AU2012227350B2 - HLA-A*1101-Restricted WT1 peptide and pharmaceutical composition comprising the same - Google Patents

Abstract

C XNRPonhnD)C(AARW 2"'>_1 I)OC-9'252 1.' -38 A BST RA CT The present invention relates to an lLA-A*l 101-restricted WTI peptide, specifically, a peptide comprising an amino acid ;equencc consisting of 9 contiguous amino acids from a WT1 protein, wherein the peptide has an ability to bind to an HlLA-A*l 101 molecule, and has an ability to induce a CTL. The present invention also relates to a peptide dimer having an ability to bind to an HlIA-A*I 101 molecule and having an ability to induce a CTL, in which two peptide monomers each comprising an amino acid sequence consisting of 9 contiguous amino acids from a WTI protein and comprising at least one cysteine residue are bound to each other through a disulfide bond. Furthermore, the present invention relates to a polynuclcotide encoding the peptide, a pharmaceutical composition for the treatment and/or prevention of a cancer comprising the same and the like.

Description

P/00/0 11 R regulation 32 A U STR A L I A Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT (ORIGINAL) Name of Applicant: International Institute of Cancer Immunology, Inc. of 13-9, Enoki-cho, Suita-shi, Osaka, 5640053, Japan Actual Inventors: Haruo SUGIYAMA Address for Service: DAVIES COLLISON CAVE. Patent & Trademark Attorneys, of I Nicholson Street, Melbourne, 3000, Victoria, Australia Ph: 03 9254 2777 Fax: 03 9254 2770 Attorney Code: DM Invention Title: LA-A*1 101-Restricted WTI peptide and pharmaceutical composition comprising the same The following statement is a full description of this invention, including the best method of' performing it known to us:- CONRPonhRDCOAARU327(1 I DOC--/25/2112 - I DESCRIPTION IILA-A* 1101 -RESTRICTED WTI PEPTIDE AND PHARMACEUTICAL COMPOSITION COMPRISING Ti-lE SAME 5 This application is a divisional of Australian Application No. 2007340679, the entire contents of which are incorporated herein by reference. Technical Field 10 100011 The present invention relates to an 11LA-A* 1101-restricted WTlI peptide, specifically, a peptide comprising an amino acid sequence consisting of' 9 contiguous amino acids from a WTI protein, wherein the peptide has an ability to bind to an H-lLA-A*1101 molecule, and has an ability to induce a CTI.. The present 15 invention also relates to a peptide dimer having an ability to bind to an H-1 LA-A*I 101 molecule, and having an ability to induce a CTL, wherein two peptide monomers each comprising an amino acid sequence consisting of 9 contiguous amino acids from a WT1I protein and comprising at least one cysteine residue are bound to each other through a disulfide bond. Furthermore, the present invention relates to a polynucleotide encoding 20 the pcptide, a pharmaceutical composition for the treatment and/or prevention of' a cancer comprising the same and the like. Background C \N~nDCCA~ r i 1 IXll,/25'2,I 2 -2 [00021 WTI gene (Wilms' tumor I gene) was identified as a gene responsible for Wilms tumor which is a renal cancer in children (Non-patent Documents I and 2). WTI is a transcription factor having a zinc Finger structure. At the beginning, the WTl 5 gene was considered to be a tumor suppressor gene. However, subsequent studies (Non-patent Documents 3, 4, 5 and 6) showed that the WTI gene rather functions as an oncogene in hematopoictic tumors and solid cancers. 10003] The WTI gene is expressed at high levels in many types of malignant 10 tumors. Then, it has been examined whether or not the WTI gene product free of mutations, which is an autologous protein, has immunogenicity in a living body. The results revealed that the protein derived from the WTI gene which is expressed at high levels in tumor cells is fragmented through intracellular processing, the resulting peptides form complexes with MIC class I molecules, and the complexes are presented 15 on the surfaces of cells, and that CTILs recognizing such complexes can be induced by peptide vaccination (Non-patent Documents 7, 8 and 9). It was also shown that in a mouse immunized with a WTI peptide or a WTI cDNA, transplanted tumor cells expressing a WT1 gene are rejected with a high probability (Non-patent Documents 7 and 10), while normal tissues expressing physiologically the WTI gene are not 20 damaged by the induced CTLs (Non-patent Document 7). It was shown in in vitro experiments using human cells that when Dbl26 peptide or WI-187 peptide (amino acids 187-195 of SEQ ID No: 1, SLGEQQYSV) having a high ability to bind to an HLA-A*0201 molecule, which is one of human MIC class I molecules, is used to % NRPonhD 0 AR1'27l_ DO .12r I11 stimulate human peripheral blood mononuclear cells having HLA-A 0201, WTlI specific CTLs are induced, the induced CTLs have a cytotoxic activity specific for tumor cells expressing endogenously a WTl gene at high levels, and the cytotoxic activity of such CTLs is -LA-A2-restricted (Non-patent Document 11). It was shown 5 in in vitro experiments in human cells using WTI peptide that matches -ILA-A'2402 (which is found most frequently in Japanese people among IILA-A alleles) (WT1235; amino acids 235-243 of SEQ ID No: 1, CMTWNQMNI..) that WTI-specific CTLs (TAK-I) are induced (Non-patent Document 12), and the induced CTLs do not suppress the colony-forming activity of normal hematopoietic stem cells which partially express 10 physiologically a WTI gene (Non-palcnt Documents 12 and 13). These reports strongly suggest that not only in mice but also in human beings, WTI-specilic CTLs can be induced, such CTLs have a cytotoxic activity against tumor cells expressing a WTl gene at high levels, but do not have a cytotoxic activity against normal cells expressing physiologically a WTl gene (Non-patent Documents 7, 10, 11, 12 and 13). 15 100041 The WTi gene product is present as a nuclear protein, and is processed by proteasomes in cytoplasm to be fragmented into peptides. The fragmented peptides are transported into endoplasmic reticulum lumen by TAP (transporter associated with antigen processing) molecules, form complexes with MIC class I molecules, and are 20 presented on the surfaces of cells. WTl-specific CTLs are induced as a result of' recognition of WT1 peptide-M-IC class I molecule complexes by CTL precursor cells via TCR, thereby exerting a cytotoxic effect on tumor cells presenting a WTI gene product through MHC class I molecules (Non-patent Documents 7, 8 and 9). Then, it is 2.\NR2~ont~d)CCAAR~l(.27( .DO ICC-'?2'21'12 -4 required at least that a WT1 peptide used in cancer immunotherapy targeting a WTI gene product is in the form that binds to an MI-IC class I molecule in a living body. However, MIC class I molecules are diverse and amino acid sequences of the WTI peptides binding to respective MIC class I molecules are different from each other. 5 Therefore, it is required to provide a peptide matching each subtype of MIC class I. However, only -ILA-A'2402 molecule-. -ILA-A'0201 molecule-, -ILA-A*2601 molecule- and HLA-A'3303 molecule-restricted peptides are known as HLA molecule restricted WTI peptides to date (Patent Document 1, Non-patent Document 11, Patent Document 2 and Patent Document 3, respectively). Thus, there is a need to find an 10 ILA-A*I 101-restricted WTI peptide. Patent Document 1: WO 2003/106682 Patent Document 2: WO 2005/095598 Patent Document 3: Japanese Patent Application No. 2006-45287 Non-patent Document 1: Daniel A. Haber et al., Cell. 1990 Jun 29: 15 6](7):1257-69. Non-patent Document 2: Call KM et al., Cell. 1990 Feb 9: 60(3):509-20. Non-patent Document 3: Menke AL et al., Int Rev Cytol. 1998; 181:151-212. Review. 20 Non-patent Document 4: Yamagami T et al., Blood. 1996 Apr 1; 87(7):2878-84. Non-patent Document 5: Inoue K et al., Blood. 1998 Apr 15; 91(8):2969-76.

C NRPonblDC1AARw127'_. DOC-'/)1212nl2 -5 Non-patent Document 6: Tsuboi A et al., Leuk Res. 1999 May; 23(5):499-505. Non-patent Document 7: Oka Y et al., .1 Immunol. 2000 Feb 15: 164(4):1873-80. 5 Non-patent Document 8: Melief CJ ct al., Immunol Rev. 1995 .un: 145:167-77. Non-patent Document 9: Ritz J, .1 Clin Oncol. 1994 Feb; 12(2):237 8. Non-patent Document 10: Tsuboi A et al., J Clin Immunol. 2000 10 May; 20(3):195-202. Non-patent Document 11: Oka Y et al., Immunogenetics. 2000 Feb; 51(2):99-107. Non-patent Document 12: Ohminami -1 et al.. Blood. 2000 Jan 1; 95(1):286-93. 15 Non-patent Document 13: Gao L et al., Blood. 2000 Apr 1: 95(7):2198-203. Disclosure of Invention Problems to be Solved by the Invention 20 [00051 The problems to be solved by the present invention arc to provide a peptide that is an HILA-A*1101 molecule-restricted and comprises an amino acid sequence from a WTI protein, and a polynucleotide encoding the same, as well as a -6 pharmaceutical composition for the treatment and/or prevention of a cancer, comprising the same, and the like. Means to Solve the Problems S [00061 As a result of intensive studies in view of the situation as described above, the present inventor has found that among peptides each comprising an amino acid sequence consisting of 9 contiguous amino acids from a WTI protein, peptides each having an ability to bind to an HlLA-A*1 101 molecule can induce a WTl -specific 10 CTL with a high rate. Thus, the present invention has been completed. 100071 The present invention provides: (1) a peptide comprising an amino acid sequence consisting of 9 contiguous amino acids from a WT1 protein, wherein the peptide has an ability to bind 15 to an H Ll.,A-A*1 101 molecule, and has an ability to induce a CTL; (2) the peptide according to (1), wherein the amino acid at position 9 of the amino acid sequence is Lys or Arg; (3) the peptide according to (1), wherein the amino acid sequence is selected from the group consisting ot': 20 Ala Ala Gly Ser Ser Ser Ser Val Lys (SEQ 11) No: 2), Pro lie Leu Cys Gly Ala Gin Tyr Arg (SEQ I) No: 3), Arg Ser Ala Ser Glu Thr Ser Glu Lys (SEQ 11) No: 4), Scr Ala Ser Glu Thr Ser Glu Lys Arg (SEQ I) No: 5), C \NRPorl\ID(CC\AAR 'M27C' _1 DOC-220212 -7 Ser His Lcu Gin Met His Ser Arg Lys (SEQ ID No: 6), Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ ID No: 7), Lys Thr Cys Gin Arg Lys Phe Ser Arg (SEQ ID No: 8), Ser Cys Arg Trp Pro Ser Cys Gin Lys (SEQ ID No: 9), and 5 Asn Met His Gin Arg Asn Met Thr Lyls (SEQ I) No: 10); (4) the peptide according to (3), wherein the amino acid sequence is Ala Ala Gly Ser Ser Ser Ser Val Lys (SEQ ID No: 2); (5) a peptide dimer having an ability to bind to an HLA-A*1101 molecule and having an ability to induce a CTL, in which two peptide monomers each 10 comprising an amino acid sequence consisting of 9 contiguous amino acids from a WT l protein, and comprising at least one cysteine residue are bound to each other through a disulfide bond; (6) the peptide dimer according to (5), wherein the amino acid sequence of the peptide monomer is selected from the group consisting of: 15 Pro lle Lcu Cys Gly Ala Gin Tyr Arg (SEQ 11) No: 3), Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ ID No: 7), Lys Thr Cys GIn Arg Lys Phe Ser Arg (SEQ I) No: 8), and Scr Cys Arg Trp Pro Ser Cys Gin Lys (SEQ ID No: 9); (7) a pharmaceutical composition for the treatment or prevention of a 20 cancer, comprising the peptide according to (1) and/or the peptide dimer according to (5); (8) a method for the treatment or prevention of a cancer, comprising administering an effective amount of the peptide according to (1) and/or the peptide .\NRPonbl\DC(\A A R\27_1 LXXC 5012-117 -8 dimer according to (5) to an HILA-A* 1101 -positive subject; (9) a polynucleotide encoding the peptide according to (1); (10) an expression vector comprising the polynucleotide according to (9); 5 (11) a pharmaceutical co)mposition for the treatment or prevention of a cancer, comprising the polynucleotide according to (9) or the vector according to (10): (12) a method for the treatment or prevention of a cancer, comprising administering an effective amount of the polynucleotide according to (9) or the vector according to (10) to an H LA-A*I 101-positive subject; 10 (13) a WTI-specific CTL., which is induced by the peptide according to (1) and/or the peptide dimer according to (5); (14) a method for the induction of' a WTl-specific CTIL, comprising culturing a peripheral blood mononuclear cell in the presence ol' the peptide according to (1) and/or the peptide dimer according to (5) to induce the WTl-specific CTL from is the peripheral blood mononuclear cell; (15) a kit for the induction of a WTI-specific CTL, comprising the peptide according to (1) and/or the pcptidc dimer according to (5) as an essential component; (16) an antigen-presenting cell presenting a WTI peptide. which is 20 induced by the peptide according to I' 1) and/or the peptide dimer according to (5); (17) a method for the induction of an antigen-presenting cell presenting a WTI peptide, comprising culturing an immature antigen-presenting cell in the presence of the peptide according to (1) and/or the peptide dimer according to (5) to induce the C\NR'onblDCC\AAR 12MIlX)C-"2 /2-112 -9 antigen-presenting cell presenting a WTI peptide from the immature antigen-presenting cell; (18) a kit for the induction of an antigen-presenting cell presenting a WTI peptide, comprising the peptide according to (1) and/or the peptide dimer 5 according to (5) as an essential component; and (19) a method for the diagnosis of' a cancer. comprising using the CTL according to (1-3) or the antigen-presenting cell according to (16). Effects of the Invention 10 [00081 The present invention provides a peptide that is H-1 LA-A* I101-restricted and comprises an amino acid sequence consisting of 9 contiguous amino acids from a WTI protein, and a polynucleotide encoding the same, as well as a pharmaceutical composition for the treatment and/or prevention of a cancer, comprising the same, and 15 the like. Therefore, it is possible to induce in vivo and in vitro WTI-specific CTLs in subjects having HlLA-A*1101. Because the rate of' H-LA-A* I101-positive in Japanese people is high (about 17.9%), WTl-specific CTLs can be induced in a wide range of subjects. 20 Brief lDescription of Drawings 100091 Fig. I represents the cytotoxic activity of the CTL induced with WTI 251. Fig. 2 represents the cytotoxic activity of the CTI. induced with WTI 27m.

C\2NRPonhD\t(AARV102 _I )DOC."/2121112 - 10 Fig. 3 represents the cytotoxic activity of the CTL induced with WTl m. Fig. 4 represents the cytotoxic activity of the CTL induced with WT m1. Fig. 5 represents the cytotoxic activity of the CT.L induced with WT 1338. Fig. 6 represents the cytotoxic activity of the CTL induced with WT 37 8 . 5 Fig. 7 represents the cytotoxic activity of the CTL induced with WTI 1386. Fig. 8 represents the cytotoxic activity of the CTL induced with WTI m. Fig. 9 represents the cytotoxic activity of the CTL induced with W[T10. Fig. 10 represents the cytotoxic activity of the CTL induced with WT137 8 peptide (a, b and c represent the cytotoxic activities observed using P3MCs from HLLA 10 A*1 101-positive healthy donors 1, 2 and 3, respectively). Fig. I I represents the cytotoxic activity of the CTL induced with W'l'137 8 peptide dimer (a and b represent the cytotoxic activities observed using PBMCs from - LA-A*I 101-positive healthy donors I and 2, respectively). Fig. 12 represents the cytotoxic activity of' the CTL induced with 15 modified WT1379 peptide (G - 1) (a. b and c represent the cytotoxic activities observed using P3MCs from HLA-A*I 101-positive healthy donors 1, 2 and 3, respectively). Fig. 13 represents the cytotoxic activity of the CTL induced with modified WT1 378 peptide (G -> V) (a, b and c represent the cytotoxic activities observed using PBMCs from HLA-A*1 101-positive healthy donors 1, 2 and 3. 20 respectively). Fig. 14 represents the cytotoxic activity of the CITL induced with WTl 3 7 N peptide (a, b and c represent the cytotoxic activities observed using PBMCs from Hl-A A*l 101-positive healthy donors 1,2 and 3, respectively).

C\NRPnorPDCC\AARW',27-.1.DOC-'25/21|2 - 11 B3est Mode for Carrying Out the Invention [01 In one aspect, the present invention relates to a peptide comprising an 5 amino acid sequence consisting of 9 contiguous amino acids from a WTl protein, wherein the peptide has an ability to bind to an HILA-A*1101 molecule, and has an ability to induce a CTL (herein also referred to as a "WTI peptide"). The amino acid sequence of the human WTI protein is shown in SEQ I) No: 1. The peptide of the present invention comprises an amino acid sequence consisting of 9 contiguous amino 10 acids in the amino acid sequence shown in SEQ ID No: 1. When the peptide of the present invention comprises an amino acid sequence comprising cystein(s) such as the amino acid sequence of SEQ I) No: S, 7, 8 or 9 as described below, the stability may be increased by substituting the cystein(s) in the amino acid sequence with another substance such as another amino acid (for example, shrine, alanine and a-aminobutyric 15 acid) or by modifying the SH group of the cystein(s) with a protecting group known in the art (for example, carboxymethyl group or pyridylethyl group). The peptide of the present invention is a cancer antigen peptide that can induce a CTL as a result of presentation, by an antigen-presenting cell, of a peptide generated by processing the peptide of the present invention in a cell. 20 [0011]l As described above, it is an object of the present invention to obtain an I-LA- A* 1101-restricted peptide. Thus, the peptide of the present invention has an ability to bind to an H LA-A* I101 molecule. The ability to bind can be determined by a C \N~,h\)0A R4,2- I MYC-9 221r 2 - 12 method known in the art. Examples of such methods include a computer-based method such as Rankpep, BIMAS or SYFPEITII, and a competitive binding test with a known peptide having an ability to bind to an H-lLA-A*1 101 molecule. For example. the determined ability to bind can be compared with that of a known H.LA-A*1101 5 restricted peptide to judge whether or not the peptide of the present invention has an ability to bind. Examples of peptides having an ability to bind according to the present invention include a peptide of whicl the affinity score to an -1 LA-A* 110 1 molecule as determined by the method described in example I is 4 or more, preferably 5 or more, more preferably 6 or more. 10 [0012] The peptide of the present invention further has an ability to induce a CTL. The WTl gene is expressed in its native form at high levels, for example, in hematopoictic tumors such as leukemia, myclodysplastic syndrome, multiple myeloma or malignant lymphoma and solid cancers such as gastric cancer, colon cancer, lung 15 cancer, breast cancer, germ cell cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer or ovarian cancer. Therefore, the peptide of the present invention can induce a CTL with a high rate in a subject suffering from such a disease. The ability to induce a CTL refers to an ability to induce a CTL in vivo or in vitro. Such an ability can be determined using a general method such as a 20 method in which a cytotoxic activity of a CTL is determined using a Cr - release assay. 10013] The peptide of the present invention may have Lys or Arg at position 9 of the amino acid sequence. It is considered that by having such an amino acid, the C1 XPorthl\DC(1AAR (,27W-_ I DOC./25121 12 - 13 ability of the peptide to bind to an HALA-A*I 101 molecule becomes higher. [0014] The amino acid sequence consisting of 9 amino acids comprised in the peptide of the present invention is preferably, Ala Ala Gly Ser Scr Ser Scr Val Lys 5 (SEQ ID No: 2), Pro lie Leu Cys Gly Ala Gln Tyr Arg (SEQ I) No: 3), Arg Ser Ala Ser Glu Thr Ser Glu Lys (SEQ ID No: 4), Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ I) No: 5), Ser His Leu GIn Met His Ser Arg Lys (SEQ I) No: 6), Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ ID No: 7), Lys Thr Cys Gin Arg Lys Phe Ser Arg (SEQ ID No: 8), Ser Cys Arg Trp Pro Ser Cys Gin Lys (SEQ I) No: 9) or Asn Met I us Gin Arg Asn 10 Met Thr Lys (SEQ ID No: 10). Most preferably, it is Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ ID No: 7). Furthermore, it may have a substitution of one to several, preferably one to five amino acids with other amino acids in the nine amino acids of any of SEQ ID Nos: 2-10. Any one of the 9 amino acids or other substituted amino acids may be appropriately modified. In any cases, the peptide of the present invention 15 retains an ability to bind to an H LA-A*l 101 molecule. [0015] As described above, the peptide of the present invention may be any one as long as it comprises an amino acid sequence that is derived from a WTl protein and consists of' 9 contiguous amino acids. Thus, the peptide of' the present invention may 20 be, for example, a peptide consisting of only the amino acid sequence shown in any of SEQ ID Nos: 2-10, or a WTI protein or a part thereof comprising the amino acid sequence shown in any of SEQ ID Nlos: 2-10. The number of amino acids comprised in the peptide of the present invention is not specifically limited, and the number is, for CNRPorthl\DCC\AARU 2.3?J(' I IDOC./25201-1 - 14 example, 9-500, 9-300, 9-200, 9-100, 9-50, 9-30 and 9-12 amino acids. Various substances may be attached at the N-terminus and/or the C-terminus of the amino acid sequence consisting of 9 contiguous amino acids in the peptide of'the present invention. For example, an amino acid, a peptide or an analog thereof may be attached. If such a 5 substance is attached to the peptide of the present invention, the substance can be processed, for example, by an enzyme in a living body or through a process such as intracellular processing, and finally the amino acid sequence consisting of 9 contiguous amino acids can be produced and! presented as a complex with an H-lLA-A*1101 molecule on the surface of a cell, thereby resulting in the effect of inducing a CTL. The 10 substance may be a substance that modulates the solubility of the peptide of the present invention, or increases its stability (resistance to protease, etc.). Alternatively, it may be a substance that delivers the peptide of the present invention specifically, for example, to a given tissue or organ, or it may have an action to increase the efficiency of uptake by an antigen-presenting cell or the like. The substance may be a substance that 15 increases an ability to induce a CTL, such as a helper peptide or the like. 10016] The peptide of the present invention can be synthesized by methods generally used in the art or modifications thereof. Such methods are described, for example, in Peptide Synthesis, Interscience, New York, 1966 ; The Proteins, Vol 2. 20 Academic Press Inc., New York, i976 ; Peptide-Gosei, Maruzen Co., Ltd., 1975; Peptide-Gosei No Kiso To Jikken, Maruzen Co., Ltd., 1985; and lyakuhin No Kaihatsu (Zoku), Vol. 14, Peptide-Gosei, Hirokawa - Book store, 1991. 100171 C-\NRPor1N\D)CC\AAR\4t127f,_IDO)CC-/25/2012 - 15 The peptide of the present invention can also be prepared using genetic engineering techniques based on the information about the nucleotide sequence that encodes the peptide of the present invention. Such genetic engineering techniques are well known to a skilled person in the art. 5 10018] In a further aspect, the present invention relates to a peptide dimer having an ability to bind to an HILA-A*l101 molecule and having an ability to induce a CTL., in which two peptide monomers each comprising an amino acid sequence consisting of 9 contiguous amino acids from a WTI protein and comprising at least one 10 cystein residue are bound to each other though a disulfide bond (hereinafter also referred to as a "WTI peptide dimer"). The stability of the peptide dimer of the present invention is increased as compared with that of the peptide monomer by forming a dimer. The peptide dimer of the present invention is a tumor antigen peptide dimer that can induce a CTL as a result of presentation, by an antigen-presenting cell, of a peptide 15 generated by processing the peptide of the present invention in a cell. [0019] The peptide dimer of .he present invention is formed by binding two peptide monomers through a disulfide bond between cystein residues present in the monomers. Thus. each of the peptide monomers comprised in the WTI peptide dimer 20 of the present invention is the WT1 peptide as described above and comprises at least one cystein residue. The WT1 peptide dimer of the present invention may be a homodimer or a heterodimer. 10020] C R PonblIDCC'\AAR\1 82)_ 1 DOC-W125/20 l - 16 In the WTI peptide dimer of the present invention, the amino acid sequence comprised by the peptide monomer comprises is preferably, Pro lie Leu Cys Gly Ala Gin Tyr Arg (SEQ I) No: 3), Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ I) No: 7), Lys Thr Cys Gin Arg Lys Phe Ser Arg (SEQ ID No: 8) or Ser Cys Arg Trp Pro 5 Ser Cys Gin Lys (SEQ ID No: 9). Most preferably, it is Thr Gly Val Lys Pro Phe Gin Cys Lys (SEQ ID No: 7). 1.0021] The WTI peptide dimer of' the preset invention can be prepared using a method known in the art. For example, if the peptide monomers comprise one pair of 10 cystein residues, the WTI peptide dimer of' the present invention can be prepared, for example, by removing all the protecting groups including the ones on the cystein side chains, and then subjecting the resulting monomer solution to air-oxidation under alkaline conditions, or adding an oxidant under alkaline or acidic conditions to form a disulfide bond. Examples of the oxidants include iodine, dimethylsulfoxide (DMSO) 15 and potassium ferricyanide. [0022] When each of' the peptide monomers comprises two or more cystein residues, the WTl peptide dimer of the present invention can also be prepared by the method as described above. In this case, isomers are obtained due to different types of' 20 disulfide bonds. Alternatively, the WTl peptide dimer of'the present invention can be prepared by selecting a combination of' protecting groups for cystein side chains. Examples of the combinations of the protecting groups include combinations of Me3zl (methylbenzyl) group and Acm (acetamidemethyl) group, Trt (trityl) group and Acm - 17 group, Npys (3-nitro-2-pyridylthio) group and Acm group, and S-Bu-t (S-tert-butyl) group and Acm group. For example. in the case of the combination of McBzl group and Acm group, the WTI peptide dimer can be prepared by removing protecting groups other than the MeBzl group and the protecting group on the cystein side chain, 5 subjecting the resulting monomer solution to air-oxidation to form a disulfide bond between the protected cystein residues, and then deprotecting and oxdizing using iodine to form a disulfide bond between the cystein residues previously protected by Acm. [0023] In another aspect, the present invention relates to a pharmaceutical 10 composition for the treatment or prevention ofa cancer comprising the HlLA-A*l 101 restricted WTI peptide and/or the WTI peptide dimer. The WTI gene is expressed at high levels in various cancers and tumors including hematopoictic tumors such as leukemia, myelodysplastic syndrome, multiple myeloma or malignant lymphoma and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, germ cell 15 cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer. uterine cancer. cervical cancer or ovarian cancer. Therefore, the pharmaceutical composition of the present invention can be used for ihe treatment or prevention of a cancer. When the pharmaceutical composition of the present invention is administered to an HLA A*1101-positive subject, WTI-speciic CTLs are induced by the H-lLA-A*1101 20 restricted WTl peptide or the WTi peptide dimer comprised in the pharmaceutical composition, and cancer cells in the subject are damaged by such CTLs. [00241 The pharmaceutical composition of the present invention may comprise C-\NRPonbhD')CC\AAR\40(27(1'_ .DOC.'/25121 2 - 18 in addition to the HLA-A*l 101-restricted WTl peptide and/or the WTI peptide dimer as an active ingredient, for example, a carrier, an excipient or the like. The HLA A*1101-restricted WTI peptide or the WTI peptide dimer comprised in the pharmaceutical composition of the present invention induces a WTl-speciFic CTL. 5 Thus, the pharmaceutical composition of the present invention may comprise an appropriate adjuvant, or may be administered together with an appropriate adjuvant in order to enhance the induction efficiency. Examples of preferable adjuvants include. but are not limited to, complete or incomplete Freund's adjuvant and aluminum hydroxide. 10 [0025] The method of the administration of the pharmaceutical composition of the present invention can be appropriately selected depending on conditions such as the type of disease, the condition of the subject or the target site. Examples of such methods include, but are not limited to, intradermal administration, subcutaneous is administration, intramuscular administration, intravenous administration, nasal administration and oral administration. The amount of the peptide or the peptide dimer comprised in the pharmaceutical composition of the present invention, as well as the dosage form, the number of times of the administration and the like of' the pharmaceutical composition of the present invention can be appropriately selected 20 depending on conditions such as the type of'disease, the condition of the subject or the target site. The single dose of the peptide is usually, 0.0001 mg - 1000 mg, preferably, 0.00 1 mg - 1000 mg. [00261 C.\NRPothrDCC\AAR\(1276 .DOC /912211 - 19 In another aspect, the present invention relates to a method for the treatment or prevention of a cancer, comprising administering an effective amount of the WTI peptide and/or the WTI peptide dimer to an HLA-A* I101-positive subject. The cancer to be treated or prevented may be any one, and examples thereof include 5 hematopoictic tumors such as leukemia. myclodysplastic syndrome, multiple myeloma or malignant lymphoma and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer. uterine cancer, cervical cancer or ovarian cancer. [00271 10 In a further aspect, !he present invention relates to a method for the determination of the presence or amount of a WTI-specific CTI. in an HLA-A*I 101 positive subject, comprising: (a) reacting a complex of a WTl peptide and an HLA-A*I 101 molecule with a sample from the subject; and 15 (b) determining the presence or amount of a CTL recognizing the complex contained in the sample. The sample from a subject may be any one as long as there is a possibility that it contains a lymphocyte. Examples of the samples include body fluid such as blood or lymph and a tissue. The complex of a WTI peptide and an H-1LA-A*l 101 molecule may be prepared, for example, as a tetramer or pentamer using a method 20 known to a skilled person in the art such as biotin-streptavidin method. The presence or amount of the CTL recognizing such a complex can be measured by a method known to a skilled person in the art. In this aspect of the present invention, the complex may be labeled. A known label such as a fluorescent label or a radioactive label can be used as CXNR~nhIDCLIAA"612(4II I)OC--1/2512II12 -20 a label. Labeling makes the determination of the presence or amount of a CTL easy and rapid. 100281 Thus, the present invention also provides a composition for the 5 determination of the presence or amount of a WTl-specific CTL in an Hl.LA-A*l 101 positive subject comprising an IlLA-A*l 101-restricted WTI peptide. [00291 Furthermore, the present invention provides a kit for the determination of the presence or amount of a WT1-specific CTL in an ILA-A* 101-positive subject, 10 comprising an llLA-A*l 101-restricted WTI peptide. [0030.1 In a further aspect, the present invention relates to a method for the production of a WTI-specific CTL using a complex of a WTI peptide and an HLA A*I 101 molecule, comprising: is (a) reacting the complex with a sample; and (b) obtaining a CTL recognizing the complex contained in the sample. The complex of a WTl peptide and an H-lLA-A*I 101 molecule is described above. The sample may be any one as long as there is a possibility that it contains a lymphocyte. Examples of the samples include a sample from a subject such as blood, and a cell culture. The CTL 20 recognizing the complex can be obtained using a method known to a skilled person in the art such as FACS or MACS. The present invention allows to culture the obtained WTI-specific CTL and use it for the treatment or prevention of various cancers. 10031] C.NRPonhN)CC\AAR"U.27(._JI DOC-/25/2012 -21 Thus, the present invention also relates to a WTI-specific CTL obtainable by a method for the production of a WTI -specifi C TL using a complex of a WTI peptide and an IHLA-A*I 101 molecule. 10032] 5 In another aspect, the present invention relates to a polynucleotide encoding the ILA-A*l 101-restricted WTI peptide. The polynucleotide of the present invention may be DNA or RNA. The base sequence of the polynucleotide of the present invention can be determined based on the amino acid sequence of the HLA A*1101-restricted WTI peptide. The polynucleotide can be prepared by a known 10 method For the synthesis of DNA or RNA (for example, chemical synthetic method). PCR method or the like. 100331 In another aspect, the present invention relates to an expression vector comprising the polynucleotide. The type of the expression vector, the comprised 15 sequence other than the sequence of the polynucleotide and the like can be appropriately selected depending on the type of a host into which the expression vector of' the present invention is introduced, the purpose of use, or the like. It is possible to treat or prevent hematopoictic tumors or solid cancers by administering the expression vector of the present invention to an H-ILA-A*1101-positive subject to produce an HLA-A*1101 20 restricted WTl peptide in a living body and induce a WTI-specific CTL, and damaging hematopoictic tumor cells or solid cancer cells in the subject. 100341 In a further aspect, the present invention relates to a pharmaceutical C-\NRPorbllCC\AR"027h _ I DOC-W25/2.I12 -22 composition for the treatment or prevention of a cancer, comprising the polynucleotide or the expression vector. The composition, method of the administration and the like of the pharmaceutical composition of the present invention in this aspect are described above. 5 100351 In another aspect, the present invention relates to a method f1or the treatment or prevention of a cancer, comprising administering an effective amount of the polynucleotide or the expression vector to an HILA-A*1 101-positive subject. Examples of cancers to be treated or prevented include hematopoictic tumors such as 10 leukemia, myclodysplastic syndrorne, multiple myeloma or malignant lymphoma and solid cancers such as gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, hepatic cancer, skin cancer. bladder cancer, prostate cancer, uterine cancer, cervical cancer or ovarian cancer. [00361 is In another aspect, the present invention relates to a cell comprising the expression vector. The cell of the present invention can be prepared, for example, by transforming a host cell such as E. coli, yeast, insect cell or animal cell with the expression vector. The method for the introduction of the expression vector into a host cell can be appropriately selected from various methods. By culturing the transformed 20 cell, and recovering and purifying the produced WTI peptide, the peptide of the present invention can be prepared. {0037] In a further aspect, the present invention relates to a WTl -specilic CTL.

C:\NR1unbN)CC\kAARWO2W..I )OC-9/2/2,12 -23 which is induced by the H-LA-A*l 101-restriceted WTl peptide and/or the WTI peptide dimer. The CTL of the present invention recognizes a complex of a WTI peptide and an HLA-A* I101 molecule. Thus, the CTL of the present invention can be used to damage specifically a tumor cell positive for H-lLA-A*1 101 and expressing WTI at a 5 high level. 10038] In another aspect, the present invention relates to a method for the treatment or prevention of a cancer, comprising administering a WTl-specific CTL to an H-lLA-A*1101-positive subject. The method of the administration of' the WTl 10 specific CTL. can be appropriately selected depending on conditions such as the type of the disease, the condition of the subject or the target site. Examples of such methods include, but are not limited to, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, nasal administration and oral administration. is [00391 In another aspect, the present invention relates to a method for the induction of a WTI-specific CTL, comprising culturing a peripheral blood mononuclear cell in the presence of the H lLA-A*l IC I-restricted WT1 peptide and/or the WTI peptide dimer to induce the WTI-specific CTIL form the peripheral blood mononuclear cell. 20 The subject from which the peripheral blood mononuclear cell is derived may be any one as long as it is positive for H LAx-A*1 101. By culturing the peripheral blood mononuclear cells in the presence of the H-lLA-A*l 101-restricted WTI peptide and/or the WTI peptide dimer, WTI-specific CTLs arc induced from CTL precursor cells \NRPonh1DCXAA RMU7? _ 1X)C.5/2 12 -24 contained in the peripheral blood mononuclear cells. It is possible to treat or prevent hematopoictic tumors or solid cancers in an HILA-A*l 101-positive subject by administering the WT1-specific CTL obtained according to the present invention to the subject. 5 [0040] In another aspect, the present invention relates to a kit for the induction of a WTI-specific CTL, comprising an 11 LA-A*I 101-restricted WT I peptide and/or the WT peptide dimer as an essential component. Prelrably, the kit is used in the method for the induction of a WTI-specific CTL. The kit of the present invention may 10 comprise in addition to the H-lLA-A*1101-restriceted WTI peptide and/or the WTI peptide dimer, for example, a means of obtaining a peripheral blood mononuclear cell. an adjuvant, a reaction vessel or the like. In general, an instruction manual is attached to the kit. By using the kit of the present invention, WTI -specific C TLs can be induced efficiently. 15 10041] In a further aspect, the present invention relates to an antigen-presenting cell (such as a dendritic cell) presenting a WTI peptide through an H-fLA-A*1 101 molecule, which is induced by the HL.A-A*1101-restricted WTl peptide and/or the WTI peptide dimer. By using the antigen-presenting cell of the present invention. 20 WTI -specific CTLs are induced efficiently. [0042] In another aspect, the present invention relates to a method for the treatment or prevention of a cancer, comprising administering the antigen-presenting C 1NRIonhM)CC\AARol,2%0_ 1 II W2/2OC- 12 2 -25 cclI presenting a WT1 peptide through an lILA-A* 101 molecule to an H lLA-A*1 101 positive subject. The method of the administration of the antigen-presenting cell can be appropriately selected depending on conditions such as the type of the disease, the condition of the subject or the target site. Examples of such methods include, but are 5 not limited to, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, nasal administration and oral administration. 100431 In another aspect, the present invention relates to a method for the 10 induction of an antigen-presenting cell presenting a WTI peptide through an HLIA A*1101 molecule, comprising culturing an immature antigen-presenting cell in the presence of the HILA-A* I101-restricted WTI peptide and/or the WTI peptide dimer to induce the antigen-presenting cell presenting a WTI peptide through an HLA-A*I 101 molecule from the immature antigen-presenting cell. The immature antigen-presenting is cell refers a cell such as an immature dendritic cell that can be matured into an antigen presenting cell. A subject from which the immature antigen-presenting cell is derived may be any one as long as it is positive flor H-1LA-A* I101. Because the immature antigen-presenting cells are contained, for example, in peripheral blood mononuclear cells, such cells may be cultured in the presence of the WT peptide and/or the WTI 20 peptide diemr. [0044] In another aspect, the present invention relates to a kit for the induction of an antigen-presenting cell presenting a WTl peptide through an HILA-A* 1101 C\NRPO1h\DC AAR\M27- )OC-9/2./2|12 -26 molecule, comprising the ILA-A*1 101-restricted WTI peptide and/or the WTI peptide dimer as an essential component. Preferably, the kit is used in the method for the induction of an antigen-presenting cell. Another component to be comprised in the kit of the present invention and the like are described above. The kit of the present 5 invention can be used to induce efficiently an antigen-presenting cell presenting a WTI peptide through an HlLA-A*l 101 molecule. 100451 In another aspect, the present invention relates to an antibody against an HLA-A* 1101-restricted WTI peptide or a polynucleotide encoding the peptide. The 10 antibody of the present invention may be a polyclonal antibody or monoclonal antibody. 100461 In a further aspect, the present invention relates to a method for the diagnosis of a cancer, comprising using the WTI -specific CTL, the antigen-presenting cell presenting a WTI peptide through an H-lLA-A*1101 molecule, or the antibody 15 against an H-LA-A-restricted WTI peptide or a polynucleotide encoding the peptide. Preferably, the WTI -specific CTL is used in the method for the diagnosis of the present invention. For example, it is possible to diagnose a cancer by incubating the CTL. the antigen-presenting cell or the antibody with a sample from an HLA-A*l 101-positive subject, or administering it to an HILA-A*I 101-positive subject, and determining, for 20 example, the position, site or amount thereof. The CTL, the antigen-presenting cell or the antibody may be labeled. By attaching a label, the method for the diagnosis of' the present invention can be practiced efficiently.

C \NRPouhrD)C(AARW.1279A.-I DOC-925/20112 -27 Examples 100471 The following examples illustrate the present invention in more detail, but are not to be construed to limit the scope thereof. 5 10048] Example 1: Selection of WTl peptide RANKPEP (http://bio.dfci.harvard.edu/Tools/rankpep.html) was used to select WI11251, WT1279, WT1312, WT1 313 , WT 338, WT378, WTiI 8, WTlI s and WT1 4 3 6 having a high ability to bind to an HL.A-A*1101 molecule derived from 10 peptides from a WTI protein (SEQ I) No: 1). Amino acid sequences, amino acid numbers in SEQ I) No: I and affinity scores to an H-1LA-A*l 101 molecule of these peptides are shown in Table 1. [0049]1 [Table 1] Peptide Amino acid Amino acid Affinity Number sequence score WT1 2 5 1 251-259 AAGSSSSVK 15.18 (SEQ ID No: 2) WT1 2 79 279-287 PlILGAQYR 11.47 (SEQ I) No: 3) WT 1312 312-320 RSASETSEK 14.96 (SEQ ID No: 4) WTI 313 313-321 SASE'ISEKR 6.87 (SEQ I) No: 5) WT1 3 3 8 338-346 SILQMI-ISRK 13.72 (SEQ ID No: 6) WTI 37 8 378-386 TGVKPFQCK 1.33 (SEQ I) No: 7) WT 13 8 6 386-394 KTCQRKFSR 1 3.82 (SEQ I) No: 8) WTlI 4 15 415-423 SCRWPSCQK 10.29 C \NRPonlhhIDCC\AAR2U3271'l I DOC-9f25/2017 -28 (SEQ ID No: 9) WT1 4 36 436-444 NMI-IQRNMTK 14.19 (SEQ ID No: 10) 10050] Preparation of B-ICL cell Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll Hypaque gradient density centrifugation method from peripheral blood that had been 5 collected from an H-lLA-A* 1101 -positive healthy donor. The P3MCs were then seeded to a 24-well cell culture plate at the density of about I x 107 in RPMI 1640 medium containing 10% FCS, and a culture supernatant of 1395-8 cells (cells producing EB virus) were added. They were cultured at 370C with 5% CO 2 for about I month. B LCL cells transformed with EB virus, which are 13-cell tumor cells, were obtained. It 10 was confirmed that the resulting 13-LCL cells did not express WTl gene. B-LCL cells were pulsed by incubating them with 20 ptg/ml of WT1 25 1 , WT279, WT1 3 1 2 , WT1 3 13 , WT1338, WT1 3 78 , WT1386, WTl1s 15 or WT1 43 for 2 hours, and irradiated with 80 Gy of radiation. The resulting B-LCL cells (hereinafter referred to as B-LCL cells pulsed with a WTl peptide) were used as antigen-presenting cells for the following experiments. 15 [00511 Induction of WTI-spccific CTL 3 x 106 of autologous P3MCs were cultured in a 24-well cell culture plate in complete medium (45% RPMI, 45% AMI-V medium and 10% human AB serum) containing 20 ptg/ml of' Wi 1251, WTl 27 9, WTI 3 1 2 , WT, 3 13 WTI 339, WI'] 378, 20 WT1 3 8 6 , WT1 4 15 or WT1 4 36 at 37C with 5% CO 2 for I week to obtain responding cells. 2 x 106 of the resulting responding cells were cocultured with 1 x 106 of the B-LCI C \NRf'o bl)C(\AAR.t,'27'n_.I IX)C-9/2/21) -29 cells pulsed with the same WTI peptide in complete medium for I week (first stimulation). The PBMCs were coculutured with the 13-LCL cells pulsed with the WTI peptide three more times (second to fourth stimulations) under the conditions under which 20 IU/ml (final concentration) of' IL2 was added as follows: second stimulation: 5 two times every other day from 3 days after the initiation of stimulation; third and fourth stimulations: three times at intervals of one day from the day after the initiation of stimulation. The resulting cells were concentrated using Negative Selection Columns Gravity Feed Kit (StemSp) so that the ratio of CD8-positive T cells became about 80%, and cocultured with the B-LCL cells pulsed with the WT1 peptide (fifth stimulation). 10 CD8-positive T cells (CTLs) obtained 5 days after the final stimulation were used for measurement of the cytotoxic activity. [0052] Cytotoxic activity of' CTIl The cytotoxic activity of CTLs was measured using i'Cr release assay. 15 CTL cells (hereinafter referred to as effector cells) were mixed at the ratio (EI/T ration) of 1:1 to 30:1 in 200 pl of medium with target cells into which 5 1 Cr had been incorporated, and cultured in a 96-well cell culture plate at 37TC with 5% CO, for 4 hours. B-LCL cells pulsed with the same WTI peptide as that used for induction of' CTLs (BLCL-Ps), and B-LCL cells without pulsing with a WT peptide (BLCL-Ns) 20 were used as target cells. After the culture, the supernatants were collected by centrifugation. The amounts of 5 'Cr released into the supernatants were measured using a liquid scintillation counter. The cytotoxic activity (%) was determined using the following formula: -30 ( 5 1 Cr release in supernatant of sample - Spontaneous 5 1 Cr release) / (Maximum 51Cr release - Spontaneous 5 1Cr release) x 100 wherein Spontaneous "Cr release is 5Cr release observed when the target cells into which 5'Cr had been incorporated were cultured alone under the same condition. and 5 Maximum 5'Cr release is 5'Cr release observed when the target cells into which 5'Cr had been incorporated were completely lysed using 1% Triton X-100. Results arc shown in Figs. 1-9. In the figures, longitudinal axes represent specific lysis (%), and horizontal axes represent EIT ratios. BLCL-Ps are represented using full lines, and 3LCL-NI's are represented using dotted lines. It was confirmed that CTLs induced 10 with WTl251, WT1279, W Iz312, WTII3, WTI338. WT1378, W'iss8, WTl ,s or WIl 3 damage specifically BLCL-Ps presening the WTI peptide as a complex with an ILA A*] 101 molecule as compared with BILCL-NPs. CTLs induced with WT1 2 5 1 , Wfl 2 79, WT1 3 13 , WT 1338 or WT1 38 6 were used for additional experiments below. [0053] 15 Cytotoxic activity of CTL against cell expressing WTl endogenously The cytotoxic activity of CTLs induced with W'lFI 25 1 , WT1 2 79, WTI 31 3 , WT1 338 or WT1 3 8 6 against B-LCI s expressing WTI was determined using the method described above. A cell expressing Wl'l refers to a B-LCL into which a human WTI gene is introduced, and that expresses a WTl protein in the cell, and presents a peptide 20 consisting of about 9 amino acids resulting from processing on an HIlLA-A*l 101 molecule. Results are shown in Figs. 1, 2, 4, 5 and 7. In the figures, B-LCLs expressing WT1 are represented using dashed lines. It was confirmed that CTlIs induced with WT1 251 , WT1279, WTi(is, WT1338 or WTl1386 have a cytotoxic activity UNRPonhADCCAAR" 27W1_I DOC.'I22tIl2 -31 against cells expressing WT1 gene endogenously. 10054] Preparation of WTI peptide dimer A mixture of 227.5 mg of WT1378 peptide monomer, 227.5 mg of N 5 methylglucarnine (NMG) and 23 ml of water was air-oxidized by stirring at room temperature for about 2 days. To the resulting mixture, an aqueous solution of 2g of' sodium acetate in 5 ml of water was added and the mixture was stirred at room temperature for about 20 minutes. To the resulting solution, 200 ml of water and about 200 ml of acetonitrile were added, and the mixture was filtered through Kiriyama funnel 10 (filter paper No. 5C) and washed with water (about 50 ml x 3). To the residue, about 200 ml of water was added and the residue was lyophilized to obtain 158 mg of crude WT1 378 peptide dimer. 100551 Purification of crude WTlI peptide dimer 15 158 mg of the crude WT1 378 peptide dimer was dissolved in 9 ml of DMSO and injected into ODS C 1 ,, column (5 cm (1) x 50 cm L, YMC Co.. LTD.) attached to HPLC (Shimadzu, LC8A) type) and equilibrated with solution 1 (120/1% AcOH) using a HPLC pump. The column was left for about 30 minutes, and eluted with concentration gradient of 0% to 40% of solution 2 (C- 3 CN/l% AcOH) over 360 20 minutes. The fractions containing WTl peptide dimer were collected using an automatic fraction collector while monitoring UV absorbance at 220 nmn. The collected fractions were combined, injected into ODS C 18 column (4.6 mm 1 x 25 cm L. YMC Co., LTD.) attached to HPLC (1-litachi, L-4000 type) and equilibrated with 17% of S\NR PorthhtCC\A A R\ 7r_1 1. C xXW2 12112 -32 solution 2, and eluted with concentration gradient of 0% to 47% of solution 2 over 30 minutes to obtain 46.6 mg of the purified WT 8 peptide dimer at retention time of 20.51 minutes. FA B.MS 2365.0 (theoretical value: 2342.70) Na F= 0.25% 5 100561 Induction of CTL by WTI peptide dimer Abilities of the resulting WT1 378 peptide dimer, WT1 37 8 peptide. modified WT137 peptide(G - I) (SEQ I) No: 11) and modified W1l1 37 8 peptide (G V) (SEQ ID No: 12) as well as WT1 379 peptide (SEQ ID No: 13, as disclosed in WO 10 2002/28414) to induce a CTI, were examined using PBMCs from ILA-A*I 101 positive healthy donors 1-3 according to the method as described above. Results are shown in Figs. 10-14. In the figs, longitudinal axes represent specific lysis (%), and horizontal axes represent E/T ratios. BLCL-Ps are represented using full lines, and BLCL-NPs are represented using dotted lines. It was confirmed that WT1378 peptide 15 dimer has an ability to induce a CTL Furthermore, it was found that the ability of each W1379 peptide of which the amino acid sequence is different from that of WT1 3 7 8 peptide by one amino acid in the amino acid sequence of WTIl protein to induce a CTL is much lower than that of WT1 378 peptide and, thus, the WTI peptide of the present invention has an excellent and unexpected effect as compared with the known peptide. 20 Industrial Applicability [0057] The present invention provides an H- lA-A*l 101-restricted WTl peptide.

C.\NRPortbilDCC\(AAR\4M-27,1_.1.DOC- 0 -33 a polynuclcotide encoding the peptide, a pharmaceutical composition comprising the same and the like. Therefore, the present invention can be used in the fields of medicine and the like, for example, in the fields of' development and preparation of a pharmaceutical composition for the prevention or treatment of various hematopoictic 5 tumors and solid cancers that express WTI gene at high levels. Sequence listing free text 10058] SEQ ID NO: 11: Modified WTIl peptide 10 SEQ ID NO: 12: Modified WTl peptide Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of' a stated element or integer or method step or group of elements 15 or integers or method steps but not the exclusion of any element or integer or method step or group of elements or integers or method steps. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common 20 general knowledge in any country.

Claims (12)

1. A method for the treatment or prevention of a cancer, comprising administering an effective amount of a peptide to an HLA-A* 1101-positive subject wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO:5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL.

2. A pharmaceutical composition when used for the treatment or prevention of a cancer in an HLA-A*1101-positive subject according to claim 1, comprising a peptide consisting of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO:5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL.

3. A method for the treatment or prevention of a cancer, comprising administering an effective amount of a polynucleotide encoding a peptide or a vector comprising the polynucleotide encoding the peptide to an HLA-A*1 101-positive subject, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO:5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), H:\fminIcmen \ cNRPonbl\DCC'!,m7465268 .DOC-13/ 2/2015 - 35 wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL.

4. A pharmaceutical composition when used for the treatment or prevention of a cancer in an HLA-A* 1101-positive subject according to claim 3, comprising a polynucleotide encoding a peptide or a vector comprising the polynucleotide encoding the peptide, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO:5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL.

5. A WT1-specific CTL, which is induced from a peripheral blood mononuclear cell by use of a peptide, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO:5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL, and wherein the peripheral blood mononuclear cell is derived from an HLA-A* 1101 positive subject.

6. A method for the induction of a WT1-specific CTL, comprising culturing a peripheral blood mononuclear cell in the presence of a peptide to induce the WT1-specific CTL from the peripheral blood mononuclear cell, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO:5); and H: \aar\lnterwoven\NRPortbl\DCC\AAR\7320669_1.doc-23/12/2014 - 36 (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL-, and wherein the peripheral blood mononuclear cell is derived from an HLA-A* 1101 positive subject.

7. A kit when used for the induction of a WT1-specific CTL according to Claim 6, comprising a peptide as an essential component, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO: 5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL-, and wherein the WT1-specific CTL is induced from a peripheral blood mononuclear cell which is derived from an HLA-A* 1101-positive subject.

8. An antigen-presenting cell presenting a WT1 peptide, which is induced from an immature antigen-presenting cell by use of a peptide, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO: 5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL-, and wherein the immature antigen-presenting cell is derived from an HLA-A*1101 positive subject. H: \aar\lnterwoven\NRPortbl\DCC\AAR\7320669_1.doc-23/12/2014 - 37

9. A method for the induction of an antigen-presenting cell presenting a WT1 peptide, comprising culturing an immature antigen-presenting cell in the presence of a peptide to induce the antigen-presenting cell presenting a WT1 peptide from the immature antigen presenting cell, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO: 5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL-, and wherein the immature antigen-presenting cell is derived from an HLA-A*1101 positive subject.

10. A kit when used for the induction of an antigen-presenting cell presenting a WT1 peptide according to Claim 9, comprising a peptide as an essential component, wherein the peptide consists of an amino acid sequence selected from: (1) Pro Ile Leu Cys Gly Ala Gln Tyr Arg (SEQ ID NO:3); (2) Ser Ala Ser Glu Thr Ser Glu Lys Arg (SEQ ID NO: 5); and (3) an amino acid sequence selected from SEQ ID NO:3 and 5, wherein one to five amino acids are substituted with other amino acid(s), wherein the peptide has an ability to bind to an HLA-A* 1101 molecule, and has an ability to induce a CTL-, and wherein the antigen-presenting cell presenting a WT1 peptide is induced from a immature antigen-presenting cell which is derived from an HLA-A* 1101-positive subject.

11. A method for the diagnosis of a cancer, comprising using the CTL according to Claim 5 or the antigen-presenting cell according to Claim 8. H:\aar\Interwoven\NRPortbl\DCC\AAR\7320669 Ldoc-23/12/2014 - 37a

12. A pharmaceutical composition according to Claims 1 or 3 or a method according to any one of Claims 2, 4, 6, 9 or 11 or a WT1-specific CTL of Claim 5 or a kit according to Claim 7 or 10 or an antigen-presenting cell of Claim 8 substantially as herein described with reference to the Figures and/or Examples.