells (non-fused cells) except required hybridoma to die. Then, by standard limiting dilution (standard limiting dilution), screen and clone the hybridoma cell that generates required antibody.

[0432] In addition to the above-mentioned method of preparing hybridomas by immunizing non-human animals with antigens, human lymphocytes, such as those infected with Epstein-Barr virus, can also be immunized in vitro with peptides, cells expressing the peptides, or lysates thereof. The immunized lymphocytes can then be fused with immortalized human-derived myeloma cells, such as U266, to produce the desired hybridomas (JPS63-17688) that produce human antibodies capable of binding to the peptides.

[0433] The resulting hybridoma is then transplanted into the peritoneal cavity of a mouse, and ascites is extracted. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or affinity column coupled with the peptide of the present invention.

[0434] Alternatively, antibody-producing immune cells (such as immune lymphocytes) can be immortalized by oncogenes and used to prepare monoclonal antibodies.

[0435] The monoclonal antibodies thus obtained can also be recombinantly prepared using genetic engineering techniques (see, for example, Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in the UK by MacMillan Publishers LTD (1990)). For example, DNA encoding the antibody can be cloned from immune cells such as hybridomas or immune lymphocytes, the DNA can be inserted into a suitable vector, and introduced into a host cell to prepare a recombinant antibody. The present invention also provides recombinant antibodies prepared as described above.

[0436] In addition, the antibodies of the present invention may be antibody fragments or modified antibodies, as long as they bind to the peptides of the present invention. For example, it is desirable that the antibody fragment contains the antigen-binding site of the antibody. Specifically, the antibody fragment may be Fab, F(ab')2, Fv, or a single-chain Fv (scFv) (Huston et al., Proc Natl Acad Sci USA 85:5879-83 (1988)) formed by connecting Fv fragments from the H chain and the L chain via a suitable linker. More specifically, antibody fragments can be generated by treating the antibody with enzymes such as papain or pepsin. Alternatively, genes encoding antibody fragments can be constructed, inserted into expression vectors, and expressed in appropriate host cells (see, e.g., Co et al., J Immunol 152:2968-76 (1994); Better and Horwitz, Methods Enzymol 178:476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178:497-515 (1989); Lamoyi, Methods Enzymol 121:652-63 (1986); Rousseaux et al., Methods Enzymol 121:663-9 (1986); Bird and Walker, Trends Biotechnol 9:132-7 (1991)).

[0437] Antibodies can be modified by conjugation with various molecules such as polyethylene glycol (PEG). The present invention provides antibodies that have undergone such modifications. Modified antibodies can be obtained by chemically modifying antibodies. These modification methods are routine in the art.

[0438] Alternatively, the antibodies of the present invention can be obtained in the form of chimeric antibodies derived from the variable region of a non-human antibody and the constant region derived from a human antibody, or humanized antibodies comprising complementary determining regions (CDRs) derived from non-human antibodies, framework regions (FRs) derived from human antibodies, and constant regions. Such antibodies can be prepared according to known techniques. Humanization can be carried out by replacing the corresponding sequences of human antibodies with non-human antibody CDR sequences (see, for example, Verhoeyen et al., Science 239: 1534-1536 (1988)). Thus, such humanized antibodies are chimeric antibodies in which a small portion of the human variable domain has been replaced by the corresponding sequence from a non-human species.

[0439] Completely human antibodies can also be used, which contain human variable regions in addition to human framework regions and constant regions. Such antibodies can be prepared using a variety of techniques known in the art. For example, in vitro methods include using recombinant libraries of human antibody fragments displayed on phage (e.g., Hoogenboom & Winter, J. Mol. Biol. 227: 381 (1991)). Similarly, human antibodies can be generated by introducing human immunoglobulin loci into transgenic animals, such as mice in which endogenous immunoglobulin genes are partially or completely inactivated. This method is described in, for example, U.S. Patent Nos. 6,150,584; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425 and 5,661,016.

[0440] The antibodies obtained as above can be purified to homogeneity. For example, the separation and purification of antibodies can be carried out according to the separation and purification methods for general proteins. For example, column chromatography such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis and isoelectric focusing can be used to separate and purify antibodies (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)) by appropriate selection and combination, but are not limited to this. Protein A columns and protein G columns can be used as affinity columns. Exemplary usable protein A columns include, for example, Hyper D, POROS and Sepharose FF (Pharmacia).

[0441] In addition to affinity chromatography, exemplary chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed. Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996)). Chromatographic procedures can be performed by liquid chromatography, such as HPLC and FPLC.

[0442] For example, the antigen-binding activity of the antibody of the present invention can be measured by using absorbance measurement, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and / or immunofluorescence (IF). In ELISA, the antibody of the present invention is immobilized on a plate, the peptide of the present invention is applied to the plate, and then a sample containing the desired antibody, such as the culture supernatant of cells generating the antibody or the purified antibody, is applied. Then a second antibody labeled with an enzyme (such as alkaline phosphatase) that recognizes the first antibody is applied, and the plate is incubated. Then, after washing, an enzyme substrate, such as p-nitrophenyl phosphate, is added to the plate, and the antigen-binding activity of the sample is assessed by measuring absorbance. In order to assess the binding activity of the antibody, a peptide fragment such as a C-terminal or N-terminal fragment can be used as the antigen. The activity of the antibody of the present invention can be assessed using BIAcore (Pharmacia).

[0443] The peptide of the present invention using the above method can be detected or measured by exposing the antibody of the present invention to a sample presumed to contain the peptide of the present invention, and detecting or measuring the immune complex formed between the antibody and the peptide.

[0444] For example, the antibodies of the present invention can be used to detect the peptides of the present invention present in a blood sample (e.g., a serum sample) of a subject. Alternatively, the peptides of the present invention can also be used to detect the antibodies of the present invention present in a blood sample (e.g., a serum sample) of a subject. The results of measuring the peptides of the present invention or the antibodies of the present invention in a blood sample of a subject can be used to select a subject for administering a pharmaceutical composition of the present invention or to monitor the efficacy of a pharmaceutical composition. In addition, it has been reported that patients with antibodies directed against peptides administered as vaccines may have high responsiveness to the vaccine. Therefore, when administering the peptides of the present invention as a vaccine using the patient's antibodies as an index, the peptides of the present invention can be used as immunoassay antigens for selecting patients with high responsiveness.

[0445] XII. Vectors and Host Cells

[0446] The present invention provides vectors comprising polynucleotides encoding the peptides of the present invention and host cells into which the vectors have been introduced. The vectors of the present invention can be used to maintain the polynucleotides of the present invention in host cells, to express the peptides of the present invention in host cells, or to administer the polynucleotides of the present invention for gene therapy.

[0447] When E. coli is used as the host cell and the vector is amplified and produced in large quantities in E. coli (e.g., JM109, DH5-alpha, HB101, or XL1-Blue), the vector needs to have a "replication origin" for amplification in E. coli and a marker gene for selecting transformed E. coli (e.g., a drug resistance gene selected by drugs such as ampicillin, tetracycline, kanamycin, chloramphenicol, etc.). For example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, etc. can be used. In addition, pGEM-T, pDIRECT, and pT7 can be used for cloning as well as the above vectors. When the vector is used to produce the peptide of the present invention, an expression vector can be used. For example, an expression vector to be expressed in E. coli should have the above-mentioned characteristics of amplification in E. coli. When using Escherichia coli such as JM109, DH5-alpha, HB101, or XL1-Blue as a host cell, the vector should contain a promoter capable of efficiently expressing the desired gene in E. coli, such as the lacZ promoter (Ward et al., Nature 341:544-6 (1989); FASEB J 6:2422-7 (1989)), the araB promoter (Better et al., Science 240:1041-3 (1988)), the T7 promoter, and the like. In this regard, for example, pGEX-5X-1 (Pharmacia), the "QIAexpress system" (Qiagen), pEGFP, and pET (in this case, the host cell is preferably BL21 expressing T7 RNA polymerase) can be used instead of the above vectors. In addition, the vector may also contain a signal sequence for peptide secretion. An exemplary signal sequence for directing peptide secretion to the periplasm of E. coli is the pelB signal sequence (Lei et al., J Bacteriol 169:4379 (1987)). Means for introducing the vector into target host cells include, for example, the calcium chloride method and electroporation.

[0448] In addition to E. coli, the polypeptides of the present invention can also be produced using, for example, expression vectors derived from mammals (e.g., pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18(17):5322 (1990)), pEF, pCDM8), expression vectors derived from insect cells (e.g., "Bac-to-BAC Baculovirus Expression System" (GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g., pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression vectors derived from retroviruses (e.g., pZIpneo), expression vectors derived from yeast (e.g., "Pichia Expression Kit" (Invitrogen), pNV11, SP-Q01), and expression vectors derived from Bacillus subtilis (e.g., pPL608, pKTH50).

[0449] In order to express the vector in animal cells such as CHO, COS or NIH3T3 cells, the vector should have a promoter necessary for expression in the cells, for example, SV40 promoter (Mulligan et al., Nature 277: 108 (1979)), MMLV-LTR promoter, EF1-alpha promoter (Mizushima et al., Nucleic Acids Res 18: 5322 (1990)), CMV promoter, etc., and preferably a marker gene for selecting transformants (for example, a drug resistance gene selected by drugs (e.g., neomycin, G418)). Examples of known vectors having these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

[0450] The following exemplifies embodiments of the present invention based on the above description; however, the present invention is not limited to these embodiments.

[0451] [1] A peptide of less than 15 amino acids having cytotoxic T cell (CTL) inducibility, comprising an amino acid sequence selected from the following group:

[0452] (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41; and

[0453] (b) an amino acid sequence in which 1, 2 or several amino acids are substituted, deleted, inserted and / or added in an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41.

[0454] [2] The peptide of [1], which is selected from the amino acid sequences of the following groups (i) to (iv):

[0455] (i) A peptide comprising an amino acid sequence, wherein one or more substitutions selected from the following groups (a) to (d) are introduced into an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30 and 32:

[0456] (a) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine and tyrosine;

[0457] (b) substituting the third amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, phenylalanine, tyrosine, isoleucine and alanine;

[0458] (c) substituting the seventh amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, isoleucine, tyrosine, valine and phenylalanine; and

[0459] (d) substituting the amino acid at the C-terminus with an amino acid selected from the group consisting of lysine and arginine;

[0460] (ii) a peptide comprising an amino acid sequence, wherein one or more substitutions selected from the following groups (a) to (c) are introduced into an amino acid sequence selected from the following group: SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85:

[0461] (a) substituting the first amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;

[0462] (b) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of phenylalanine, tyrosine, alanine, isoleucine, leucine and valine;

[0463] (c) substituting the amino acid at the C-terminus with an amino acid selected from the group consisting of arginine and lysine;

[0464] (iii) A peptide comprising an amino acid sequence, wherein one or more substitutions selected from the following groups (a) to (b) are introduced into an amino acid sequence selected from the group consisting of SEQ ID NOs: 27, 30 and 52:

[0465] (a) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, methionine, valine, alanine, isoleucine, serine and threonine; and

[0466] (b) substituting the C-terminal amino acid with an amino acid selected from the group consisting of arginine, lysine, tyrosine and phenylalanine; and

[0467] (iv) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the following groups (a) to (c) are introduced into an amino acid sequence selected from the following group: SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41:

[0468] (a) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of threonine and serine;

[0469] (b) substituting the third amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;

[0470] (c) Substitution of the C-terminal amino acid with tyrosine.

[0471] [3] The peptide of [1], which consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41.

[0472] [4] A polynucleotide encoding the peptide of any one of [1] to [3].

[0473] [5] A composition comprising a pharmaceutically acceptable carrier and at least one component selected from the following groups (a) to (e):

[0474] (a) one or more types of peptides according to any one of [1] to [3];

[0475] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0476] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on their cell surface;

[0477] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface; and

[0478] (e) CTLs targeting the peptide of any one of [1] to [3].

[0479] [6] The composition of [5], which is a composition for inducing CTL, wherein the component is at least one component selected from the following groups (a) to (d):

[0480] (a) one or more types of peptides according to any one of [1] to [3];

[0481] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0482] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on their cell surface;

[0483] (d) An exosome presenting a complex of the peptide of any one of [1] to [3] and an HLA antigen on its cell surface.

[0484] [7][5] The composition of [7][5] is a pharmaceutical composition.

[0485] [8][7] The composition of [8], which is used for one or more uses selected from the group consisting of: (i) cancer treatment, (ii) cancer prevention (prophylaxis) and (iii) prevention (prophylaxis) of cancer recurrence after surgery.

[0486] [9][7] A composition for inducing an immune response against cancer.

[0487]

[10] The composition of [8] or [9], wherein the cancer is selected from the group consisting of bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors and testicular cancer.

[0488]

[11] The composition of any one of [5] to

[10] , formulated for administration to a subject positive for at least one HLA selected from the group consisting of HLA-A11, HLA-A33, HLA-A03, and HLA-A01.

[0489]

[12] A method for inducing APCs having CTL inducibility, comprising a step selected from the following groups (a) and (b):

[0490] (a) contacting APCs with the peptide described in any one of [1] to [3] in vitro, ex vivo or in vivo; and

[0491] (b) A polynucleotide encoding the peptide of any one of [1] to [3] is introduced into APC.

[0492]

[13] A method for inducing CTL, comprising a step selected from the group consisting of:

[0493] (a) co-culturing CD8-positive T cells with APCs presenting a complex of an HLA antigen and the peptide of any one of [1] to [3] on its surface;

[0494] (b) co-culturing CD8-positive T cells with exosomes presenting a complex of an HLA antigen and the peptide of any one of [1] to [3] on its surface;

[0495] (c) introducing a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to a peptide of any one of [1] to [3] presented by an HLA antigen on the cell surface into a CD8-positive T cell.

[0496]

[14] APC that presents a complex of an HLA antigen and the peptide of any one of [1] to [3] on its surface.

[0497]

[15]

[14] APCs, which were induced by the method of

[12] .

[0498]

[16] CTL targeting the peptide of any one of [1] to [3].

[0499]

[17]

[16] CTLs, which were induced by the method of

[13] .

[0500]

[18] A method for inducing an immune response against cancer, comprising administering to a subject at least one component selected from the following groups (a) to (e):

[0501] (a) one or more types of peptides according to any one of [1] to [3];

[0502] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0503] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on their cell surface;

[0504] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on its cell surface; and

[0505] (e) CTLs targeting the peptide of any one of [1] to [3].

[0506]

[19] A method for treating and / or preventing cancer, and / or preventing its recurrence after surgery, comprising administering to a subject at least one component selected from the following groups (a) to (e):

[0507] (a) one or more types of peptides according to any one of claims 1 to 3;

[0508] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0509] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on their cell surface;

[0510] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on its cell surface; and

[0511] (e) CTLs targeting the peptide of any one of [1] to [3].

[0512]

[20] An antibody that binds to the peptide of any one of [1] to [3].

[0513]

[21] A method for screening peptides having CTL inducibility, comprising the following steps:

[0514] (a) generating a candidate sequence consisting of an amino acid sequence, wherein one, two or several amino acid residues are substituted, deleted, inserted and / or added to an original amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41;

[0515] (b) selecting, from the candidate sequences generated in (a), candidate sequences that do not have significant homology (sequence identity) with any known human gene product other than KOC1;

[0516] (c) contacting the APC with a peptide consisting of the candidate sequence selected in (b);

[0517] (d) contacting the APC of (c) with CD8-positive T cells; and

[0518] (e) Selecting peptides having equal or higher CTL inducibility than the peptide consisting of the original amino acid sequence.

[0519]

[22] Use of at least one component selected from the following groups (a) to (e) in the preparation of a composition for inducing an immune response against cancer:

[0520] (a) one or more types of peptides according to any one of [1] to [3];

[0521] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0522] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;

[0523] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface; and

[0524] (e) CTLs targeting the peptide of any one of [1] to [3].

[0525]

[23] Use of at least one component selected from the following groups (a) to (e) in the preparation of a pharmaceutical composition for treating and / or preventing cancer and / or preventing its postoperative recurrence:

[0526] (a) one or more types of peptides according to any one of [1] to [3];

[0527] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0528] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;

[0529] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface; and

[0530] (e) CTLs targeting the peptide of any one of [1] to [3].

[0531]

[24] Use of at least one component selected from the following groups (a) to (e) in inducing an immune response against cancer:

[0532] (a) one or more types of peptides according to any one of [1] to [3];

[0533] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0534] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;

[0535] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface; and

[0536] (e) CTLs targeting the peptide of any one of [1] to [3].

[0537]

[25] At least one ingredient selected from the following groups (a) to (e), for use in treating and / or preventing cancer and / or preventing its recurrence after surgery:

[0538] (a) one or more types of peptides according to any one of [1] to [3];

[0539] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0540] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;

[0541] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface; and

[0542] (e) CTLs targeting the peptide of any one of [1] to [3].

[0543]

[26] A method for inducing cytotoxic activity against cells expressing KOC1, comprising the step of administering to a subject at least one component selected from the following groups (a) to (e):

[0544] (a) one or more types of peptides according to any one of [1] to [3];

[0545] (b) one or more types of polynucleotides encoding the peptide of any one of [1] to [3] in an expressible form;

[0546] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface;

[0547] (d) an exosome that presents a complex of the peptide of any one of [1] to [3] and an HLA antigen on the cell surface; and

[0548] (e) CTLs targeting the peptide of any one of [1] to [3].

[0549]

[27] A freeze-dried preparation comprising one or more types of the peptide of any one of [1] to [3].

[0550]

[28] A pharmaceutical composition prepared by a method comprising dissolving one or more types of peptides according to any one of [1] to [3] in a water-soluble carrier and performing filtration sterilization.

[0551]

[29] A filter-sterilized aqueous solution comprising one or more types of the peptide of any one of [1] to [3] and a water-soluble carrier.

[0552]

[30] An emulsion comprising one or more types of peptides according to any one of [1] to [3], a water-soluble carrier and an oil adjuvant.

[0553]

[31] A kit comprising a container containing the pharmaceutical composition of any one of [7] to

[11] and a container containing an adjuvant.

[0554]

[32] A kit comprising a container for storing a freeze-dried preparation comprising the peptide of any one of [1] to [3], a container for storing an adjuvant, and a container for storing the freeze-dried preparation in a dissolving solution.

[0555] The present invention is explained in detail herein with reference to its specific embodiments. However, it should be understood that the above explanation is actually illustrative and explanatory explanation, and is intended to explain the present invention and its preferred embodiments. Through routine experiments, those skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited to the above description, but is intended to be limited by the appended claims and their equivalents.

[0556] Hereinafter, the present invention will be described in more detail with reference to the examples. However, although the following materials, methods and examples can be used to help those of ordinary skill in the art to carry out and use certain embodiments of the present invention, these are merely intended to illustrate various aspects of the present invention and are not intended to limit the scope of the present invention. As one of ordinary skill in the art will readily recognize, methods and materials similar or equivalent to those described herein can be used for implementation or testing of the present invention.

[0557] All prior art documents cited herein are incorporated into this specification by reference. Example

[0558] [Example 1]

[0559] Materials and methods

[0560] cell lines

[0561] C1R, HLA-A negative and HLA-B negative human B lymphoblastoid cell lines, and COS7, African green monkey kidney cell line were purchased from ATCC.

[0562] Generation of stimulator cells with stable HLA-A*1101 expression

[0563] C1R cells stably expressing HLA-A*1101 (C1R-A11) were used as stimulator cells. The cDNA encoding the open reading frame of HLA-A*1101 was amplified by PCR and cloned into an expression vector. C1R cells were transfected with the expression vector and then selected with G418 (Invitrogen) for two weeks. Cells selected with G418 were seeded into wells of a 96-well plate containing medium supplemented with G418 and cultured for a further 30 days. Flow cytometry analysis confirmed exogenous HLA-A*1101 expression in C1R cells.

[0564] Selection of candidate KOC1-derived peptides

[0565] Using the combined prediction server "NetMHC3.2" ( www.cbs.dtu.dk / services / NetMHC / )(Buus et al., Tissue Antigens. 2003 Nov, 62(5):378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17, Bioinformatics. 2004 Jun 12;20(9):1388-97) predicted KOC1-derived 9-mer and 10-mer peptides that bind to HLA-A*1101 molecules.

[0566] Peptide synthesis

[0567] The peptide was synthesized by Biosynthesis (Lewisville, Texas) according to standard solid-phase synthesis methods and purified by reverse-phase high-performance liquid chromatography (HPLC). The purity (>90%) and identity of the peptide were analyzed by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethyl sulfoxide at 20 mg / ml and stored at -80°C.

[0568] In vitro CTL induction

[0569] Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells to induce cytotoxic T lymphocyte (CTL) responses against peptides presented on human leukocyte antigens (HLA). DCs were prepared in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003, 63(14):4112-8). Specifically, peripheral blood mononuclear cells isolated from healthy volunteers (HLA-A*1101 positive) using Ficoll-Paque Plus (Pharmacia) solution were separated by adherence to plastic tissue culture plates (Becton Dickinson) and concentrated as a mononuclear cell fraction. The monocyte-enriched population was cultured in AIM-V medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS) in the presence of 1000 IU / ml of granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml of interleukin (IL)-4 (R&D System). After 7 days of culture, the cytokine-induced DCs were pulsed with 20 μg / ml of each synthetic peptide in the presence of 3 μg / ml β2-microglobulin in AIM-V medium at 37°C for 3 hours. The generated cells showed expression of DC-associated molecules such as CD80, CD83, CD86 and HLA class II on their cell surfaces (data not shown). Subsequently, these peptide-pulsed DCs were inactivated by X-ray irradiation (20 Gy) and incubated at a ratio of 1:20 with autologous CD8 obtained by positive selection using a CD8 positive isolation kit (Dynal). + These culture products were inoculated into 48-well plates (Corning). Each well was prepared to contain 1.5 x 10 T cells in 0.5 ml of AIM-V / 2% AS medium. 4 Peptide-pulsed DCs, 3x 10 5 CD8 +T cells and 10 ng / ml IL-7 (R&D System). After 3 days, IL-2 (CHIRON) was added to these cultures at a final concentration of 20 IU / ml. On days 7 and 14, these T cells were further stimulated with autologous DCs pulsed with peptides. DCs were prepared in the same manner as described above. On day 21, after the third peptide stimulation, CTLs against peptide-pulsed C1R-A11 cells were detected by human interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0570] CTL expansion procedure

[0571] CTLs were expanded in culture using a method similar to that disclosed by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16):1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2):216-23). ​​CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines in a total of 25 ml of AIM-V medium (5 x 10 6 Cells / flask) were cultured in a medium containing 5% AS (AIM-V / 5% AS) and 40 ng / ml anti-CD3 antibody. One day after the start of culture, 120 IU / ml IL-2 was added to the culture. On days 5, 8, and 11, fresh AIM-V / 5% AS medium containing 30 IU / ml IL-2 was added to the culture (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0572] Establishment of CTL clones

[0573] CTLs were diluted to 0.3, 1, and 3 cells / well in 96 round-bottom microtiter plates (Nalge Nunc International). CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines (1 x 10 4 cells / well). After 10 days, 50 μl / well of IL-2 was added to the culture medium to a final concentration of 125 IU / ml. CTL activity was tested on day 14, and CTL clones were expanded using the same method as described above (Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0574] Specific CTL activity

[0575] To examine specific CTL activity, IFN-gamma ELISPOT assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A11 (1×10 4 Cells / well) were used as stimulator cells. Induced CTLs (i.e., CTL lines and CTL clones) were used as responder cells. IFN-gamma ELISPOT assays and IFN-gamma ELISA were performed according to the manufacturer's instructions.

[0576] Establishment of target cells forced to express target genes and HLA-A*1101

[0577] cDNA encoding the open reading frame of the target gene or HLA-A*1101 was amplified by PCR. The PCR amplification product was cloned into an expression vector. One or both of the target gene-expressing vector and the HLA-A*1101-expressing vector were transfected into COS7 (a cell line negative for the target gene and HLA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Two days after transfection, the transfected cells were harvested with Versene (Invitrogen) and used as target cells for CTL activity assays (5 x 10 4 cells / well).

[0578] result

[0579] Prediction of KOC1-derived HLA-A*1101-binding peptides

[0580] Tables 1a and 1b show, in descending order of binding affinity, KOC1-derived 9-mer and 10-mer peptides predicted to bind to HLA-A*1101. A total of 60 peptides with potential HLA-A*1101 binding ability were selected and examined to determine epitope peptides.

[0581] [Table 1a]

[0582] HLA-A11-binding 9-mer peptide derived from KOC1

[0583] Starting position Amino acid sequence Kd(nM) SEQ ID NO 258 KSILEIMHK 16 1 121 VVNVTYSSK 18 2 465 KAQGRIYGK 32 3 517 SSAEVVVPR 45 4 415 LSVGAIIGK 76 5 28 PVSGPFLVK 85 6 52 KAIEALSGK 92 7 142 FQLENFTLK 150 8 215 GATIRNITK 293 9 338 KAEEEIMKK 297 10 497 FAAGRVIGK 405 11 15 PSDLESIFK 451 12 536 VVKITGHFY 757 13 479 FVSPKEEVK 791 14 493 RVPSFAAGR 1100 15 272 KFTEEIPLK 1108 16 226 QSKIDVHRK 1125 17 330 KGNVETCAK 1835 18 544 YACQVAQRK 1841 19 220 NITKQTQSK 2617 20 301 KIEQDTDTK 2631 21 553 IQEILTQVK 3008 22 182 QGSPGSVSK 3293 23 559 QVKQHQQQK 3313 24 58 SGKIELHGK 4377 25 205 QVVGAIIGK 4472 26

[0584] [Table 1b]

[0585] HLA-A11-binding 10-mer peptide derived from KOC1

[0586] Starting position Amino acid sequence Kd(nM) SEQ ID NO 120 AVVNVTYSSK 13 27 414 ALSVGAIIGK 33 28 456 ITGPPEAQFK 73 29 204 TQFVGAIIGK 86 30 73 SVPKRQRIRK 103 31 14 APSDLESIFK 152 32 183 GSPGSVSKQK 165 33 496 SFAAGRVIGK 168 34 516 LSSAEVVVPR 169 35 552 KIQEILTQVK 214 36 431 LSRFAGASIK 342 37 181 RQGSPGSVSK 368 38 124 VTYSSKDQAR 437 39 225 TQSKIDVHRK 478 40 535 VVVKITGHFY 678 41 224 QTQSKIDVHR 711 42 57 LSGKIELHGK 904 43 141 GFQLENFTLK 911 44 568 ALQSGPPQSR 1065 45 312 ISPLQELTLY 1262 46 441 IAPAEAPDAK 1570 47 27 IPVSGPFLVK 1622 48 570 QSGPPQSRRK 1777 49 95 EVLDSLLVQY 2124 50 336 CAKAEEEIMK 2194 51 281 ILAHNNFVGR 2264 52 466 AQGRIYGKIK 2595 53 160 AQQNPLQQPR 2689 54 420 IIGKQGQHIK 2763 55 558 TQVKQHQQQK 2862 56 257 CKSILEIMHK 3467 57 291 LIGKEGRNLK 3795 58 263 IMHKEAQDIK 4083 59 569 LQSGPPQSRR 4107 60

[0587] The starting position indicates the number of amino acid residues from the N-terminus of KOC1, and the dissociation constant [Kd (nM)] is derived from "NetMHC3.2".

[0588] Induction of CTLs by predicted KOC1-derived HLA-A*1101-restricted peptides

[0589] CTLs against KOC1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured by IFN-gamma ELISPOT assay ( Figure 1 Compared to the control, CTLs in well #6 (a) with KOC1-A11-9-415 (SEQ ID NO: 5), well #4 (b) with KOC1-A11-10-414 (SEQ ID NO: 28), well #5 (c) with KOC1-A11-10-204 (SEQ ID NO: 30), and well #8 (d) with KOC1-A11-10-14 (SEQ ID NO: 32) showed strong IFN-gamma production. Meanwhile, although other peptides shown in Tables 1a and 1b potentially have HLA-A*1101 binding activity, no specific CTL activity was detected as a result of stimulation with these peptides. An example of typical negative data is the absence of specific IFN-gamma production observed in CTLs stimulated with KOC1-A11-9-258 (SEQ ID NO: 1) (e). As a result, four types of KOC1-derived peptides were selected as peptides capable of inducing effective CTLs.

[0590] Establishment of CTL lines and clones targeting KOC1-derived HLA-A*1101-restricted peptides

[0591] CTL lines were established by proliferating CTLs in well #6 (a) with KOC1-A11-9-415 (SEQ ID NO: 5), in well #5 (b) with KOC1-A11-10-204 (SEQ ID NO: 30), and in well #8 (c) with KOC1-A11-10-14 (SEQ ID NO: 32), which showed peptide-specific CTL activity in IFN-gamma ELISPOT assay. The CTL activity of these CTL lines was measured by IFN-gamma ELISA ( Figure 2 ). These CTL lines showed strong IFN-gamma production against target cells pulsed with the corresponding peptides compared to target cells not pulsed with the peptides. In addition, CTL clones were established by limiting dilution from the CTL lines as described in the "Materials and Methods" section above, and IFN-gamma production from CTL clones against peptide-pulsed C1R-A11 was measured by IFN-gamma ELISA. Strong IFN-gamma production was observed in CTL clones stimulated with KOC1-A11-9-415 (SEQ ID NO: 5) (a) and KOC1-A11-10-14 (SEQ ID NO: 32) (b) ( Figure 3 ).

[0592] Specific CTL activity against target cells expressing KOC1 and HLA-A*1101

[0593] CTL clones against KOC1-A11-10-14 (SEQ ID NO: 32) were established to study their ability to recognize target cells expressing KOC1 and HLA-A*1101 molecules. COS7 cells transfected with both full-length KOC1 and HLA-A*1101 genes (a specific model of target cells expressing KOC1 and HLA-A*1101 genes) were prepared as target cells. COS7 cells transfected with full-length KOC1 or HLA-A*1101 were prepared as controls. CTL clones stimulated with KOC1-A11-10-14 (SEQ ID NO: 32) showed strong CTL activity against COS7 cells expressing both KOC1 and HLA-A*1101 ( Figure 4 On the other hand, no significant specific CTL activity was detected against the control. These data clearly demonstrate that KOC1-A11-10-14 (SEQ ID NO: 32) is a peptide produced by endogenous processing of KOC1, presented on target cells with HLA-A*1101 molecules, and recognized by CTLs. These results suggest the possibility that KOC1-A11-10-14 (SEQ ID NO: 32) may be suitable as a cancer vaccine for patients with KOC1-expressing cancers.

[0594] Homology analysis of antigenic peptides

[0595] CTLs stimulated with KOC1-A11-9-415 (SEQ ID NO: 5), KOC1-A11-10-414 (SEQ ID NO: 28), KOC1-A11-10-204 (SEQ ID NO: 30), or KOC1-A11-10-14 (SEQ ID NO: 32) showed significant specific CTL activity. These results are likely due to the fact that the sequences of KOC1-A11-9-415 (SEQ ID NO: 5), KOC1-A11-10-414 (SEQ ID NO: 28), KOC1-A11-10-204 (SEQ ID NO: 30), and KOC1-A11-10-14 (SEQ ID NO: 32) are homologous to peptides derived from other molecules known to sensitize the human immune system. To rule out this possibility, homology analysis was performed by querying the sequences of these peptides using the BLAST algorithm (blast.ncbi.nlm.nih.gov / Blast.cgi). This result showed no sequences with significant homology to the sequences of KOC1-A11-9-415 (SEQ ID NO: 5), KOC1-A11-10-414 (SEQ ID NO: 28), KOC1-A11-10-204 (SEQ ID NO: 30), and KOC1-A11-10-14 (SEQ ID NO: 32). On the other hand, KOC1-A11-10-204 (SEQ ID NO: 30) was identical to a peptide sequence identified in IMP-2, another member of the IMP family. IMP-2 has previously been reported to be unexpressed in normal organs other than testis and fetal liver (Hammer NA et al., Reproduction. 2005; 130(2): 203-12). It is implicated in the proliferation of glioblastoma cancer stem cells (Janiszewska M et al., Genes Dev. 2012; 26(17): 1926-44). IMP-2 is expected to be a target antigen for cancer immunotherapy. Therefore, to the best of the present inventors' knowledge, these peptides are unlikely to trigger unintended immune responses against other unrelated molecules. In summary, novel KOC1-derived HLA-A11-restricted epitope peptides were identified. This demonstrates that KOC1-derived epitope peptides are suitable for cancer immunotherapy.

[0596] [Example 2]

[0597] Materials and methods

[0598] cell lines

[0599] C1R, HLA-A negative and HLA-B negative human B lymphoblastoid cell lines, and COS7, African green monkey kidney cell line were purchased from ATCC.

[0600] Generation of stimulator cells with stable HLA-A*3303 expression

[0601] C1R cells stably expressing HLA-A*3303 (C1R-A33) were used as stimulator cells. The cDNA encoding the open reading frame for HLA-A*3303 was amplified by PCR and cloned into an expression vector. C1R cells were transfected with the expression vector and then selected with G418 (Invitrogen) for two weeks. Cells selected with G418 were seeded into wells of a 96-well plate containing medium supplemented with G418 and cultured for a further 30 days. Flow cytometry analysis confirmed exogenous HLA-A*3303 expression in C1R cells.

[0602] Selection of candidate KOC1-derived peptides

[0603] KOC1-derived 9-mer and 10-mer peptides that bind to HLA-A*3303 molecules were predicted using the binding prediction server “NetMHCpan2.8” (www.cbs.dtu.dk / services / NetMHCpan / ) (Nielsen et al., PLoS One. 2007; 29; 2(8): e796; Hoof et al., Immunogenetics. 2009; 61(1): 1-13).

[0604] Peptide synthesis

[0605] These peptides were synthesized by Biosynthesis (Lewisville, Texas) according to standard solid-phase synthesis methods and purified by reverse-phase high-performance liquid chromatography (HPLC). The purity (>90%) and identity of the peptides were analyzed by analytical HPLC and mass spectrometry, respectively. The peptides were dissolved in dimethyl sulfoxide at 20 mg / ml and stored at -80°C.

[0606] In vitro CTL induction

[0607] Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells to induce cytotoxic T lymphocyte (CTL) responses against peptides presented on human leukocyte antigens (HLA). DCs were prepared in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003, 63(14):4112-8). Specifically, peripheral blood mononuclear cells isolated from healthy volunteers (HLA-A*3303 positive) using Ficoll-Paque Plus (Pharmacia) solution were separated by adherence to plastic tissue culture plates (Becton Dickinson) and concentrated as a mononuclear cell fraction. The monocyte-enriched population was cultured in AIM-V medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS) in the presence of 1000 IU / ml of granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml of interleukin (IL)-4 (R&D System). After 7 days of culture, the DC induced by cytokines was pulsed with 20 micrograms / ml of each synthetic peptide at 37°C in the presence of 3 micrograms / ml β2-microglobulin in AIM-V culture medium for 3 hours. The generated cells apparently express DC-related molecules such as CD80, CD83, CD86 and HLA II class on their cell surface (data not shown). Then, these DCs pulsed with peptides were inactivated by X-ray radiation (20 Gy) and mixed with autologous CD8+ T cells obtained by positive selection using a CD8 positive separation kit (Dynal) at a ratio of 1:20. These culture products were inoculated in 48-well plates (Corning). Each well was prepared to contain 1.5 x 104 DCs pulsed with peptides, 3 x 105 CD8+ T cells and 10 ng / ml IL-7 (R&D System) in 0.5 ml AIM-V / 2% AS culture medium. After 3 days, IL-2 (CHIRON) was added to these cultures at a final concentration of 20 IU / ml. On days 7 and 14, these T cells were further stimulated with autologous DC pulsed with peptides. DC were prepared in the same manner as described above each time.On day 21, after the third peptide stimulation, CTLs against peptide-pulsed C1R-A33 cells were detected by human interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0608] CTL expansion procedure

[0609] CTLs were expanded in culture using a method similar to that disclosed by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16):1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2):216-23). ​​CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines in a total of 25 ml of AIM-V medium (5 x 10 6 Cells / flask) were cultured in a medium containing 5% AS (AIM-V / 5% AS) and 40 ng / ml anti-CD3 antibody. One day after the start of culture, 120 IU / ml IL-2 was added to the culture. On days 5, 8, and 11, fresh AIM-V / 5% AS medium containing 30 IU / ml IL-2 was added to the culture (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0610] Establishment of CTL clones

[0611] CTLs were diluted to 0.3, 1, and 3 cells / well in 96 round-bottom microtiter plates (Nalge Nunc International). CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines (1 x 10 4 cells / well). After 10 days, 50 μl / well of IL-2 was added to the culture medium to a final concentration of 125 IU / ml. CTL activity was tested on day 14, and CTL clones were expanded using the same method as described above (Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0612] Specific CTL activity

[0613] To examine specific CTL activity, IFN-gamma ELISPOT assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A33 (1×10 4 Cells / well) were used as stimulator cells. Induced CTLs (i.e., CTL lines and CTL clones) were used as responder cells. IFN-gamma ELISPOT assays and IFN-gamma ELISA were performed according to the manufacturer's instructions.

[0614] Establishment of target cells with forced expression of target genes and HLA-A*3303

[0615] cDNA encoding the open reading frame of the target gene or HLA-A*3303 was amplified by PCR. The PCR amplification product was cloned into an expression vector. One or both of the target gene- and HLA-A*3303-expressing vectors were transfected into COS7 (a cell line negative for the target gene and HLA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Two days after transfection, the transfected cells were harvested with Versene (Invitrogen) and used as target cells for CTL activity assays (5 x 10 4 cells / well).

[0616] result

[0617] Prediction of KOC1-derived HLA-A*3303 binding peptides

[0618] Tables 2a and 2b show KOC1-derived 9-mer and 10-mer peptides predicted to bind to HLA-A*3303 in descending order of binding affinity. A total of 37 peptides with potential HLA-A*3303 binding ability were selected and examined to identify epitope peptides. [Table 2a]

[0619] HLA-A33-binding 9-mer peptide derived from KOC1

[0620] Starting position Amino acid sequence Kd(nM) SEQ ID NO 543 FYACQVAQR 98 61 517 SSAEVVVPR 105 4 282 LAHNNFVGR 140 62 493 RVPSFAAGR 182 15 317 ELTLYNPER 260 63 485 EVKLEAHIR 278 64 125 TYSSKDQAR 467 65 461 EAQFKAQGR 555 66 286 NFVGRLIGK 933 67 225 TQSKIDVHR 1039 68 69 EVEHSVPKR 1453 69 161 QQNPLQQPR 1485 70 406 ETVHLFIPA 2340 71 73 SVPKRQRIR 2969 72 570 QSGPPQSRR 3001 73 121 VVNVTYSSK 3272 2 465 KAQGRIYGK 3308 3 142 FQLENFTLK 4063 8 34 LVKTGYAFV 4107 74 211 IGKEGATIR 4197 75 307 DTKITISPL 4676 76

[0621] [Table 2b]

[0622] HLA-A33-binding 10-mer peptide derived from KOC1

[0623] Starting position Amino acid sequence Kd(nM) SEQ ID NO 542 HFYACQVAQR 16 77 124 VTYSSKDQAR 201 39 224 QTQSKIDVHR 296 42 281 ILAHNNFVGR 320 52 72 HSVPKRQRIR 428 78 516 LSSAEVVVPR 467 35 496 SFAAGRVIGK 1710 34 543 FYACQVAQRK 2635 79 424 QGQHIKQLSR 2752 80 190 KQKPCDLPLR 3492 81 431 LSRFAGASIK 3726 37 568 ALQSGPPQSR 4478 45 210 IIGKEGATIR 4831 82 478 NFVSPKEEVK 4831 83 77 RQRIRKLQIR 4883 84 285 NNFVGRLIGK 4883 85

[0624] The starting position indicates the number of amino acid residues from the N-terminus of KOC1, and the dissociation constant [Kd (nM)] is derived from "NetMHCpan2.8".

[0625] Induction of CTLs by predicted KOC1-derived HLA-A*3303-restricted peptides

[0626] CTLs against KOC1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured by IFN-gamma ELISPOT assay ( Figure 5Compared to the control, well #1 with KOC1-A33-9-543 (SEQ ID NO: 61) (a), well #2 with KOC1-A33-9-282 (SEQ ID NO: 62) (b), well #3 with KOC1-A33-9-317 (SEQ ID NO: 63) (c), well #8 with KOC1-A33-9-485 (SEQ ID NO: 64) (d), well #3 with KOC1-A33-9-286 (SEQ ID NO: 67) (e), well #8 with KOC1-A33-9-34 (SEQ ID NO: 74), well #4 with KOC1-A33-10-542 (SEQ ID NO: 77), well #5 with KOC1-A33-10-281 (SEQ ID NO: 78) (f). Well #3 (h) with KOC1-A33-10-543 (SEQ ID NO: 52), well #6 (i) with KOC1-A33-10-424 (SEQ ID NO: 80), and well #5 (k) with KOC1-A33-10-285 (SEQ ID NO: 85) showed strong IFN-gamma production. Meanwhile, although other peptides shown in Tables 2a and 2b potentially have HLA-A*3303 binding activity, no specific CTL activity was detected as a result of stimulation with these peptides. An example of typical negative data is that no specific IFN-gamma production was observed in CTLs stimulated with KOC1-A33-9-517 (SEQ ID NO: 4) (l). As a result, 11 types of KOC1-derived peptides were selected as peptides capable of inducing potent CTLs.

[0627] Establishment of CTL lines and clones targeting KOC1-derived HLA-A*3303-restricted peptides

[0628] CTL lines were established by proliferating CTLs in well #1 with KOC1-A33-9-543 (SEQ ID NO: 61) (a), well #2 with KOC1-A33-9-282 (SEQ ID NO: 62) (b), well #8 with KOC1-A33-9-485 (SEQ ID NO: 64) (c), well #3 with KOC1-A33-9-286 (SEQ ID NO: 67) (d), well #4 with KOC1-A33-10-542 (SEQ ID NO: 77) (e), and well #3 with KOC1-A33-10-281 (SEQ ID NO: 52) (f), which showed peptide-specific CTL activity in IFN-gamma ELISPOT assay. The CTL activity of these CTL lines was measured by IFN-gamma ELISA ( Figure 6 These CTL lines exhibited potent IFN-gamma production against target cells pulsed with the corresponding peptides compared to target cells not pulsed with the peptides. Furthermore, CTL clones were established by limiting dilution from the CTL lines described in the "Materials and Methods" section above, and IFN-gamma production from the CTL clones against peptide-pulsed C1R-A33 was measured by IFN-gamma ELISA. Strong IFN-gamma production was observed in CTL clones stimulated with KOC1-A33-9-543 (SEQ ID NO: 61) (a), KOC1-A33-9-282 (SEQ ID NO: 62) (b), KOC1-A33-9-485 (SEQ ID NO: 64) (c), KOC1-A33-9-286 (SEQ ID NO: 67) (d), KOC1-A33-10-542 (SEQ ID NO: 77) (e), and KOC1-A33-10-281 (SEQ ID NO: 52) (f). Figure 7 ).

[0629] Specific CTL activity against target cells expressing KOC1 and HLA-A*3303

[0630] CTL lines and clones were established against KOC1-A33-9-485 (SEQ ID NO: 64) (a), KOC1-A33-9-286 (SEQ ID NO: 67) (b), and KOC1-A33-10-542 (SEQ ID NO: 77) (c) to investigate their ability to recognize target cells expressing KOC1 and HLA-A*3303 molecules. COS7 cells transfected with both full-length KOC1 and HLA-A*3303 genes (a specific model for target cells expressing both KOC1 and HLA-A*3303 genes) were prepared as target cells. COS7 cells transfected with either full-length KOC1 or HLA-A*3303 were prepared as controls. CTL clones stimulated with KOC1-A33-9-485 (SEQ ID NO: 64) (a), KOC1-A33-9-286 (SEQ ID NO: 67) (b), or KOC1-A33-10-542 (SEQ ID NO: 77) (c) showed potent CTL activity against COS7 cells expressing both KOC1 and HLA-A*3303 ( Figure 8On the other hand, no significant specific CTL activity was detected against the control. These data clearly demonstrate that KOC1-A33-9-485 (SEQ ID NO: 64), KOC1-A33-9-286 (SEQ ID NO: 67), and KOC1-A33-10-542 (SEQ ID NO: 77) are peptides produced by endogenous processing of KOC1, presented on target cells with HLA-A*3303 molecules, and recognized by CTLs. These results suggest the possibility that KOC1-A33-9-485 (SEQ ID NO: 64), KOC1-A33-9-286 (SEQ ID NO: 67), and KOC1-A33-10-542 (SEQ ID NO: 77) may be suitable as cancer vaccines for patients with KOC1-expressing cancers.

[0631] Homology analysis of antigenic peptides

[0632] Use KOC1-A33-9-543(SEQ ID NO:61),KOC1-A33-9-282(SEQ ID NO:62),KOC1-A33-9-317(SEQ ID NO:63),KOC1-A33-9-485(SEQ ID NO:64),KOC1-A33-9-286(SEQ ID NO:67),KOC1-A33-9-34(SEQ ID NO:74),KOC1-A33-10-542(SEQ ID NO:77),KOC1-A33-10-281(SEQ ID NO:52),KOC1-A33-10-543(SEQ ID NO:79),KOC1-A33-10-424(SEQ ID NO:80), or KOC1-A33-10-285 (SEQ ID NO:85) stimulated CTLs showed significant specific CTL activity. These results may be due to the fact that KOC1-A33-9-543 (SEQ ID NO:61), KOC1-A33-9-282 (SEQ ID NO:62), KOC1-A33-9-317 (SEQ ID NO:63), KOC1-A33-9-485 (SEQ ID NO:64), KOC1-A33-9-286 (SEQ ID NO:67), KOC1-A33-9-34 (SEQ ID NO:74), KOC1-A33-10-542 (SEQ ID NO:77), KOC1-A33-10-281 (SEQ ID NO:52), KOC1-A33-10-543 (SEQ ID NO:79), KOC1-A33-10-424 (SEQ ID NO:80), and KOC1-A33-10-285 (SEQ ID NO:81). The peptide sequences of KOC1-A33-9-543 (SEQ ID NO: 61), KOC1-A33-9-317 (SEQ ID NO: 63), KOC1-A33-9-485 (SEQ ID NO: 64), KOC1-A33-9-34 (SEQ ID NO: 74), KOC1-A33-10-542 (SEQ ID NO: 77), KOC1-A33-10-543 (SEQ ID NO: 79), and KOC1-A33-10-424 (SEQ ID NO: 80) were found to be unique.On the other hand, KOC1-A33-9-282 (SEQ ID NO: 62), KOC1-A33-9-286 (SEQ ID NO: 67), KOC1-A33-10-281 (SEQ ID NO: 52), and KOC1-A33-10-285 (SEQ ID NO: 85) are identical to peptide sequences identified in IMP-1, another IMP family member. KOC1-A33-9-485 (SEQ ID NO: 64) is identical to a peptide sequence identified in IMP-2. IMP-1 has previously been reported to be not expressed in normal organs other than testis, fetal liver, and placenta (Hammer NA et al., Reproduction. 2005; 130(2): 203-12). It has also been reported to be associated with tumor progression in lung cancer patients (Kato T et al., Clin Cancer Res. 2007; 13: 434-42). It has been previously reported that IMP-2 is not expressed in normal organs other than testis and fetal liver (Hammer NA et al., Reproduction. 2005; 130(2): 203-12). It is involved in the proliferation of glioblastoma cancer stem cells (Janiszewska M et al., Genes Dev. 2012; 26(17): 1926-44). IMP-1 and IMP-2 are expected to be target antigens for cancer immunotherapy. Therefore, according to the present inventors' knowledge, these peptides are unlikely to trigger unexpected immune responses against other unrelated molecules. In summary, new KOC1-derived HLA-A33-restricted epitope peptides were identified. It was demonstrated that KOC1-derived epitope peptides are suitable for cancer immunotherapy.

[0633] [Example 3]

[0634] Materials and methods

[0635] cell lines

[0636] C1R, HLA-A negative and HLA-B negative human B lymphoblastoid cell lines, and COS7, African green monkey kidney cell line were purchased from ATCC.

[0637] Generation of stimulator cells with stable HLA-A*0301 expression

[0638] C1R cells stably expressing HLA-A*0301 (C1R-A03) were used as stimulator cells. The cDNA encoding the open reading frame of HLA-A*0301 was amplified by PCR and cloned into an expression vector. C1R cells were transfected with the expression vector and then selected with G418 (Invitrogen) for two weeks. Cells selected with G418 were seeded into wells of a 96-well plate containing G418-supplemented medium and cultured for a further 30 days. Flow cytometry analysis confirmed exogenous HLA-A*0301 expression in C1R cells.

[0639] Selection of derived KOC1 peptide candidates

[0640] KOC1-derived 9-mer and 10-mer peptides that bind to HLA-A*0301 molecules were predicted using the binding prediction server "NetMHC 3.2" (www.cbs.dtu.dk / services / NetMHC / ) (Buus et al., Tissue Antigens. 2003 Nov, 62(5):378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17; Bioinformatics. 2004 Jun 12:20(9):1388-97).

[0641] Peptide synthesis

[0642] These peptides were synthesized by Biosynthesis (Lewisville, Texas) according to standard solid-phase synthesis methods and purified by reverse-phase high-performance liquid chromatography (HPLC). The purity (>90%) and identity of the peptides were analyzed by analytical HPLC and mass spectrometry, respectively. The peptides were dissolved in dimethyl sulfoxide at 20 mg / ml and stored at -80°C.

[0643] In vitro CTL induction

[0644] Monocyte-derived dendritic cells (DCs) were used as antigen-presenting cells to induce cytotoxic T lymphocyte (CTL) responses against peptides presented on human leukocyte antigens (HLA). DCs were prepared in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003, 63(14):4112-8). Specifically, peripheral blood mononuclear cells isolated from healthy volunteers (HLA-A*0301 positive) using Ficoll-Paque Plus (Pharmacia) solution were separated by adherence to plastic tissue culture plates (Becton Dickinson) and concentrated as a mononuclear cell fraction. The monocyte-enriched population was cultured in AIM-V medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS) in the presence of 1000 IU / ml of granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml of interleukin (IL)-4 (R&D System). After 7 days of culture, the cytokine-induced DCs were pulsed with 20 micrograms / ml of each synthetic peptide at 37°C in the presence of 3 micrograms / ml β2-microglobulin in AIM-V culture medium for 3 hours. The generated cells apparently expressed DC-associated molecules such as CD80, CD83, CD86, and HLA class II on their cell surfaces (data not shown). Next, these peptide-pulsed DCs were inactivated by X-ray radiation (20 Gy) and mixed with autologous CD8+ T cells obtained by positive selection using a CD8 positive separation kit (Dynal) at a ratio of 1:20. These culture products were inoculated in 48-well plates (Corning). Each well was prepared to contain 1.5 x 10 4Peptide-pulsed DCs, 3x105 CD8+ T cells, and 10 ng / ml IL-7 (R&D System) were added to the cultures. After 3 days, IL-2 (CHIRON) was added to the cultures at a final concentration of 20 IU / ml. On days 7 and 14, the T cells were further stimulated with peptide-pulsed autologous DCs. DCs were prepared in the same manner as described above. On day 21, after the third peptide stimulation, CTLs against peptide-pulsed C1R-A03 cells were detected by human interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0645] CTL expansion procedure

[0646] CTLs were expanded in culture using a method similar to that disclosed by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16):1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2):216-23). ​​CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines in a total of 25 ml of AIM-V medium (5 x 10 6Cells / flask) were cultured in a medium containing 5% AS (AIM-V / 5% AS) and 40 ng / ml anti-CD3 antibody. One day after the start of culture, 120 IU / ml IL-2 was added to the culture. On days 5, 8, and 11, fresh AIM-V / 5% AS medium containing 30 IU / ml IL-2 was added to the culture (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0647] Establishment of CTL clones

[0648] CTLs were diluted to 0.3, 1, and 3 cells / well in 96 round-bottom microtiter plates (Nalge Nunc International). CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines (1 x 10 4 cells / well). After 10 days, 50 μl / well of IL-2 was added to the culture medium to a final concentration of 125 IU / ml. CTL activity was tested on day 14, and CTL clones were expanded using the same method as described above (Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0649] Specific CTL activity

[0650] In order to examine the specific CTL activity, IFN-gamma ELISPOT assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A03 (1×10 4Cells / well) were used as stimulator cells. Induced CTLs (i.e., CTL lines and CTL clones) were used as responder cells. IFN-gamma ELISPOT assays and IFN-gamma ELISA were performed according to the manufacturer's instructions.

[0651] Establishment of target cells forced to express target genes and HLA-A*0301

[0652] cDNA encoding the open reading frame of the target gene or HLA-A*0301 was amplified by PCR. The PCR amplification product was cloned into an expression vector. One or both of the target gene-expressing vector and the HLA-A*0301-expressing vector were transfected into COS7 (a cell line negative for the target gene and HLA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Two days after transfection, the transfected cells were harvested with Versene (Invitrogen) and used as target cells for CTL activity assays (5 x 10 4 cells / well).

[0653] result

[0654] Prediction of KOC1-derived HLA-A*0301 binding peptides

[0655] Tables 3a and 3b show KOC1-derived 9-mer and 10-mer peptides predicted to bind to HLA-A*0301 in descending order of binding affinity. A total of 23 peptides with potential HLA-A*0301 binding ability were selected and examined to determine epitope peptides.

[0656] [Table 3a]

[0657] HLA-A3-binding 9-mer peptide derived from KOC1

[0658] Starting position Amino acid sequence Kd(nM) SEQ ID NO 121 VVNVTYSSK 84 2 258 KSILEIMHK 124 1 52 KAIEALSGK 496 7 28 PVSGPFLVK 613 6 465 KAQGRIYGK 650 3 142 FQLENFTLK 752 8 415 LSVGAIIGK 764 5 497 FAAGRVIGK 927 11 559 QVKQHQQQK 1152 24

[0659] [Table 3b]

[0660] HLA-A3-binding 10-mer peptide derived from KOC1

[0661] Starting position Amino acid sequence Kd(nM) SEQ ID NO 120 AVVNVTYSSK 17 27 414 ALSVGAIIGK 37 28 431 LSRFAGASIK 69 37 181 RQGSPGSVSK 104 38 456 ITGPPEAQFK 211 29 204 TQFVGAIIGK 211 30 496 SFAAGRVIGK 215 34 552 KIQEILTQVK 365 36 281 ILAHNNFVGR 459 52 73 SVPKRQRIRK 521 31 535 VVVKITGHFY 682 41 420 IIGKQGQHIK 858 55 141 GFQLENFTLK 1112 44 14 APSDLESIFK 1275 32

[0662] The starting position indicates the number of amino acid residues from the N-terminus of KOC1, and the dissociation constant [Kd (nM)] is derived from "NetMHC3.2".

[0663] Induction of CTLs by predicted KOC1-derived HLA-A*0301-restricted peptides

[0664] CTLs against KOC1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured by IFN-gamma ELISPOT assay ( Figure 9 Compared to the control, well #5 (a) containing KOC1-A03-10-120 (SEQ ID NO: 27), well #3 (b) containing KOC1-A03-10-204 (SEQ ID NO: 30), and well #5 (c) containing KOC1-A03-10-281 (SEQ ID NO: 52) showed strong IFN-gamma production. Meanwhile, although the other peptides shown in Tables 3a and 3b potentially have HLA-A*0301 binding activity, no specific CTL activity was detected as a result of stimulation with these peptides. An example of typical negative data is that no specific IFN-gamma production was observed in CTLs stimulated with KOC1-A03-10-414 (SEQ ID NO: 28) (d). As a result, three types of KOC1-derived peptides were selected as peptides capable of inducing potent CTLs.

[0665] Establishment of CTL lines and clones targeting KOC1-derived HLA-A*0301-restricted peptides

[0666] CTL lines were established by proliferating CTLs in well #5 with KOC1-A03-10-120 (SEQ ID NO: 27), which showed peptide-specific CTL activity in IFN-gamma ELISPOT assay. The CTL activity of these CTL lines was measured by IFN-gamma ELISA ( Figure 10 ). These CTL lines showed strong IFN-gamma production against target cells pulsed with the corresponding peptides compared to target cells not pulsed with the peptides. In addition, CTL clones were established by limiting dilution of the CTL lines as described in the "Materials and Methods" section above, and IFN-gamma production from CTL clones against peptide-pulsed C1R-A03 was measured by IFN-gamma ELISA. Strong IFN-gamma production was observed in CTL clones stimulated with KOC1-A03-10-120 (SEQ ID NO: 27) ( Figure 11 ).

[0667] Specific CTL activity against target cells expressing KOC1 and HLA-A*0301

[0668] CTL clones against KOC1-A03-10-120 (SEQ ID NO: 27) were established to study their ability to recognize target cells expressing KOC1 and HLA-A*0301 molecules. COS7 cells transfected with both full-length KOC1 and HLA-A*0301 genes (a specific model of target cells expressing KOC1 and HLA-A*0301 genes) were prepared as target cells. COS7 cells transfected with full-length KOC1 or HLA-A*0301 were prepared as controls. CTL clones stimulated with KOC1-A03-10-120 (SEQ ID NO: 27) showed strong CTL activity against COS7 cells expressing both KOC1 and HLA-A*0301 ( Figure 12 On the other hand, no significant specific CTL activity was detected against the control. These data clearly demonstrate that KOC1-A03-10-120 (SEQ ID NO: 27) is a peptide produced by endogenous processing of KOC1, presented on target cells with HLA-A*0301 molecules, and recognized by CTLs. These results suggest the possibility that KOC1-A03-10-120 (SEQ ID NO: 27) may be suitable as a cancer vaccine for patients with KOC1-expressing cancers.

[0669] Homology analysis of antigenic peptides

[0670] CTLs stimulated with KOC1-A03-10-120 (SEQ ID NO: 27), KOC1-A03-10-204 (SEQ ID NO: 30), or KOC1-A03-10-281 (SEQ ID NO: 52) exhibited significant specific CTL activity. These results may be due to the fact that the sequences of KOC1-A03-10-120 (SEQ ID NO: 27), KOC1-A03-10-204 (SEQ ID NO: 30), and KOC1-A03-10-281 (SEQ ID NO: 52) are homologous to peptides derived from other molecules known to sensitize the human immune system. To rule out this possibility, homology analysis was performed by querying the sequences of these peptides using the BLAST algorithm (blast.ncbi.nlm.nih.gov / Blast.cgi). This result revealed no sequences with significant homology to the KOC1-A03-10-120 (SEQ ID NO: 27) sequence. On the other hand, KOC1-A03-10-204 (SEQ ID NO: 30) is identical to a peptide sequence identified in IMP-2, another IMP family member. KOC1-A03-10-281 (SEQ ID NO: 52) is identical to a peptide sequence identified in IMP-1, another IMP family member. IMP-1 has been previously reported to be not expressed in normal organs other than testis, fetal liver, and placenta (Hammer NA et al., Reproduction. 2005; 130(2): 203-12). It has also been reported to be associated with tumor progression in lung cancer patients (Kato T et al., Clin Cancer Res. 2007; 13: 434-42). IMP-2 has been previously reported to be not expressed in normal organs other than testis and fetal liver (Hammer NA et al., Reproduction. 2005; 130(2): 203-12). It is implicated in the proliferation of glioblastoma cancer stem cells (Janiszewska M et al., Genes Dev. 2012; 26(17): 1926-44). IMP-1 and IMP-2 are promising target antigens for cancer immunotherapy. Therefore, to the best of the present inventors' knowledge, these peptides are unlikely to elicit unintended immune responses against other unrelated molecules. In summary, novel KOC1-derived HLA-A03-restricted epitope peptides were identified. This demonstrates that KOC1-derived epitope peptides are suitable for cancer immunotherapy.

[0671] [Example 4]

[0672] Materials and methods

[0673] cell lines

[0674] C1R, HLA-A negative and HLA-B negative human B lymphoblastoid cell lines, and COS7, African green monkey kidney cell line were purchased from ATCC.

[0675] Generation of stimulator cells with stable HLA-A*0101 expression

[0676] C1R cells stably expressing HLA-A*0101 (C1R-A01) were used as stimulator cells. The cDNA encoding the open reading frame of HLA-A*0101 was amplified by PCR and cloned into an expression vector. C1R cells were transfected with the expression vector and then selected with G418 (Invitrogen) for two weeks. Cells selected with G418 were seeded into wells of a 96-well plate containing G418-supplemented medium and cultured for a further 30 days. Flow cytometry analysis confirmed exogenous HLA-A*0101 expression in C1R cells.

[0677] Selection of derived KOC1 peptide candidates

[0678] KOC1-derived 9-mer and 10-mer peptides that bind to HLA-A*0101 molecules were predicted using the binding prediction server "NetMHC 3.2" (www.cbs.dtu.dk / services / NetMHC / ) (Buus et al., Tissue Antigens. 2003 Nov, 62(5):378-84; Nielsen et al., Protein Sci. 2003 May, 12(5):1007-17; Bioinformatics. 2004 Jun 12:20(9):1388-97).

[0679] Peptide synthesis

[0680] These peptides were synthesized by Biosynthesis (Lewisville, Texas) according to standard solid-phase synthesis methods and purified by reverse-phase high-performance liquid chromatography (HPLC). The purity (>90%) and identity of the peptides were analyzed by analytical HPLC and mass spectrometry, respectively. The peptides were dissolved in dimethyl sulfoxide at 20 mg / ml and stored at -80°C.

[0681] In vitro CTL induction

[0682] Monocyte-derived dendritic cells (DCs) were used as antigen presenting cells to induce cytotoxic T lymphocyte (CTL) responses against peptides presented on human leukocyte antigens (HLA). DCs were prepared in vitro as described elsewhere (Nakahara S et al., Cancer Res 2003, 63(14):4112-8). Specifically, peripheral blood mononuclear cells isolated from healthy volunteers (HLA-A*0101 positive) were separated using Ficoll-Paque Plus (Pharmacia) solution, separated by adherence to plastic tissue culture plates (Becton Dickinson), and concentrated as a mononuclear cell fraction. The monocyte-enriched population was cultured in AIM-V medium (Invitrogen) containing 2% heat-inactivated autologous serum (AS) in the presence of 1000 IU / ml of granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml of interleukin (IL)-4 (R&D System). System). After 7 days of culture, the cytokine-induced DCs were pulsed with 20 micrograms / ml of each synthetic peptide at 37°C in the presence of 3 micrograms / ml β2-microglobulin in AIM-V culture medium for 3 hours. The generated cells apparently express DC-related molecules such as CD80, CD83, CD86 and HLAII class on their cell surfaces (data not shown). Then, these peptide-pulsed DCs were inactivated with X-ray radiation (20 Gy) and mixed with autologous CD8+ T cells obtained by positive selection using a CD8 positive separation kit (Dynal) at a ratio of 1:20. These culture products were inoculated in 48-well plates (Corning). Each well was prepared to contain 1.5x 10 4 Peptide-pulsed DCs, 3x10 5CD8+ T cells and 10 ng / ml IL-7 (R&D System). After 3 days, IL-2 (CHIRON) was added to these cultures at a final concentration of 20 IU / ml. On days 7 and 14, these T cells were further stimulated with autologous DCs pulsed with peptides. DCs were prepared in the same manner as described above. CTL activity was detected against peptide-pulsed C1R-A01 cells on day 21 after the third peptide stimulation (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0683] CTL expansion procedure

[0684] CTLs were expanded in culture using a method similar to that disclosed by Riddell et al. (Walter EA et al., N Engl J Med 1995 Oct 19, 333(16):1038-44; Riddell SR et al., Nat Med 1996 Feb, 2(2):216-23). ​​CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines in a total of 25 ml of AIM-V medium (5 x 10 6 Cells / flask) were cultured in a medium containing 5% AS (AIM-V / 5% AS) and 40 ng / ml anti-CD3 antibody. One day after the start of culture, 120 IU / ml IL-2 was added to the culture. On days 5, 8, and 11, fresh AIM-V / 5% AS medium containing 30 IU / ml IL-2 was added to the culture (Tanaka H et al., Br J Cancer 2001, 84(1):94-9; Umano Y et al., Br J Cancer 2001, 84(8):1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0685] Establishment of CTL clones

[0686] CTLs were diluted to make 1 or 10 cells / well in 96 round-bottom microtiter plates (Nalge Nunc International). CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblastoid cell lines (1 x 10 4 cells / well). After 10 days, 50 μl / well of IL-2 was added to the culture medium to a final concentration of 125 IU / ml. CTL activity was tested on day 14, and CTL clones were expanded using the same method as described above (Uchida N et al., Clin Cancer Res 2004, 10(24):8577-86; Suda T et al., Cancer Sci 2006, 97(5):411-9; Watanabe T et al., Cancer Sci 2005, 96(8):498-506).

[0687] Specific CTL activity

[0688] To examine specific CTL activity, IFN-gamma ELISPOT assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-pulsed C1R-A01 (1×10 4 Cells / well) were used as stimulator cells. Induced CTLs (i.e., CTL lines and CTL clones) were used as responder cells. IFN-gamma ELISPOT assays and IFN-gamma ELISA were performed according to the manufacturer's instructions.

[0689] Establishment of target cells forced to express target genes and HLA-A*0101

[0690] cDNA encoding the open reading frame of the target gene or HLA-A*0101 was amplified by PCR. The PCR amplification product was cloned into an expression vector. One or both of the target gene-expressing vector and the HLA-A*0101-expressing vector were transfected into COS7 (a cell line negative for the target gene and HLA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Two days after transfection, the transfected cells were harvested with Versene (Invitrogen) and used as target cells for CTL activity assays (5 x 10 4 cells / well).

[0691] result

[0692] Prediction of KOC1-derived HLA-A*0101-binding peptides

[0693] Tables 4a and 4b show KOC1-derived 9-mer and 10-mer peptides predicted to bind to HLA-A*0101 in descending order of binding affinity. A total of 27 peptides with potential HLA-A*0101 binding ability were selected and examined to determine epitope peptides.

[0694] [Table 4a]

[0695] HLA-A1-binding 9-mer peptide derived from KOC1

[0696] Starting position Amino acid sequence Kd(nM) SEQ ID NO 96 VLDSLLVQY 56 86 118 ETAVVNVTY 57 87 114 NTDSETAVV 453 88 273 FTEEIPLKI 528 89 404 ETETVHLFI 1528 90 536 VVKITGHFY 5589 13 15 PSDLESIFK 5882 12 143 QLENFTLKV 11578 91 402 QSETETVHL 13101 92 305 DTDTKITIS 13406 93 313 SPLQELTLY 21305 94 338 KAEEEIMKK 23866 10 301 KIEQDTDTK 24016 21 69 EVEHSVPKR 24734 69

[0697] [Table 4b]

[0698] HLA-A1-binding 10-mer peptide derived from KOC1

[0699] Starting position Amino acid sequence Kd(nM) SEQ ID NO 312 ISPLQELTLY 146 46 95 EVLDSLLVQY 498 50 402 QSETETVHLF 1737 95 9 LSENAAPSDL 2164 96 535 VVVKITGHFY 3359 41 273 FTEEIPLKIL 4478 97 462 AQFKAQGRIY 6263 98 342 EIMKKIRESY 7665 99 252 GTSAACKSIL 9689 100 30 SGPFLVKTGY 17794 101 114 NTDSETAVVN 22411 102 305 DTDTKITISP 29728 103 17 DLESIFKDAK 35078 104

[0700] The starting position indicates the number of amino acid residues from the N-terminus of KOC1, and the dissociation constant [Kd (nM)] is derived from "NetMHC 3.2".

[0701] Induction of CTLs by predicted KOC1-derived HLA-A*0101-restricted peptides

[0702] CTLs against KOC1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured by IFN-gamma ELISPOT assay ( Figure 13Compared to the control, well #2 (a) with KOC1-A01-9-96 (SEQ ID NO: 86), well #4 (b) with KOC1-A01-9-118 (SEQ ID NO: 87), well #3 (c) with KOC1-A01-9-404 (SEQ ID NO: 90), well #4 (d) with KOC1-A01-9-402 (SEQ ID NO: 92), well #7 (e) with KOC1-A01-10-312 (SEQ ID NO: 46), well #1 (f) with KOC1-A01-10-402 (SEQ ID NO: 95), and well #1 (g) with KOC1-A01-10-535 (SEQ ID NO: 41) showed potent IFN-gamma production. Meanwhile, although other peptides shown in Tables 4a and 4b potentially possess HLA-A*0101 binding activity, no specific CTL activity was detected as a result of stimulation with these peptides. A typical example of negative data is the lack of specific IFN-gamma production (h) observed in CTLs stimulated with KOC1-A01-9-536 (SEQ ID NO: 13). Consequently, seven types of KOC1-derived peptides were selected as peptides capable of inducing potent CTLs.

[0703] Establishment of CTL lines and clones against KOC1-derived peptides

[0704] CTL lines were established by proliferating CTLs in well #2 (a) with KOC1-A01-9-96 (SEQ ID NO: 86) and well #4 (b) with KOC1-A01-9-118 (SEQ ID NO: 87), which showed peptide-specific CTL activity in IFN-gamma ELISPOT assay. The CTL activity of these CTL lines was measured by IFN-gamma ELISA ( Figure 14 ). These CTL lines showed strong IFN-gamma production against target cells pulsed with the corresponding peptides compared to target cells not pulsed with the peptides. In addition, CTL clones were established by limiting dilution of the CTL lines as described in the "Materials and Methods" section above, and IFN-gamma production from CTL clones against peptide-pulsed C1R-A01 was measured by IFN-gamma ELISA. Strong IFN-gamma production was observed in CTL clones stimulated with KOC1-A01-9-96 (SEQ ID NO: 86) ( Figure 15 ).

[0705] Specific CTL activity against target cells expressing KOC1 and HLA-A*0101

[0706] CTL lines and CTL clones against KOC1-A01-9-96 (SEQ ID NO: 86) were established to study their ability to recognize target cells expressing KOC1 and HLA-A*0101 molecules. COS7 cells transfected with both full-length KOC1 and HLA-A*0101 genes (a specific model of target cells expressing KOC1 and HLA-A*0101 genes) were prepared as target cells. COS7 cells transfected with full-length KOC1 or HLA-A*0101 were prepared as controls. CTL clones stimulated with KOC1-A01-9-96 (SEQ ID NO: 86) showed strong CTL activity against COS7 cells expressing both KOC1 and HLA-A*0101 ( Figure 16 On the other hand, no significant specific CTL activity was detected against the control. These data clearly demonstrate that KOC1-A01-9-96 (SEQ ID NO: 86) is a peptide produced by endogenous processing of KOC1, presented on target cells with HLA-A*0101 molecules, and recognized by CTLs. These results suggest the possibility that KOC1-A01-9-96 (SEQ ID NO: 86) may be suitable as a cancer vaccine for patients with KOC1-expressing cancers.

[0707] Homology analysis of antigenic peptides

[0708] CTLs stimulated with KOC1-A01-9-96 (SEQ ID NO:86), KOC1-A01-9-118 (SEQ ID NO:87), KOC1-A01-9-404 (SEQ ID NO:90), KOC1-A01-9-402 (SEQ ID NO:92), KOC1-A01-10-312 (SEQ ID NO:46), KOC1-A01-10-402 (SEQ ID NO:95), or KOC1-A01-10-535 (SEQ ID NO:41) showed significant specific CTL activity. These results may be due to the fact that the sequences of KOC1-A01-9-96 (SEQ ID NO: 86), KOC1-A01-9-118 (SEQ ID NO: 87), KOC1-A01-9-404 (SEQ ID NO: 90), KOC1-A01-9-402 (SEQ ID NO: 92), KOC1-A01-10-312 (SEQ ID NO: 46), KOC1-A01-10-402 (SEQ ID NO: 95), and KOC1-A01-10-535 (SEQ ID NO: 41) are homologous to peptides derived from other molecules known to sensitize the human immune system. To rule out this possibility, homology analysis was performed by querying the sequences of these peptides using the BLAST algorithm (blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that there were no sequences with significant homology to KOC1-A01-9-96 (SEQ ID NO: 86), KOC1-A01-9-118 (SEQ ID NO: 87), KOC1-A01-9-404 (SEQ ID NO: 90), KOC1-A01-9-402 (SEQ ID NO: 92), KOC1-A01-10-312 (SEQ ID NO: 46), KOC1-A01-10-402 (SEQ ID NO: 95), and KOC1-A01-10-535 (SEQ ID NO: 41). On the other hand, KOC1-A01-9-118 (SEQ ID NO: 87) was identical to peptide sequences identified in IMP-1 and IMP-2, which are other members of the IMP family. IMP-1 has been previously reported to be not expressed in normal organs except testis, fetal liver, and placenta (Hammer NA et al., Reproduction. 2005; 130(2): 203-12). It has also been reported to be associated with tumor progression in lung cancer patients (Kato T et al., Clin Cancer Res. 2007; 13: 434-42).It has been previously reported that IMP-2 is not expressed in normal organs other than testis and fetal liver (Hammer NA et al., Reproduction. 2005; 130(2): 203-12). It is implicated in the proliferation of glioblastoma cancer stem cells (Janiszewska M et al., Genes Dev. 2012; 26(17): 1926-44). IMP-1 and IMP-2 are promising target antigens for cancer immunotherapy. Therefore, according to the present inventors' knowledge, these peptides are unlikely to induce unexpected immune responses against other unrelated molecules. In summary, new KOC1-derived HLA-A01-restricted epitope peptides were identified. It was demonstrated that KOC1-derived epitope peptides are suitable for cancer immunotherapy.

[0709] [Example 5]

[0710] Preparation of emulsion formulations

[0711] The peptide is dissolved in an injection solvent or sterile saline to 1.0 mg / ml to 10.0 mg / ml and collected in a syringe. This is connected to a syringe equipped with an IFA equivalent to an injection solvent or sterile saline via a connector, and then mixed by alternately pushing the syringe plungers of two connected syringes. After mixing for a few minutes, the completion of the emulsion is evaluated by a drop test method. The drop test method can be performed by dropping a drop of mixed sample on water. When the sample dropped on the water does not diffuse in water immediately, the emulsion is evaluated to be complete; and when the sample dropped on the water diffuses in water immediately, the emulsion is evaluated to be incomplete. When the emulsion is evaluated to be incomplete, further mixing is performed to complete the emulsion. The emulsion completed can be administered to cancer patients by subcutaneous injection. Cancer patients to be administered can be selected from patients affected by the following cancers: bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, testicular cancer, etc.

[0712] Preparation of freeze-dried preparations

[0713] The peptide is dissolved in an injection solvent to a concentration of 1.0 mg / ml to 10.0 mg / ml and sterilized by filtration. It is placed in a sterilized vial and semi-enclosed with a sterile rubber stopper. After the vial is freeze-dried, it is completely enclosed and sutured with an aluminum cap to produce a freeze-dried preparation. When used, an injection solvent or sterile physiological saline is injected into the vial to redissolve the freeze-dried powder. A syringe is used to collect the redissolved solution in the vial, and the syringe is connected to a syringe filled with the same amount of IFA as the amount of the collected redissolved solution via a connector. The redissolved solution and IFA are mixed by alternately pushing the syringe plungers of the two connected syringes. After mixing for a few minutes, the completion of the emulsion is evaluated by a drop test method. The completed emulsion can be administered to cancer patients by subcutaneous injection. Cancer patients to be administered can be selected from patients affected by the following cancers: bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, testicular cancer, etc.

[0714] Industrial Applicability

[0715] The present invention provides novel HLA-A11-restricted, HLA-A33-restricted, HLA-A03-restricted, and HLA-A01-restricted epitope peptides derived from KOC1 that induce strong and specific anti-tumor immune responses and are therefore applicable to a wide range of cancer types. The peptides, compositions, APCs, and CTLs of the present invention can be used as peptide vaccines against cancers expressing KOC1, such as bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, renal cancer, head and neck cancer, soft tissue tumors, and testicular cancer.

[0716] Although the present invention has been described in detail and with respect to specific embodiments thereof, it should be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the present invention and its preferred embodiments. Through routine experimentation, those skilled in the art will readily recognize that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, the metes and bounds of which are defined by the appended claims.

[0717] The present invention provides

[0718] 1. A peptide of less than 15 amino acids having cytotoxic T cell (CTL) inducibility, comprising an amino acid sequence selected from the group consisting of:

[0719] (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41; and

[0720] (b) an amino acid sequence in which 1, 2 or several amino acids are substituted, deleted, inserted and / or added in an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41.

[0721] 2. The peptide of item 1, which is selected from the following groups (i) to (iv):

[0722] (i) A peptide comprising an amino acid sequence, wherein one or more substitutions selected from the following groups (a) to (d) are introduced into an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30 and 32:

[0723] (a) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine and tyrosine;

[0724] (b) substituting the third amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, phenylalanine, tyrosine, isoleucine and alanine;

[0725] (c) substituting the seventh amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, isoleucine, tyrosine, valine, and phenylalanine; and

[0726] (d) substituting the amino acid at the C-terminus with an amino acid selected from the group consisting of lysine and arginine;

[0727] (ii) a peptide comprising an amino acid sequence, wherein one or more substitutions selected from the following groups (a) to (c) are introduced into an amino acid sequence selected from the following group: SEQ ID NOs: 61, 62, 63, 64, 67, 74, 77, 52, 79, 80 and 85:

[0728] (a) substituting the first amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;

[0729] (b) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of phenylalanine, tyrosine, alanine, isoleucine, leucine and valine;

[0730] (c) substituting the amino acid at the C-terminus with an amino acid selected from the group consisting of arginine and lysine;

[0731] (iii) a peptide comprising an amino acid sequence, wherein one or more substitutions selected from the following groups (a) to (b) are introduced into an amino acid sequence selected from the following group: SEQ ID NOs: 27, 30 and 52:

[0732] (a) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, methionine, valine, alanine, isoleucine, serine, and threonine; and

[0733] (b) substituting the amino acid at the C-terminus with an amino acid selected from the group consisting of arginine, lysine, tyrosine, and phenylalanine;

[0734] (iv) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the following groups (a) to (c) are introduced into an amino acid sequence selected from the following group: SEQ ID NOs: 86, 87, 90, 92, 46, 95 and 41:

[0735] (a) substituting the second amino acid from the N-terminus with an amino acid selected from the group consisting of threonine and serine;

[0736] (b) substituting the third amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;

[0737] (c) Substitution of the C-terminal amino acid with tyrosine.

[0738] 3. The peptide of claim 1, which consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95, and 41.

[0739] 4. A polynucleotide encoding the peptide of any one of items 1 to 3.

[0740] 5. A composition comprising a pharmaceutically acceptable carrier and at least one component selected from the following groups (a) to (e):

[0741] (a) one or more types of peptides according to any one of items 1 to 3;

[0742] (b) one or more types of polynucleotides encoding the peptide of any one of items 1 to 3 in an expressible form;

[0743] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of items 1 to 3 and an HLA antigen on their cell surface;

[0744] (d) an exosome that presents a complex of the peptide of any one of items 1 to 3 and an HLA antigen on its cell surface; and

[0745] (e) CTLs targeting the peptide of any one of items 1 to 3.

[0746] 6. The composition of item 5, which is a composition for inducing CTL, wherein the component is at least one component selected from the following groups (a) to (d):

[0747] (a) one or more types of peptides according to any one of items 1 to 3;

[0748] (b) one or more types of polynucleotides encoding the peptide of any one of items 1 to 3 in an expressible form;

[0749] (c) antigen-presenting cells (APCs) that present a complex of the peptide of any one of items 1 to 3 and an HLA antigen on their cell surface; and

[0750] (d) An exosome that presents a complex of the peptide of any one of items 1 to 3 and an HLA antigen on its cell surface.

[0751] 7. The composition of claim 5, which is a pharmaceutical composition.

[0752] 8. The composition of claim 7, for use in one or more uses selected from the group consisting of: (i) cancer treatment, (ii) cancer prevention (prophylaxis), and (iii) prevention (prophylaxis) of cancer recurrence after surgery.

[0753] 9. The composition of claim 7, for use in inducing an immune response against cancer.

[0754] 10. The composition of claim 8 or 9, wherein the cancer is selected from the group consisting of bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colorectal cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head and neck cancer, soft tissue tumors, and testicular cancer.

[0755] 11. The composition of any one of items 5 to 10, formulated for administration to a subject positive for at least one HLA selected from the group consisting of HLA-A11, HLA-A33, HLA-A03, and HLA-A01.

[0756] 12. A method for inducing APCs having CTL inducibility, comprising the steps selected from the group consisting of:

[0757] (a) contacting APC with the peptide of any one of items 1 to 3 in vitro, ex vivo or in vivo; and

[0758] (b) A polynucleotide encoding the peptide of any one of items 1 to 3 is introduced into APC.

[0759] 13. A method for inducing CTL, comprising a step selected from the group consisting of:

[0760] (a) co-culturing CD8-positive T cells with APCs presenting a complex of an HLA antigen and the peptide of any one of items 1 to 3 on its surface;

[0761] (b) co-culturing CD8-positive T cells with exosomes presenting a complex of an HLA antigen and the peptide of any one of items 1 to 3 on its surface;

[0762] (c) introducing a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding to a peptide of any one of items 1 to 3 presented by an HLA antigen on a cell surface into a CD8-positive T cell.

[0763] 14. APC that presents a complex of an HLA antigen and the peptide according to any one of items 1 to 3 on its surface.

[0764] 15. The APC of item 14, which is induced by the method of item 12.

[0765] 16. CTL targeting the peptide of any one of items 1 to 3.

[0766] 17. The CTL of item 16, which is induced by the method of item 13.

[0767] 18. A method for inducing an immune response against cancer, comprising administering to a subject at least one component selected from the following groups (a) to (e):

[0768] (a) one or more types of peptides according to any one of items 1 to 3;

[0769] (b) one or more types of polynucleotides encoding the peptide of any one of items 1 to 3 in an expressible form;

[0770] (c) APCs that present a complex of the peptide of any one of items 1 to 3 and an HLA antigen on their cell surface;

[0771] (d) an exosome that presents a complex of the peptide of any one of items 1 to 3 and an HLA antigen on its cell surface; and

[0772] (e) CTLs targeting the peptide of any one of items 1 to 3.

[0773] 19. A method for treating and / or preventing cancer, and / or preventing its recurrence after surgery, comprising administering to a subject at least one component selected from the following groups (a) to (e):

[0774] (a) one or more types of peptides according to any one of items 1 to 3;

[0775] (b) one or more types of polynucleotides encoding the peptide of any one of items 1 to 3 in an expressible form;

[0776] (c) APCs that present a complex of the peptide of any one of items 1 to 3 and an HLA antigen on their cell surface;

[0777] (d) an exosome that presents a complex of the peptide of any one of items 1 to 3 and an HLA antigen on its cell surface; and

[0778] (e) CTLs targeting the peptide of any one of items 1 to 3.

[0779] 20. An antibody that binds to the peptide of any one of items 1 to 3.

[0780] 21. A method for screening a peptide having CTL inducibility, comprising the following steps:

[0781] (a) generating a candidate sequence consisting of an amino acid sequence, wherein one, two or several amino acid residues are substituted, deleted, inserted and / or added to an original amino acid sequence consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 28, 30, 32, 61, 62, 63, 64, 67, 74, 77, 52, 79, 80, 85, 27, 86, 87, 90, 92, 46, 95 and 41;

[0782] (b) selecting, from the candidate sequences generated in (a), candidate sequences other than KOC1 that do not have significant homology (sequence identity) to any known human gene product;

[0783] (c) contacting the APC with a peptide consisting of the candidate sequence selected in (b);

[0784] (d) contacting the APC of (c) with CD8-positive T cells; and

[0785] (e) Selecting peptides having equal or higher CTL inducibility than that of peptides consisting of the original amino acid sequence.

[0786] 22. An emulsion comprising one or more types of peptides according to any one of items 1 to 3, a water-soluble carrier and an oil adjuvant.

[0787] 23. A kit comprising a container containing the composition of any one of items 5 to 11 and a container containing an adjuvant. Sequence Listing <110> Oncotherapy Sciences, Inc. <120> KOC1-derived peptides and vaccines containing them <130> ONC-A1501P <150> JP 2014-158919 <151> 2014-08-04 <150> JP 2014-158920 <151> 2014-08-04 <150> JP 2014-158921 <151> 2014-08-04 <160> 111 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 1 Lys Ser Ile Leu Glu Ile Met His Lys 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 2 Val Val Asn Val Thr Tyr Ser Ser Lys 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 3 Lys Ala Gln Gly Arg Ile Tyr Gly Lys 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 4 Ser Ser Ala Glu Val Val Val Pro Arg 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 5 Leu Ser Val Gly Ala Ile Ile Gly Lys 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 6 Pro Val Ser Gly Pro Phe Leu Val Lys 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 7 Lys Ala Ile Glu Ala Leu Ser Gly Lys 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 8 Phe Gln Leu Glu Asn Phe Thr Leu Lys 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 9 Gly Ala Thr Ile Arg Asn Ile Thr Lys 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 10 Lys Ala Glu Glu Glu Ile Met Lys Lys 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 11 Phe Ala Ala Gly Arg Val Ile Gly Lys 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 12 Pro Ser Asp Leu Glu Ser Ile Phe Lys 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 13 Val Val Lys Ile Thr Gly His Phe Tyr 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 14 Phe Val Ser Pro Lys Glu Glu Val Lys 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 15 Arg Val Pro Ser Phe Ala Ala Gly Arg 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 16 Lys Phe Thr Glu Glu Ile Pro Leu Lys 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 17 Gln Ser Lys Ile Asp Val His Arg Lys 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 18 Lys Gly Asn Val Glu Thr Cys Ala Lys 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 19 Tyr Ala Cys Gln Val Ala Gln Arg Lys 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 20 Asn Ile Thr Lys Gln Thr Gln Ser Lys 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> twenty one Lys Ile Glu Gln Asp Thr Asp Thr Lys 1 5 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> twenty two Ile Gln Glu Ile Leu Thr Gln Val Lys 1 5 <210> twenty three <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> twenty three Gln Gly Ser Pro Gly Ser Val Ser Lys 1 5 <210> twenty four <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> twenty four Gln Val Lys Gln His Gln Gln Gln Lys 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial sequence <220> <223> SGKIELHGK <400> 25 Ser Gly Lys Ile Glu Leu His Gly Lys 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 26 Gln Phe Val Gly Ala Ile Ile Gly Lys 1 5 <210> 27 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 27 Ala Val Val Asn Val Thr Tyr Ser Ser Lys 1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 28 Ala Leu Ser Val Gly Ala Ile Ile Gly Lys 1 5 10 <210> 29 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 29 Ile Thr Gly Pro Pro Glu Ala Gln Phe Lys 1 5 10 <210> 30 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 30 Thr Gln Phe Val Gly Ala Ile Ile Gly Lys 1 5 10 <210> 31 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 31 Ser Val Pro Lys Arg Gln Arg Ile Arg Lys 1 5 10 <210> 32 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 32 Ala Pro Ser Asp Leu Glu Ser Ile Phe Lys 1 5 10 <210> 33 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 33 Gly Ser Pro Gly Ser Val Ser Lys Gln Lys 1 5 10 <210> 34 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 34 Ser Phe Ala Ala Gly Arg Val Ile Gly Lys 1 5 10 <210> 35 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 35 Leu Ser Ser Ala Glu Val Val Val Pro Arg 1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 36 Lys Ile Gln Glu Ile Leu Thr Gln Val Lys 1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 37 Leu Ser Arg Phe Ala Gly Ala Ser Ile Lys 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 38 Arg Gln Gly Ser Pro Gly Ser Val Ser Lys 1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 39 Val Thr Tyr Ser Ser Lys Asp Gln Ala Arg 1 5 10 <210> 40 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 40 Thr Gln Ser Lys Ile Asp Val His Arg Lys 1 5 10 <210> 41 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 41 Val Val Val Lys Ile Thr Gly His Phe Tyr 1 5 10 <210> 42 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 42 Gln Thr Gln Ser Lys Ile Asp Val His Arg 1 5 10 <210> 43 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 43 Leu Ser Gly Lys Ile Glu Leu His Gly Lys 1 5 10 <210> 44 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 44 Gly Phe Gln Leu Glu Asn Phe Thr Leu Lys 1 5 10 <210> 45 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 45 Ala Leu Gln Ser Gly Pro Pro Gln Ser Arg 1 5 10 <210> 46 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 46 Ile Ser Pro Leu Gln Glu Leu Thr Leu Tyr 1 5 10 <210> 47 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 47 Ile Ala Pro Ala Glu Ala Pro Asp Ala Lys 1 5 10 <210> 48 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 48 Ile Pro Val Ser Gly Pro Phe Leu Val Lys 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 49 Gln Ser Gly Pro Pro Gln Ser Arg Arg Lys 1 5 10 <210> 50 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 50 Glu Val Leu Asp Ser Leu Leu Val Gln Tyr 1 5 10 <210> 51 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 51 Cys Ala Lys Ala Glu Glu Glu Ile Met Lys 1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 52 Ile Leu Ala His Asn Asn Phe Val Gly Arg 1 5 10 <210> 53 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 53 Ala Gln Gly Arg Ile Tyr Gly Lys Ile Lys 1 5 10 <210> 54 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 54 Ala Gln Gln Asn Pro Leu Gln Gln Pro Arg 1 5 10 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 55 Ile Ile Gly Lys Gln Gly Gln His Ile Lys 1 5 10 <210> 56 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 56 Thr Gln Val Lys Gln His Gln Gln Gln Lys 1 5 10 <210> 57 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 57 Cys Lys Ser Ile Leu Glu Ile Met His Lys 1 5 10 <210> 58 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 58 Leu Ile Gly Lys Glu Gly Arg Asn Leu Lys 1 5 10 <210> 59 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 59 Ile Met His Lys Glu Ala Gln Asp Ile Lys 1 5 10 <210> 60 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 60 Leu Gln Ser Gly Pro Pro Gln Ser Arg Arg 1 5 10 <210> 61 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 61 Phe Tyr Ala Cys Gln Val Ala Gln Arg 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 62 Leu Ala His Asn Asn Phe Val Gly Arg 1 5 <210> 63 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 63 Glu Leu Thr Leu Tyr Asn Pro Glu Arg 1 5 <210> 64 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 64 Glu Val Lys Leu Glu Ala His Ile Arg 1 5 <210> 65 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 65 Thr Tyr Ser Ser Lys Asp Gln Ala Arg 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 66 Glu Ala Gln Phe Lys Ala Gln Gly Arg 1 5 <210> 67 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 67 Asn Phe Val Gly Arg Leu Ile Gly Lys 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 68 Thr Gln Ser Lys Ile Asp Val His Arg 1 5 <210> 69 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 69 Glu Val Glu His Ser Val Pro Lys Arg 1 5 <210> 70 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 70 Gln Gln Asn Pro Leu Gln Gln Pro Arg 1 5 <210> 71 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 71 Glu Thr Val His Leu Phe Ile Pro Ala 1 5 <210> 72 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 72 Ser Val Pro Lys Arg Gln Arg Ile Arg 1 5 <210> 73 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 73 Gln Ser Gly Pro Pro Gln Ser Arg Arg 1 5 <210> 74 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 74 Leu Val Lys Thr Gly Tyr Ala Phe Val 1 5 <210> 75 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 75 Ile Gly Lys Glu Gly Ala Thr Ile Arg 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 76 Asp Thr Lys Ile Thr Ile Ser Pro Leu 1 5 <210> 77 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 77 His Phe Tyr Ala Cys Gln Val Ala Gln Arg 1 5 10 <210> 78 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 78 His Ser Val Pro Lys Arg Gln Arg Ile Arg 1 5 10 <210> 79 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 79 Phe Tyr Ala Cys Gln Val Ala Gln Arg Lys 1 5 10 <210> 80 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 80 Gln Gly Gln His Ile Lys Gln Leu Ser Arg 1 5 10 <210> 81 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 81 Lys Gln Lys Pro Cys Asp Leu Pro Leu Arg 1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 82 Ile Ile Gly Lys Glu Gly Ala Thr Ile Arg 1 5 10 <210> 83 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 83 Asn Phe Val Ser Pro Lys Glu Glu Val Lys 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 84 Arg Gln Arg Ile Arg Lys Leu Gln Ile Arg 1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 85 Asn Asn Phe Val Gly Arg Leu Ile Gly Lys 1 5 10 <210> 86 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 86 Val Leu Asp Ser Leu Leu Val Gln Tyr 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 87 Glu Thr Ala Val Val Asn Val Thr Tyr 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 88 Asn Thr Asp Ser Glu Thr Ala Val Val 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 89 Phe Thr Glu Glu Ile Pro Leu Lys Ile 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 90 Glu Thr Glu Thr Val His Leu Phe Ile 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 91 Gln Leu Glu Asn Phe Thr Leu Lys Val 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 92 Gln Ser Glu Thr Glu Thr Val His Leu 1 5 <210> 93 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 93 Asp Thr Asp Thr Lys Ile Thr Ile Ser 1 5 <210> 94 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptides derived from KOC1 <400> 94 Ser Pro Leu Gln Glu Leu Thr Leu Tyr 1 5 <210> 95 <211> 10 <...

Claims

1. A peptide having cytotoxic T cell (CTL) inducibility, which consists of the amino acid sequence of SEQ ID NO:

27.

2. A polynucleotide encoding the peptide of claim 1.

3. A composition comprising a pharmaceutically acceptable carrier and at least one component selected from the following groups (a) to (e): (a) one or more types of the peptide of claim 1; (b) one or more types of polynucleotides encoding the peptide of claim 1 in an expressible form; (c) an antigen presenting cell (APC) which presents a complex of the peptide of claim 1 and an HLA-A*0301 antigen on its cell surface; (d) an exosome presenting a complex of the peptide of claim 1 and an HLA-A*0301 antigen on its cell surface; and (e) CTL, which is induced by an in vitro method for inducing CTL, the method comprising a step selected from the group consisting of: (i) co-culturing CD8-positive T cells with APCs presenting a complex of the HLA-A*0301 antigen and the peptide of claim 1 on its surface; and (ii) co-culturing CD8-positive T cells with exosomes presenting a complex of the HLA-A*0301 antigen and the peptide of claim 1 on its surface.

4. The composition of claim 3, which is a composition for inducing CTL, wherein the component is at least one component selected from the following groups (a) to (d): (a) one or more types of the peptide of claim 1; (b) one or more types of polynucleotides encoding the peptide of claim 1 in an expressible form; (c) an antigen presenting cell (APC) which presents a complex of the peptide of claim 1 and an HLA-A*0301 antigen on its cell surface; and (d) An exosome presenting a complex of the peptide of claim 1 and an HLA-A*0301 antigen on its cell surface.

5. The composition of claim 3, which is a pharmaceutical composition.

6. An ex vivo or in vitro method for inducing APCs with CTL inducibility, comprising a step selected from the group consisting of: (a) contacting APC with the peptide of claim 1 in vitro or ex vivo; and (b) introducing a polynucleotide encoding the peptide of claim 1 into APC.

7. An in vitro method for inducing CTLs, comprising a step selected from the group consisting of: (a) co-culturing CD8-positive T cells with APCs presenting a complex of the HLA-A*0301 antigen and the peptide of claim 1 on its surface; and (b) co-culturing CD8-positive T cells with exosomes presenting a complex of the HLA-A*0301 antigen and the peptide of claim 1 on its surface.

8. APC which presents a complex of HLA-A*0301 antigen and the peptide of claim 1 on its surface.

9. The APC of claim 8, which is induced by a method comprising a step selected from the group consisting of: (a) contacting APC with the peptide of claim 1 in vitro or ex vivo; and (b) introducing a polynucleotide encoding the peptide of claim 1 into APC.

10. CTLs targeting the peptide of claim 1, wherein the CTLs are induced by an in vitro method comprising a step selected from the group consisting of: (a) co-culturing CD8-positive T cells with APCs presenting a complex of the HLA-A*0301 antigen and the peptide of claim 1 on its surface; and (b) co-culturing CD8-positive T cells with exosomes presenting a complex of the HLA-A*0301 antigen and the peptide of claim 1 on its surface.

11. Use of at least one component selected from the following groups (a) to (e) in the preparation of a medicament for a method for inducing an immune response against a cancer expressing KOC1, wherein the method comprises administering to a subject at least one component selected from the following groups (a) to (e): (a) one or more types of the peptide of claim 1; (b) one or more types of polynucleotides encoding the peptide of claim 1 in an expressible form; (c) APCs which present a complex of the peptide of claim 1 and an HLA-A*0301 antigen on their cell surface; (d) an exosome presenting a complex of the peptide of claim 1 and an HLA-A*0301 antigen on its cell surface; and (e) CTLs induced by the method of claim 7, wherein the cancer is selected from the group consisting of bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head and neck cancer, soft tissue tumors and testicular cancer.

12. Use of at least one component selected from the following groups (a) to (e) in the preparation of a medicament for a method of treating a cancer expressing KOC1, wherein the method comprises administering to a subject at least one component selected from the following groups (a) to (e): (a) one or more types of the peptide of claim 1; (b) one or more types of polynucleotides encoding the peptide of claim 1 in an expressible form; (c) APCs which present a complex of the peptide of claim 1 and an HLA-A*0301 antigen on their cell surface; (d) an exosome presenting a complex of the peptide of claim 1 and an HLA-A*0301 antigen on its cell surface; and (e) CTLs induced by the method of claim 7, wherein the cancer is selected from the group consisting of bladder cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), colon cancer, rectal cancer, esophageal cancer, diffuse gastric cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, osteosarcoma, ovarian cancer, kidney cancer, head and neck cancer, soft tissue tumors and testicular cancer.

13. An emulsion comprising one or more types of the peptide of claim 1, a water-soluble carrier and an oil adjuvant.

14. A kit comprising a container containing the composition of any one of claims 3 to 5 and a container containing an adjuvant.

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