Peptides derived from cdca1 and vaccines containing them

Abstract

The present invention provides CDCA1-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 of inducing antigen-presenting cells or CTLs. The present invention also provides compositions and pharmaceutical compositions containing them as active ingredients. Furthermore, the present invention provides methods of using the peptides, polynucleotides, antigen-presenting cells, cytotoxic T cells, or pharmaceutical compositions of the present invention to treat and / or prevent cancer, and / or prevent postoperative recurrence thereof. Methods of inducing an immune response against cancer are also provided.

Description

[0001] This application is a divisional application of Chinese patent application No. 201580052500.2, filed on July 31, 2015, entitled "CDCA1-derived peptides and vaccines containing them". Technical Field

[0002] This invention relates to the field of bioscience, and more specifically to the field of cancer treatment. In particular, the invention relates to novel peptides effective as cancer vaccines, methods of using said peptides to treat and / or prevent tumors, and pharmaceutical compositions comprising said peptides.

[0003] This application claims the benefit of Japanese patent applications filed on August 4, 2014 (Japanese Patent Application Nos. 2014-158922, 2014-158923, and 2014-158924), the entire contents of which are incorporated herein by reference. Background Technology

[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 through 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 under clinical development as targets for immunotherapy.

[0005] Among these TAAs, epitope peptides that can be recognized by CTLs were identified, and their applications in immunotherapy for various types of cancer are anticipated (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 have shown 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, it is still necessary to identify new CTL epitopes that can be used for cancer immunotherapy.

[0006] CDCA1 (cell cycle-associated protein 1; also described as NUF2, NDC80 kinetochore complex component: Nuf2; reference sequence: GeneBank accession number NM_145697 (SEQ ID NO: 81) or GeneBank accession number NM_031423 (SEQ ID NO: 83)) has been identified as a member of the gene co-expressed with CDC2, cyclins, topoisomerase II and other cell cycle genes (NPL14: Walker et al., Curr Cancer Drug Targets 2001 May; 1(1): 73-83). CDCA1 has been found to be associated with the centromere of HeLa cells undergoing mitosis and is considered a functional homolog of yeast Nuf2 (NPL15: Wigge PA et al., J Cell Biol 2001 Jan 22; 152(2): 349-60). Meanwhile, CDCA1 has been identified as an upregulated gene in non-small cell lung cancer using gene expression profiling analysis of a whole-genome cDNA microarray containing 23,040 genes (NPL16: Hayama et al., CancerRes 2006 Nov 1; 66(21):10339-48; PTL1:WO2007 / 013480; PTL2:WO2005 / 089735). CDCA1 expression is upregulated in tumors and tumor cell lines and is undetectable in normal organs except the testes (NPL16; PTL1). Furthermore, siRNA-mediated downregulation of CDCA1 expression leads to inhibition of cell proliferation in lung cancer cell lines expressing CDCA1.

[0007] Recently, HLA-A2-restricted CTL epitopes derived from CDCA1 (NPL17: Harao et al., Int J Cancer. 2008:123(11):2616-25; PTL3:WO2009 / 025117) and HLA-A24-restricted CTL epitopes (PTL4:WO2009 / 153992) have been identified. These peptides are effective in cancer patients with HLA-A2 or HLA-A24 genotypes, but are not expected to be effective in cancer patients without these HLA genotypes.

[0008] [List of Citations]

[0009] [Patent Literature]

[0010] [PTL 1]WO2007 / 013480

[0011] [PTL 2]WO2005 / 089735

[0012] [PTL 3]WO2009 / 025117

[0013] [PTL 4]WO2009 / 153992

[0014] [Non-patent literature]

[0015] [NPL 1]Boon T,Int J Cancer 1993 May 8,54(2):177-80

[0016] [NPL 2]Boon T&van der Bruggen P,J Exp Med 1996 Mar 1,183(3):725-9

[0017] [NPL 3]Harris CC,J Natl Cancer Inst 1996 Oct 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 1999 May 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] Walker et al., Curr Cancer Drug Targets 2001 May, 1(1):73-83

[0029] [NPL 15] Wigge PA et al., J Cell Biol 2001 Jan 22, 152(2):349-60

[0030] [NPL 16]Hayama et al., Cancer Res 2006 Nov 1, 66(21):10339-48

[0031] [NPL 17] Harao et al., Int J Cancer 2008 Dec 1, 123(11):2616-25 Summary of the Invention

[0032] This invention relates to peptides that can induce cytotoxic T cells (CTLs) specific to cells expressing CDCA1. When these peptides are presented on antigen-presenting cells (APCs) via human leukocyte antigens (HLA), they induce CTLs exhibiting specific cytotoxic activity against cells expressing CDCA1. The CDCA1-derived peptides with CTL-inducing capacity (CTL inducibility) identified to date are HLA-A2-restricted or HLA-A24-restricted peptides, which cannot induce CTLs against cells that do not express these HLAs. Therefore, conventional peptides are unsuitable for immunotherapy in subjects who do not possess these HLAs. HLA-A11 and HLA-A33 are common alleles in Asians (Sette A, Sidney J., Immunogenetics 1999, 50:201-12), while HLA-A03 is common 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, and HLA-A03-restricted peptides to HLA-A03-positive subjects. Therefore, this invention relates to CDCA1-derived peptides possessing CTL-inducing ability against HLA-A11, HLA-A33, or HLA-A03-restricted peptides. Based on the results disclosed herein, the peptides of this invention have been demonstrated to be epitope peptides capable of inducing strong and specific immune responses against cells expressing CDCA1 and HLA-A11, HLA-A33, or HLA-A03.

[0033] Therefore, one object of the present invention is to provide CDCA1-derived peptides capable of inducing CTLs in an HLA-A11-, HLA-A33-, or HLA-A03-restricted manner. These peptides can be used to induce CTLs in vitro, ex vivo, or in vivo, or can be administered to subjects for the purpose of inducing an immune response against cancer cells expressing CDCA1. Preferred peptides are peptides comprising the amino acid sequences selected from the following SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69 and 47; more preferred peptides are nonapeptides or decapeptides; and even more preferred peptides are peptides composed of the following amino acid sequences SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69 and 47.

[0034] The peptides of the present invention comprise peptides in which one, two or more amino acids are substituted, deleted, inserted and / or added, provided that the resulting modified peptide retains the CTL-inducible ability of the original peptide.

[0035] 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-inducible activity and can be administered to subjects to induce an immune response against cancer cells expressing CDCA1.

[0036] The present invention also provides compositions 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, exogenous bodies presenting the peptides of the present invention, and / or CTLs of the present invention. The compositions of the present invention are preferably pharmaceutical compositions. The pharmaceutical compositions of the present invention can be used to treat and / or prevent cancer, and to prevent its recurrence after surgery. 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 an APC, resulting in the induction of CTLs targeting the peptides. Therefore, another object of the present invention is to provide compositions for inducing CTLs, wherein the compositions comprise 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 exogenous bodies presenting the peptides of the present invention.

[0037] Another object of the present invention is to provide a method for inducing APCs with CTL inducibility, wherein the method includes 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 of the peptides of the present invention into APCs.

[0038] 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 HLA antigen and the peptide of the present invention on their surface, co-culturing CD8-positive T cells with exogenous organisms presenting a complex of HLA antigen and the peptide of the present invention on their surface, or introducing a vector into CD8-positive T cells, said vector comprising a polynucleotide encoding each subunit of a T cell receptor (TCR) capable of binding the peptide of the present invention presented by the HLA antigen on the cell surface. Preferred HLA antigens in the present invention are HLA-A11, HLA-A33, or HLA-A03.

[0039] 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 expressing CDCA1. In the present invention, the cancer subjected to immunotherapy is, for example, cancer present in homozygous or heterozygous patients having HLA-A11, HLA-A33, or HLA-A03. That is, the present invention provides immunotherapy for cancers expressing CDCA1 and at least one HLA antigen selected from HLA-A11, HLA-A33, and HLA-A03.

[0040] Another object of the present invention is to provide a method for inducing an immune response against cancer in a subject, wherein the method includes administering to the subject a composition comprising a peptide of the present invention, or a polynucleotide encoding said peptide, an APC of the present invention, an exogenous body 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 recurrence after surgery in a subject, wherein the method includes administering to the subject a peptide of the present invention, a polynucleotide encoding said peptide, an APC of the present invention, an exogenous body presenting the peptide of the present invention, and / or a CTL of the present invention.

[0041] This invention provides the following:

[0042] 1. A peptide of fewer than 15 amino acids with cytotoxic T cell (CTL) induction ability, comprising an amino acid sequence selected from the following group:

[0043] (a) An amino acid sequence selected from the group consisting of: SEQ ID NOs:3,5-7,9,10,12-14,17,19,21,30,35,38-40,45,53,56,58,27,60,28,67,69 and 47; and

[0044] (b) An amino acid sequence selected from the group consisting of substituted, deleted, inserted and / or added amino acids of one, two or more amino acids: SEQ ID NOs:3,5-7,9,10,12-14,17,19,21,30,35,38-40,45,53,56,58,27,60,28,67,69 and 47.

[0045] 2. The peptide of item 1, selected from the following groups (i) to (iii):

[0046] (i) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the group(a) to (d) are introduced into an amino acid sequence selected from the group: SEQ ID NOs:3,5-7,9,10,12-14,17,19,21,30,35,38-40,45,53,56 and 58:

[0047] (a) Replace 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.

[0048] (b) Replace the third amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, phenylalanine, tyrosine, isoleucine and alanine.

[0049] (c) Replace 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

[0050] (d) Replace the C-terminal amino acid with an amino acid selected from the group consisting of lysine and arginine;

[0051] (ii) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the group(a) to (c) are introduced into an amino acid sequence selected from the group: SEQ ID NOs:27,3,60,28,5,67 and 69:

[0052] (a) Replace the first amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;

[0053] (b) Replace 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.

[0054] (c) Replace the C-terminal amino acid with an amino acid selected from the group consisting of arginine and lysine; and

[0055] (iii) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the group(a) to (b) are introduced into an amino acid sequence selected from the group: SEQ ID NOs:7,38 and 47:

[0056] (a) Replace 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

[0057] (b) Replace the C-terminal amino acid with an amino acid selected from the group consisting of arginine, lysine, tyrosine and phenylalanine.

[0058] 3. The peptide of item 1, which consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs:3,5-7,9,10,12-14,17,19,21,30,35,38-40,45,53,56,58,27,60,28,67,69 and 47.

[0059] 4. Polynucleotide, a peptide that encodes any one of terms 1 to 3.

[0060] 5. A composition comprising a pharmaceutically acceptable carrier and at least one ingredient selected from groups (a) to (e) below:

[0061] (a) A peptide of one or more types from item 1 to 3;

[0062] (b) One or more types of polynucleotides that encode a peptide of any one of items 1 to 3 in an expressible form;

[0063] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides 1 to 3 with HLA antigens on their cell surface.

[0064] (d) An exogenous body that presents a complex of any one of the peptides from items 1 to 3 with an HLA antigen on its cell surface; and

[0065] (e)CTL, targeting any one of the peptides in terms of item 1 to 3.

[0066] 6. The composition of item 5, which is a composition for inducing CTL, wherein said component is at least one component selected from the following groups (a) to (d):

[0067] (a) A peptide of one or more types from item 1 to 3;

[0068] (b) One or more types of polynucleotides that encode a peptide of any one of items 1 to 3 in an expressible form;

[0069] (c) Antigen-presenting cells (APCs) that present complexes of any one of peptides 1 to 3 with HLA antigens on their cell surface; and

[0070] (d) Exogenous organisms that present a complex of any one of the peptides from item 1 to 3 with an HLA antigen on their cell surface.

[0071] 7. The composition of item 5, which is a pharmaceutical composition.

[0072] 8. The composition of item 7, for use in one or more of the following groups: (i) cancer treatment, (ii) cancer prevention (prophylaxis) and (iii) prevention of cancer recurrence after surgery (prophylaxis).

[0073] 9. The composition of item 7, which is used to induce an immune response against cancer.

[0074] 10. The composition of item 8 or 9, wherein the cancer is selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, and colorectal cancer.

[0075] 11. The composition of any one of claims 5 to 10, formulated for administration to at least one HLA-positive subject selected from the group consisting of HLA-A11, HLA-A33 and HLA-A03.

[0076] 12. A method for inducing APCs with CTL induction ability, comprising steps selected from the group consisting of:

[0077] (a) Contacting APC with any of the peptides in items 1 to 3 in vitro, ex vivo, or in vivo; and

[0078] (b) Introduce a polynucleotide of the peptide encoding any one of terms 1 to 3 into the APC.

[0079] 13. A method for inducing CTLs, comprising steps selected from the group consisting of:

[0080] (a) CD8 positive T cells are co-cultured with APCs, wherein the APCs present a complex of HLA antigen and any one of the peptides in items 1 to 3 on their surface.

[0081] (b) Co-culturing CD8 positive T cells with exogenous bodies, said exogenous bodies presenting on their surface a complex of an HLA antigen and a peptide of any one of items 1 to 3;

[0082] (c) Introducing a polynucleotide into CD8-positive T cells, the polynucleotide encoding each subunit of a T-cell receptor (TCR) capable of binding to a peptide on the cell surface presented by an HLA antigen of any one of items 1 to 3.

[0083] 14. APC, which presents on its surface a complex of an HLA antigen and a peptide of any one of items 1 to 3.

[0084] 15. The APC of item 14, which is induced by the method of item 12.

[0085] 16. CTL, targeting any one of the peptides in items 1-3.

[0086] 17. The CTL of item 16, which is induced by the method of item 13.

[0087] 18. A method for inducing an immune response against cancer, comprising administering to a subject at least one component selected from groups (a) to (e) below:

[0088] (a) A peptide of one or more types from item 1 to 3;

[0089] (b) One or more types of polynucleotides that encode a peptide of any one of items 1 to 3 in an expressible form;

[0090] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides 1 to 3 with HLA antigens on their cell surface.

[0091] (d) An exogenous body that presents a complex of any one of the peptides from items 1 to 3 with an HLA antigen on its cell surface; and

[0092] (e)CTL, targeting any one of the peptides in terms of item 1 to 3.

[0093] 19. A method for treating and / or preventing cancer, and / or preventing its recurrence after surgery, said method comprising administering to a subject at least one ingredient selected from groups (a) to (e) below:

[0094] (a) A peptide of one or more types from item 1 to 3;

[0095] (b) One or more types of polynucleotides that encode a peptide of any one of items 1 to 3 in an expressible form;

[0096] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides 1 to 3 with HLA antigens on their cell surface.

[0097] (d) An exogenous body that presents a complex of any one of the peptides from items 1 to 3 with an HLA antigen on its cell surface; and

[0098] (e)CTL, targeting any one of the peptides in terms of item 1 to 3.

[0099] 20. An antibody, which binds to any one of terms 1 to 3.

[0100] 21. A method for screening peptides with CTL-inducible ability, comprising the following steps:

[0101] (a) Generate a candidate sequence consisting of an amino acid sequence wherein one, two, or more amino acid residues are substituted, deleted, inserted, and / or added to the original amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69, and 47;

[0102] (b) Select candidate sequences, other than CDCA1, from the candidate sequences generated in (a) that do not have significant homology (sequence identity) with any known human gene product;

[0103] (c) Contact the APC with a peptide consisting of the candidate sequences selected in (b);

[0104] (d) Expose the APCs from (c) to CD8-positive T cells; and

[0105] (e) Select peptides that have equal or higher CTL induction capacity compared to peptides composed of the original amino acid sequence.

[0106] 22. An emulsion comprising one or more types of peptides, water-soluble carriers, and oil adjuvants of any one of items 1 to 3.

[0107] 23. A kit comprising a container for containing the composition of any one of items 5 to 11 and a container for containing the adjuvant.

[0108] In addition to the foregoing, other objects and features of the invention will become more apparent when read in conjunction with the accompanying drawings and embodiments in the following detailed description. However, it should be understood that the foregoing overview and the following detailed description of the invention are exemplary embodiments and are not restrictive of the invention or other alternative embodiments thereof. In particular, although the invention has been described herein with reference to several specific embodiments, it should be understood that this description is illustrative and not a limitation thereof. Various modifications and applications will occur to those skilled in the art without departing from the spirit and scope of the invention as set forth in the appended claims. Similarly, other objects, features, benefits, and advantages of the invention will become apparent from the foregoing and certain embodiments described below, and will be obvious to those skilled in the art. Such objects, features, benefits, and advantages will be apparent from the foregoing in conjunction with the appended embodiments, data, drawings, and all reasonable inferences drawn therefrom, alone or in conjunction with the references incorporated herein. Attached Figure Description

[0109] Figure 1-1 : Figure 1-1Composed of photographs (a) to (v), which show the results of interferon (IFN)-gamma enzyme-linked immunosorbent assay (ELISPOT) in CTLs induced by peptides derived from CDCA1. Compared with the control, the following are examples: hole #7 (a) having CDCA1-A11-9-123 (SEQ ID NO:3), hole #8 (b) having CDCA1-A11-9-105 (SEQ ID NO:5); hole #1 (c) having CDCA1-A11-9-419 (SEQ ID NO:6), hole #4 (d) having CDCA1-A11-9-219 (SEQ ID NO:7), hole #2 (e) having CDCA1-A11-9-439 (SEQ ID NO:9), hole #2 (f) having CDCA1-A11-9-343 (SEQ ID NO:10), hole #8 (g) having CDCA1-A11-9-21 (SEQ ID NO:12), and hole #8 having CDCA1-A11-9-157 (SEQ ID NO:3). Hole #3 (h) with CDCA1-A11-9-244 (SEQ ID NO:14) , Hole #7 (i) with CDCA1-A11-9-213 (SEQ ID NO:17) , Hole #6 (j) with CDCA1-A11-9-335 (SEQ ID NO:19) , Hole #7 (k) with CDCA1-A11-9-25 (SEQ ID NO:21) , Hole #4 (l) with CDCA1-A11-10-339 (SEQ ID NO:30) , Hole #2 (n) with CDCA1-A11-10-452 (SEQ ID NO:35) , Hole #3 with CDCA1-A11-10-400 (SEQ ID NO:13) , Hole #7 (i) with CDCA1-A11-9-244 (SEQ ID NO:14) , Hole #6 (j) with CDCA1-A11-9-213 (SEQ ID NO:17) , Hole #7 (k) with CDCA1-A11-9-335 (SEQ ID NO:19) , Hole #4 (l) with CDCA1-A11-9-25 (SEQ ID NO:21) , Hole #3 (m) with CDCA1-A11-10-339 (SEQ ID NO:30) , Hole #2 (n) with CDCA1-A11-10-400 (SEQ ID NO:13) , Hole #7 (i) with CDCA1-A11-9-213 (SEQ ID NO:17) , Hole #6 (j) with CDCA1-A11-9-335 (SEQ ID NO:19) , Hole #7 (k) with CDCA1-A11-9-25 (SEQ ID NO:21) , Hole #3 (m) with CDCA1-A11- CTLs in well #7 (o) with CDCA1-A11-10-289 (SEQ ID NO:39), well #6 (p) with CDCA1-A11-10-203 (SEQ ID NO:40), well #5 (q) with CDCA1-A11-10-156 (SEQ ID NO:45), well #2 (r) with CDCA1-A11-10-340 (SEQ ID NO:53), well #4 (t) with CDCA1-A11-10-106 (SEQ ID NO:56), and well #2 (u) with CDCA1-A11-10-358 (SEQ ID NO:58) showed strong IFN-gamma production. In these photographs, the squares on the wells indicate the cells propagated from the corresponding wells for establishing CTL lines.In contrast, CDCA1-A11-10-391 (SEQ ID NO:33)(v) is shown as an example of typical negative data where no specific IFN-gamma production was observed. In the figure, "+" indicates IFN-gamma production for target cells pulsed with an appropriate peptide; and "-" indicates IFN-gamma production for target cells that have not yet been pulsed with any peptide.

[0110] Figure 1-2 Showing Figure 1-1 The continuation of.

[0111] Figure 2-1 : Figure 2-1 The series of line graphs (a) to (i) shows the results of an IFN-gamma enzyme-linked immunosorbent assay (ELISA) confirming the production of IFN-gamma in the following CTL lines stimulated in sequence: CDCA1-A11-9-123 (SEQ ID NO:3) (a), CDCA1-A11-9-105 (SEQ ID NO:5) (b), CDCA1-A11-9-419 (SEQ ID NO:6) (c), CDCA1-A11-9-219 (SEQ ID NO:7) (d), CDCA1-A11-9-439 (SEQ ID NO:9) (e), CDCA1-A11-9-157 (SEQ ID NO:13) (f), CDCA1-A11-9-25 (SEQ ID NO:21) (g), CDCA1-A11-10-339 (SEQ ID NO:13) (f), CDCA1-A11-9-25 (SEQ ID NO:21) (g), CDCA1-A11-10-339 (SEQ ID NO:13) (i). NO:30)(h) or CDCA1-A11-10-358 (SEQ ID NO:58)(i). These results confirm that the CTL lines established by stimulation with each peptide exhibit strong IFN-gamma production compared to the control. In the figure, "+" indicates IFN-gamma production for target cells shocked with the appropriate peptide; and "-" indicates IFN-gamma production for target cells not yet shocked 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).

[0112] Figure 2-2 Showing Figure 2-1 The continuation of.

[0113] Figure 3Comprising a series of line graphs (a) through (c), this study illustrates IFN-gamma production in CTL clones established from limiting dilutions of CTL lines stimulated with the following peptides: CDCA1-A11-9-105 (SEQ ID NO:5) (a), CDCA1-A11-9-419 (SEQ ID NO:6) (b), or CDCA1-A11-9-219 (SEQ ID NO:7) (c). These results confirm that CTL clones established with stimulation by each peptide exhibit strong IFN-gamma production compared to controls. In the figures, "+" indicates IFN-gamma production in target cells bombarded with the appropriate peptide; and "-" indicates IFN-gamma production in target cells not yet bombarded 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).

[0114] Figure 4 This is a line graph showing specific CTL activity against target cells expressing both CDCA1 and HLA-A*1101. COS7 cells transfected with either HLA-A*1101 or the full-length CDCA1 gene were prepared as controls. The CTL clone established using CDCA1-A11-9-219 (SEQ ID NO:7) exhibited specific CTL activity against COS7 cells transfected with both CDCA1 and HLA-A*1101 (black diamonds). On the other hand, no significant specific CTL activity was shown against target cells transfected with either HLA-A*1101 (triangles) or CDCA1 (white circles).

[0115] Figure 5Composed of photographs (a) to (h), which show the results of IFN-gamma ELISPOT assays in CTLs induced by peptides derived from CDCA1. Compared with the control, the CTLs in well #2 (a) with CDCA1-A33-9-43 (SEQ ID NO:27), well #1 (b) with CDCA1-A33-9-123 (SEQ ID NO:3), well #3 (c) with CDCA1-A33-9-108 (SEQ ID NO:60), well #2 (d) with CDCA1-A33-9-261 (SEQ ID NO:28), well #6 (e) with CDCA1-A33-9-105 (SEQ ID NO:5), well #8 (f) with CDCA1-A33-10-10 (SEQ ID NO:67), and well #5 (g) with CDCA1-A33-10-260 (SEQ ID NO:69) showed strong IFN-gamma production. In these images, the squares on the wells indicate the proliferation of cells from the corresponding wells used to establish CTL lines. In contrast, CDCA1-A33-10-122 (SEQ ID NO:68)(h) is shown as an example of typical negative data where no specific IFN-gamma production was observed. In the figures, "+" indicates IFN-gamma production for target cells bombarded with a suitable peptide; and "-" indicates IFN-gamma production for target cells that have not yet been bombarded with any peptide.

[0116] Figure 6 A series of line graphs (a) through (d) show the results of the IFN-gamma ELISA, confirming IFN-gamma production in CTL lines stimulated sequentially with the following peptides: CDCA1-A33-9-43 (SEQ ID NO:27)(a), CDCA1-A33-9-123 (SEQ ID NO:3)(b), CDCA1-A33-10-10 (SEQ ID NO:67)(c), or CDCA1-A33-10-260 (SEQ ID NO:69)(d). These results confirm that the CTL lines established by stimulation with each peptide exhibit strong IFN-gamma production compared to the control. In the figures, "+" indicates IFN-gamma production in target cells shocked with the appropriate peptide; and "-" indicates IFN-gamma production in target cells not yet shocked 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).

[0117] Figure 7Comprising a series of line graphs (a) to (b), this study illustrates IFN-gamma production in CTL clones established from limiting dilutions of CTL lines stimulated with either CDCA1-A33-9-43 (SEQ ID NO:27) (a) or CDCA1-A33-9-123 (SEQ ID NO:3) (b). These results confirm that CTL clones established with stimulation by each peptide exhibit strong IFN-gamma production compared to controls. In the figures, "+" indicates IFN-gamma production in target cells bombarded with the appropriate peptide; and "-" indicates IFN-gamma production in target cells not yet bombarded 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).

[0118] Figure 8 This is a line graph showing specific CTL activity against target cells expressing both CDCA1 and HLA-A*3303. COS7 cells transfected with either HLA-A*3303 or the full-length CDCA1 gene were used as controls. CTL clones established using CDCA1-A33-9-43 (SEQ ID NO:27) demonstrated specific CTL activity against COS7 cells transfected with both CDCA1 and HLA-A*3303 (black diamonds). On the other hand, no significant specific CTL activity was shown against target cells transfected with either HLA-A*3303 (white triangles) or CDCA1 (white circles).

[0119] Figure 9 Photographs (a) through (d) show the results of IFN-gamma ELISPOT assays in CTLs induced by peptides derived from CDCA1. Compared to the control, CTLs in well #3 (a) with CDCA1-A03-9-219 (SEQ ID NO:7), well #1 (b) with CDCA1-A03-10-400 (SEQ ID NO:38), and well #2 (c) with CDCA1-A03-10-257 (SEQ ID NO:47) showed strong IFN-gamma production. In these photographs, the squares on the wells indicate the cells proliferating from the corresponding wells used to establish the CTL line. Conversely, CDCA1-A03-9-343 (SEQ ID NO:10) (d) is shown as an example of a typical negative result in which no specific IFN-gamma production was observed. In the figure, "+" indicates IFN-gamma production for target cells bombarded with a suitable peptide; and "-" indicates IFN-gamma production for target cells that have not yet been bombarded with any peptide.

[0120] Figure 10 A series of line graphs (a) through (c) show the results of the IFN-gamma ELISA, confirming IFN-gamma production in CTL lines stimulated with the following peptides: CDCA1-A03-9-219 (SEQ ID NO:7) (a), CDCA1-A03-10-400 (SEQ ID NO:38) (b), or CDCA1-A03-10-257 (SEQ ID NO:47) (c). These results confirm that CTL lines established by stimulation with each peptide exhibit strong IFN-gamma production compared to controls. In the graphs, "+" indicates IFN-gamma production in target cells shocked with the appropriate peptide; and "-" indicates IFN-gamma production in target cells not yet shocked 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).

[0121] Figure 11 Comprising a series of line graphs (a) through (c), this study illustrates IFN-gamma production in CTL clones established from limiting dilutions of CTL lines stimulated with the following peptides: CDCA1-A03-9-219 (SEQ ID NO:7) (a), CDCA1-A03-10-400 (SEQ ID NO:38) (b), or CDCA1-A03-10-257 (SEQ ID NO:47) (c). These results confirm that CTL clones established with stimulation by each peptide exhibit strong IFN-gamma production compared to controls. In the figures, "+" indicates IFN-gamma production in response to target cells bombarded with the appropriate peptide; and "-" indicates IFN-gamma production in response to target cells not yet bombarded 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).

[0122] Figure 12 A series of line graphs (a) and (b) show specific CTL activity against target cells expressing both CDCA1 and HLA-A*0301. COS7 cells transfected with either HLA-A*0301 or the full-length CDCA1 gene were used as controls. CTL clones established using CDCA1-A03-9-219 (SEQ ID NO:7)(a) or CDCA1-A03-10-400 (SEQ ID NO:38)(b) showed specific CTL activity against COS7 cells transfected with both CDCA1 and HLA-A*0301 (black diamonds). On the other hand, no significant specific CTL activity was shown against target cells transfected with either HLA-A*0301 (white triangles) or CDCA1 (white circles). Detailed Implementation

[0123] Description of Detailed Implementation

[0124] Although any methods and materials similar to or equivalent to those described herein may be used in implementing or testing embodiments of the invention, preferred methods, apparatus, and materials are now described. However, before describing the materials and methods of the invention, it should be understood that the invention is not limited to the specific sizes, shapes, dimensions, materials, methodologies, schemes, etc., described herein, as these can be varied through routine experimentation and optimization. It should also be understood that the terminology used in this specification is for the purpose of describing a particular version or embodiment only and is not intended to limit the scope of the invention, which will be defined only by the appended claims.

[0125] I. Definition

[0126] Unless otherwise expressly stated, the terms “a / kind,” “the,” and “the” used herein mean “at least one / kind.”

[0127] When the terms "isolated" and "purified" are used in connection with substances (e.g., peptides, antibodies, polynucleotides, etc.), they mean that the substance is substantially free of at least one substance that can be included in natural sources. Thus, an isolated or purified peptide means that it is substantially free of another cellular material, such as carbohydrates, lipids, or other contaminating proteins of cellular or tissue origin from which the peptide is derived. When peptides are chemically synthesized, an isolated or purified peptide means a peptide that is substantially free of precursor substances or another chemical substance. The phrase "substantially free of cellular material" includes peptide preparations in which the peptide is isolated from the cellular components of the cells from which the peptide was isolated or recombined to produce the peptide. Thus, peptides that are substantially free of cellular material include peptide preparations containing less than about 30%, 20%, 10%, or 5%, 3%, 2%, or 1% (on a dry weight basis) of other cellular material. When peptides are generated in a recombinant manner, the isolated or purified peptides are substantially free of culture medium, containing a peptide preparation containing less than about 20%, 10%, or 5%, 3%, 2%, or 1% (by dry weight) of culture medium by volume. When peptides are generated by chemical synthesis, the isolated or purified peptides are substantially free of precursor substances or other chemicals, containing a peptide preparation containing less than about 30%, 20%, 10%, 5%, 3%, 2%, or 1% (by dry weight) of precursor substances or other chemicals by volume. The isolation or purification of the peptide preparation can be confirmed, for example, by the appearance of a single band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining or such gels. In a preferred embodiment, the peptides and polynucleotides of the present invention are isolated or purified.

[0128] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. In addition to naturally occurring amino acid polymers, these terms also apply to non-naturally occurring amino acid polymers that contain one or more non-naturally occurring amino acid residues. Non-naturally occurring amino acids include amino acid analogs, amino acid mimics, and the like.

[0129] As used herein, the term "amino acid" refers to naturally occurring amino acids, as well as amino acid analogs and amino acid mimics 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-translational modified (e.g., hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine, etc.). The phrase "amino acid analog" refers to a compound having the same basic chemical structure as a naturally occurring amino acid (alpha carbon bound to hydrogen, carboxyl, amino, and R groups) but with modified R groups or modified skeletons (e.g., homoserine, ortholeucine, methionine sulfoxide, methionine methylsulfonium, etc.). The phrase "amino acid mimic" refers to a compound having a different structure than a typical amino acid but with similar functions. Amino acids can be L-amino acids or D-amino acids, and the peptides of the present invention are preferably L-amino acid polymers.

[0130] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably in this document and refer to polymers of nucleotides.

[0131] As used in this specification, the term "composition" is intended to include products comprising a specified amount of a specified ingredient, and any product resulting directly or indirectly from a combination of a specified amount of the specified ingredient. When the composition is a pharmaceutical composition, the term "composition" is intended to include products comprising an active ingredient and an inert ingredient, and any product resulting directly or indirectly from a combination, complexation or aggregation, dissociation of one or more ingredients, or other type of reaction or interaction of one or more ingredients. Therefore, 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. Not limited thereto, 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 pharmaceutically or physiologically acceptable materials, compositions, substances, or media.

[0132] Unless otherwise stated, the term "cancer" refers to cancer that overexpresses the CDCA1 gene; and examples include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, colorectal cancer, etc. In an exemplary embodiment, "cancer" is cancer that expresses CDCA1 and HLA-A11, HLA-A33, and / or HLA-A03.

[0133] Unless otherwise stated, the terms “cytotoxic T lymphocytes” and “cytotoxic T cells” and “CTL” are used interchangeably in this document. Unless otherwise explicitly stated, 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.

[0134] Unless otherwise stated, the term "HLA-A11" refers to the HLA-A11 type, which includes subtypes such as HLA-A*1101, HLA-A*1102, HLA-A*1103, and HLA-A*1104.

[0135] Unless otherwise stated, the term "HLA-A33" refers to the HLA-A33 type, which includes subtypes such as HLA-A*3303, HLA-A*3301, and HLA-A*3304.

[0136] Unless otherwise stated, the term "HLA-A03" refers to the HLA-A03 type, which includes subtypes such as HLA-A*0301, HLA-A*0302, and HLA-A*0305.

[0137] In the context of the subject or patient, the phrase "the subject (or patient) has HLA-A11" means that the subject or patient possesses 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 the subject's or patient's cells. Similarly, the phrases "the subject (or patient) has HLA-A33" and "the subject (or patient) has HLA-A03" as used in this article respectively mean that the subject or patient possesses a homozygous or heterozygous HLA-A33 antigen gene as an MHC (major histocompatibility complex) class I molecule, and that the HLA-A33 antigen is expressed as an HLA antigen in the subject's or patient's cells; and that the subject or patient possesses a homozygous or heterozygous HLA-A03 antigen gene as an MHC (major histocompatibility complex) class I molecule, and that the HLA-A03 antigen is expressed as an HLA antigen in the subject's or patient's cells.

[0138] The methods and compositions of this invention are considered "effective" when they achieve clinical advantage, provided they are useful in the context of cancer "treatment," such treatment reduces the size, spread, or metastasis of cancer in subjects, delays cancer progression, alleviates clinical symptoms of cancer, prolongs survival, or inhibits postoperative recurrence. When the treatment is administered prophylactically, "effective" means that the treatment delays or prevents cancer formation, or prevents or alleviates clinical symptoms of cancer. Effectiveness is determined relative to any known method for diagnosing or treating a particular type of tumor.

[0139] Wherever the methods and compositions of this invention are used in the context of cancer “prevention,” the term “prevention” herein includes any work aimed at reducing the burden of cancer-related mortality or morbidity. Prevention can be carried out at “primary, secondary, and tertiary levels of prevention.” While primary prevention avoids the development of disease, prevention at secondary and tertiary levels includes preventing disease progression and the onset of symptoms, as well as aiming to reduce the adverse effects of existing disease by restoring function and reducing disease-related complications. Alternatively, prevention may include mitigating the severity of specific conditions, such as extensive preventative treatments aimed at reducing tumor growth and metastasis.

[0140] In the context of this invention, cancer treatment and / or prevention (protection) and / or postoperative recurrence prevention (protection) includes any event such as inhibition of cancer cell proliferation, tumor regression or regression, mitigation and inhibition of cancer-inducing development, tumor regression, and reduction or inhibition of metastasis, inhibition of postoperative cancer recurrence, and prolongation of survival. Effective cancer treatment and / or prevention (protection) reduces mortality, improves the prognosis of individuals with cancer, reduces blood levels of tumor markers, and alleviates detectable symptoms associated with cancer. For example, symptom relief or improvement constitutes effective treatment and / or prevention (protection), and includes patients whose symptoms are stable or reduced by 10%, 20%, 30%, or more.

[0141] In the context of this invention, the term "antibody" refers to an immunoglobulin or fragment thereof that specifically reacts with a specified protein or its peptide. Antibodies may include human antibodies, primate-derived antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused with other proteins or radiolabeled substances, and antibody fragments. Furthermore, the term "antibody" is used herein in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies, multispecific antibodies formed from two or more intact antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity. "Antibody" can be of all classes of antibodies (e.g., IgA, IgD, IgE, IgG, and IgM).

[0142] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0143] II. Peptides

[0144] HLA-A11 and HLA-A33 are common alleles in Asians (Sette A, Sidney J., Immunogenetics 1999, 50:201-12), and HLA-A03 is a common allele 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 CDCA1 could be provided by offering CDCA1-derived CTL-inducible peptides restricted to HLA-A11, HLA-A33, or HLA-A03. Therefore, this invention provides CDCA1-derived peptides that can induce CTLs in an HLA-A11-, HLA-A33-, or HLA-A03-restricted manner.

[0145] The peptides of the present invention are CDCA1-derived peptides capable of inducing CTLs in an HLA-A11-, HLA-A33-, or HLA-A03-restricted manner. Peptides capable of inducing CTLs in an HLA-A11-restricted manner include peptides having amino acid sequences selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58. Peptides capable of inducing CTLs in an HLA-A33-restricted manner include peptides having amino acid sequences selected from SEQ ID NOs:27,3,60,28,5,67, and69. Peptides capable of inducing CTLs in an HLA-A03-restricted manner include peptides having amino acid sequences selected from SEQ ID NOs:7,38, and47.

[0146] CTLs exhibiting specific cytotoxic activity against these peptides can be established by in vitro stimulation of T cells with dendritic cells (DCs) pulsed with these peptides. The established CTLs showed specific cytotoxic activity against target cells pulsed with each peptide.

[0147] The CDCA1 gene is overexpressed in cancer cells, such as cancer cells in bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, colorectal cancer, etc., but is not expressed in most normal organs. Therefore, it is an excellent target for immunotherapy. Thus, the peptides of the present invention can be suitably 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 peptides are peptides selected from the group consisting of the following amino acid sequences: SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69, and 47. For example, peptides having the amino acid sequence of SEQ ID NO:7 are suitable for inducing CTLs exhibiting specific cytotoxic activity against HLA-A11 and CDCA1 cells, and may be suitable for cancer immunotherapy in HLA-A11-positive patients. Furthermore, for example, peptides having the amino acid sequence of SEQ ID NO:27 are suitable for inducing CTLs exhibiting specific cytotoxic activity against HLA-A33 and CDCA1 cells, and may be suitable for cancer immunotherapy in HLA-A33-positive patients. Furthermore (for example), peptides having an amino acid sequence selected from SEQ ID NOs:7 and 38 are suitable for inducing CTLs exhibiting specific cytotoxic activity against HLA-A03 and CDCA1 cells, and may be suitable for cancer immunotherapy in HLA-A03-positive patients. In a more preferred embodiment, the peptides of the present invention are peptides selected from the amino acid sequences of SEQ ID NOs:7, 27, and 38.

[0148] For the peptides of the present invention, additional amino acid residues may be added to the amino acid sequence of the peptide of the present invention, as long as the resulting peptide retains the CTL-inducible ability of the original peptide. The additional amino acid residues may consist of any type of amino acids, as long as they do not impair the CTL-inducible ability of the original peptide. Therefore, the peptides of the present invention comprise peptides with CTL-inducible ability, comprising amino acid sequences selected from the following: SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69, and 47. Such peptides are, for example, 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 comprise peptides 10 amino acids long or 11-40 amino acids long, which are produced by adding additional amino acids to said peptide. Furthermore, if the original peptide is a decapeptide, the peptides of the present invention comprise peptides 11-40 amino acids long. Such peptides can be, for example, peptides of 11-20 amino acids or peptides of 11-15 amino acids. Another preferred example of amino acid residues is an amino acid residue adjacent to the amino acid sequence of the peptide of the present invention in the full-length amino acid sequence of CDCA1 (e.g., SEQ ID NO: 82 or 84). Therefore, the peptides of the present invention comprise peptides containing amino acid sequences selected from: SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69 and 47, and wherein said peptide is a peptide fragment of CDCA1 and has CTL-inducing activity.

[0149] Generally, modifications of one, two, or more amino acids in certain peptides do not affect the peptide's function, 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 composed of amino acid sequences modified (i.e., substituted, added, deleted, and / or inserted) by one, two, or more amino acid residues 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 of the amino acid sequences selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56,58,27,60,28,67,69 and 47 is present, two or more amino acid residues are substituted, deleted, inserted and / or added, and has CTL-inducing activity.

[0150] Those skilled in the art will recognize that individual substitutions of an amino acid sequence (which alter a single amino acid or a small percentage of amino acids) tend to result in the preservation of the properties of the original amino acid side chains. Therefore, these are commonly referred to as "conservative substitutions" or "conservative modifications"; and modifications of proteins by "conservative substitutions" or "conservative modifications" can result in the modified protein having functions similar to the original protein. Conservative representations that provide 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); base-containing side chains (R, K, H); and aromatic side chains (H, F, Y, W). Additionally, the following eight groups each contain amino acids that are recognized in the art as conserved substitutes for each other:

[0151] 1) Alanine (A), glycine (G);

[0152] 2) Aspartic acid (D), glutamic acid (E);

[0153] 3) Asparagine (N), glutamine (Q);

[0154] 4) Arginine (R), Lysine (K);

[0155] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0156] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0157] 7) Serine (S), threonine (T); and

[0158] 8) Cysteine ​​(C), Methionine (M) (see, for example, Creighton, Proteins 1984).

[0159] Such conserved 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-inducible ability of the original peptide. In addition, modified peptides do not exclude CTL-inducible peptides derived from polymorphic variants of CDCA1, interspecies homologs, and alleles.

[0160] Provided the peptide retains the CTL-inducible ability of the original peptide, it can be modified (i.e., substituted, deleted, inserted, and / or added) by a few (e.g., one, two, or several) or a small percentage of amino acids. Here, the term "several" means five or fewer amino acids, such as four, three, or fewer. The percentage of modified amino acids is preferably 20% or less, more preferably 15% or less, and even more preferably 10% or less, for example, 1 to 5%.

[0161] When used in the context of cancer immunotherapy, the peptides of the present invention are presented on the surface of cells or exogenous bodies, preferably as complexes with HLA antigens. Therefore, it is preferred that the peptides of the present invention possess a high binding affinity for 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 exhibited by binding 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.

[0162] 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 usually involved in binding to anchor residues of HLA class I (Rammensee HG, et al., Immunogenetics. 1995; 41(4):178-228.). For example, for HLA-A11, threonine, valine, isoleucine, leucine, phenylalanine, and tyrosine as the second amino acid from the N-terminus, and lysine and arginine as the C-terminal amino acid are considered to be anchor residues with high binding affinity to HLA-A11 (Falk, et al., Immunogenetics 1994, 40:232-41; Chujoh, et al., Tissue Antigens 1998:52:501-9). Furthermore, in HLA-A11, there are auxiliary anchoring residues at positions 3 and 7 of the N-terminus; and it is known that leucine, phenylalanine, tyrosine, isoleucine, and alanine are preferably the third amino acid from the N-terminus, and leucine, isoleucine, tyrosine, valine, and phenylalanine are preferably 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, in order to maintain or enhance the binding affinity of HLA-A11, it may be desirable to replace the second amino acid from the N-terminus with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine, and / or to replace the C-terminal amino acid with lysine or arginine. Furthermore, it may be necessary to replace the third amino acid from the N-terminus with leucine, phenylalanine, tyrosine, isoleucine, or alanine, and / or replace the seventh amino acid from the N-terminus with leucine, isoleucine, tyrosine, valine, or phenylalanine. Therefore, peptides with CTL-inducible ability comprising amino acid sequences selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58 are included in the peptides of the present invention where the second amino acid from the N-terminus is replaced with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine; the third amino acid from the N-terminus is replaced with leucine, phenylalanine, tyrosine, isoleucine, or alanine; the seventh amino acid from the N-terminus is replaced with leucine, isoleucine, tyrosine, valine, or phenylalanine; and / or the C-terminal amino acid is replaced with lysine or arginine.In a preferred embodiment, the peptide of the present invention may be a peptide composed of an amino acid sequence having CTL inducibility, wherein in the amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58, 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. That is, the peptides of the present invention comprise peptides containing an amino acid sequence having CTL inducibility, wherein the amino acid sequence has one or more substitutions selected from the amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58, selected from the following (a) to (d): (a) the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine;

[0163] (b) The third amino acid from the N-terminus is replaced with leucine, phenylalanine, tyrosine, isoleucine or alanine.

[0164] (c) The seventh amino acid from the N-terminus is replaced with leucine, isoleucine, tyrosine, valine, or phenylalanine; and

[0165] (d) The C-terminal amino acid is replaced with lysine or arginine.

[0166] In a preferred embodiment, the peptide of the present invention may be a peptide composed of amino acids having CTL-inducible ability, wherein one or more substitutions selected from (a) to (d) above are introduced into an amino acid sequence selected from SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56 and 58. In the present invention, the preferred number of substitutions is 1, 2, 3 or 4 substitutions selected from (a) to (d) above.

[0167] The peptides of the present invention may be peptides comprising an amino acid sequence having CTL inducibility, wherein in the amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58, 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 can be a peptide composed of an amino acid sequence having CTL inducibility, wherein in the amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58, 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 can be a peptide comprising an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from (a) and (b) below are introduced into the amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58:

[0168] (a) The second amino acid from the N-terminus is replaced with threonine, valine, isoleucine, leucine, phenylalanine, or tyrosine; and

[0169] (b) The C-terminal amino acid is replaced with lysine or arginine.

[0170] In a preferred embodiment, the peptide of the present invention may be a peptide composed of an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from (a) to (b) above are introduced into an amino acid sequence selected from SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56 and 58. In a more preferred embodiment, the second amino acid from the N-terminus is substituted with threonine, valine, isoleucine, or leucine.

[0171] In HLA-A33, phenylalanine, tyrosine, alanine, isoleucine, leucine, and valine, the second amino acid from the N-terminus, and lysine and arginine, the C-terminal amino acids, are known to be anchoring residues with high binding affinity to 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 anchoring residue, and aspartic acid and glutamic acid are known to be 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 replace 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 with CTL-inducible ability comprising the following amino acid sequences are included in the peptides of the present invention: the amino acid sequence of the peptide is selected from SEQ ID NO: 27, 3, 60, 28, 5, 67, and 69, wherein the first amino acid from the N-terminus is replaced with aspartic acid or glutamic acid, the second amino acid from the N-terminus is replaced with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine, and / or the C-terminal amino acid is replaced with lysine or arginine. In a preferred embodiment, the peptide of the present invention may be a peptide composed of amino acids having CTL-inducible ability, wherein in the amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69, 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. That is, the peptide of the present invention comprises a peptide comprising an amino acid sequence having CTL-inducible ability, wherein one or more substitutions selected from (a) to (c) below are introduced into the amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69:

[0172] (a) The first amino acid from the N-terminus is replaced with either aspartic acid or glutamic acid;

[0173] (b) The second amino acid from the N-terminus is replaced with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine; and

[0174] (c) The C-terminal amino acid is replaced with arginine or lysine.

[0175] In a preferred embodiment, the peptide of the present invention may be a peptide composed of an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from (a) to (c) above are introduced into an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69. In the present invention, the preferred number of substitutions is 1, 2 or 3 substitutions selected from (a) to (c) above.

[0176] Furthermore, the peptide of the present invention can be a peptide comprising an amino acid sequence with CTL inducibility, wherein in the amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69, 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 can be a peptide composed of an amino acid sequence with CTL inducibility, wherein in the amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69, 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 can be a peptide containing an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from (a) and (b) below are introduced into the amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69:

[0177] (a) The second amino acid from the N-terminus is replaced with phenylalanine, tyrosine, alanine, isoleucine, leucine, or valine; and

[0178] (b) The C-terminal amino acid is replaced with arginine or lysine.

[0179] In a preferred embodiment, the peptide of the present invention may be a peptide composed of an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from (a) and (b) above are introduced into an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69. In a more preferred embodiment, the second amino acid from the N-terminus is substituted with phenylalanine or tyrosine.

[0180] In HLA-A03, alanine, isoleucine, serine, and threonine, the second amino acid from the N-terminus, and arginine, lysine, tyrosine, and phenylalanine, the C-terminal amino acids, are known to be anchoring residues with high binding affinity to 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 the binding affinity of HLA-A03, it is possible to preferably replace the second amino acid from the N-terminus with leucine, methionine, valine, alanine, isoleucine, serine, or threonine, and / or replace the C-terminal amino acid with arginine, lysine, tyrosine, or phenylalanine. Therefore, peptides with CTL-inducible ability comprising the following amino acid sequences are included in the peptides of the present invention: wherein, in the amino acid sequences selected from SEQ ID NOs:7,38 and 47, 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. In a preferred embodiment, the peptides of the present invention may be peptides composed of amino acids with CTL-inducible ability, wherein, in the amino acid sequences selected from SEQ ID NOs:7,38 and 47, 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 comprise peptides comprising amino acid sequences with CTL-inducible ability, wherein one or more substitutions selected from (a) and (b) below are introduced into the amino acid sequences selected from SEQ ID NOs:7,38 and 47:

[0181] (a) The second amino acid from the N-terminus is replaced with leucine, methionine, valine, alanine, isoleucine, serine, or threonine; and

[0182] (b) The C-terminal amino acid is replaced with arginine, lysine, tyrosine or phenylalanine.

[0183] In a preferred embodiment, the peptide of the present invention may be a peptide composed of an amino acid sequence having CTL inducibility, wherein one or more substitutions selected from (a) and (b) above are introduced into an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47. In the present invention, the preferred number of substitutions is one or two substitutions selected from (a) and (b) above.

[0184] In a more preferred embodiment, the second amino acid from the N-terminus is substituted with leucine, methionine, or valine. Substitution can be introduced not only at the anchoring site but also at the potential T-cell receptor (TCR) recognition site of the peptide. Several studies have demonstrated that peptides with amino acid substitutions can have equivalent or better functions than their original functions, such as CAP1 and p53. (264-272) Her-2 / neu (369-377) or gp100 (209-217) (Zaremba et al. Cancer Res. 57, 4570-4577, 1997; TKHoffmann 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).

[0185] The present invention also contemplates the addition of one, two, or more amino acids to the N-terminus and / or C-terminus of the peptides of the present invention (e.g., peptides composed of amino acid sequences selected from the following: SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69, and 47). Such modified peptides that retain CTL-inducing ability are also included in the present invention. For example, when a peptide is contacted with an APC (one, two, or more amino acids of the peptide are added to the N-terminus and / or C-terminus of a peptide composed of the amino acid sequence of SEQ ID NO: 7), it is incorporated into the APC and processed into a peptide composed of the amino acid sequence of SEQ ID NO: 7. It can then induce CTLs by being presented on the cell surface of the APC via an antigen-presentation pathway. More specifically, the peptides of the present invention can be peptides in which one, two, or more amino acids are added to either or both of the N-terminus and C-terminus.

[0186] However, when the amino acid sequence of a peptide is identical to a portion of the amino acid sequence of an endogenous or exogenous protein with a different function, it can induce side effects such as autoimmune diseases and / or allergic symptoms targeting that specific substance. Therefore, it is preferable to use available databases for homology searches to avoid situations where the amino acid sequence of a peptide matches that of another protein. When a homology search clearly indicates that no peptide differs from the target peptide by only one or two amino acids, the target peptide can be modified to increase its binding affinity to HLA antigens and / or enhance its CTL-inducing ability without any risk of such side effects.

[0187] It is predicted that peptides in which one, two, or more amino acids of the peptides of the present invention are modified will retain the CTL-inducible ability of the original peptide; however, it is preferred to verify the CTL-inducible ability of the modified peptides. Here, "peptide with CTL inducibility" refers to a peptide that induces CTLs by APCs stimulated with the peptide. "CTL induction" includes inducing differentiation into CTLs, inducing CTL activation, inducing CTL proliferation, inducing CTL cytotoxic activity, inducing CTL-mediated target cell lysis, and inducing an increase in CTL IFN-gamma production.

[0188] The inducibility of CTLs can be confirmed as follows: APCs (e.g., B lymphocytes, macrophages, and dendritic cells) retaining HLA antigens are induced and stimulated with peptides and then mixed with CD8-positive cells; the IFN-gamma released by the CTLs against the target cells is then measured. For APCs, dendritic cells derived from human peripheral blood mononuclear cells are preferred. As a reaction system, transgenic animals already prepared to express HLA antigens can be used. Alternatively, for example, [the following can be used]. 51 Cr or similar radiolabeled target cells can be used, and the cytotoxic activity of peptide-induced CTLs can be calculated from the radioactive release from the target cells. Alternatively, in the presence of peptide-stimulated APCs, CTL induction capacity can be assessed by measuring IFN-gamma generated and released by CTLs and displaying the inhibition zone on the culture medium using an anti-IFN-gamma monoclonal antibody.

[0189] In addition to the modifications described above, the peptides of the present invention can be linked to other peptides, provided that the resulting linked peptide retains CTL inducibility. Examples of suitable peptides for linking to the peptides of the present invention include TAA-derived CTL-inducible peptides. Furthermore, the peptides of the present invention can also be linked to each other. Suitable adapters for linking peptides are known in the art, and for example, adapters such as AAY (PM Daftarian et al., J Trans Med 2007, 5:26), AAA, NKRK (SEQ ID NO:85) (RPMSutmuller et al., J Immunol. 2000, 165:7308-15), or K (S. Ota et al., Can Res. 62, 1471-6, KSKawamura et al., J Immunol. 2002, 168:5709-15) can be used. Peptides can be linked in various arrangements (e.g., chain-like, repeating, etc.), and three or more peptides can also be linked.

[0190] The peptides of the present invention can also be linked to other substances, as long as the resulting linked peptide retains CTL inducibility. Examples of suitable substances for linking to the peptides of the present invention include, for example, peptides, lipids, sugars or glycans, acetyl groups, and natural or synthetic polymers. The peptides of the present invention can be modified by glycosylation, side-chain oxidation, or phosphorylation, etc., as long as their CTL inducibility is not impaired. This type of modification can also be performed to impart additional functions (e.g., targeting and delivery functions) or to stabilize the peptides.

[0191] For example, to improve the in vivo stability of peptides, it is known in the art to introduce D-amino acids, amino acid mimics, or non-natural amino acids; this concept can also be applied 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).

[0192] Furthermore, as described above, among the modified peptides with substitutions, deletions, insertions, and / or additions of one, two, or more amino acid residues, those with the same or higher activity compared to the original peptide can be screened or selected. Therefore, the present invention also provides a method for screening or selecting modified peptides with the same or higher activity compared to the original peptide. Specifically, the present invention provides a method for screening peptides with CTL-inducible activity, wherein the method includes the following steps:

[0193] (a) Generate a candidate sequence consisting of an amino acid sequence obtained by modifying an original amino acid sequence by substitution, deletion, insertion and / or addition of one, two or more amino acid residues, wherein the original amino acid sequence is selected from the amino acid sequences of SEQ IDNOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56,58,27,60,28,67,69 and 47;

[0194] (b) Select candidate sequences from the candidate sequences generated in (a) that have no significant homology (or sequence homology) with peptides of any known human gene product other than CDCA1;

[0195] (c) Contact the peptide composed of the candidate sequences selected in (b) with APCs;

[0196] (d) Contact the APCs from (c) with CD8-positive T cells; and

[0197] (e) Select peptides that have equal or greater CTL induction ability than peptides composed of the original amino acid sequence.

[0198] In this document, the peptides of the present invention are also described as "CDCA1 peptide" or "CDCA1 polypeptide".

[0199] III. Preparation of the peptides of the present invention

[0200] The peptides of the present invention can be prepared using known techniques. For example, recombinant DNA technology or chemical synthesis can be used to prepare the peptides of the present invention. The peptides of the present invention can be synthesized independently or synthesized as longer polypeptides comprising two or more peptides. The peptides of the present invention can be generated in host cells using recombinant DNA technology 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 to make them substantially free of other host cell proteins and their fragments, or any other chemical substances.

[0201] The peptides of the present invention may contain modifications such as glycosylation, side-chain oxidation, or phosphorylation, provided that such modifications do not impair the biological activity of the original peptide. Other exemplary modifications include incorporation of one or more D-amino acids or other available amino acid mimics to, for example, increase the serum half-life of the peptide.

[0202] The peptides of this invention can be obtained by chemical synthesis based on a selected amino acid sequence. Examples of conventional peptide synthesis methods suitable for synthesis include those described in the following literature:

[0203] (i) Peptide Synthesis, Interscience, New York, 1966;

[0204] (ii)The Proteins,Vol.2,Academic Press,New York,1976;

[0205] (iii) “Peptide Synthesis” (Japanese), Maruzen Co., 1975;

[0206] (iv) "Basics and Experiment of Peptide Synthesis" (in Japanese), MaruzenCo., 1985;

[0207] (v) "Development of Pharmaceuticals" (Japanese), Continued Vol.14 (peptide synthesis), Hirokawa, 1991;

[0208] (vi)WO99 / 67288; and

[0209] (vii) Barany G. & Merrifield RB, Peptides Vol. 2, Solid Phase Peptide Synthesis, Academic Press, New York, 1980, 100-118.

[0210] Alternatively, any known genetically engineered peptide production method can be used 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 corresponding to a promoter sequence) is prepared and transformed into a suitable host cell. Such vectors and host cells are also provided in this invention. The host cells are then cultured to generate the peptide of interest. The peptides of the present invention can also be produced in vitro using an in vitro translation system.

[0211] IV. Polynucleotides

[0212] This invention also provides polynucleotides encoding any of the peptides of this invention. These include polynucleotides derived from the naturally occurring CDCA1 gene (e.g., GenBank accession number NM_145697 (SEQ ID NO: 81) or GenBank accession number NM_031423 (SEQ ID NO: 83)) and polynucleotides having their conserved modified nucleotide sequences. Hereinafter, the phrase “conserved modified nucleotide sequence” refers to a sequence encoding the same or substantially the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at any position specified by the codon for alanine, that codon can be changed to any corresponding codon without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and are a type of conserved modified variation. Each nucleic acid sequence encoding a peptide herein also describes each possible silent variation of that nucleic acid. Those skilled in the art will recognize that every codon in a nucleic acid (except AUG and TGG, where AUG is the only codon for methionine under normal circumstances and TGG is the only codon for tryptophan under normal circumstances) can be modified to produce a functionally identical molecule. Therefore, every silencing variant of a nucleic acid encoding a peptide is implicitly described in every published sequence.

[0213] The polynucleotides of the present invention can be composed of DNA, RNA, and derivatives thereof. DNA is suitably composed of bases such as A, T, C, and G, and in RNA, T is substituted with U.

[0214] The polynucleotides of this invention can encode multiple peptides of this invention, with or without an intervening amino acid sequence. 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. Furthermore, the polynucleotide can include any additional sequence encoding the coding sequence of the peptide of this invention. For example, the polynucleotide can be a recombinant polynucleotide including regulatory sequences required for peptide expression, or it can be an expression vector (e.g., a plasmid) containing a marker gene, etc. Generally, such recombinant polynucleotides can be prepared by manipulating polynucleotides using conventional recombinant techniques, such as by using polymerases and endonucleases.

[0215] 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 into a suitable vector that can be expressed after transfection into competent cells. Alternatively, the polynucleotides can be amplified using PCR techniques or by expression 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 the polynucleotides, as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48:2223-311; Mattes et al., EMBO J 1984, 3:801-5.

[0216] V. Exosomes

[0217] The present invention further provides intracellular vesicles called exogenous bodies, which present a complex formed between the peptide of the present invention and the HLA antigen on their surface. The exogenous bodies can be prepared by, for example, methods described in detail in JPH11-510507 and WO99 / 03499, and can be prepared from APCs obtained from patients as subjects of treatment and / or prevention. The exogenous bodies of the present invention can be administered as vaccines in a manner similar to that of the peptide of the present invention.

[0218] The types of HLA antigens contained in the aforementioned complexes must match the type of the subject requiring treatment and / or prophylaxis (protection). For example, HLA-A11 (e.g., HLA-A*1101) and HLA-A33 (e.g., HLA-A*3303) are alleles widely and prevalent in Asian populations, and these HLA antigen types are considered suitable for treatment in Asian patients. Furthermore, HLA-A03 (e.g., HLA-A*0301) is an allele widely and prevalent in Caucasian populations and is considered suitable for treatment in Caucasian patients. Typically, in clinical practice, by prior research into the HLA antigen types of patients requiring treatment, suitable peptides can be selected that have a high level of binding affinity for a specific HLA antigen, or that possess CTL-induced ability through antigen presentation mediated by a specific HLA antigen.

[0219] The foreign body of the present invention presents a complex of the peptide of the present invention with HLA-A11, HLA-A33, or HLA-A03 on its surface. When the HLA forming the complex 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:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58, or a peptide modified thereto, and more preferably a peptide or a peptide modified thereto composed of an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58. Furthermore, when the HLA forming the 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:27,3,60,28,5,67 and69, or a peptide modified therefrom, and more preferably a peptide having an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69, or a peptide modified therefrom. Furthermore, when the HLA forming the 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:7,38 and47, or a peptide modified therefrom, and more preferably a peptide having an amino acid sequence selected from SEQ ID NOs:7,38 and47, or a peptide modified therefrom.

[0220] VI. Antigen-presenting cells (APCs)

[0221] The present invention also provides APCs that present a complex formed between an HLA antigen and the peptide of the present invention on their surface. Alternatively, the present invention provides APCs having a complex formed between an HLA antigen and the peptide of the present invention on their surface. The APCs of the present invention can be isolated APCs. When used in the context of cells (APCs, CTLs, etc.), the term "isolated" means that the cell is isolated from another type of cell. The APCs of the present invention can be APCs induced from APCs derived from patients undergoing treatment and / or prevention (protection), and can be administered as a vaccine on their own or in combination with other drugs (including the peptides, exogens, or CTLs of the present invention).

[0222] The APCs of the present invention are not limited to specific cell types, but include cells that inhibit the presentation of protein-like antigens on their cell surface for recognition by lymphocytes (e.g., dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells). Since DCs are representative APCs with the strongest CTL-inducing activity, they are preferably used as the APCs of the present invention. In the present invention, preferred DCs are isolated DCs derived from humans. Furthermore, the APCs of the present invention can be a mixture of multiple cell types with antigen-presenting function, and can be a mixture of APCs wherein each APC presents a different type of peptide of the present invention.

[0223] 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 presenting 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 the subject with the peptides of the present invention.

[0224] To induce an immune response against cancer cells expressing CDCA1 in subjects, the APC of the present invention can be administered to subjects alone or in combination with other drugs (including the peptides, exogenous bodies, or CTLs of the present invention). For example, ex vivo administration may include the following steps:

[0225] (a) APCs were collected from the first subject;

[0226] (b) Contacting the APCs of step (a) with the peptides of the present invention; and

[0227] (c) Administer the APCs of step (b) to the second subject.

[0228] The first and second subjects can be the same individual or different individuals. When the first and second subjects are different individuals, it is preferable that their HLA types are the same. The APC obtained from step (b) above can be a vaccine for cancer treatment and / or prevention (protection).

[0229] The APCs of the present invention, obtained from the methods described above, possess CTL-inducible ability. In the context of APCs, the term "CTL-inducible ability (CTL inducibility)" refers to the ability of APCs to induce CTLs upon contact with CD8-positive cells. Furthermore, "CTL-inducible ability (CTL inducibility)" includes the ability of APCs to induce CTL activation, the ability of APCs to induce CTL proliferation, the ability of APCs to promote CTL-mediated lysis of target cells, and the ability of APCs to enhance CTL-mediated IFN-gamma production. The CTLs induced by the APCs of the present invention are CDCA1-specific CTLs and exhibit specific cytotoxic activity against cells expressing CDCA1.

[0230] In addition to the methods described above, APCs of the present invention can be prepared by introducing polynucleotides encoding the peptides of the present invention into APCs in vitro. The polynucleotides to be introduced can be in the form of DNA or RNA. The method of introduction is not particularly limited, and examples include various methods conventionally practiced in the art, such as liposome transfection, electroporation, and calcium phosphate methods. More specifically, the methods described in Cancer Res 1996, 56:5672-7; J Immunol 1998, 161:5607-13; J ExpMed 1996, 184:465-72, and JP2000-509281 can be used. By introducing polynucleotides encoding the peptides of the present invention into APCs, the polynucleotides are transcribed and translated in the cells, and the resulting peptides are then processed by MHC class I and presented via a presentation pathway to present the peptides of the present invention on the cell surface of the APCs.

[0231] In a preferred embodiment, the APC of the present invention presents on its cell surface the peptide of the present invention and a complex formed by HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), or HLA-A03 (more preferably HLA-A*0301). When the HLA forming the complex 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:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58, or a peptide modified thereto, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58. When the HLA forming the 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:27,3,60,28,5,67 and69, or a modified peptide thereof, and more preferably a peptide composed of an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69. When the HLA forming the 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:7,38 and47, or a modified peptide thereof, and more preferably a peptide composed of an amino acid sequence selected from SEQ ID NOs:7,38 and47.

[0232] The APC of the present invention is preferably an APC induced by a method including the steps of (a) or (b) below:

[0233] (a) Contacting an APC with the peptide of the present invention, wherein the APC expresses at least one selected from HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), and HLA-A03 (more preferably HLA-A*0301); or

[0234] (b) Introducing a polynucleotide encoding the peptide of the present invention into an APC, wherein the APC expresses at least one selected from HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303) and HLA-A03 (more preferably HLA-A*0301).

[0235] The peptide of the present invention in contact with an APC expressing HLA-A11 is preferably a peptide having an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58, or a peptide modified thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58.

[0236] The peptide of the present invention in contact with an APC expressing HLA-A33 is preferably a peptide having an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69.

[0237] The peptide of the present invention in contact with an APC expressing HLA-A03 is preferably a peptide having an amino acid sequence selected from SEQ ID NOs:7,38 and 47 or a modified peptide thereof, and more preferably a peptide consisting of an amino acid sequence selected from SEQ ID NOs:7,38 and 47.

[0238] This invention provides the use of the peptide of the invention for preparing pharmaceutical compositions that induce APCs with CTL-inducible ability. Furthermore, this invention provides a method or process for preparing pharmaceutical compositions that induce APCs with CTL-inducible ability. Further, this invention provides the peptide of the invention for inducing APCs with CTL-inducible ability.

[0239] VII. Cytotoxic T lymphocytes (CTLs)

[0240] CTLs induced by the peptides of the present invention can be used as vaccines in a similar manner to the peptides of the present invention to enhance the immune response of cells targeting CDCA1 expression 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 an HLA antigen. The binding of the CTL to the complex is mediated via T cell receptors (TCRs) 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 can be a mixture of CTLs, each targeting a different type of peptide of the present invention.

[0241] 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 in vitro with the peptide of the present invention, (3) contacting CD8-positive T cells or PBMCs in vitro with APCs or exogenous bodies that present a complex of HLA antigens and the peptide of the present invention on their surfaces, or (4) introducing a vector containing a polynucleotide encoding a subunit of each T cell receptor (TCR) into CD8-positive T cells that are capable of binding the peptide of the present invention presented by HLA antigens on the cell surface. The exogenous bodies and APCs used in methods (2) or (3) above can be prepared by the methods described in sections “V. Exogenous Bodies” and “VI. Antigen-Presenting Cells (APCs)”, respectively, and details of the method in section (4) above will be described in section “VIII. T Cell Receptors (TCRs)”.

[0242] The CTLs of the present invention can be administered alone to patients undergoing treatment and / or prophylaxis, or in combination with other drugs (including the peptides, APCs, or exogenous substances of the present invention) for the purpose of modulating efficacy. Further, the CTLs of the present invention can be CTLs induced from CD8-positive T cells derived from patients receiving the CTLs. The CTLs of the present invention specifically act on target cells that present the peptides of the present invention, for example, the same peptides used to induce the CTLs of the present invention. Target cells can be cells endogenously expressing CDCA1, such as cancer cells, or cells transfected with the CDCA1 gene. Cells that present the peptides of the present invention on their cell surface due to peptide stimulation can also be targets of the CTLs of the present invention. The cells targeted by the CTLs of the present invention are preferably HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), and HLA-A03 (more preferably HLA-A*0301).

[0243] In a preferred embodiment, the CTLs of the present invention specifically target cells expressing CDCA1 and at least one of HLA selected from HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), and HLA-A03 (more preferably HLA-A*0301). In the present invention, the cells targeted by the CTLs can be homozygous or heterozygous cells possessing any one of the alleles of HLA-A11, HLA-A33, and HLA-A03.

[0244] In this document, the term "targeting" CTL refers to the recognition of cells on which the CTL presents a complex of HLA and the peptide of the present invention on its cell surface, and the expression of cytotoxic activity against those cells. Further, "specific targeting" means that the CTLs exhibit cytotoxic activity against these cells, but do not show destructive activity against other cells. The expression "recognizing cell" used in the context of CTLs refers to the binding via its TCR to the complex of HLA and the peptide of the present invention presented on the cell surface, and the expression of specific cytotoxic activity against that cell. Therefore, the CTLs of the present invention are preferably CTLs capable of binding via their TCR to the complex presented on the cell surface, 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), or HLA-A03 (more preferably HLA-A*0301).

[0245] Furthermore, the CTLs of the present invention are preferably CTLs induced by a method comprising the steps described in (a) or (b) below:

[0246] (a) In vitro, CD8-positive T cells are contacted with APCs or exogenous organisms that present a complex on their surface, said complex being a complex of the peptide of the present invention and HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), or HLA-A03 (more preferably HLA-A*0301); or

[0247] (b) Introducing a polynucleotide containing a subunit encoding each TCR into CD8-positive T cells, wherein the TCR is capable of binding to the peptides of the present invention presented on the cell surface via HLA-A11 (more preferably HLA-A*1101), HLA-A33 (more preferably HLA-A*3303), or HLA-A03 (more preferably HLA-A*0301).

[0248] VIII. T cell receptor (TCR)

[0249] The present invention also provides compositions and methods of using the compositions, the compositions comprising polynucleotides encoding each TCR subunit, the TCRs being capable of binding the peptides of the present invention presented on the cell surface via HLA antigens. The polynucleotides confer specificity of CD8-positive T cells against cancer cells expressing CDCA1, which is achieved by expressing TCRs on the cell surface capable of binding the peptides of the present invention presented on the cell surface via HLA antigens. Polynucleotides encoding the alpha and beta chains of the TCR subunits in CTLs induced by the peptides of the present invention can be identified using methods known in the art (WO2007 / 032255 and Morgan et al., J Immunol, 171, 3288 (2003)). For example, PCR methods are preferred for TCR analysis. Not limited thereto, the PCR primers used for analysis can be, for example, primer sets for amplification by combining the following 5' side primers and 3' side primers:

[0250] 5' side primer:

[0251] 5'-R primer (5'-gtctaccaggcattcgcttcat-3') (SEQ ID NO:77)

[0252] 3' side primer

[0253] TCR-alpha chain C-region specificity

[0254] 3-TRa-C primer (5'-tcagctggaccacagccgcagcgt-3') (SEQ ID NO:78)

[0255] TCR-beta chain C1 region specificity

[0256] 3-TRb-C1 primer (5'-tcagaaatcctttctcttgac-3') (SEQ ID NO:79) or

[0257] TCR-beta chain C2 region specificity

[0258] 3-TR-beta-C2 primer (5'-ctagcctctggaatcctttctctt-3') (SEQ ID NO:80)

[0259] TCRs formed by introducing identified polynucleotides into CD8-positive T cells can bind to target cells presenting the peptides of the present invention with high affinity and mediate highly efficient killing of target cells presenting the peptides of the present invention in vivo and in vitro.

[0260] Polynucleotides encoding each TCR subunit can be incorporated into suitable vectors, such as retroviral vectors. These vectors are well known in the art. The polynucleotides or vectors containing them can be introduced into CD8-positive T cells, such as CD8-positive T cells derived from a patient. This invention provides a ready-to-use composition that allows for the 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 superior cancer cell killing properties.

[0261] In this text, a specific TCR is a TCR that, when presented on the surface of CD8-positive T cells, confers target-cell-specific cytotoxic activity by specifically recognizing the complex of the peptide of the present invention and the HLA antigen presented on the surface of target cells. The specific recognition of the aforementioned complex can be confirmed by any known method, with preferred examples including HLA multimer staining analysis using HLA molecules and the peptide of the present invention, and the ELISPOT assay. By performing the ELISPOT assay, the recognition mediated by the specific TCR of the T cells introduced with the aforementioned polynucleotide and the target cells undergoing intracellular signal transduction can be confirmed. When the aforementioned TCR is presented on the surface of CD8-positive T cells, it can also be confirmed by known methods whether the TCR confers target-cell-specific cytotoxic activity against CD8-positive T cells. Preferred methods include, for example, measuring cytotoxic activity against HLA-positive target cells using a chromium release assay.

[0262] In the context of HLA-A11, the present invention provides CTLs prepared by converting CD8-positive T cells with a polynucleotide encoding each TCR subunit, wherein the TCR binds, for example, a peptide having an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58.

[0263] In the context of HLA-A33, the present invention provides CTLs prepared by converting CD8-positive T cells with a polynucleotide encoding each TCR subunit, wherein the TCR binds, for example, a peptide having an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69.

[0264] In the context of HLA-A03, the present invention provides CTLs prepared by converting CD8-positive T cells with a polynucleotide encoding each TCR subunit, wherein the TCR binds, for example, a peptide having an amino acid sequence selected from SEQ ID NOs:7,38 and 47.

[0265] The transformed CTLs are capable of homing in vivo and can be amplified using known in vitro culture methods (e.g., Kawakami et al., J Immunol., 142, 3452-3461 (1989)). The CTLs of this invention can be used to form immunogenic compositions for the treatment or prevention of disease in patients requiring treatment and / or prevention (see reference WO2006 / 031221, the contents of which are incorporated herein by reference).

[0266] IX. Pharmaceutical Compositions

[0267] The present invention also provides compositions or pharmaceutical compositions comprising at least one active ingredient selected from the following:

[0268] (a) The peptide of the present invention;

[0269] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0270] (c) The APC of the present invention;

[0271] (d) the foreign body of the present invention; and

[0272] (e) The CTL of the present invention.

[0273] In addition to the active ingredients described above, the pharmaceutical compositions of the present invention may include, as needed, a carrier, excipient, or such commonly used in pharmaceuticals without particular limitation. Examples of carriers that can be used in the pharmaceutical compositions of the present invention include sterile water, physiological saline, phosphate buffer, culture medium, etc. Therefore, the present invention also provides pharmaceutical compositions comprising at least one active ingredient selected from (a) to (e) below and a pharmaceutically acceptable carrier:

[0274] (a) The peptide of the present invention;

[0275] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0276] (c) The APC of the present invention;

[0277] (d) the foreign body of the present invention; and

[0278] (e) The CTL of the present invention.

[0279] Furthermore, the pharmaceutical compositions of the present invention may, as needed, contain stabilizers, suspending agents, preservatives, surfactants, solubilizers, pH adjusters, aggregation inhibitors, etc.

[0280] Compared to normal tissues, CDCA1 expression is significantly upregulated in cancer cells. Therefore, the peptides or polynucleotides encoding said peptides of the present invention 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 the 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 exogenous bodies or APCs for use as pharmaceutical compositions. Furthermore, CTLs of the present invention targeting any of the peptides of the present invention can also be used as active ingredients in the pharmaceutical compositions of the present invention. The pharmaceutical compositions of the present invention may contain a therapeutically effective amount or a pharmaceutically effective amount of the above-described active ingredients.

[0281] The pharmaceutical compositions of the present invention can also be used as vaccines. In the context of this invention, the term "vaccine" (also referred to as "immunogenic composition") refers to a composition that, when administered to an animal, has the function of inducing an immune response resulting in an antitumor effect. Therefore, the pharmaceutical compositions of the present invention can be used to induce an immune response resulting in an antitumor effect. There are no particular limitations on the immune response induced by the peptides, polynucleotides, APCs, CTLs, and pharmaceutical compositions of the present invention, as long as it is an immune response resulting in an antitumor effect, and examples include inducing cancer cell-specific CTLs and inducing cancer cell-specific cytotoxic activity.

[0282] The pharmaceutical compositions of the present invention can be used in human subjects or patients to treat and / or prevent cancer, and / or prevent its postoperative recurrence. The pharmaceutical compositions of the present invention are preferably used in subjects who are positive for at least one HLA selected from HLA-A11, HLA-A33, and HLA-A03. Furthermore, the pharmaceutical compositions of the present invention are preferably used to treat and / or prevent cancers expressing CDCA1 and at least one HLA selected from HLA-A11, HLA-A33, and HLA-A03 and / or prevent their postoperative recurrence.

[0283] 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:

[0284] (a) The peptide of the present invention;

[0285] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0286] (c) An APC that presents the peptide of the present invention on its surface;

[0287] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0288] (e) The CTL of the present invention.

[0289] Alternatively, the present invention further provides an active ingredient selected from the following for treating or preventing cancer:

[0290] (a) The peptide of the present invention;

[0291] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0292] (c) An APC that presents the peptide of the present invention on its surface;

[0293] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0294] (e) The CTL of the present invention.

[0295] 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 includes the step of formulating at least one active ingredient selected from the following active ingredients using a pharmaceutically or physiologically acceptable carrier:

[0296] (a) The peptide of the present invention;

[0297] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0298] (c) An APC that presents the peptide of the present invention on its surface;

[0299] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0300] (e) The CTL of the present invention.

[0301] 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 includes the step of mixing an active ingredient selected from the group consisting of a pharmaceutically or physiologically acceptable carrier:

[0302] (a) The peptide of the present invention;

[0303] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0304] (c) An APC that presents the peptide of the present invention on its surface;

[0305] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0306] (e) The CTL of the present invention.

[0307] 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:

[0308] (a) The peptide of the present invention;

[0309] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0310] (c) An APC that presents the peptide of the present invention on its surface;

[0311] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0312] (e) The CTL of the present invention.

[0313] In this invention, peptides having an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58 are identified as HLA-A11-restricted epitope peptides capable of inducing 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:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58 are particularly suitable for administration to subjects having 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., the polynucleotides of the present invention), APCs or exogenous bodies presenting these peptides (i.e., the APCs or exogenous bodies of the present invention), or CTLs targeting these peptides (i.e., the 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:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58 are suitable for administration to subjects having 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:7.

[0314] Similarly, in this invention, peptides having an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67, and69 are identified as HLA-A33-restricted epitope peptides capable of inducing 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:27,3,60,28,5,67, and69 are particularly suitable for administration to subjects having HLA-A33 (e.g., HLA-A*3303) as an HLA antigen. This is equally applicable to pharmaceutical compositions comprising: polynucleotides encoding any of these peptides (i.e., the polynucleotides of the present invention), APCs or exogens presenting these peptides (i.e., the APCs or exogens of the present invention), or CTLs targeting these peptides (i.e., the CTLs of the present invention). In other words, a pharmaceutical composition comprising an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and69 is 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 a peptide having an amino acid sequence having SEQ ID NO:27.

[0315] Similarly, in this invention, peptides having amino acid sequences selected from SEQ ID NOs:7,38, and 47 are identified as HLA-A03-restricted epitope peptides capable of inducing 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:7,38, and 47 are particularly suitable for administration to subjects having HLA-A03 (e.g., HLA-A*0301) as an HLA antigen. This is equally applicable to pharmaceutical compositions comprising: polynucleotides encoding any of these peptides (i.e., the polynucleotides of the present invention); APCs or exogens presenting these peptides (i.e., the APCs or exogens of the present invention); or CTLs targeting these peptides (i.e., the 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:7,38, and 47 are suitable for administration to subjects having 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 an amino acid sequence having SEQ ID NO: 7 or 38.

[0316] There are no particular limitations on the cancers that can be treated and / or prevented by the pharmaceutical compositions of the present invention, as long as they are cancers expressing CDCA1, including various cancers such as bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, colorectal cancer, etc. The pharmaceutical compositions of the present invention are preferably used in subjects who are homozygous or heterozygous for HLA alleles selected from HLA-A11, HLA-A33, and HLA-A03.

[0317] In addition to the active ingredients described above, the pharmaceutical compositions of the present invention may contain other peptides (e.g., other TAA-derived CTL-inducing peptides) that have the ability to induce CTLs against cancer cells, other polynucleotides encoding other peptides, other cells that present other peptides, etc.

[0318] The pharmaceutical compositions of the present invention may optionally contain other therapeutic substances as active ingredients, provided that they do not inhibit the antitumor activity of the aforementioned active ingredients (e.g., the peptides of the present invention). For example, the pharmaceutical compositions of the present invention may optionally contain anti-inflammatory compositions, analgesics, chemotherapeutic agents, etc. In addition to including other therapeutic substances in the pharmaceutical compositions of the present invention themselves, the pharmaceutical compositions of the present invention may be administered sequentially or simultaneously with one or more other pharmaceutical compositions. The dosage of the pharmaceutical compositions 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 the scheduling and route of administration.

[0319] It should be understood that, considering the type of formulation, the pharmaceutical compositions of the present invention may contain other components conventional in the art, in addition to those explicitly mentioned herein.

[0320] This invention also provides articles or kits comprising the pharmaceutical compositions of the present invention. The articles or kits of the present invention may include containers containing the pharmaceutical compositions of the present invention. Examples of suitable containers include, but are not limited to, bottles, vials, or test tubes. Containers may be made of a variety of materials, such as glass or plastic. Labels may be affixed to the containers, and the label may describe the disease or disease condition to which the pharmaceutical compositions of the present invention are applied. The label may also indicate instructions regarding administration, etc.

[0321] In addition to the container holding the pharmaceutical composition of the present invention, the articles or kits of the present invention may optionally include a second container holding a pharmaceutically acceptable diluent. The articles or kits of the present invention may also include other materials required from a commercial and user perspective, such as other buffers, diluents, filters, injection needles, syringes, and packaging inserts with instructions for use.

[0322] Where appropriate, the pharmaceutical compositions of the present invention may be provided in a package or dispensing device containing one or more unit dosage forms containing the active ingredient. For example, the package may include, for instance, metal or plastic foil, such as a blister pack. Instructions for use may be included with the package or dispensing device.

[0323] (1) Pharmaceutical compositions containing peptides as active ingredients

[0324] Pharmaceutical compositions containing the peptides of the present invention can be formulated as needed using conventional formulation methods. In addition to the peptides of the present invention, the pharmaceutical compositions of the present invention may contain carriers, excipients, etc., commonly used in pharmaceuticals, without particular limitation. Examples of carriers that can be used in the pharmaceutical compositions 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, etc. Furthermore, the pharmaceutical compositions of the present invention may contain stabilizers, suspending agents, preservatives, surfactants, solubilizers, pH adjusters, aggregation inhibitors, etc., as needed. The pharmaceutical compositions of the present invention can induce specific immunity against cancer cells expressing CDCA1, and therefore can be used for cancer treatment or prevention (protection).

[0325] For example, the pharmaceutical composition of the present invention can be prepared by dissolving the peptide solution 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, as needed, stabilizers, suspending agents, preservatives, surfactants, solubilizers, pH adjusters, aggregation inhibitors, etc., and then sterilizing the peptide solution. The method of sterilizing the peptide solution is not particularly limited, but filtration sterilization is preferred. Filtration sterilization can be performed using, for example, a filtration sterilization filter with a pore size of 0.22 μm or less. The filtration sterilized peptide solution can be administered to the subject, for example, as an injection, but is not limited thereto. The pharmaceutical composition of the present invention can be prepared as a lyophilized formulation by freeze-drying the above-described peptide solution. The lyophilized formulation can be prepared by filling the peptide solution prepared as described above into a suitable container, such as an ampoule, vial, or plastic container, followed by freeze-drying and sealing it in the container after pressure recovery using a washed and sterilized rubber stopper, etc. Freeze-dried formulations can be administered to subjects after being redissolved in a pharmaceutically or physiologically acceptable water-soluble carrier, such as sterile water (e.g., water for injection), physiological saline, phosphate-buffered saline, phosphate-buffered saline, and Tris-buffered saline. Preferred examples of pharmaceutical compositions of the present invention include injectable formulations of such filtered sterile peptide solutions and freeze-dried formulations obtained by lyophilizing such peptide solutions. The present invention also includes kits containing such freeze-dried formulations and redissolved solutions. The present invention also includes kits containing containers for freeze-dried formulations (which are pharmaceutical compositions of the present invention) and containers for redissolved solutions.

[0326] The pharmaceutical compositions 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, 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 by HLA antigens, and then CTLs that specifically react with the complex formed between the displayed peptide and the HLA antigen 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 displaying any of the peptides of the present invention on their cell surface. Re-administering these APCs to a subject to induce CTLs in the subject results in increased attack against cancer cells expressing CDCA1.

[0327] The pharmaceutical compositions of the present invention may also contain adjuvants known for effectively establishing cellular immunity. An adjuvant is a compound that, when administered together with (or sequentially with) an antigen, enhances the immune response against an immunologically active antigen. Known adjuvants described in the literature, such as Clin Microbiol Rev 1994, 7:277-89, may be used. Examples of suitable adjuvants include aluminum salts (aluminum phosphate, aluminum hydroxide, aluminum hydroxide, 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 a CpG motif (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 derivatives thereof (poliIC, etc.), bacterial DNA, imidazoquinone (Imiquimod, R848, etc.), C-type lectin ligands (trehalose-6,6'-dibenzyl ester (TDB), etc.), CD1d ligands (alpha-galactosylceramide, etc.), squalene emulsions (MF59, AS03, AF03, etc.), PLGA, etc., but not limited thereto. In a kit containing the pharmaceutical composition of the present invention, the adjuvant may be contained in a separate container from the pharmaceutical composition containing the peptide of the present invention. In this case, the adjuvant and the pharmaceutical composition may be administered to the subject continuously or mixed together immediately before administration to the subject. The present invention also provides such kits containing the pharmaceutical composition containing the peptide of the present invention and the adjuvant. When the pharmaceutical composition of the present invention is a lyophilized formulation, the kit may also contain a reconstitution solution. Furthermore, 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 be further included, if needed, as a container for storing the redissolved solution.

[0328] When an oil adjuvant is used as an adjuvant, the pharmaceutical compositions of the present invention can be prepared as emulsions. For example, an emulsion can be prepared by mixing and stirring a peptide solution and an oil adjuvant prepared as described above. The peptide solution can be a solution that has been lyophilized and then reconstituted. The emulsion can be a W / O type emulsion or an O / W type emulsion, with W / O type emulsions being preferred for achieving a high immune response enhancement effect. IFA can preferably be used as an oil adjuvant, but is not limited thereto. The preparation of the emulsion can be performed immediately before administration to the subject, and in this case, the pharmaceutical compositions of the present invention can be provided as a kit containing the peptide solution and the oil adjuvant of the present invention. When the pharmaceutical compositions of the present invention are lyophilized formulations, the kit may further contain a reconstituted solution.

[0329] Furthermore, the pharmaceutical compositions of the present invention may be liposome formulations in which the peptides of the present invention are encapsulated, particulate formulations in which the peptides are bound to beads having a diameter of a few micrometers, or formulations in which lipids are bound to peptides.

[0330] In another embodiment of the invention, the peptides of the invention may also be administered in the form of pharmaceutically acceptable salts. 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.). The phrase "pharmaceutically acceptable salt" as used herein refers to a salt that retains the biological, physiological, pharmacological, and pharmaceutical efficacy and properties of the compound. Therefore, pharmaceutical compositions comprising pharmaceutically acceptable salts of the peptides of the invention are also included within the scope of this invention. Furthermore, in addition to free peptides, "peptides of the invention" also include their pharmaceutically acceptable salts.

[0331] In some embodiments, the pharmaceutical compositions of the present invention may further include components that initiate CTLs. Lipids have been identified as components capable of initiating CTLs against viral antigens in vivo. For example, palmitic acid residues may be linked to the ε- and α-amino groups of lysine residues, and then to the peptides of the present invention. The lipotropic peptides can then be administered directly in micelles or particles, incorporated into liposomes, or emulsified in an adjuvant. As other examples of lipid-initiated CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerocysteylserine-serine (P3CSS), can be used to initiate CTLs when covalently linked to a suitable peptide (see, for example, Deres et al., Nature 1989, 342:561-4).

[0332] Examples of suitable methods for administering the peptides or pharmaceutical compositions of the present invention include, but are not limited to, oral, intradermal, subcutaneous, intramuscular, intraosseous, intraperitoneal, and intravenous injection, as well as systemic or local administration near the target site. Preferred administration methods include subcutaneous injection near lymph nodes such as the armpit or groin. Administration can be performed by a single dose or enhanced by multiple doses. 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 against cancer cells expressing CDCA1 (more specifically, CTLs). The dosage of the peptides of the present invention can be appropriately adjusted according to the disease to be treated, the patient's age, weight, method of administration, etc. For each peptide of the present invention, the dosage is typically 0.001 mg to 1000 mg, for example 0.01 mg to 100 mg, for example 0.1 mg to 30 mg, for example 0.1 mg to 10 mg, for example 0.5 mg to 5 mg. The dosing interval can be from every few days to every few months, and for example, it can be administered at a weekly interval. Those skilled in the art can appropriately select the appropriate dose.

[0333] 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 contain 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, more preferably 0.1 mg to 30 mg, and even more preferably 0.1 mg to 10 mg, for example 0.5 mg to 5 mg of the peptide of the present invention. When the pharmaceutical composition of the present invention is an injectable, it may contain the following concentrations of the peptide of the present invention: 0.001 mg / ml to 1000 mg / ml, preferably 0.01 mg / ml to 100 mg / ml, more preferably 0.1 mg / ml to 30 mg / ml, and even more preferably 0.1 mg / ml to 10 mg / ml, for example 0.5 mg / ml to 5 mg / ml. In this case, for example, 0.1 to 5 ml, preferably 0.5 ml to 2 ml, of the pharmaceutical composition of the present invention may be administered to a subject by injection.

[0334] Furthermore, the present invention provides a method for treating and / or preventing cancer and / or preventing its postoperative recurrence, comprising administering to a subject a therapeutically effective amount of the peptide of the present invention or a pharmaceutical composition of the present invention. As described above, the peptide of the present invention can be administered to a subject in a single dose typically 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 the subject together with an adjuvant. Furthermore, the dosing interval can be from every few days to every few months, preferably from every few days to once a month, for example, once a week or once every two weeks.

[0335] (2) Pharmaceutical compositions containing polynucleotides as active ingredients

[0336] The pharmaceutical compositions of the present invention may also contain polynucleotides encoding the peptides disclosed herein in an expressible form. In this document, the phrase “in an expressible form” means that the polynucleotide is expressed as the peptide of the present invention upon introduction into cells. In exemplary embodiments, 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 to achieve stable insertion into the genome of target cells (see, for example, Thomas KR & Capecchi MR, Cell 1987, 51:503-12 for a description of homologous recombination cassette vectors). 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, for example, U.S. Patent No. 5,922,687).

[0337] 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 vaccinia or fowlpox. For example, vaccinia virus can be used as a vector for expressing the peptides of the present invention. After introduction into a host, recombinant vaccinia virus expresses the immunogenic peptide, thereby triggering an immune response. Vaccine vectors and methods for use in immunization programs are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al., Nature 1991, 351:456-60. Various other vectors that can be used for therapeutic administration or immunization are readily apparent, such as adenovirus and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and so on. See, for example, 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.

[0338] The polynucleotides of the present invention can be delivered to patients directly, in which case the patient can be directly exposed to a carrier carrying the polynucleotides of the present invention, or indirectly, in which case cells are first transformed in vitro with a carrier carrying the polynucleotides of the present invention, and then the cells are transplanted into the patient. These two methods are respectively referred to as in vivo and in vitro gene therapy.

[0339] 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. Known methods applicable to the recombinant DNA technology of this 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.

[0340] Similar to peptide administration, polynucleotide administration can be performed orally, intradermally, subcutaneously, intravenously, intramuscularly, intraosseously, or intraperitoneally, etc. Administration can be systemic or localized near the target site. Administration can be performed as a single dose or intensified through multiple doses. The polynucleotides of the present invention can be administered to subjects in therapeutically or pharmaceutically effective amounts to induce immunity against cancer cells expressing CDCA1 (more specifically, CTLs), or to treat cancer in therapeutically or pharmaceutically effective amounts. The dosage of the polynucleotide in a suitable carrier or in cells transformed with the polynucleotide encoding the peptides 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 to 1000 mg, for example, 0.01 mg to 100 mg, for example, 0.1 mg to 30 mg, for example, 0.1 mg to 10 mg, or for example, 0.5 mg to 5 mg. Dosing intervals can range from every few days to every few months; for example, dosing can be performed weekly. Those skilled in the art can appropriately select the appropriate dose.

[0341] X. Methods using peptides, exosomes, APCs, and CTLs

[0342] The peptides and polynucleotides of the present invention can be used to induce APCs and CTLs. CTLs can also be induced using the exogenous forms and APCs of the present invention. The peptides, polynucleotides, exogenous forms, and APCs can be used in combination with any other compound, provided that their CTL-inducing ability is not inhibited. Therefore, pharmaceutical compositions comprising any peptide, polynucleotide, APC, and exogenous form of the present invention can be used to induce the CTLs of the present invention. Furthermore, pharmaceutical compositions comprising the peptides or polynucleotides of the present invention can be used to induce the APCs of the present invention.

[0343] (1) Methods for inducing APCs

[0344] This invention provides a method for inducing APCs with CTL induction ability using the peptides or polynucleotides of this invention.

[0345] The method of the present invention includes the step of contacting APC with the peptide of the present invention in vitro, ex vivo, or in vivo. For example, a method of contacting APC with the peptide in vitro may include the following steps:

[0346] (a) Collecting APCs from subjects; and

[0347] (b) Contact the APCs from step (a) with the peptides of the present invention.

[0348] The aforementioned APCs are not limited to specific cell types and can utilize dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells that are known to present protein antigens on their cell surface for recognition by lymphocytes. DCs exhibit the strongest CTL-inducing ability among APCs and are therefore preferred. Any peptide of the present invention can be used alone or in combination with other peptides of the present invention. Furthermore, the peptides of the present invention can be used in combination with other CTL-inducing peptides (e.g., other TAA-derived CTL-inducing peptides).

[0349] Simultaneously, when the peptide of the present invention is administered to a subject, APCs come into contact with the peptide in vivo, resulting in the induction of APCs with high CTL induction capacity in the subject's body. Therefore, the method of the present invention may include the step of administering the peptide of the present invention to a subject. Similarly, when an expressible form of the polynucleotide of the present invention is administered to a subject, the peptide of the present invention is expressed in vivo, and the expressed peptide comes into contact with APCs in vivo, resulting in the induction of APCs with high CTL induction capacity in the subject's body. Therefore, the present invention may further include the step of administering the polynucleotide of the present invention to a subject.

[0350] To induce APCs with CTL inducibility, the present invention further includes the step of introducing the polynucleotide of the present invention into the APCs. For example, the method may include the following steps:

[0351] (a) Collect APCs from the subjects; and

[0352] (b) Introduce the polynucleotide encoding the peptide of the present invention into the APCs of step (a).

[0353] Step (b) can be performed as described in section “VI. Antigen Presenting Cells (APCs)” above.

[0354] Therefore, in one embodiment, the present invention provides a method for inducing APCs with CTL induction capability, comprising the steps of (a) or (b):

[0355] (a) Contacting APCs with the peptides of the present invention; and

[0356] (b) Introducing a polynucleotide encoding the peptide of the present invention into APCs.

[0357] Furthermore, the present invention provides a method for preparing APCs with CTL induction ability, comprising the steps of (a) or (b):

[0358] (a) Contacting APCs with the peptides of the present invention; or

[0359] (b) Introducing a polynucleotide encoding the peptide of the present invention into APCs.

[0360] The methods described above can be performed in vitro, ex vivo, or in vivo, and are preferably performed in vitro or ex vivo. The APCs used in the methods described above can be derived from the subject to whom 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 whom the administration is planned are used, the administering subject and the donor must have the same HLA type. In the methods of the present invention, when a peptide or a modified peptide thereof having an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58 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 methods described above 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:27,3,60,28,5,67 and69, or a modified peptide thereof, is used as the peptide of the present invention, the HLA type in the administering subject and 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:7,38 and47, or a modified peptide thereof, is used as the peptide of the present invention, the HLA type in the administering subject and 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). After separating PBMCs from blood collected from the donor by gravity centrifugation or the like, APCs can be prepared from PBMCs using known methods.

[0361] 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-inducible ability.

[0362] Alternatively, the present invention also provides the use of the peptide of the present invention or the polynucleotide encoding the peptide in the preparation of a pharmaceutical composition for inducing APCs with CTL-inducible ability.

[0363] Alternatively, the present invention also provides the peptide of the present invention or the polynucleotide encoding the peptide for inducing APCs with CTL inducibility.

[0364] Alternatively, the present invention further provides a method or process for preparing a pharmaceutical composition for inducing APC, wherein the method or process includes the step of formulating the peptide or polynucleotide of the present invention with a pharmaceutically or physiologically acceptable carrier.

[0365] In another embodiment, the present invention further provides a method or process for preparing a pharmaceutical composition for inducing APCs with CTL-inducible capabilities, wherein the method or process comprises the step of mixing the peptide or polynucleotide of the present invention with a pharmaceutically or physiologically acceptable carrier.

[0366] APCs induced by the method of the present invention can induce CTLs specific to CDCA1 (i.e., the CTLs of the present invention).

[0367] (2) Methods for inducing CTL

[0368] The present invention also provides methods for inducing CTLs using the peptides, polynucleotides, exogens, or APCs of the present invention. The present invention further provides methods for inducing CTLs using one or more polynucleotides encoding polypeptides (i.e., TCR subunits) capable of forming a T cell receptor (TCR), wherein the TCR recognizes a complex of the peptides and HLA antigens of the present invention. Preferably, the method for inducing CTLs comprises at least one step selected from the following:

[0369] (a) Contacting CD8 positive T cells with antigen-presenting cells, the antigen-presenting cells having a complex of HLA antigen and the peptide of the present invention presented on their surface;

[0370] (b) Contacting CD8-positive T cells with an exogenous body, the exogenous body having a complex of HLA antigen and the peptide of the present invention presented on its surface; and

[0371] (c) Introducing one or more polynucleotides encoding a polypeptide capable of forming a TCR into CD8-positive T cells, wherein the TCR is capable of recognizing a complex of the peptide and HLA antigen of the present invention.

[0372] When the peptides, polynucleotides, APCs, or exogenes 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 cancer cells expressing CDCA1 is enhanced. Therefore, the method of the present invention may include the step of administering the peptides, polynucleotides, APCs, or exogenes of the present invention to a subject.

[0373] Alternatively, they can be used in vitro or ex vivo to induce CTLs. For example, the method of the present invention may include the following steps:

[0374] (a) APCs were collected from the subjects;

[0375] (b) Contacting the APCs from step (a) with the peptides of the present invention; and

[0376] (c) Co-culture the APCs from step (b) with CD8-positive T cells.

[0377] The induced CTL can then be returned to the subject.

[0378] The 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 the APC, as described in section “VI. Antigen-presenting cells (APCs)” above. However, the APCs used in the methods of the present invention are not limited thereto, and any APC that presents a complex of HLA antigen and the peptide of the present invention on its surface can be used.

[0379] In the method of the present invention, in addition to such APCs, exogenous bodies that present a complex of HLA antigen and the peptide of the present invention on their surface can also be used. That is, the method of the present invention may include a step of co-culturing with exogenous bodies that present a complex of HLA antigen and the peptide of the present invention on their surface. Such exogenous bodies can be prepared by the method described in the "V. Exogenous Bodies" section above.

[0380] Furthermore, CTLs can be induced by introducing a vector containing a polynucleotide encoding each subunit of a TCR into CD8-positive T cells, the TCRs being able to bind the peptides of the present invention presented via HLA antigens on the cell surface. Such conversion can be carried out as described in the "VIII. T Cell Receptor (TCR)" section above.

[0381] Therefore, in one embodiment, the present invention provides a method for inducing CTLs, comprising steps selected from the following:

[0382] (a) CD8 positive T cells are co-cultured with APCs, the APCs presenting a complex of HLA antigen and the peptide of the present invention on their surface;

[0383] (b) Co-culturing CD8-positive T cells with exogenous bodies, said exogenous bodies presenting a complex of HLA antigen and the peptide of the present invention on their surface; and

[0384] (c) Introducing a vector containing a polynucleotide encoding each subunit of a TCR into CD8-positive T cells, wherein the TCR is capable of binding the peptide of the present invention presented via an HLA antigen on the cell surface.

[0385] The methods described above can be performed in vitro, ex vivo, or in vivo, and are preferably performed in vitro or ex vivo. The APCs or exogenous and CD8-positive T cells used in the methods described above can be derived from the subject to whom the planned administration of induced CTLs is performed, or they can be derived from different subjects. When using APCs or exogenous and CD8-positive T cells from a subject (donor) different from the subject to whom the administration is planned, the subject and the donor must have the same HLA type. For example, when a peptide or a modified peptide thereof having an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56, and 58 is used as the peptide of the present invention, the HLA type in both the subject and the donor is preferably HLA-A11 (more preferably HLA-A*1101). Alternatively, the APC or exogenous organism used in the above methods is preferably an APC or exogenous organism that presents 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: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56 and 58 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 CDCA1). Alternatively, for example, when a peptide having an amino acid sequence selected from SEQ ID NOs: 27, 3, 60, 28, 5, 67 and 69 or a modified peptide thereof is used as the peptide of the present invention, the HLA in the target and the donor are preferably both HLA-A33 (more preferably HLA-A*3303). Alternatively, the APC or exogenous organism used in the above methods is preferably an APC or exogenous organism that presents 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: 27, 3, 60, 28, 5, 67 and 69 or a modified peptide thereof) on its 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 CDCA1). Alternatively, for example, when a peptide having an amino acid sequence selected from SEQ ID NOs: 7, 38 and 47 or a modified peptide thereof is used as the peptide of the present invention, the HLA in the target and the donor are preferably both HLA-A03 (more preferably HLA-A*0301). Alternatively, the APC or exogenous substance used in the above methods is preferably an APC or exogenous substance that presents 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: 7, 38 and 47 or a modified peptide thereof) on its surface.In this configuration, the induced CTLs exhibit cytotoxic activity against cells presenting a complex of HLA-AO3 and the peptide of the present invention (e.g., HLA-AO3-positive cells expressing CDCA1). In another embodiment, the present invention also provides compositions or pharmaceutical compositions for inducing CTLs, comprising at least one active ingredient selected from:

[0386] (a) The peptide of the present invention;

[0387] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0388] (c) An APC that presents the peptides of the invention on its surface; and

[0389] (d) Presenting the exogenous form of the peptide of the present invention on its surface.

[0390] In another embodiment, the present invention also provides the use of an active ingredient selected from the group consisting of:

[0391] (a) The peptide of the present invention;

[0392] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0393] (c) An APC that presents the peptides of the invention on its surface; and

[0394] (d) Presenting the exogenous form of the peptide of the present invention on its surface.

[0395] Alternatively, the present invention further provides an active ingredient selected from the following for inducing CTLs:

[0396] (a) The peptide of the present invention;

[0397] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0398] (c) An APC that presents the peptides of the invention on its surface; and

[0399] (d) Presenting the exogenous form of the peptide of the present invention on its surface.

[0400] Alternatively, the present invention further provides a method or process for preparing a composition or pharmaceutical composition for inducing CTLs, which includes the step of formulating an active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier:

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

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

[0403] (c) An APC that presents the peptides of the invention on its surface; and

[0404] (d) Presenting the exogenous form of the peptide of the present invention on its surface.

[0405] In another embodiment, the present invention further provides a method or process for preparing a composition or pharmaceutical composition for inducing CTLs, comprising the steps of mixing an active ingredient selected from the following with a pharmaceutically or physiologically acceptable carrier:

[0406] (a) The peptide of the present invention;

[0407] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0408] (c) An APC that presents the peptides of the invention on its surface; and

[0409] (d) Presenting the exogenous form of the peptide of the present invention on its surface.

[0410] XI. Methods for inducing immune responses

[0411] This invention also provides a method for inducing an immune response against cancers expressing CDCA1. Applicable cancers include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, renal cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, and colorectal cancer. Preferably, the cancer expression is selected from at least one HLA group chosen from HLA-A11, HLA-A33, and HLA-A03.

[0412] This invention also provides a method for inducing an immune response against cancer cells expressing CDCA1. CDCA1 is believed to be overexpressed in the various types of cancers described above. Therefore, when an immune response is induced against cancer cells expressing CDCA1, the proliferation of cancer cells is inhibited. Therefore, this invention also provides a method for inhibiting the proliferation of cancer cells expressing CDCA1. The method of this invention is particularly suitable for inhibiting the proliferation of cancer cells expressing CDCA1 and at least one HLA selected from HLA-A11, HLA-A33, and HLA-A03.

[0413] The method of the present invention may include the step of administering a composition comprising any peptide of the present invention or a polynucleotide encoding said peptide. The method of the present invention also covers the administration of an APC or exogenous body that presents any peptide of the present invention. For details, see Section IX. Pharmaceutical Compositions, particularly the section describing the use of the pharmaceutical compositions of the present invention as vaccines. Additionally, exogenous bodies and APCs that can be used in the methods of inducing immune responses of the present invention are described in detail in items (1) and (2) of Section V. Exogenous Bodies, Section VI. Antigen Presenting Cells (APCs), and Section X. Methods Using Peptides, Exogenous Bodies, APCs, and CTLs above.

[0414] In another embodiment, the present invention provides a pharmaceutical composition or vaccine for inducing an immune response against cancers expressing CDCA1, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from:

[0415] (a) The peptide of the present invention;

[0416] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0417] (c) An APC that presents the peptide of the present invention on its surface;

[0418] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0419] (e) The CTL of the present invention.

[0420] Alternatively, the present invention also provides a pharmaceutical composition or vaccine for inducing an immune response against cancer cells expressing CDCA1, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from:

[0421] (a) The peptide of the present invention;

[0422] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0423] (c) An APC that presents the peptide of the present invention on its surface;

[0424] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0425] (e) The CTL of the present invention.

[0426] Alternatively, the present invention also provides a pharmaceutical composition or vaccine for inhibiting the proliferation of cancer cells expressing CDCA1, wherein the pharmaceutical composition or vaccine comprises an active ingredient selected from:

[0427] (a) The peptide of the present invention;

[0428] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0429] (c) An APC that presents the peptide of the present invention on its surface;

[0430] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0431] (e) The CTL of the present invention.

[0432] In another embodiment, the present invention provides the use of an active ingredient selected from the group consisting of:

[0433] (a) The peptide of the present invention;

[0434] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0435] (c) An APC that presents the peptide of the present invention on its surface;

[0436] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0437] (e) The CTL of the present invention.

[0438] Alternatively, the present invention also provides the use of an active ingredient selected from the group consisting of:

[0439] (a) The peptide of the present invention;

[0440] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0441] (c) An APC that presents the peptide of the present invention on its surface;

[0442] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0443] (e) The CTL of the present invention.

[0444] Alternatively, the present invention further provides the 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 CDCA1:

[0445] (a) The peptide of the present invention;

[0446] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0447] (c) An APC that presents the peptide of the present invention on its surface;

[0448] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0449] (e) The CTL of the present invention.

[0450] The present invention also provides a method or process for preparing a pharmaceutical composition for inducing an immune response against cancers expressing CDCA1, wherein the method may include the step of mixing or formulating the peptide or polynucleotide of the present invention with a pharmaceutically acceptable carrier.

[0451] Alternatively, the present invention provides a method for inhibiting the proliferation of cancer cells expressing CDCA1 or for inducing an immune response against cancers expressing CDCA1, comprising the step of administering to a subject a vaccine or pharmaceutical composition comprising an active ingredient selected from:

[0452] (a) The peptide of the present invention;

[0453] (b) A polynucleotide encoding the peptide of the present invention in an expressible form;

[0454] (c) An APC that presents the peptide of the present invention on its surface;

[0455] (d) Presenting exogenous forms of the peptides of the present invention on their surface; and

[0456] (e) The CTL of the present invention.

[0457] In the context of this invention, cancers expressing CDCA1 can be treated by administering the peptides, polynucleotides, APCs, exogenous bodies, and / or CTLs of this invention. Alternatively, administering the peptides, polynucleotides, APCs, exogenous bodies, and / or CTLs of this invention can induce an immune response against cancers expressing CDCA1. Examples of such cancers include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, colorectal cancer, etc., but are not limited thereto. Furthermore, administering the peptides, polynucleotides, APCs, exogenous bodies, and / or CTLs of this invention can induce an immune response against cancers expressing CDCA1. Therefore, before administering a vaccine or pharmaceutical composition containing the above-described active ingredients, it is preferable to confirm whether the expression level of CDCA1 at the disease site is increased in the subject to be treated.

[0458] Therefore, in one embodiment, the present invention provides a method for treating cancers expressing CDCA1 in patients requiring cancer treatment, wherein the method includes the following steps:

[0459] (i) Measure the level of CDCA1 expression in biological samples collected from diseased sites in subjects with cancer;

[0460] (ii) Identify subjects with cancer expressing CDCA1 based on CDCA1 expression levels measured in (i); and

[0461] (iii) Administer at least one ingredient selected from the group consisting of (a) to (e) above to cancer patients who overexpressed CDCA1 compared with normal controls.

[0462] Alternatively, the present invention further provides a vaccine and pharmaceutical composition comprising at least one active ingredient selected from the group consisting of (a) to (e) above, for administration to a cancer patient expressing CDCA1. 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:

[0463] (i) Measure the level of CDCA1 expression in biological samples collected from diseased sites in subjects with cancer;

[0464] (ii) Identify subjects with cancer expressing CDCA1 based on CDCA1 expression levels measured in (i); and

[0465] (iii) Identify or select the subjects identified in (ii) as subjects who can be treated with at least one active ingredient selected from the group consisting of (a) to (e).

[0466] In the above methods, there are no particular limitations on the biological samples collected from the subjects for measuring CDCA1 expression levels, and for example, tissue samples containing cancer cells collected through biopsy or similar methods are preferred. The CDCA1 expression level in the biological sample can be measured by known methods, such as methods that detect the transcription product of the CDCA1 gene using probes or PCR methods (e.g., cDNA microarray, Northern blotting, RT-PCR, etc.), methods that detect the translation product of the CDCA1 gene using antibodies (e.g., Western blotting, immunostaining, etc.), and so on. Furthermore, the biological sample can be a blood sample, in which case the blood level of antibodies against CDCA1 or fragments thereof is measured, and the CDCA1 expression level at the disease site can be assessed based on the blood level. Known methods can be used to measure the blood level of antibodies against CDCA1, and for example, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), etc., using the CDCA1 protein or the peptide of the present invention as an antigen can be used.

[0467] Typically, CDCA1 transcription and translation products are barely detectable in tissues and cells that do not express CDCA1. Therefore, when CDCA1 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 CDCA1. In blood samples from subjects with cancer that does not express CDCA1, antibodies against CDCA1 or its fragments are barely detectable. Therefore, when antibodies against CDCA1 or its fragments are detected in blood samples collected from a subject, it can be determined that the subject's cancer expresses CDCA1. Whether a subject's cancer expresses CDCA1 can also be determined by comparing measurements with those from the same type of biological material collected from non-cancer sites of the subject or with the same type of biological material collected from subjects without cancer (normal control samples). That is, in comparison with the measured target level (normal control level) in the normal control sample, the subject's cancer is assessed as expressing CDCA1 when the level in the tested subject's biological sample is elevated. For example, when the detected amount of the measured substance increases by at least 10% or more compared to the normal control level, the subject's cancer can be assessed as expressing CDCA1. Ideally, the amount of the detected target should preferably be 25% or higher than the normal control level, more preferably 50% or higher. Furthermore, the amount of the detected CDCA1 transcript or translation product can be evaluated by normalizing it relative to the amount of a known housekeeping gene (e.g., β-actin, glyceraldehyde-3-phosphate dehydrogenase, or ribosomal protein P1).

[0468] In a preferred embodiment, the subject's HLA type 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 an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58, HLA-A11 positive subjects are preferred. For subjects to be administered an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs:27,3,60,28,5,67 and 69, HLA-A33 positive subjects are preferred. For subjects to be administered an active ingredient associated with a peptide having an amino acid sequence selected from SEQ ID NOs:7,38 and 47, HLA-A03 positive subjects are preferred. The present invention also provides complexes of the peptides and HLA of the present invention. The complexes of the present invention described above can be monomers or polymers. When the complex of the present invention is a polymer, the number of polymers is not particularly limited and can be any number of polymers. Examples include, but are not limited to, tetramers, pentamers, hexamers, etc. The polymers of the present invention also include dextramers (WO2002 / 072631) and streptamers (Knabel M et al., Nat Med. 2002 Jun; 8(6):631-7.). The peptide and HLA complex of the present invention 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 to the peptide of the present invention. For example, blood samples are collected from subjects who have been given the pharmaceutical composition of the present invention, and CD4 negative cells are prepared after PBMC isolation and contacted with the fluorescent dye-conjugated complex of the present invention. The percentage of CTLs specific to the peptides 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 specific CTLs against the peptides of the present invention before, during, and / or after administration of the pharmaceutical composition of the present invention.

[0469] XII. Antibody

[0470] 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) peptides that are not of the present invention. In another embodiment, such antibodies may include antibodies that recognize peptides in the context of HLA molecules, i.e., antibodies that bind to peptide-MHC complexes. The binding specificity of the antibody can be confirmed by an inhibition assay. That is, if the binding between the antibody to be analyzed and the full-length CDCA1 peptide is inhibited in the presence of the peptide of the present invention, the antibody exhibits 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 subject selection and monitoring of the pharmaceutical compositions of the present invention.

[0471] The present invention also provides various immunoassays for detecting and / or quantifying the peptides or fragments thereof of the present invention. Such immunoassays include, but are not limited to, radioimmunoassays, immunochromatography, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescence assays (ELIFA), etc., and are performed within the range of various immunoassay formats well known in the art.

[0472] The antibodies of this invention can be used in immunological imaging methods that can detect diseases expressing CDCA1, and examples include, but are not limited to, radioscintigraphy using the labeled antibodies of this invention. Such assays are used clinically to detect, monitor, and predict cancers expressing CDCA1; examples of such cancers include, but are not limited to, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, colorectal cancer, etc.

[0473] The antibodies of the present invention can be used in any form, such as monoclonal or polyclonal antibodies, and may also include antiserum 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 generated by genetic recombination.

[0474] The peptides or fragments thereof of the present invention used as antigens for obtaining antibodies can be obtained by chemical synthesis or genetic engineering techniques based on the amino acid sequences disclosed herein.

[0475] The peptide used as an immunogenic antigen can be the peptide of the present invention or a fragment of the peptide of the present invention. Furthermore, the peptide can be bound to or conjugated to a carrier to increase immunogenicity. Keyhole hemocyanin (KLH) is known as a carrier. Methods for binding KLH to the peptide are also known in the art.

[0476] Any mammal can be immunized with the aforementioned antigens, and compatibility with the parental cells used for cell fusion is preferred when generating monoclonal antibodies. Typically, animals of the orders Rodentia, Lagomorpha, or Primate can be used. Rodents include, for example, mice, rats, and hamsters. Lagomorphs include, for example, domestic rabbits. Primates include, for example, old-world monkeys such as cynomolgus monkeys (Macacafascicularis), rhesus monkeys, baboons, and chimpanzees.

[0477] Methods for immunizing animals with antigens are known in the art. Intraperitoneal and subcutaneous injection of antigens are standard methods for immunizing mammals. More specifically, antigens can be diluted and suspended in appropriate amounts of phosphate-buffered saline (PBS), physiological saline, etc. If desired, the antigen suspension can be mixed with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, to form an emulsion, which is then administered to the mammal. Preferably, antigens mixed with an appropriate amount of Freund's incomplete adjuvant are then administered several times every 4 to 21 days. A suitable carrier can be used for immunization. After immunization as described above, the increase in the amount of desired antibodies in the serum can be detected using standard methods.

[0478] Polyclonal antibodies against the peptides of the present invention can be prepared as follows: blood is collected from a mammal (where an increase in the desired antibody serum level has been confirmed after immunization), and serum is separated from the blood using any conventional method. The polyclonal antibody can be a serum containing the polyclonal antibody, or a fraction containing the polyclonal antibody that can be separated from said serum. Immunoglobulin G or M can be prepared from the fraction that only recognizes the peptides of the present invention using, for example, an affinity column conjugated to the peptides of the present invention, and the fraction can be further purified using a protein A or protein G column.

[0479] To prepare monoclonal antibodies, immune cells are collected from mammals and fused when serum levels of the desired antibody increase after confirmatory immunization. The immune cells used for cell fusion are preferably derived from the spleen. For other parental cells to be fused with the aforementioned immune cells, mammalian myeloma cells can be used, preferably myeloma cells with drug-selective properties from which fusion has been obtained.

[0480] The aforementioned immune cells can be fused with myeloma cells using known methods, such as those of Milstein et al. (Galfre and Milstein, Methods Enzymol 73:3-46 (1981)).

[0481] For hybridomas derived from cell fusion, selection can be performed by culturing in standard selection media such as HAT medium (containing hypoxanthine, aminopterin, and thymidine). Typically, cells are cultured in HAT medium for a sufficient time (e.g., several days to several weeks) to allow all other cells (non-fusion cells) except the desired hybridoma to die. Hybridoma cells generating the desired antibody are then screened and cloned using standard limiting dilutions.

[0482] In addition to the methods described above for preparing hybridomas by immunizing non-human animals with antigens, human lymphocytes, such as those infected with Epstein-Barr virus, can be immunized in vitro with peptides, peptide-expressing cells, or their lysates. The immunized lymphocytes can then be fused with immortalized human-derived myeloma cells, such as U266, to generate the desired hybridoma (JPS63-17688) that produces human antibodies capable of binding to the peptide.

[0483] The resulting hybridoma was then transplanted into the peritoneal cavity of mice, and ascites fluid was aspirated. 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 conjugated with the peptide of the present invention.

[0484] Alternatively, immune cells (such as immune lymphocytes) can be generated through oncogene immortalization antibodies and used to prepare monoclonal antibodies.

[0485] 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 instance, recombinant antibodies can be prepared by inserting the DNA encoding the antibody from immune cells such as hybridomas or immune lymphocyte clones into a suitable vector and introducing it into host cells. This invention also provides recombinant antibodies prepared as described above.

[0486] Furthermore, the antibodies of the present invention can 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 fragments contain the antigen-binding site of the antibody. Specifically, the antibody fragments can be Fab, F(ab')2, Fv, or single-chain Fv (scFv) formed by linking Fv fragments from the H and L chains through a suitable linker (Huston et al., ProcNatl Acad Sci USA 85:5879-83 (1988)). More specifically, antibody fragments can be generated by treating the antibody with an enzyme such as papain or pepsin. Alternatively, a gene encoding an antibody fragment can be constructed, inserted into an expression vector, and expressed in a suitable host cell (see, for example, 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)).

[0487] Antibodies can be modified by conjugating various molecules such as polyethylene glycol (PEG). This invention provides antibodies modified in this way. Modified antibodies can be obtained by chemically modifying them. These modification methods are conventional in the art.

[0488] Alternatively, the antibodies of the present invention can be obtained as chimeric antibodies between a variable region derived from a non-human antibody and a constant region derived from a human antibody, or as humanized antibodies comprising a complementarity-determining region (CDR) derived from a non-human antibody, a framework region (FR) derived from a human antibody, and a constant region. Such antibodies can be prepared according to known techniques. Humanization can be performed by replacing the corresponding sequence of the human antibody with a non-human antibody CDR sequence (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 a corresponding sequence from a non-human species.

[0489] Complete human antibodies can also be used, such antibodies containing human variable regions in addition to the human framework 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 bacteriophages (e.g., Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991)). Similarly, human antibodies can be generated by introducing human immunoglobulin gene loci into transgenic animals, such as mice with partially or completely inactivated endogenous immunoglobulin genes. Such methods are described, for example, in 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.

[0490] The antibodies obtained as described above can be purified to homogeneity. For example, antibodies can be separated and purified using methods generally used for the separation and purification of proteins. For instance, antibodies can be separated and purified by appropriately selecting and combining column chromatography techniques such as affinity chromatography, filtration, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but are not limited to these methods. Protein A columns and protein G columns can be used as affinity columns. Illustrative examples of usable protein A columns include, for example, Hyper D, POROS, and Sepharose FF (Pharmacia).

[0491] Besides affinity chromatography, exemplary chromatographic procedures include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, adsorption chromatography, etc. (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 using liquid chromatography, such as HPLC and FPLC.

[0492] For example, the antigen-binding activity of the antibodies of the present invention can be measured 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 culture supernatant of antibody-generating cells or purified antibody, is applied. A second antibody labeled with an enzyme (such as alkaline phosphatase) that recognizes the first antibody is then applied, and the plate is incubated. Next, after washing, an enzyme substrate, such as p-nitrobenzene phosphate, is added to the plate, and the antigen-binding activity of the sample is assessed by measuring absorbance. To assess antibody binding activity, peptide fragments such as C-terminal or N-terminal fragments can be used as antigens. The activity of the antibodies of the present invention can be assessed using BIAcore (Pharmacia).

[0493] The peptides of the present invention can be detected or measured using the above method by exposing the antibody of the present invention to a sample presumably containing the peptide of the present invention and detecting or measuring the immune complex formed between the antibody and the peptide.

[0494] For example, the antibodies of the present invention can be used to detect the peptides of the present invention present in a subject's blood sample (e.g., a serum sample). Alternatively, the peptides of the present invention can also be used to detect the antibodies of the present invention present in a subject's blood sample (e.g., a serum sample). The results of measuring the peptides of the present invention or the antibodies of the present invention in a subject's blood sample can be used for the subject to select for administration of the pharmaceutical composition of the present invention or to monitor the efficacy of the pharmaceutical composition. Furthermore, it has been reported that patients with antibodies (which target peptides administered as vaccines) may have a high response to vaccines. Therefore, when using a patient's antibody as an index for administration of the peptides of the present invention as vaccines, the peptides of the present invention can be used as immunoassay antigens for selecting patients with high responses.

[0495] XII. Vector and Host Cell

[0496] This invention provides a vector comprising a polynucleotide encoding the peptide of the invention and a host cell into which said vector is introduced. The vector of the invention can be used to hold the polynucleotide of the invention in a host cell to express the peptide of the invention in the host cell, or to administer the polynucleotide of the invention for gene therapy.

[0497] When *E. coli* is used as the host cell and vectors are generated through mass amplification in *E. coli* (e.g., JM109, DH5-alpha, HB101, or XL1-Blue), the vector needs to have an "origin of replication" 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. Additionally, pGEM-T, pDIRECT, and pT7 can be used for cloning and the above vectors. When the vector is used to produce the peptides of the present invention, an expression vector can be used. For example, an expression vector intended for expression in *E. coli* should possess the characteristics described above for amplification in *E. coli*. When using *E. coli* strains such as JM109, DH5-alpha, HB101, or XL1-Blue as host cells, the vector should contain promoters 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)), and the T7 promoter. In this regard, vectors such as pGEX-5X-1 (Pharmacia), the "QIAexpress system" (Qiagen), pEGFP, and pET (in this case, the host preferably expresses BL21, a T7 RNA polymerase) can be used instead of the aforementioned vectors. Additionally, the vector may contain a signal sequence for peptide secretion. An exemplary signal sequence guiding peptide secretion into the periplasm of *E. coli* is the pelB signal sequence (Lei et al., *J Bacteriol* 169:4379 (1987)). Methods for introducing vectors into target host cells include, for example, the calcium chloride method and electroporation.

[0498] In addition to *Escherichia coli*, expression vectors derived from mammals (e.g., pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids Res 18(17):5322(1990)), pEF, pCDM8), insect cells (e.g., “Bac-to-BAC Baculovirus Expression System” (GIBCO BRL), pBacPAK8), plants (e.g., pMH1, pMH2), animals (e.g., pHSV, pMV, pAdexLcw), retroviruses (e.g., pZIpneo), yeast (e.g., “Pichia pastoris Expression Kit” (Invitrogen), pNV11, SP-Q01), and Bacillus subtilis (e.g., pPL608, pKTH50) can also be used to generate the polypeptides of the present invention.

[0499] To express the vector in animal cells such as CHO, COS, or NIH3T3 cells, the vector should possess the promoters necessary for expression in said cells, such as the SV40 promoter (Mulligan et al., Nature 277:108 (1979)), the MMLV-LTR promoter, the EF1-alpha promoter (Mizushima et al., Nucleic Acids Res 18:5322 (1990)), the CMV promoter, etc., and preferably a marker gene for selecting transformants (e.g., a drug resistance gene selected by a drug (e.g., neomycin, G418)). Examples of known vectors with these characteristics include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0500] The following embodiments of the present invention are illustrated based on the above description; however, the present invention is not limited to these embodiments.

[0501] [1] A peptide of fewer than 15 amino acids that has the ability to induce cytotoxic T cells (CTLs), comprising an amino acid sequence selected from the following group:

[0502] (a) The amino acid sequence selected from the group consisting of: SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56,58,27,60,28,67,69 and 47; and

[0503] (b) An amino acid sequence selected from the group consisting of substituted, deleted, inserted and / or added amino acids of one, two or more amino acids: SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56,58,27,60,28,67,69 and 47.

[0504] [2][1] peptides, whose amino acid sequences are selected from the following groups (i) to (iv):

[0505] (i) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the group(a) to (d) are introduced into an amino acid sequence selected from the group: SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56 and 58:

[0506] (a) Replace 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.

[0507] (b) Replace the third amino acid from the N-terminus with an amino acid selected from the group consisting of leucine, phenylalanine, tyrosine, isoleucine and alanine.

[0508] (c) Replace the seventh amino acid starting from the N-terminus with an amino acid selected from the group consisting of leucine, isoleucine, tyrosine, valine, and phenylalanine; and

[0509] (d) Replace the C-terminal amino acid with an amino acid selected from the group consisting of lysine and arginine;

[0510] (ii) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the group(a) to (c) are introduced into an amino acid sequence selected from the group: SEQ ID NOs:27,3,60,28,5,67 and 69:

[0511] (a) Replace the first amino acid from the N-terminus with an amino acid selected from the group consisting of aspartic acid and glutamic acid;

[0512] (b) Replace 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.

[0513] (c) Replace the C-terminal amino acid with an amino acid selected from the group consisting of arginine and lysine; and

[0514] (iii) A peptide comprising an amino acid sequence wherein one or more substitutions selected from the group(a) to (b) are introduced into an amino acid sequence selected from the group: SEQ ID NOs:7,38 and 47:

[0515] (a) Replace 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

[0516] (b) Replace the C-terminal amino acid with an amino acid selected from the group consisting of arginine, lysine, tyrosine and phenylalanine;

[0517] [3][1] peptides, which consist of amino acid sequences selected from the group consisting of: SEQ ID NOs:3,5 to 7,9,10,12 to 14,17,19,21,30,35,38 to 40,45,53,56,58,27,60,28,67,69 and 47.

[0518] [4] Polynucleotides that encode a peptide of any one of [1] to [3].

[0519] [5] A composition comprising a pharmaceutically acceptable carrier and at least one component selected from the groups (a) to (e) below:

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

[0521] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0522] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0523] (d) an exogenous body that presents a complex of a peptide from [1] to [3] with an HLA antigen on the cell surface; and

[0524] (e)CTL, which targets any of the peptides in [1] to [3].

[0525] [6][5] compositions, which are compositions for inducing CTLs, wherein the component is at least one component selected from the following groups (a) to (d):

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

[0527] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0528] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0529] (d) Exogenous organisms that present a complex of a peptide of any one of [1] to [3] with an HLA antigen on their cell surface.

[0530] [7][5] The composition is a pharmaceutical composition.

[0531] [8][7] The composition is a pharmaceutical composition for one or more uses selected from the group consisting of: (i) cancer treatment, (ii) cancer prevention (prophylaxis) and (iii) prevention of postoperative cancer recurrence (prophylaxis).

[0532] [9][7] compositions for inducing an immune response against cancer.

[0533] The composition of

[10] [8] or [9], wherein the cancer is selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors and colorectal cancer.

[0534] The composition of any one of

[11] [5] to

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

[0535]

[12] A method for inducing APCs with CTL induction capability, comprising steps selected from the group consisting of:

[0536] (a) Contacting APC with any one of the peptides described in [1] to [3] in vitro, ex vivo, or in vivo; and

[0537] (b) Introduce a polynucleotide encoding any one of the peptides in [1] to [3] into the APC.

[0538]

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

[0539] (a) CD8 positive T cells are co-cultured with APCs, wherein the APCs present a complex of HLA antigen and any one of the peptides in [1] to [3] on their surface;

[0540] (b) CD8 positive T cells are co-cultured with exogenous bodies that present on their surface a complex of HLA antigen and a peptide of any one of [1] to [3];

[0541] (c) Introducing a polynucleotide into CD8-positive T cells, the polynucleotide encoding each subunit of a T-cell receptor (TCR) that is capable of binding to any one of the peptides presented on the cell surface by an HLA antigen [1] to [3].

[0542]

[14] APC, which presents on its surface a complex of HLA antigen with any of the peptides in [1] to [3].

[0543]

[15]

[14] APC, which is induced by the method of

[12] .

[0544]

[16] CTL, which targets any of the peptides in [1] to [3].

[0545]

[17]

[16] CTL, which is induced by the method of

[13] .

[0546]

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

[0547] (a) One or more types of peptides from any of [1] to [3];

[0548] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0549] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0550] (d) an exogenous body that presents a complex of a peptide from any one of [1] to [3] with an HLA antigen on its cell surface; and

[0551] (e)CTL, which targets any of the peptides in [1] to [3].

[0552]

[19] Methods for treating and / or preventing cancer, and / or preventing its recurrence after surgery, comprising administering to a subject at least one ingredient selected from groups (a) to (e) below:

[0553] (a) A peptide of one or more types as claimed in any one of claims 1 to 3;

[0554] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0555] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0556] (d) an exogenous body that presents a complex of a peptide from any one of [1] to [3] with an HLA antigen on its cell surface; and

[0557] (e)CTL, which targets any of the peptides in [1] to [3].

[0558]

[20] Antibody that binds to any one of the peptides in [1] to [3].

[0559]

[21] A method for screening peptides with CTL-inducible ability, comprising the following steps:

[0560] (a) Generate a candidate sequence consisting of an amino acid sequence wherein one, two, or more amino acid residues are substituted, deleted, inserted, and / or added to the original amino acid sequence consisting of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 3, 5 to 7, 9, 10, 12 to 14, 17, 19, 21, 30, 35, 38 to 40, 45, 53, 56, 58, 27, 60, 28, 67, 69, and 47;

[0561] (b) Select candidate sequences from the candidate sequences generated in (a) that do not have significant homology (sequence identity) with any known human gene product other than CDCA1;

[0562] (c) Contact the APC with a peptide consisting of the candidate sequences selected in (b);

[0563] (d) Expose the APCs from (c) to CD8-positive T cells; and

[0564] (e) Select peptides that have equal or higher CTL induction capacity compared to peptides composed of the original amino acid sequence.

[0565]

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

[0566] (a) One or more types of peptides from any of [1] to [3];

[0567] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0568] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0569] (d) an exogenous body that presents a complex of a peptide from [1] to [3] with an HLA antigen on the cell surface; and

[0570] (e)CTL, which targets any of the peptides in [1] to [3].

[0571]

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

[0572] (a) One or more types of peptides from [1] to [3];

[0573] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0574] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0575] (d) an exogenous body that presents a complex of a peptide from any one of [1] to [3] with an HLA antigen on the cell surface; and

[0576] (e)CTL, which targets any of the peptides in [1] to [3].

[0577]

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

[0578] (a) One or more types of peptides from [1] to [3];

[0579] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0580] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0581] (d) an exogenous body that presents a complex of a peptide from any one of [1] to [3] with an HLA antigen on the cell surface; and

[0582] (e)CTL, which targets any of the peptides in [1] to [3].

[0583]

[25] At least one ingredient selected from the following groups (a) to (e) is used for the treatment and / or prevention of cancer and / or prevention of its recurrence after surgery:

[0584] (a) One or more types of peptides from [1] to [3];

[0585] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0586] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0587] (d) an exogenous body that presents a complex of a peptide from [1] to [3] with an HLA antigen on the cell surface; and

[0588] (e)CTL, which targets any of the peptides in [1] to [3].

[0589]

[26] A method for inducing cytotoxic activity against cells expressing CDCA1, comprising the step of administering to a subject at least one component selected from the following groups (a) to (e):

[0590] (a) One or more types of peptides from any of [1] to [3];

[0591] (b) One or more types of polynucleotides that encode a peptide of any one of [1] to [3] in an expressible form;

[0592] (c) Antigen-presenting cells (APCs) that present complexes of any one of the peptides [1] to [3] with HLA antigens on their cell surface;

[0593] (d) an exogenous body that presents a complex of a peptide from [1] to [3] with an HLA antigen on the cell surface; and

[0594] (e)CTL, which targets any of the peptides in [1] to [3].

[0595]

[27] A freeze-dried formulation comprising one or more peptides of any one of [1] to [3].

[0596]

[28] A pharmaceutical composition prepared by means of dissolving one or more peptides of any one of [1] to [3] in a water-soluble carrier and then filtration sterilizing.

[0597]

[29] A filtered sterilized aqueous solution, which is an aqueous solution containing one or more types of peptides from [1] to [3] and a water-soluble carrier.

[0598]

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

[0599]

[31] A kit comprising a container for containing a composition of any one of [5] to

[11] and a container for containing an adjuvant.

[0600]

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

[0601] This document explains the invention in detail with reference to its specific embodiments. However, it should be understood that the above explanation is illustrative and explanatory in nature, and is intended to explain the invention and its preferred embodiments. Through conventional experimentation, 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 invention. Therefore, the invention is not limited to the above description, but is intended to be defined by the appended claims and their equivalents.

[0602] The invention is described in more detail below with reference to embodiments. However, while the materials, methods, and embodiments described below can help those skilled in the art to make and use certain embodiments of the invention, these are merely illustrative of various aspects of the invention and are in no way limiting its scope. Those skilled in the art will readily recognize that similar or equivalent methods and materials to those described herein can be used for the implementation or testing of the invention.

[0603] All prior art references cited herein are incorporated herein by reference.

[0604] Example

[0605] [Example 1]

[0606] Materials and methods

[0607] cell lines

[0608] C1R, HLA-A negative and HLA-B negative human B lymphoblast cell lines, and COS7, African green monkey kidney cell line were purchased from ATCC.

[0609] The generation of cells with stable HLA-A*1101 expression

[0610] C1R cells stably expressing HLA-A*1101 (C1R-A11) were used as stimulatory 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 using G418 (Invitrogen) for two weeks. Cells selected with G418 were seeded into wells of 96-well plates containing medium supplemented with G418 and cultured for another 30 days. Flow cytometry analysis confirmed the expression of exogenous HLA-A*1101 in C1R cells.

[0611] Selection of candidate CDCA1-derived peptides

[0612] 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 the CDCA1-derived 9-mer and 10-mer peptides that bind to the HLA-A*1101 molecule.

[0613] peptide synthesis

[0614] These peptides were synthesized via standard solid-phase synthesis at Biosynthesis (Lewisville, Texas) and purified by reversed-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.

[0615] In vitro CTL induction

[0616] 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 monocytes isolated from healthy volunteers (HLA-A*1101 positive) using Ficoll-Paque Plus (Pharmacia) solution were separated by adhesive plastic tissue culture discs (Becton Dickinson) and concentrated as a monocyte 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 granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml interleukin (IL)-4 (R&D System). After 7 days of culture, cytokine-induced dendritic cells (DCs) were shocked for 3 hours at 37°C with 20 μg / ml of each synthetic peptide in the presence of 3 μg / ml β2-microglobulin in AIM-V medium. The resulting cells exhibited expression of DC-related molecules such as CD80, CD83, CD86, and HLA class II (data not shown) on their cell surface. Subsequently, these peptide-shocked DCs were inactivated by X-ray irradiation (20 Gy) and compared 1:20 with autologous CD8+ obtained through positive selection using a CD8+ positive isolation kit (Dynal). + T cell mixture. These culture products were seeded into 48-well plates (Corning). Each well was prepared to contain 1.5 x 10⁻⁶ cells in 0.5 ml AIM-V / 2% AS medium. 4 DC stimulated by peptide, 3x10 5 CD8 +T cells and 10 ng / ml IL-7 (R&D System). After 3 days, IL-2 (CHIRON) was added to these cultures to a final concentration of 20 IU / ml. On days 7 and 14, these T cells were further stimulated with peptide-emerged autologous dendritic cells (DCs). The DCs were prepared each time in the same manner as described above. On day 21, after the third peptide stimulation, CTLs targeting peptide-stimulated 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).

[0617] CTL amplification program

[0618] CTLs were amplified in culture using a method similar to that described 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 lymphoblastocyte lines in a total of 25 ml of AIM-V medium (5 x 10⁻⁶ cells / mL). 6 Cells / flask), the culture medium contained 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).

[0619] Establishment of CTL clones

[0620] CTLs were diluted in 96 round-bottom microtiter plates (Nalge Nunc International) to prepare 0.3, 1, and 3 cells / well. CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblast cell lines (1 x 10⁻⁶ cells / well) in 150 μL / well AIM-V / 5% AS medium containing a total of 30 ng / ml anti-CD3 antibody and 125 IU / ml IL-2. 4 Cells / well). After 10 days, 50 μL / well of IL-2 was added to the culture medium to reach a final concentration of 125 IU / ml. CTL activity was tested on day 14, and CTL clones were amplified 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).

[0621] Specific CTL activity

[0622] To examine specific CTL activity, IFN-gamma ELISPOT assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-stimulated C1R-A11 (1x10⁻¹⁰) was prepared. 4 Cells / wells were used as stimulating cells. Induced CTLs (i.e., CTL lines and CTL clones) were used as responding cells. IFN-gamma ELISPOT assay and IFN-gamma ELISA were performed according to the manufacturer's instructions.

[0623] Establishment of target cells with forced expression of target genes and HLA-A*1101

[0624] cDNA encoding the target gene or the open reading frame of HLA-A*1101 was amplified by PCR. The PCR amplification product was cloned into an expression vector. Using Lipofectamine 2000 (Invitrogen), one or both of the vectors expressing the target gene and expressing HLA-A*1101 were transfected into COS7 cells (a target gene and HLA-negative cell line) according to the manufacturer's instructions. Two days after transfection, the transfected cells were harvested with Versene (Invitrogen) and used as target cells (5 x 10⁻⁶) for CTL activity assay. 4 (cells / well).

[0625] result

[0626] Prediction of CDCA1-derived HLA-A*1101 binding peptide

[0627] Tables 1a and 1b show the CDCA1-derived 9- and 10-meric peptides predicted to bind to HLA-A*1101 in descending order of binding affinity. A total of 58 peptides with potential HLA-A*1101 binding ability were selected and examined to determine epitope peptides.

[0628] [Table 1a]

[0629] HLA-A11-binding 9-peptide derived from CDCA1

[0630]

[0631] [Table 1b]

[0632] HLA-A11-binding decapeptide derived from CDCA1

[0633] Starting position amino acid sequence Kd(nM) SEQ ID NO 353 ATAQFKINKK 27 29 339 NSFKRLMIVK 50 30 423 FLNLKTALEK 53 31 418 KSQEIFLNLK 56 32 391 VTTINQEIQK 57 33 192 QSLNQDFHQK 79 34 452 TAELKRKMFK 87 35 241 KTKIVDSPEK 110 36 352 LATAQFKINK 131 37 400 KIKLGIQQLK 146 38 289 CQLEVQLYQK 172 39 203 IVLQEGNSQK 201 40 136 ETYMEFLWQY 237 41 137 TYMEFLWQYK 254 42 243 KIVDSPEKLK 317 43 447 KIDEKTAELK 360 44 156 NAAHQEALMK 386 45 333 KLKTEENSFK 420 46 257 KMKDTVQKLK 438 47 323 QIESDESELK 465 48 232 SLKEIQESLK 518 49 393 TINQEIQKIK 555 50 210 SQKKSNISEK 599 51 204 VLQEGNSQKK 629 52 340 SFKRLMIVKK 811 53 225 ELKLSVVSLK 969 54 246 DSPEKLKNYK 1354 55 106 TADILCPKAK 1476 56 290 QLEVQLYQKK 1566 57 358 KINKKHEDVK 3452 58

[0634] The start position indicates the number of amino acid residues starting from the N-terminus of CDCA1, and the dissociation constant [Kd(nM)] is derived from "NetMHC3.2".

[0635] Inducing CTLs via predicted CDCA1-derived HLA-A*1101-restricted peptides

[0636] CTLs targeting CDCA1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured using an IFN-gamma ELISPOT assay. Figure 1-1 and Figure 1-2Compared with the control well, well #7(a) has CDCA1-A11-9-123 (SEQ ID NO:3), well #8(b) has CDCA1-A11-9-105 (SEQ ID NO:5), well #1(c) has CDCA1-A11-9-419 (SEQ ID NO:6), well #4(d) has CDCA1-A11-9-219 (SEQ ID NO:7), well #2(e) has CDCA1-A11-9-439 (SEQ ID NO:9), well #2(f) has CDCA1-A11-9-343 (SEQ ID NO:10), well #8(g) has CDCA1-A11-9-21 (SEQ ID NO:12), and well #8 has CDCA1-A11-9-157 (SEQ ID NO:3). Hole #3(h) with CDCA1-A11-9-244 (SEQ ID NO:14), Hole #7(i) with CDCA1-A11-9-213 (SEQ ID NO:17), Hole #6(j) with CDCA1-A11-9-335 (SEQ ID NO:19), Hole #4(l) with CDCA1-A11-9-25 (SEQ ID NO:21), Hole #3(m) with CDCA1-A11-10-339 (SEQ ID NO:30), Hole #2(n) with CDCA1-A11-10-452 (SEQ ID NO:35), Hole #7(o) with CDCA1-A11-10-400 (SEQ ID NO:38), Hole #7(o) with CDCA1-A11-10-289 (SEQ ID NO:13). CTLs in well #6(p) with CDCA1-A11-10-203 (SEQ ID NO:39), well #5(q) with CDCA1-A11-10-156 (SEQ ID NO:45), well #2(r) with CDCA1-A11-10-340 (SEQ ID NO:53), well #4(t) with CDCA1-A11-10-106 (SEQ ID NO:56), and well #2(u) with CDCA1-A11-10-358 (SEQ ID NO:58) showed strong IFN-gamma generation. Meanwhile, although other peptides shown in Tables 1a and 1b potentially possess HLA-A*1101 binding activity, no specific CTL activity was detected as a result of stimulation by these peptides. An example of typical negative data is the lack of specific IFN-gamma production (v) observed in CTLs stimulated with CDCA1-A11-10-391 (SEQ ID NO:33).The results showed that 21 types of CDCA1-derived peptides were selected as peptides capable of inducing effective CTLs.

[0637] Establishment of CTL lines and cloning of HLA-A*1101-derived restriction peptides from CDCA1

[0638] By propagating wells #7(a) with CDCA1-A11-9-123 (SEQ ID NO:3), wells #8(b) with CDCA1-A11-9-105 (SEQ ID NO:5), wells #1(c) with CDCA1-A11-9-419 (SEQ ID NO:6), wells #4(d) with CDCA1-A11-9-219 (SEQ ID NO:7), wells #4(e) with CDCA1-A11-9-439 (SEQ ID NO:9), wells #3(f) with CDCA1-A11-9-157 (SEQ ID NO:13), wells #4(g) with CDCA1-A11-9-25 (SEQ ID NO:21), and wells #4 with CDCA1-A11-10-339 (SEQ ID NO:3), the results were obtained. CTL lines were established using CTLs in well #3(h) containing NO:30 and well #2(i) containing CDCA1-A11-10-358 (SEQ ID NO:58), which exhibited peptide-specific CTL activity in an IFN-gamma ELISPOT assay. The CTL activity of these CTL lines was measured by IFN-gamma ELISA. Figure 2-1 and Figure 2-2 Compared to target cells not subjected to peptide shock, these CTL lines exhibited strong IFN-gamma generation against target cells shocked with the corresponding peptides. Furthermore, CTL clones were established by limiting dilutions of the CTL lines described in the "Materials and Methods" section above, and IFN-gamma generation from the CTL clones against C1R-A11 shock was measured by IFN-gamma ELISA. Strong IFN-gamma generation was observed in CTL clones stimulated with CDCA1-A11-9-105 (SEQ ID NO:5)(a), CDCA1-A11-9-419 (SEQ ID NO:6)(b), or CDCA1-A11-9-219 (SEQ ID NO:7)(c). Figure 3 ).

[0639] Specific CTL activity against target cells expressing CDCA1 and HLA-A*1101

[0640] A CTL clone targeting CDCA1-A11-9-219 (SEQ ID NO:7) was established to investigate its ability to recognize target cells expressing both CDCA1 and HLA-A*1101 molecules. COS7 cells transfected with both full-length CDCA1 and HLA-A*1101 genes (a specific model of target cells expressing both CDCA1 and HLA-A*1101 genes) were prepared as target cells. COS7 cells transfected with either full-length CDCA1 or HLA-A*1101 were prepared as controls. The CDCA1-A11-9-219 (SEQ ID NO:7)-stimulated CTL clone exhibited strong CTL activity against COS7 cells expressing both CDCA1 and HLA-A*1101. Figure 4 On the other hand, no significant specific CTL activity was detected in the control group. These data clearly demonstrate that CDCA1-A11-9-219 (SEQ ID NO:7) is a peptide derived from endogenously processed CDCA1, presented on target cells with the HLA-A*1101 molecule, and recognized by CTLs. These results suggest the possibility that CDCA1-A11-9-219 (SEQ ID NO:7) could be suitable as a cancer vaccine for patients with cancers expressing CDCA1.

[0641] Homology analysis of antigenic peptides

[0642] CDCA1-A11-9-123(SEQ ID NO:3),CDCA1-A11-9-105(SEQ ID NO:5),CDCA1-A11-9-419(SEQ ID NO:6),CDCA1-A11-9-219(SEQ ID NO:7),CDCA1-A11-9-439(SEQ IDNO:9),CDCA1-A11-9-343(SEQ ID NO:10),CDCA1-A11-9-21(SEQ ID NO:12),CDCA1-A11-9-157(SEQ ID NO:13),CDCA1-A11-9-244(SEQ ID NO:14),CDCA1-A11-9-213(SEQ ID NO:14). NO:17),CDCA1-A11-9-335(SEQ ID NO:19),CDCA1-A11-9-25(SEQ ID NO:21),CDCA1-A11-10-339(SEQ ID NO:30),CDCA1-A11-10-452(SEQ ID NO:35),CDCA1-A11-10-400(SEQ ID NO:35). NO:38),CDCA1-A11-10-289(SEQ ID NO:39),CDCA1-A11-10-203(SEQ ID NO:40),CDCA1-A11-10-156(SEQ ID NO:45),CDCA1-A11-10-340(SEQ ID NO:53),CDCA1-A11-10-106(SEQ IDNO:56), strain CDCA1-A11-10-358(SEQ ID NO:58) encodes CTLs.These results may be due to the following sequences: CDCA1-A11-9-123 (SEQ ID NO:3), CDCA1-A11-9-105 (SEQ ID NO:5), CDCA1-A11-9-419 (SEQ ID NO:6), CDCA1-A11-9-219 (SEQ ID NO:7), CDCA1-A11-9-439 (SEQ ID NO:9), CDCA1-A11-9-343 (SEQ ID NO:10), CDCA1-A11-9-21 (SEQ ID NO:12), CDCA1-A11-9-157 (SEQ ID NO:13), CDCA1-A11-9-244 (SEQ ID NO:14), CDCA1-A11-9-213 (SEQ ID NO:17), CDCA1-A11-9-335 (SEQ ID NO:19), and CDCA1-A11-9-25 (SEQ ID NO:10). The sequences NO:21), CDCA1-A11-10-339 (SEQ ID NO:30), CDCA1-A11-10-452 (SEQ ID NO:35), CDCA1-A11-10-400 (SEQ ID NO:38), CDCA1-A11-10-289 (SEQ ID NO:39), CDCA1-A11-10-203 (SEQ ID NO:40), CDCA1-A11-10-156 (SEQ ID NO:45), CDCA1-A11-10-340 (SEQ ID NO:53), CDCA1-A11-10-106 (SEQ ID NO:56), and CDCA1-A11-10-358 (SEQ ID NO:58) 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 shows that there is no correlation with CDCA1-A11-9-123 (SEQ ID NO:3), CDCA1-A11-9-105 (SEQ ID NO:5), CDCA1-A11-9-419 (SEQ ID NO:6), CDCA1-A11-9-219 (SEQ ID NO:7), CDCA1-A11-9-439 (SEQ ID NO:9), CDCA1-A11-9-343 (SEQ ID NO:10), CDCA1-A11-9-21 (SEQ ID NO:12), CDCA1-A11-9-157 (SEQ ID NO:13), CDCA1-A11-9-244 (SEQ ID NO:14), CDCA1-A11-9-213 (SEQ ID NO:17), CDCA1-A11-9-335 (SEQ ID NO:19), or CDCA1-A11-9-25 (SEQ ID NO:10). The sequences NO:21), CDCA1-A11-10-339 (SEQ ID NO:30), CDCA1-A11-10-452 (SEQ ID NO:35), CDCA1-A11-10-400 (SEQ ID NO:38), CDCA1-A11-10-289 (SEQ ID NO:39), CDCA1-A11-10-203 (SEQ ID NO:40), CDCA1-A11-10-156 (SEQ ID NO:45), CDCA1-A11-10-340 (SEQ ID NO:53), CDCA1-A11-10-106 (SEQ ID NO:56), and CDCA1-A11-10-358 (SEQ ID NO:58) are sequences with significant homology. Therefore, according to the inventors' knowledge, these peptides are virtually impossible to elicit unintended immune responses against other unrelated molecules. In summary, novel CDCA1-derived HLA-A11-restricted epitope peptides were identified. These demonstrate the potential of CDCA1-derived epitope peptides for cancer immunotherapy.

[0643] [Example 2]

[0644] Materials and methods

[0645] cell lines

[0646] C1R, HLA-A negative and HLA-B negative human B lymphoblast cell lines, and COS7, African green monkey kidney cell line were purchased from ATCC.

[0647] The generation of cells with stable HLA-A*3303 expression

[0648] C1R cells stably expressing HLA-A*3303 (C1R-A33) were used as stimulatory cells. The cDNA encoding the open reading frame of HLA-A*3303 was amplified by PCR and cloned into an expression vector. C1R cells were transfected with the expression vector and then selected using G418 (Invitrogen) for two weeks. Cells selected with G418 were seeded into wells of 96-well plates containing medium supplemented with G418 and cultured for another 30 days. Flow cytometry analysis confirmed the expression of exogenous HLA-A*3303 in C1R cells.

[0649] Selection of candidate CDCA1-derived peptides

[0650] 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) predicted the binding of CDCA1-derived 9-mer and 10-mer peptides to the HLA-A*3303 molecule.

[0651] peptide synthesis

[0652] These peptides were synthesized via standard solid-phase synthesis at Biosynthesis (Lewisville, Texas) and purified by reversed-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.

[0653] In vitro CTL induction

[0654] 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 monocytes isolated from healthy volunteers (HLA-A*3303 positive) using Ficoll-Paque Plus (Pharmacia) solution were separated by adhesive plastic tissue culture discs (Becton Dickinson) and concentrated as a monocyte 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 granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml interleukin (IL)-4 (R&D System). After 7 days of culture, cytokine-induced dendritic cells (DCs) were shocked for 3 hours at 37°C with 20 μg / ml of each synthetic peptide in the presence of 3 μg / ml β2-microglobulin in AIM-V medium. The resulting cells epigenetically expressed DC-related molecules such as CD80, CD83, CD86, and HLA class II (data not shown) on their cell surface. These peptide-shocked DCs were then inactivated by X-ray irradiation (20 Gy) and mixed at a 1:20 ratio with autologous CD8+ T cells obtained through positive selection using a CD8+ isolation kit (Dynal). These culture products were seeded into 48-well plates (Corning). Each well was prepared to contain 1.5 x 10⁻⁶ cells in 0.5 ml AIM-V / 2% AS medium. 4One peptide-emergent dendritic cell (DC), 3 x 10⁵ CD8+ T cells, and 10 ng / ml IL-7 (R&D System) were added to these cultures after 3 days. IL-2 (CHIRON) was then added to these cultures to a final concentration of 20 IU / ml. On days 7 and 14, these T cells were further stimulated with peptide-emergent autologous DCs. The DCs were prepared each time using the same method described above. On day 21, after the third peptide stimulation, CTLs targeting peptide-stimulated 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).

[0655] CTL amplification program

[0656] CTLs were expanded in culture using a method similar to that described 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 lymphoblastocyte lines in a total of 25 ml of AIM-V medium (5 x 10⁻⁶ cells / mL). 6Cells / flask), the culture medium contained 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).

[0657] Establishment of CTL clones

[0658] CTLs were diluted in 96 round-bottom microtiter plates (Nalge Nunc International) to prepare 0.3, 1, and 3 cells / well. CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblast cell lines (1 x 10⁻⁶ cells / well) in 150 μL / well AIM-V / 5% AS medium containing a total of 30 ng / ml anti-CD3 antibody and 125 IU / ml IL-2. 4 Cells / well). After 10 days, 50 μL / well of IL-2 was added to the culture medium to reach a final concentration of 125 IU / ml. CTL activity was tested on day 14, and CTL clones were amplified 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).

[0659] Specific CTL activity

[0660] To examine specific CTL activity, IFN-gamma ELISPOT assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-stimulated C1R-A33 (1x10⁻¹⁰) was prepared. 4Cells / wells were used as stimulating cells. Induced CTLs (i.e., CTL lines and CTL clones) were used as responding cells. IFN-gamma ELISPOT assay and IFN-gamma ELISA were performed according to the manufacturer's instructions.

[0661] Establishment of target cells forcibly expressing target genes and HLA-A*3303

[0662] cDNA encoding the target gene or the open reading frame of HLA-A*3303 was amplified by PCR. The PCR amplification product was cloned into an expression vector. Using Lipofectamine 2000 (Invitrogen), one or both of the vectors expressing the target gene and expressing HLA-A*3303 were transfected into COS7 cells (a target gene and HLA-negative cell line) according to the manufacturer's instructions. Two days after transfection, the transfected cells were harvested with Versene (Invitrogen) and used as target cells (5 x 10⁻⁶) for CTL activity assay. 4 (cells / well).

[0663] result

[0664] Prediction of CDCA1-derived HLA-A*3303 binding peptide

[0665] Tables 2a and 2b show the CDCA1-derived 9- and 10-meric peptides predicted to bind 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 determine epitope peptides.

[0666] [Table 2a]

[0667] HLA-A33-binding 9-peptide derived from CDCA1

[0668] Starting position amino acid sequence Kd(nM) SEQ ID NO 43 EVLHMIYMR 27.11 27 127 FIHFREACR 54.78 59 123 GIINFIHFR 63.05 3 108 DILCPKAKR 68.75 60 261 TVQKLKNAR 190.11 28 105 ETADILCPK 218.82 5 138 YMEFLWQYK 688.93 11 335 KTEENSFKR 1318.61 19 137 TYMEFLWQY 1567.83 20 443 DSYAKIDEK 1746.99 25 340 SFKRLMIVK 1864.16 23 11 VAEIVIHIR 1904.94 61 382 EKRGAVYER 2145.7 62 111 CPKAKRTSR 2635.42 63 354 TAQFKINKK 2664.09 8 376 DCNKVQEKR 2751.98 64 52 ALQIVYGIR 3000.81 65 299 KIQDLSDNR 3099.81 18

[0669] [Table 2b]

[0670] HLA-A33-binding decapeptide derived from CDCA1

[0671] Starting position amino acid sequence Kd(nM) SEQ ID NO 126 NFIHFREACR 39.6 66 137 TYMEFLWQYK 74.97 42 10 NVAEIVIHIR 101.5 67 122 SGIINFIHFR 228.5 68 260 DTVQKLKNAR 375.87 69 91 HLDSFLPICR 514.41 70 51 RALQIVYGIR 819.15 71 339 NSFKRLMIVK 855.38 30 225 ELKLSVVSLK 984.56 54 340 SFKRLMIVKK 1039.29 53 2 ETLSFPRYNV 1362.11 72 232 SLKEIQESLK 2077.17 49 452 TAELKRKMFK 2314.53 35 159 HQEALMKLER 2904.96 73

[0672] The start position indicates the number of amino acid residues starting from the N-terminus of CDCA1, and the dissociation constant [Kd(nM)] is derived from "NetMHCpan2.8".

[0673] Inducing CTLs via predicted CDCA1-derived HLA-A*3303-restricted peptide

[0674] CTLs targeting CDCA1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured using an IFN-gamma ELISPOT assay. Figure 5 Compared with the control, the CTLs in well #2(a) with CDCA1-A33-9-43 (SEQ ID NO: 27), well #1(b) with CDCA1-A33-9-123 (SEQ ID NO: 3), well #3(c) with CDCA1-A33-9-108 (SEQ ID NO: 60), well #2(d) with CDCA1-A33-9-261 (SEQ ID NO: 28), well #6(e) with CDCA1-A33-9-105 (SEQ ID NO: 5), well #8(f) with CDCA1-A33-10-10 (SEQ ID NO: 67), and well #5(g) with CDCA1-A33-10-260 (SEQ ID NO: 69) showed strong IFN-gamma generation. Meanwhile, although other peptides shown in Tables 2a and 2b potentially possess HLA-A*3303 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 CDCA1-A33-10-122 (SEQ ID NO: 68). As a result, seven types of CDCA1-derived peptides were selected as peptides capable of inducing effective CTLs.

[0675] Establishment of CTL lines and cloning of HLA-A*3303-restricted peptides derived from CDCA1

[0676] CTL lines were established by amplifying CTLs in well #2 (a) containing CDCA1-A33-9-43 (SEQ ID NO:27), well #1 (b) containing CDCA1-A33-9-123 (SEQ ID NO:3), well #8 (c) containing CDCA1-A33-10-10 (SEQ ID NO:67), and well #5 (d) containing CDCA1-A33-10-260 (SEQ ID NO:69). These CTL lines exhibited peptide-specific CTL activity in an IFN-gamma ELISPOT assay. The CTL activity of these CTL lines was measured by IFN-gamma ELISA. Figure 6These CTL lines exhibited strong IFN-gamma generation against target cells bombarded with the corresponding peptides, compared to target cells not bombarded with the peptides. Furthermore, CTL clones were established by limiting dilutions of the CTL lines described in the "Materials and Methods" section above, and IFN-gamma generation from the CTL clones against C1R-A33 bombarded with the peptides was measured by IFN-gamma ELISA. Strong IFN-gamma generation was observed in CTL clones stimulated with CDCA1-A33-9-43 (SEQ ID NO:27)(a) or CDCA1-A33-9-123 (SEQ ID NO:3)(b). Figure 7 ).

[0677] Specific CTL activity against target cells expressing CDCA1 and HLA-A*3303

[0678] This study investigated the ability of a CTL clone established targeting CDCA1-A33-9-43 (SEQ ID NO:27) to recognize target cells expressing both CDCA1 and HLA-A*3303 molecules. COS7 cells transfected with both full-length CDCA1 and HLA-A*3303 genes (a specific model of target cells expressing both CDCA1 and HLA-A*3303 genes) were prepared as target cells. COS7 cells transfected with either full-length CDCA1 or HLA-A*3303 were prepared as controls. The CTL clone stimulated with CDCA1-A33-9-43 (SEQ ID NO:27) exhibited strong CTL activity against COS7 cells expressing both CDCA1 and HLA-A*3303. Figure 8 On the other hand, no significant specific CTL activity was detected in the control group. These data clearly demonstrate that CDCA1-A33-9-43 (SEQ ID NO:27) is a peptide derived from endogenously processed CDCA1, presented on target cells along with the HLA-A*3303 molecule, and recognized by CTLs. These results suggest the possibility that CDCA1-A33-9-43 (SEQ ID NO:27) could be suitable as a cancer vaccine for patients with cancers expressing CDCA1.

[0679] Homology analysis of antigenic peptides

[0680] The homology may be due to the fact that the sequences CDCA1-A33-9-43 (SEQ ID NO:27), CDCA1-A33-9-123 (SEQ ID NO:3), CDCA1-A33-9-108 (SEQ ID NO:60), CDCA1-A33-9-261 (SEQ ID NO:28), CDCA1-A33-9-105 (SEQ ID NO:5), CDCA1-A33-10-10 (SEQ ID NO:67), and CDCA1-A33-10-260 (SEQ ID NO:69) 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 no significant homology with the sequences CDCA1-A33-9-43 (SEQ ID NO:27), CDCA1-A33-9-123 (SEQ ID NO:3), CDCA1-A33-9-108 (SEQ ID NO:60), CDCA1-A33-9-261 (SEQ ID NO:28), CDCA1-A33-9-105 (SEQ ID NO:5), CDCA1-A33-10-10 (SEQ ID NO:67), and CDCA1-A33-10-260 (SEQ ID NO:69). Therefore, based on the inventors' knowledge, these peptides are highly unlikely to elicit unintended immune responses against other unrelated molecules. In summary, novel CDCA1-derived HLA-A33-restricted epitope peptides were identified. The suitability of these CDCA1-derived epitope peptides for cancer immunotherapy is demonstrated.

[0681] [Example 3]

[0682] Materials and methods

[0683] cell lines

[0684] C1R, HLA-A negative and HLA-B negative human B lymphoblast cell lines, and COS7, African green monkey kidney cell line were purchased from ATCC.

[0685] The generation of cells with stable HLA-A*0301 expression

[0686] C1R cells stably expressing HLA-A*0301 (C1R-A03) were used as stimulatory 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 using G418 (Invitrogen) for two weeks. Cells selected with G418 were seeded into wells of 96-well plates containing medium supplemented with G418 and cultured for another 30 days. Flow cytometry analysis confirmed the expression of exogenous HLA-A*0301 in C1R cells.

[0687] Selection of derived CDCA1 peptide candidates

[0688] 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) predicted the binding of CDCA1-derived 9-mer and 10-mer peptides to the HLA-A*0301 molecule.

[0689] peptide synthesis

[0690] These peptides were synthesized via standard solid-phase synthesis at Biosynthesis (Lewisville, Texas) and purified by reversed-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.

[0691] In vitro CTL induction

[0692] 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 monocytes isolated from healthy volunteers (HLA-A*0301 positive) using Ficoll-Paque Plus (Pharmacia) solution were separated by adhesive plastic tissue culture discs (Becton Dickinson) and concentrated as a monocyte 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 granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU / ml interleukin (IL)-4 (R&D System). After 7 days of culture, cytokine-induced dendritic cells (DCs) were shocked for 3 hours at 37°C with 20 μg / ml of each synthetic peptide in the presence of 3 μg / ml β2-microglobulin in AIM-V medium. The resulting cells epigenetically expressed DC-related molecules such as CD80, CD83, CD86, and HLA class II (data not shown) on their cell surface. These peptide-shocked DCs were then inactivated by X-ray irradiation (20 Gy) and mixed at a 1:20 ratio with autologous CD8+ T cells obtained through positive selection using a CD8+ isolation kit (Dynal). These culture products were seeded into 48-well plates (Corning). Each well was prepared to contain 1.5 x 10⁻⁶ cells in 0.5 ml AIM-V / 2% AS medium. 4One peptide-emergent dendritic cell (DC), 3 x 10⁵ CD8+ T cells, and 10 ng / ml IL-7 (R&D System) were added to these cultures after 3 days. IL-2 (CHIRON) was then added to these cultures to a final concentration of 20 IU / ml. On days 7 and 14, these T cells were further stimulated with peptide-emergent autologous DCs. The DCs were prepared each time using the same method described above. On day 21, after the third peptide stimulation, CTLs targeting peptide-stimulated 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).

[0693] CTL amplification program

[0694] CTLs were expanded in culture using a method similar to that described 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 lymphoblastocyte lines in a total of 25 ml of AIM-V medium (5 x 10⁻⁶ cells / mL). 6Cells / flask), the culture medium contained 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).

[0695] Establishment of CTL clones

[0696] CTLs were diluted in 96 round-bottom microtiter plates (Nalge Nunc International) to prepare 0.3, 1, and 3 cells / well. CTLs were co-cultured with two types of mitomycin C-treated human B lymphoblast cell lines (1 x 10⁻⁶ cells / well) in 150 μL / well AIM-V / 5% AS medium containing a total of 30 ng / ml anti-CD3 antibody and 125 IU / ml IL-2. 4 Cells / well). After 10 days, 50 μL / well of IL-2 was added to the culture medium to reach a final concentration of 125 IU / ml. CTL activity was tested on day 14, and CTL clones were amplified 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).

[0697] Specific CTL activity

[0698] To examine specific CTL activity, IFN-gamma ELISPOT assay and IFN-gamma enzyme-linked immunosorbent assay (ELISA) were performed. Specifically, peptide-stimulated C1R-A03 (1x10⁻¹⁰) was prepared. 4Cells / wells were used as stimulating cells. Induced CTLs (i.e., CTL lines and CTL clones) were used as responding cells. IFN-gamma ELISPOT assay and IFN-gamma ELISA were performed according to the manufacturer's instructions.

[0699] Establishment of target cells for forced expression of target genes and HLA-A*0301

[0700] cDNA encoding the target gene or the open reading frame of HLA-A*0301 was amplified by PCR. The PCR amplification product was cloned into an expression vector. Using Lipofectamine 2000 (Invitrogen), one or both of the vectors expressing the target gene and expressing HLA-A*0301 were transfected into COS7 cells (a target gene and HLA-negative cell line) according to the manufacturer's instructions. Two days after transfection, the transfected cells were harvested with Versene (Invitrogen) and used as target cells (5 x 10⁻⁶) for CTL activity assay. 4 (cells / well).

[0701] result

[0702] Prediction of CDCA1-derived HLA-A*0301 binding peptide

[0703] Tables 3a and 3b show the CDCA1-derived 9- and 10-meric peptides predicted to bind to HLA-A*0301 in descending order of binding affinity. A total of 24 peptides with potential HLA-A*0301 binding ability were selected and examined to determine epitope peptides.

[0704] [Table 3a]

[0705] HLA-A03-binding 9-peptide derived from CDCA1

[0706] Starting position amino acid sequence Kd(nM) SEQ ID NO 343 RLMIVKKEK 56 10 353 ATAQFKINK 112 1 55 IVYGIRLEH 139 22 193 SLNQDFHQK 160 4 204 VLQEGNSQK 206 16 21 KILTGADGK 263 12 219 KTKRLNELK 303 7 49 YMRALQIVY 399 74 143 WQYKSSADK 432 75

[0707] [Table 3b]

[0708] HLA-A03-binding decapeptide derived from CDCA1

[0709] Starting position amino acid sequence Kd(nM) SEQ ID NO 423 FLNLKTALEK 41 31 333 KLKTEENSFK 52 46 400 KIKLGIQQLK 55 38 257 KMKDTVQKLK 67 47 250 KLKNYKEKMK 132 76 203 IVLQEGNSQK 155 40 339 NSFKRLMIVK 160 30 353 ATAQFKINKK 205 29 243 KIVDSPEKLK 261 43 447 KIDEKTAELK 274 44 232 SLKEIQESLK 373 49 418 KSQEIFLNLK 386 32 452 TAELKRKMFK 387 35 225 ELKLSVVSLK 389 54 289 CQLEVQLYQK 488 39

[0710] The start position indicates the number of amino acid residues starting from the N-terminus of CDCA1, and the dissociation constant [Kd(nM)] is derived from "NetMHC3.2".

[0711] Inducing CTLs via predicted CDCA1-derived HLA-A*0301-restricted peptides

[0712] CTLs targeting CDCA1-derived peptides were generated according to the protocol described in "Materials and Methods". Peptide-specific CTL activity was measured using an IFN-gamma ELISPOT assay. Figure 9 Compared to the control, well #3(a) with CDCA1-A03-9-219 (SEQ ID NO: 7), well #1(b) with CDCA1-A03-10-400 (SEQ ID NO: 38), and well #2(c) with CDCA1-A03-10-257 (SEQ ID NO: 47) showed strong IFN-gamma generation. Meanwhile, although other peptides shown in Tables 3a and 3b potentially possess HLA-A*0301 binding activity, no specific CTL activity was detected as a result of stimulation by these peptides. An example of typical negative data is the absence of specific IFN-gamma generation observed in CTLs stimulated with CDCA1-A03-9-343 (SEQ ID NO: 10) (d). As a result, three types of CDCA1-derived peptides were selected as peptides capable of inducing effective CTLs.

[0713] Establishment of CTL line and cloning of CDCA1-derived HLA-A*0301 restricted peptide

[0714] CTL lines were established by amplifying CTLs in well #3(a) containing CDCA1-A03-9-219 (SEQ ID NO:7), well #1(b) containing CDCA1-A03-10-400 (SEQ ID NO:38), and well #2(c) containing CDCA1-A03-10-257 (SEQ ID NO:47). These CTL lines exhibited peptide-specific CTL activity in an IFN-gamma ELISPOT assay. The CTL activity of these CTL lines was measured by IFN-gamma ELISA. Figure 10 Compared to target cells not subjected to peptide shock, these CTL lines exhibited strong IFN-gamma generation against target cells shocked with the corresponding peptides. Furthermore, CTL clones were established by limiting dilutions of the CTL lines described in the "Materials and Methods" section above, and IFN-gamma generation from CTL clones against peptide shock in C1R-A03 was measured by IFN-gamma ELISA. Strong IFN-gamma generation was observed in CTL clones stimulated with CDCA1-A03-9-219 (SEQ ID NO:7)(a), CDCA1-A03-10-400 (SEQ ID NO:38)(b), and CDCA1-A03-10-257 (SEQ ID NO:47)(c). Figure 11 ).

[0715] Specific CTL activity against target cells expressing CDCA1 and HLA-A*0301

[0716] This study investigated the ability of CTL clones established using CDCA1-A03-9-219 (SEQ ID NO:7)(a) and CDCA1-A03-10-400 (SEQ ID NO:38)(b) to recognize target cells expressing both CDCA1 and HLA-A*0301 molecules. COS7 cells transfected with both full-length CDCA1 and HLA-A*0301 genes (a specific model of target cells expressing both CDCA1 and HLA-A*0301 genes) were prepared as target cells. COS7 cells transfected with either full-length CDCA1 or HLA-A*0301 were prepared as controls. CTL clones stimulated with CDCA1-A03-9-219 (SEQ ID NO:7)(a) or CDCA1-A03-10-400 (SEQ ID NO:38)(b) exhibited strong CTL activity against COS7 cells expressing both CDCA1 and HLA-A*0301. Figure 12 On the other hand, no significant specific CTL activity was detected in the control group. These data clearly demonstrate that CDCA1-A03-9-219 (SEQ ID NO:7) and CDCA1-A03-10-400 (SEQ ID NO:38) are peptides derived from endogenously processed CDCA1, presented on target cells with the HLA-A*0301 molecule, and recognized by CTLs. These results suggest the possibility that CDCA1-A03-9-219 (SEQ ID NO:7) and CDCA1-A03-10-400 (SEQ ID NO:38) could be suitable as cancer vaccines for patients with cancers expressing CDCA1.

[0717] Homology analysis of antigenic peptides

[0718] CTLs stimulated with CDCA1-A03-9-219 (SEQ ID NO:7), CDCA1-A03-10-400 (SEQ ID NO:38), or CDCA1-A03-10-257 (SEQ ID NO:47) exhibited significant specific CTL activity. These results may be because the sequences of CDCA1-A03-9-219 (SEQ ID NO:7), CDCA1-A03-10-400 (SEQ ID NO:38), and CDCA1-A03-10-257 (SEQ ID NO:47) 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 CDCA1-A03-9-219 (SEQ ID NO:7), CDCA1-A03-10-400 (SEQ ID NO:38), and CDCA1-A03-10-257 (SEQ ID NO:47). Therefore, based on the inventors' knowledge, these peptides are highly unlikely to elicit unintended immune responses against other unrelated molecules. In summary, novel CDCA1-derived HLA-A03-restricted epitope peptides were identified. The suitability of these CDCA1-derived epitope peptides for cancer immunotherapy is demonstrated.

[0719] [Example 4]

[0720] Preparation of emulsion formulations

[0721] The peptide is dissolved in an injectable solvent or sterile saline to a concentration of 1.0 mg / ml to 10.0 mg / ml and collected in a syringe. This is connected via a connector to a syringe containing an equal volume of IFA (injectable solvent or sterile saline), and then mixed by alternately pushing the plungers of the two connected syringes. After mixing for several minutes, the completion of the emulsion is evaluated using a drop test method. The drop test method is performed by dropping a drop of the mixed sample onto water. The emulsion is assessed as complete when the sample dropped onto the water does not immediately diffuse in the water; and as incomplete when the sample dropped onto the water immediately diffuses in the water. When the emulsion is assessed as incomplete, further mixing is performed to complete the emulsion. The completed emulsion can be administered to cancer patients via subcutaneous injection. Cancer patients who receive this treatment may be selected from those affected by the following cancers: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, colorectal cancer, etc.

[0722] Preparation of freeze-dried formulations

[0723] The peptide is dissolved in an injectable solvent to a concentration of 1.0 mg / ml to 10.0 mg / ml and then filtered and sterilized. It is then placed into a sterile vial and partially sealed with a sterile rubber stopper. After lyophilizing the vial, it is completely sealed and sutured with an aluminum cap to produce the lyophilized formulation. When needed, the lyophilized powder is redissolved in the vial by injecting either the injectable solvent or sterile saline. The redissolved solution is collected using a syringe, and the syringe is connected via a connector to a syringe filled with the same amount of IFA as the collected redissolved solution. The redissolved solution and IFA are mixed by alternately pushing the plungers of the two connected syringes. After mixing for several minutes, the completion of the emulsion is evaluated using a drop test method. The completed emulsion can be administered to cancer patients via subcutaneous injection. Cancer patients who receive this treatment may be selected from those affected by the following cancers: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, colorectal cancer, etc.

[0724] Industrial application

[0725] This invention provides novel HLA-A11-restricted, HLA-A33-restricted, and HLA-A03-restricted epitope peptides derived from CDCA1 that induce strong and specific antitumor immune responses and are therefore available across a wide range of cancer types. The peptides, compositions, APCs, and CTLs of this invention can be used as peptide vaccines against CDCA1-expressing cancers, such as bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, and colorectal cancer.

[0726] Although the invention has been described in detail and with respect to specific embodiments herein, it should be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Those skilled in the art will readily recognize through conventional experimentation that various changes and modifications can be made to the invention without departing from its spirit and scope, the boundaries and scope of which are defined by the appended claims.

Claims

1. A peptide having the ability to induce cytotoxic T cells (CTLs) in an HLA-A33 restricted manner, comprising the amino acid sequence of SEQ ID NO: 27, 60, 28, 67 or 69.

2. A polynucleotide encoding the peptide of claim 1.

3. A composition comprising a pharmaceutically acceptable carrier and at least one ingredient selected from groups (a) to (e) below, said composition being formulated for administration to an HLA-A33-positive subject: (a) The peptide according to claim 1; (b) A polynucleotide that encodes the peptide of claim 1 in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide of claim 1 and the HLA-A33 antigen on their cell surface; (d) An exogenous body that presents a complex of the peptide of claim 1 and the HLA-A33 antigen on its cell surface; and (e) CTL, which targets the peptide of claim 1.

4. The composition according to claim 3, wherein the component is at least one component selected from the group consisting of (a) to (d): (a) The peptide according to claim 1; (b) A polynucleotide that encodes the peptide of claim 1 in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide of claim 1 and the HLA-A33 antigen on their cell surface; and (d) An exogenous body that presents a complex of the peptide of claim 1 and the HLA-A33 antigen on its cell surface.

5. The composition according to claim 3, wherein it is a pharmaceutical composition.

6. The composition according to claim 5, for use in one or more of the following: (i) cancer treatment, (ii) cancer prevention, and (iii) prevention of cancer recurrence after surgery.

7. The composition according to claim 5, used to induce an immune response against cancer.

8. The composition according to claim 6 or 7, wherein the cancer is selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia (CML), esophageal cancer, gastric cancer, non-small cell lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer, small cell lung cancer, head and neck cancer, soft tissue tumors, and colorectal cancer.

9. An in vitro or ex vivo method for inducing APCs with CTL-inducible ability, comprising steps selected from the group consisting of: (a) Contacting an HLA-A33-expressing APC with the peptide of claim 1 in vitro or in vitro; and (b) Introducing a polynucleotide encoding the peptide of claim 1 into an APC expressing HLA-A33.

10. An in vitro method for inducing CTLs, comprising the following (a) or (b) steps: (a) Co-culturing CD8-positive T cells with APCs, wherein the APCs present a complex of HLA-A33 antigen and the peptide of claim 1 on their surface; or (b) Co-culturing CD8 positive T cells with an exogenous body, the exogenous body presenting a complex of HLA-A33 antigen and the peptide of claim 1 on its surface.

11. An APC that presents a complex of HLA-A33 antigen and the peptide of claim 1 on its surface.

12. The APC of claim 11, induced by an in vitro or ex vivo method, said method comprising steps selected from the group consisting of: (a) Contacting an HLA-A33-expressing APC with the peptide of claim 1 in vitro or in vitro; and (b) Introducing a polynucleotide encoding the peptide of claim 1 into an APC expressing HLA-A33.

13. CTL, which targets the peptide of claim 1.

14. The CTL of claim 13, induced by an in vitro method, said method comprising the following (a) or (b) steps: (a) Co-culturing CD8-positive T cells with APCs, wherein the APCs present a complex of HLA-A33 antigen and the peptide of claim 1 on their surface; or (b) Co-culturing CD8 positive T cells with an exogenous body, the exogenous body presenting a complex of HLA-A33 antigen and the peptide of claim 1 on its surface.

15. Use of at least one component selected from groups (a) to (e) in the preparation of a medicament for inducing an immune response against cancers expressing CDCA1 in an HLA-A33-restricted manner: (a) The peptide according to claim 1; (b) A polynucleotide that encodes the peptide of claim 1 in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide of claim 1 and the HLA-A33 antigen on their cell surface; (d) An exogenous body that presents a complex of the peptide of claim 1 and the HLA-A33 antigen on its cell surface; and (e) CTL, which targets the peptide of claim 1.

16. Use of at least one component selected from groups (a) to (e) in the preparation of a medicament for treating cancers expressing CDCA1 in HLA-A33-positive subjects: (a) The peptide according to claim 1; (b) A polynucleotide that encodes the peptide of claim 1 in an expressible form; (c) Antigen-presenting cells (APCs) that present a complex of the peptide of claim 1 and the HLA-A33 antigen on their cell surface; (d) An exogenous body that presents a complex of the peptide of claim 1 and the HLA-A33 antigen on its cell surface; and (e) CTL, which targets the peptide of claim 1.

17. A method for screening peptides with CTL-inducible ability, comprising the following steps: (a) Generate a candidate sequence consisting of an amino acid sequence in which one, two, three or fewer amino acid residues are replaced in an original amino acid sequence consisting of amino acid sequences selected from the group consisting of: SEQ ID NOs: 27, 60, 28, 67 and 69; (b) Select candidate sequences from the candidate sequences generated in (a) that do not have significant homology (sequence identity) with any known human gene product other than CDCA1; (c) Contact the APC expressing HLA-A33 with a peptide composed of the candidate sequences selected in (b); (d) Expose the APCs from (c) to CD8-positive T cells; and (e) Select peptides that have equal or higher CTL induction capacity compared to peptides composed of the original amino acid sequence.

18. An emulsion comprising the peptide, water-soluble carrier, and oil adjuvant of claim 1.

19. A kit comprising a container containing the composition of any one of claims 3 to 8 and a container containing an adjuvant.

20. Use of the peptide of claim 1 or a polynucleotide encoding the peptide of claim 1 in the preparation of a composition for inducing APCs having CTL induction ability in an HLA-A33 restricted manner, wherein the induction comprises: (a) Contacting an APC expressing HLA-A33 with the peptide of claim 1 in vitro, in vitro or in vivo; and (b) Introducing a polynucleotide encoding the peptide of claim 1 into an APC expressing HLA-A33.

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