Patent Description:
The present invention further relates to a cancer vaccine composition for HLA-A*<NUM>-positive persons, comprising a modified peptide of the WT1 peptide.

The Wilms' tumor gene WT1 was isolated as a gene associated with tumorigenesis in Wilms' tumor, which is a pediatric renal tumor (see nonpatent literature <NUM>). This gene encodes a zinc finger transcription factor associated with the regulatory mechanism of cell growth and differentiation, and apoptosis and tissue development.

The WT1 gene was originally classified as a tumor suppressor gene. However, based on the recent evidences shown in the following (i) to (iii):.

it is suggested that the wild-type WT1 gene exhibits an oncogenic effect rather than a tumor suppressive effect on various malignant diseases (see patent literature <NUM>).

There is also known high expression of the WT1 gene in 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 and ovarian cancer (see patent literature <NUM>).

In general, the immune system for eliminating foreign substances comprises humoral immunity, in which macrophages, which recognize an antigen and serve as antigen presenting cells, helper T cells, which recognize the antigen presented by the macrophages and produce various lymphokines to activate other T cells, and B lymphocytes, which differentiate into antibody producing cells via the actions of the lymphokines, are involved; and cell-mediated immunity, in which cytotoxic T lymphocytes (CTLs), which are produced through differentiation in response to antigen presentation, attack and destroy target cells.

Currently, it has been considered that cancer immunity is mainly based on cell-mediated immunity in which CTLs are involved. In the CTL-based cancer immunity, precursor T cells recognize a cancer antigen presented in the form of a complex of a major histocompatibility complex (MHC) class I and the cancer antigen, and thereby differentiate and grow into CTLs, which attack and destroy cancer cells. In this case, the cancer cell presents, on the cell surface, a complex of the MHC class I antigen and the cancer antigen, which is the target of the CTLs (see nonpatent literatures <NUM> to <NUM>). MHC is called as a human leukocyte antigen (HLA) in humans.

It is considered that the above-mentioned cancer antigen, which is presented by an MHC class I antigen on the surfaces of cancer cells, i.e., target cells, is a peptide of about <NUM> to <NUM> amino acids produced through intracellular protease-mediated processing of an antigen protein synthesized in cancer cells (see nonpatent literatures <NUM> to <NUM>). Currently, search for antigen proteins of various cancers is underway, but only a few proteins have been identified as a cancer specific antigen.

The present inventor synthesized polypeptides that each consist of <NUM> to <NUM> contiguous amino acids based on the amino acid sequence of the WT1 gene expression product and each contain at least one amino acid presumably serving as an anchor amino acid for binding with HLA-A*<NUM> or HLA-A*<NUM>, confirmed that these peptides bind with HLA-A*<NUM> or HLA-A*<NUM> (these peptides are HLA-A*<NUM>- or HLA-A*<NUM>-restricted), and found that the binding of the peptides with HLA-A*<NUM> or HLA-A*<NUM> induces CTLs, resulting in cytotoxic response to target cells (hereinafter abbreviated as CTL response). From this fact, these peptides were identified as a CTL epitope derived from the WT1 gene expression product (WT protein).

At this point, WT1-specific CTL epitopes only for HLA-A*<NUM> and HLA-A*<NUM> (see patent literature <NUM>), HLA-A*<NUM> (see patent literature <NUM>) or HLA-A*<NUM> are identified (see patent literature <NUM>). It is confirmed that CTL responses induced by the polypeptides disclosed by the above literatures are restricted by HLA-A*<NUM>, HLA-A*<NUM>, HLA-A*<NUM> and HLA-A*<NUM>.

This indicates a possibility that the protein product of the tumor suppressor gene WT1 is a promising tumor rejection antigen, also called as a tumor associated antigen (TAA). In fact, high levels of WT1-specific CTLs or high-titer anti-WT1 antibodies were observed not in peripheral blood of healthy blood donors, but in that of cancer patients.

However, HLA types are diverse enough to serve as markers for identifying individuals. In the HLAs, MHC class I antigens are classified into HLA-A, HLA-B and HLA-C, and MHC class II antigens are classified into HLA-DP, HLA-DQ and HLA-DR. Each class has several types of antigens. The antigen binding site of each HLA has genetic polymorphism. For example, it is known that HLA-A, HLA-B and HLA-C have <NUM> or more, <NUM> or more, and <NUM> or more kinds of polymorphisms (alleles), respectively.

Therefore, there has been a desire to identify a cancer antigen that binds to other types of HLAs than HLA-A*<NUM>, HLA-A*<NUM>, HLA-A*<NUM> and HLA-A*<NUM> and induces a CTL response, and to thereby apply immunotherapy to a wider range of subjects.

Meanwhile, the following three of modified WT1 peptides were reported in two documents:.

Further, the following two peptides were reported in the written argument for the examination of <CIT>:
the WT1<NUM>P2L peptide (RLFPNAPYL; SEQ ID NO: <NUM>) and the WT1<NUM>F2L&P9V peptide (RLFPNAPYV; SEQ ID NO: <NUM>) (see nonpatent literature <NUM>).

However, it has never been reported whether these WT1 modified peptides serve as a cancer antigen that binds to other types of HLAs than HLA-A*<NUM>, HLA-A*<NUM>, HLA-A*<NUM> and HLA-A*<NUM> and induces a CTL response.

An object of the present invention is to apply, further to HLA-A*<NUM>-positive persons, a method of cancer treatment and/or prevention for patients with malignant tumors including leukemia, the method being based on a protein product of the tumor suppressor gene WT1 (WT1 protein) or a partial peptide thereof (WT1 peptide).

The present inventor conducted intensive studies to achieve the above-mentioned object. As a result, he found that the WT1<NUM> peptide (SLGEQQYSV) and the WT1<NUM> peptide (RMFPNAPYL) each derived from the human WT1 protein, which were known to induce HLA-A*<NUM>-restricted CTLs only, surprisingly induce HLA-A*<NUM>-restricted CTLs as well. Under the circumstances where only the peptides described in <CIT> pamphlet were known as a WT1 peptide that induces HLA-A*<NUM>-restricted CTLs, the present inventor found that a modified peptide of the WT1<NUM> peptide (also referred to as a modified WT1<NUM> peptide) and a modified peptide of the WT1<NUM> peptide (also referred to as a WT1<NUM> modified peptide) also bind to an HLA-A*<NUM> molecule. Based on these findings, the present inventor conducted further intensive studies and completed the present invention.

Namely, the present invention relates to the following (<NUM>) to (<NUM>).

The "cancer vaccine composition" as used herein refers to a medicament used for cancer prevention or treatment via inoculation or administration to an animal including a human. The "treatment" refers to, besides completely curing disease state, stopping progression of disease state by inhibiting progression and/or aggravation of symptoms to some degree even falling short of a complete cure; or improving all or a part of disease state in a direction towards a cure. The "prevention" refers to preventing, inhibiting or delaying disease development.

The following terms: peripheral blood mononuclear cells, immature dendritic cells, WT1-specific CTLs, samples etc. derived from HLA-A*<NUM>-positive or HLA-A*<NUM>-positive persons refer to peripheral blood mononuclear cells, immature dendritic cells, WTl-specific CTLs, biological specimens etc. , such as blood, which are isolated or collected from HLA-A*<NUM>-positive or HLA-A*<NUM>-positive persons, respectively. The WT1-specific CTLs derived from HLA-A*<NUM>-positive or HLA-A*<NUM>-positive persons also include CTLs induced from peripheral blood mononuclear cells, immature dendritic cells or biological specimens such as blood, which are isolated or collected from HLA-A*<NUM>-positive or HLA-A*<NUM>-positive persons.

The present invention enables in vivo and in vitro induction of WTl-specific CTLs in HLA-A*<NUM>-positive subjects. Although the subjects of immunotherapy using a vaccine comprising the WT1 protein or WT1 peptide have conventionally been limited to HLA-A*<NUM>-positive patients and HLA-A*<NUM>-positive patients, the present invention can widen the range of the subjects to HLA-A*<NUM>-positive patients. HLA-A2 , which is a serotype of HLA class I antigens, is the most frequent in Caucasians (about <NUM>%), and the large majority have HLA-A*<NUM>, while about <NUM>% of Caucasians have HLA-A*<NUM>. On the other hand, HLA-A24 is the most frequent serotype in Japanese people (about <NUM>%), and the large majority have HLA-A*<NUM>. About <NUM>% of Japanese people have HLA-A2. Among them, only about <NUM>% have HLA-A*<NUM>, and the others have HLA-A*<NUM> or HLA-A*<NUM>. In other words, about <NUM>% of Japanese people have HLA-A*<NUM>, and about <NUM>% of Japanese people have HLA-A*<NUM>. Therefore, the fact that at least an HLA-A*<NUM>-restricted CTL epitope was identified from Japanese people as well as an HLA-A*<NUM>-restricted CTL epitope is significantly useful to widen the subjects of cancer immunotherapy to HLA-A*<NUM>-positive persons. Since <NUM>% of Chinese people and <NUM>% of South Korean people have this allele, it is possible to apply the cancer vaccine composition of the present invention to a further wider range of subjects.

The cancer vaccine composition of the present invention is useful for treatment of WT1-expressing cancers such as hematopoietic tumors and solid cancers in HLA-A*<NUM>-positive persons. The cancer vaccine composition of the present invention is also useful for prevention of cancer development in HLA-A*<NUM>-positive persons.

Hereinafter, the present invention will be illustrated.

The following codes are used when amino acid residues are abbreviated in this description and drawings.

The WT1 protein as disclosed herein may be a gene product of a zinc finger-type transcription factor isolated as a causative gene of Wilms' tumor, the gene product being capable of binding to an HLA-A*<NUM> molecule and thereby serving as a target antigen of malignant tumors. More specifically, the WT1 protein as disclosed herein is preferably the human WT1 protein consisting of <NUM> amino acids (Sequence list: SEQ ID NO: <NUM>) or a protein which consists of an amino acid sequence comprising deletion, substitution or addition of one to several amino acids (preferably about <NUM> to <NUM> amino acids) in the amino acid sequence of the human WT1 protein, and which is immunogenic in HLA-A*<NUM>-positive persons. The amino acid used for addition or substitution may be a non-natural amino acid besides <NUM> gene-encoded amino acids.

The partial peptide of the WT1 protein (WT1 peptide) refers to a peptide consisting of a part of the amino acid sequence that constitutes the WT1 protein. The WT1 peptide may be a peptide which consists of <NUM> to <NUM> amino acids, preferably <NUM> to <NUM> amino acids derived from the WT1 protein and which binds to an HLA-A*<NUM> molecule and thereby induces cytotoxic T cells. Particularly preferred is the WT1<NUM> peptide (Ser Leu Gly Glu Gin Gin Tyr Ser Val; SEQ ID NO: <NUM>) or the WT1<NUM> peptide (Arg Met Phe Pro Asn Ala Pro Tyr Leu; SEQ ID NO: <NUM>), both described in the <CIT> pamphlet.

A modified peptide comprising deletion, substitution or addition of one or several amino acids of the WT1 peptide can also be used as the WT1 peptide as disclosed herein as long as it is immunogenic in HLA-A*<NUM>-positive persons. Examples of such a modified peptide include a modified WT1<NUM> peptide and a modified WT1<NUM> peptide.

The modified WT1<NUM> peptide is preferably a peptide comprising the same amino acid residues (EQQYS) at positions <NUM> to <NUM> from the N terminus as the WT1<NUM> peptide has at the corresponding positions, and more preferably a peptide comprising the same amino acid residues (EQQYSV) at positions <NUM> to <NUM> from the N terminus as the WT1<NUM> peptide has at the corresponding positions. Such a modified WT1<NUM> peptide is preferably a peptide consisting of any of the following amino acid sequences of SEQ ID NO: <NUM> to <NUM> and <NUM> to <NUM>.

The modified WT1<NUM> peptide is preferably a peptide comprising the same amino acid residues (PNAPY) at positions <NUM> to <NUM> from the N terminus as the WT1<NUM> peptide has at the corresponding positions. Such a modified WT1<NUM> peptide is preferably a peptide consisting of any of the following amino acid sequences of SEQ ID NO: <NUM> to <NUM> and <NUM> to <NUM>.

Inter alia, the modified WT1<NUM> peptide is preferably the WT1<NUM>P1F peptide (SEQ ID NO: <NUM>), the WT1<NUM>P2M peptide (SEQ ID NO: <NUM>) or the WT1<NUM>P3M peptide (SEQ ID NO: <NUM>), more preferably the WT1<NUM>P1F peptide or the WT1<NUM>P2M peptide, and still more preferably the WT1<NUM>P2M peptide. The modified WT1<NUM> peptide is preferably the WT1<NUM>P1F peptide (SEQ ID NO: <NUM>), the WT<NUM>P2L peptide (SEQ ID NO: <NUM>), the WT1<NUM>P3M peptide (SEQ ID NO: <NUM>) or the WT1<NUM>P9V peptide (SEQ ID NO: <NUM>), more preferably the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide, and still more preferably the WT1<NUM>P9V peptide.

The WT1 peptide in the cancer vaccine composition of the present invention is preferably the WT1<NUM> peptide, the WT1<NUM> peptide, the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide, the WT1<NUM>P3M peptide, the WT1<NUM>P1F peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide. More preferred is the WT1<NUM> peptide, the WT1<NUM> peptide, the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide. Even preferred is the WT1<NUM> peptide, the WT1<NUM> peptide, the WT1<NUM>P2M peptide or the WT1<NUM>P9V peptide. Particularly preferred is the WT1<NUM> peptide or the WT1<NUM> peptide.

A derivative of the WT1 peptide can also be used as the WT1 peptide. For example, the derivative of the WT1<NUM> or WT1<NUM> peptide may be formed of an amino acid sequence of the above-mentioned <NUM> contiguous amino acids and various substances bound to the N and/or C terminus thereof. The various substances may be, for example, amino acids, peptides, analogs thereof, etc. Such a substance bound to the WT1<NUM> peptide, the WT1<NUM> peptide or a modified peptide thereof undergoes, for example, in vivo enzyme treatment through intracellular processing etc., and finally the peptide consisting of the above-mentioned <NUM> amino acids is produced and presented as a complex with an HLA-A*<NUM> molecule on the cell surface. Thus, a WT1-specific CTL response can be induced in patients with HLA-A*<NUM>.

The WT1 peptide can be prepared by a method usually used in the technical field, such as a peptide synthesis method described in <NPL>; <NPL>; <NPL>; <NPL>; the <NPL>; etc..

As a method of screening for the WT1 peptide and a modified peptide thereof, for example, a method involving conducting the IFNγ assay under single stimulation of, with a peptide, PBMCs (peripheral blood mononuclear cells) of some patients having HLA-A*<NUM>, and then selecting a peptide showing a good response, is preferred because of simplicity.

In the present invention, polynucleotides, such as DNA encoding the above-mentioned WT1 protein or WT1 peptide immunogenic in HLA-A*<NUM>-positive persons, can also be used as an active ingredient of the cancer vaccine composition. Namely, by inserting a polynucleotide encoding the WT1 protein or WT1 peptide into a suitable vector, preferably an expression vector, and then administering the vector into animals including humans, cancer immunity can be produced in the living body. Examples of the polynucleotide include DNA, RNA and the like, and preferred is DNA or RNA. The base sequence of the polynucleotide can be determined based on the amino acid sequence of the WT1 protein or WT1 peptide immunogenic in HLA-A*<NUM>-positive persons. The polynucleotide can be prepared by a known DNA or RNA synthesis method, the PCR method, etc. Such a cancer vaccine composition for HLA-A*<NUM>-positive persons, comprising DNA encoding the WT1 protein or WT1 peptide is also one aspect of the present invention. The WT1 protein or WT1 peptide is preferably a WT1 peptide, more preferably the WT1<NUM> peptide, the WT1<NUM> peptide or a modified peptide thereof, and most preferably the WT1<NUM> peptide or the WT1<NUM> peptide. The expression vector used to insert the above-mentioned DNA into is not particularly limited. RNA does not have to be inserted into a vector and can be used as it is as an active ingredient of the composition.

The cancer vaccine composition of the present invention can comprise an adjuvant. The adjuvant is not limited as long as, after administered together with or separately from the WT1 protein or WT1 peptide used as an antigen, it can nonspecifically enhance immunological responses to the antigen. Examples of the adjuvant include precipitating-type adjuvants and oily adjuvants. Examples of the precipitating-type adjuvant include sodium hydroxide, aluminum hydroxide, calcium phosphate, aluminum phosphate, alum, PEPES and carboxyvinyl polymers. A preferable oily adjuvant is one that can form micelles so that oil encloses an aqueous solution of an antigen. Specific examples thereof include liquid paraffin, lanolin, Freund, Montanide ISA-763AVG, Montanide ISA-<NUM>, incomplete Freund's adjuvant and complete Freund's adjuvant. These adjuvants can also be used as a mixture of two or more kinds thereof. Preferred is an oily adjuvant.

The amount of the adjuvant in the cancer vaccine composition of the present invention is not particularly limited as long as immunological responses to antigens can be nonspecifically enhanced. The amount thereof may be suitably selected depending on the kind of the adjuvant, etc..

The cancer vaccine composition of the present invention can be administered orally or parenterally (for example, intraperitoneally, subcutaneously, intracutaneously, intramuscularly, intravenously, intranasally, etc.). In the case of parenteral administration, an active ingredient, i.e., the WT1 protein or WT1 peptide, may also be percutaneously absorbed by applying the vaccine composition to the skin, or by attaching to the skin a patch containing the vaccine composition. The vaccine composition of the present invention can also be administered via inhaling etc. The vaccine composition is administered preferably parenterally, and more preferably intracutaneously or subcutaneously. The body part for intracutaneous or subcutaneous administration is preferably the upper arm etc., for example.

The cancer vaccine composition of the present invention can be in various dosage forms depending on its administration route, and exemplary dosage forms thereof include a solid preparation and a liquid preparation. The cancer vaccine composition may be, for example, in the form of a solid or liquid preparation to be used internally for oral administration, an injection for parenteral administration, or the like.

Examples of the solid preparation to be used internally for oral administration include tablets, pills, capsules, powders and granules.

For preparation of the solid preparation to be used internally, the WT1 protein or WT1 peptide is untreated, mixed with an additive, or granulated (according to, for example, stirring granulation, fluidized bed granulation, dry granulation, rolling stirring fluidized bed granulation, etc.), and then is subjected to a usual method. For example, the capsules can be prepared by encapsulation etc. and the tablets can be prepared by tableting etc. One or two kinds or more of the additives may be appropriately incorporated into the solid preparation. Examples of the additive include excipients such as lactose, mannitol, glucose, microcrystalline cellulose and corn starch; binders such as hydrpxypropylcellulose, polyvinylpyrrolidone and magnesium aluminometasilicate; dispersing agents such as corn starch; disintegrators such as calcium carboxymethyl cellulose; lubricants such as magnesium stearate; solubilizing agents such as glutamic acid and aspartic acid; stabilizers; water soluble polymers including celluloses such as hydroxypropylcellulose, hydroxypropylmethylcellulose and methylcellulose, and synthetic polymers such as polyethylene glycol, polyvinylpyrrolidone and polyvinyl alcohol; and sweeteners such as white sugar, powder sugar, sucrose, fructose, glucose, lactose, reduced malt sugar syrup (maltitol syrup), reduced malt sugar syrup powder (maltitol syrup powder), high-glucose corn syrup, high-fructose corn syrup, honey, sorbitol, maltitol, mannitol, xylitol, erythritol, aspartame, saccharin and saccharin sodium.

The granules or tablets may be covered with a coating agent etc. if needed, and may be covered with two or more layers thereof. Examples of the coating agent include white sugar, gelatin, hydroxypropyl cellulose and hydroxypropylmethylcellulose phthalate. The capsules can be prepared by mixing the active ingredient with pranlukast hydrate and an excipient appropriately selected from the above excipients, optionally granulating the mixture, and optionally covering the resulting granules with a coating agent, followed by capsule filling. Alternatively, the capsules can be prepared by adding glycerol, sorbitol, etc. to an appropriate capsule base (gelatin etc.) to increase its plasticity, and encapsulating the active ingredient with the resulting base. To the capsule base may be added a colorant or a preservative (sulfur dioxide; and parabens such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate and propyl parahydroxybenzoate) if needed. The capsules include hard capsules and soft capsules.

Examples of the liquid preparation to be used internally for oral administration include waters, suspensions/emulsions, syrups, preparations to be dissolved before use such as dry syrups, and elixirs. For preparation of the liquid preparation to be used internally, the WT1 protein or WT1 peptide is dissolved, suspended or emulsified in a diluent generally used for liquid preparations to be used internally. Examples of the diluent include purified water, ethanol and a mixture thereof. The liquid preparation may further contain a wetting agent, a suspending agent, an emulsifier, a sweetener, a flavoring, a fragrance, a preservative or a buffering agent. The dry syrups can be prepared, for example, by mixing the active ingredient with pranlukast hydrate and an additional ingredient such as white sugar, powder sugar, sucrose, fructose, glucose and lactose. The dry syrups may also be made into granules in a usual manner.

Examples of the dosage form for parenteral administration include injections, ointments, gels, creams, patches, aerosols and sprays. Preferred are injections. For example, the injection preferably contains a conventional carrier with the WT1 protein or WT1 peptide.

The injection for parenteral administration may be an aqueous injection or an oily injection. The aqueous injection can be prepared according to a known method, for example, by appropriately adding a pharmaceutically acceptable additive to an aqueous solvent (water for injection, purified water, etc.) to make a solution, mixing the WT1 protein or WT1 peptide with the solution, filter sterilizing the resulting mixture with a filter etc., and then filling an aseptic container with the resulting filtrate. Examples of the pharmaceutically acceptable additive include the above-mentioned adjuvants; isotonizing agents such as sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, glucose and propylene glycol; buffering agents such as a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a carbonate buffer solution, a citrate buffer solution, a Tris buffer solution, a glutamate buffer solution and an epsilon-aminocaproate solution; preservatives such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid and borax; thickeners such as hydroxyethylcellulose, hydroxypropylcellulose, polyvinyl alcohol and polyethylene glycol; stabilizers such as sodium hydrogen sulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid and dibutyl hydroxy toluene; and pH adjusters such as hydrochloric acid, sodium hydroxide, phosphoric acid and acetic acid. The injection may further contain an appropriate solubilizing agent, and examples thereof include alcohols such as ethanol; polyalcohols such as propylene glycol and polyethylene glycol; and non-ionic surfactants such as polysorbate <NUM>, polyoxyethylene hydrogenated castor oil <NUM>, lysolecithin and pluronic polyols. Also, proteins such as bovine serum albumin and keyhole limpet hemocyanin; polysaccharides such as aminodextran; etc. may be contained in the injection. For preparation of the oily injection, for example, sesame oil or soybean oil is used as an oily solvent, and benzyl benzoate or benzyl alcohol may be blended as a solubilizing agent. The prepared injection is usually stored in an appropriate ampule, vial, etc. The liquid preparations, such as injections, can also be deprived of moisture and preserved by cryopreservation or lyophilization. The lyophilized preparations become ready to use by redissolving them in added distilled water for injection etc. just before use.

Another dosage form of the cancer vaccine composition of the present invention may be a liposome containing the WT1 protein or WT1 peptide and, if needed, polysaccharides and/or other ingredients that can be blended into the cancer vaccine composition.

The dose of the cancer vaccine composition of the present invention varies with the kind of the WT1 protein, WT1 peptide or DNA to be used, the age and body weight of the patient, the disease to be treated, etc. For example, in the case of the vaccine composition comprising the WT1 peptide, for example the WT1<NUM> peptide or the WT1<NUM> peptide, the daily dose is preferably about <NUM>µg/kg bw to <NUM>/kg bw as the amount of the WT1 peptide. The dose of the WT1 peptide is usually <NUM> to <NUM>, preferably <NUM> to <NUM>, and more preferably <NUM> to <NUM>. This amount is preferably administered once in several days to several months.

The cancer vaccine composition of the present invention is a cancer vaccine composition for HLA-A*<NUM>-positive persons. The HLA type, which, is a measure , for selecting HLA-A*<NUM>-positive persons, can be determined from, for example, donors' peripheral blood. Examples of the method of determining the HLA type include known methods, such as the DNA typing method, for example, the SBT (Sequencing Based Typing) method or the SSP method, and the HLA typing method. In the SBT method, the base sequence of a PCR-amplified DNA is compared with the base sequence data of the known alleles to precisely identify the HLA gene type. In the SSP method, after PCR amplification using a variety of primers specific to respective HLA alleles, subsequent electrophoresis is performed to check a positive band. Thus, the HLA gene type can be identified.

When the cancer vaccine composition of the present invention has been administered into an HLA-A*<NUM>-positive person, the HLA-A*<NUM>-restricted WT1 protein or WT1 peptide in the vaccine composition, or the WT1 protein or WT1 peptide expressed from DNA or RNA in the vaccine composition binds to an HLA-A*<NUM> molecule on the surface of an antigen presenting cell (dendritic cell) of the HLA-A*<NUM>-positive person. This induces specific antitumor immunity, i.e., WT1-specific CTLs, which destroy cancer cells in the subject (HLA-A*<NUM>-positive person). Such antitumor immunity can be checked, for example by the WT1-specific CTL response, the cytotoxicity test against cancer cells (for example, <NUM>Cr release cytotoxicity test), etc. For example, the HLA-A*<NUM>-restricted WT1<NUM> peptide and WT1<NUM> peptide each consisting of <NUM> amino acids derived from the WT1 protein, which have been reported to be capable of inducing a WT1-specific CTL response, can induce an HLA-A*<NUM>-restricted response. About <NUM>% of Japanese people are HLA-A*<NUM>-positive, while almost the same proportion are HLA-A*<NUM>-positive. In the following Examples <NUM> to <NUM>, WT1<NUM> peptide-specific CTLs were prepared from PBMCs of three HLA-A*<NUM>-positive blood donors. The induced CTLs showed the cytotoxic effect on WT1-expressing, HLA-A*<NUM>-positive leukemia cells. Since WT1<NUM> peptide- and WT1<NUM> peptide-specific CTL activity can be inhibited by an anti-HLA class I antibody, the activity is found to be exhibited by HLA class I-restricted CTLs. The WT1 protein or WT1 peptide including the WT1<NUM> peptide and/or the WT1<NUM> peptide, or a modified peptide thereof can be a vaccine for HLA-A*<NUM>-positive cancer patients as well as HLA-A*<NUM>-positive cancer patients. Therefore, the immunotherapy based on the WT1 protein or WT1 peptide for patients with malignant tumors, such as hematopoietic tumors and solid cancers, can be applied further to HLA-A*<NUM>-positive cancer patients. The method of cancer treatment and/or prevention in HLA-A*<NUM>-positive persons, comprising administering the cancer vaccine composition of the present invention into an HLA-A*<NUM>-positive person, is also disclosed herein.

In HLA-A*<NUM>-positive persons, the cancer vaccine composition of the present invention can be used for treatment and/or prevention of cancers accompanied by increased expression of the WT1 gene: for example, hematopoietic tumors such as leukemia, myelodysplastic syndrome, multiple myeloma and 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 and ovarian cancer.

An exemplary administration method of the cancer vaccine composition as disclosed herein is a method comprising collecting PBMCs from peripheral blood of an HLA-A*<NUM>-positive patient, extracting dendritic cells from the PBMCs, pulsing the dendritic cells with a peptide, for example the WT1<NUM> peptide or the WT1<NUM> peptide, or a polynucleotide, for example DNA or RNA, contained as an active ingredient in the cancer vaccine composition of the present invention, and returning the dendritic cells to the patient via subcutaneous administration etc. The conditions for pulsing dendritic cells with the WT1 peptide etc. are not particularly limited as long as the effect of the present invention is achieved, and may be ordinary conditions.

In the case where RNA encoding the WT1 protein or WT1 peptide is used for the cancer vaccine composition, it is preferable that the composition is administered so that the RNA is introduced into dendritic cells of an HLA-A*<NUM>-positive person. An exemplary method for introducing RNA into dendritic cells of an HLA-A*<NUM>-positive person is a method comprising collecting dendritic cells from an HLA-A*<NUM>-positive person in the same manner as mentioned above, and introducing RNA into the dendritic cells with an electric pulse. The WT1 protein or WT1 peptide expressed from the introduced RNA in the dendritic cells is allowed to be presented on the surface thereof. By returning the dendritic cells pulsed with the RNA into the HLA-A*<NUM>-positive person, cancer immunity can be quickly produced in the living body. Such a method of cancer treatment or prevention, comprising introducing RNA encoding the WT1 protein or WT1 peptide into dendritic cells of an HLA-A*<NUM>-positive person, is also disclosed herein. Another embodiment of the present invention relates to an ex vivo method for inducing WT1-specific CTLs, by culturing, in the presence of the WT1 protein or WT1 peptide, PBMCs derived from an HLA-A*<NUM>-positive person, to obtain WT1-specific CTLs induced therefrom. The subject from which PBMCs are derived is not particularly limited as long as the subject is HLA-A*<NUM>-positive. Examples of the WT1 protein or WT1 peptide include the WT1<NUM> peptide, the WT1<NUM> peptide and a modified peptide thereof, and preferably the WT1<NUM> peptide and the WT1<NUM> peptide. For example, WT1-specific CTLs can be induced from CTL precursor cells among PBMCs by culturing PBMCs derived from an HLA-A*<NUM>-positive person in the presence of the WT1<NUM> peptide (or WT1<NUM> peptide). The culture conditions for PBMCs derived from an HLA-A*<NUM>-positive person is not particularly limited, and may be ordinary conditions. The thus-obtained CTLs recognize a complex of the WT1<NUM> peptide (or the WT1<NUM> peptide) and an HLA-A*<NUM> molecule. Therefore, by use of WT1-specific CTLs induced according to the present invention, WT1-highly-expressing tumor cells can be specifically destroyed in an HLA-A*<NUM>-positive person, and thereby hematopoietic tumors and solid cancers in the subject, i.e., an HLA-A*<NUM>-positive person, can be treated and/or prevented. The method for administering such WT1-specific CTLs into an HLA-A*<NUM>-positive subject is not particularly limited, and for example, may be the same as the administration method of the above-mentioned cancer vaccine composition.

There is also disclosed a kit for inducing WT1-specific CTLs, comprising the HLA-A*<NUM>-restricted WT1 protein or WT1 peptide as an essential constituent. Preferably, the kit is used for the above-mentioned method for inducing WT1-specific CTLs derived from an HLA-A*<NUM>-positive person. Such a kit may comprise, for example, a means for collecting PBMCs, an adjuvant and a reaction container in addition to the HLA-A*<NUM>-restricted WT1 protein or WT1 peptide. By use of the kit, WT1-specific CTLs that recognize a complex of a cancer antigen, such as the WT1<NUM> peptide and the WT1<NUM> peptide, and an HLA-A*<NUM> molecule can be efficiently induced. Another embodiment of the present invention relates to an ex vivo method for inducing dendritic cells that present the WT1 protein or WT1 peptide, by culturing, in the presence of the WT1 protein or WT1 peptide, immature dendritic cells derived from an HLA-A*<NUM>-positive person, to obtain dendritic cells induced therefrom which present the WT1 protein or WT1 peptide. Examples of the WT1 protein or WT1 peptide include the WT1<NUM> peptide, the WT1<NUM> peptide and a modified peptide thereof, and preferably the WT1<NUM> peptide and the WT1<NUM> peptide. The subject from which immature dendritic cells are derived is not particularly limited as long as the subject is HLA-A*<NUM>-positive. Since immature dendritic cells are present among PBMCs etc., PBMCs may also be cultured in the presence of the WT1<NUM> peptide or the WT1<NUM> peptide, for example. By administration of the thus-obtained dendritic cells to an HLA-A*<NUM>-positive person, the above-mentioned WT1-specific CTLs are efficiently induced, and thereby hematopoietic tumors and solid cancers in the subject can be treated and/or prevented. The method for administering such dendritic cells into an HLA-A*<NUM>-positive subject is not particularly limited, and for example, may be the same as the administration method of the above-mentioned cancer vaccine composition.

There is also disclosed a kit for inducing dendritic cells that present the WT1 protein or WT1 peptide, comprising the HLA-A*<NUM>-restricted WT1 protein or WT1 peptide as an essential constituent. Preferably, the kit is used for the above-mentioned method for inducing dendritic cells. Such a kit may comprise, for example, a means for collecting immature dendritic cells and PBMCs, an adjuvant and a reaction container in addition to the HLA-A*<NUM>-restricted WT1 protein or WT1 peptide. By use of the kit, dendritic cells that present the WT1 protein or WT1 peptide via an HLA-A*<NUM> molecule can be efficiently induced.

Cancers in HLA-A*<NUM>-positive persons can be diagnosed by use of.

Such an in vitro method of cancer diagnosis is also one aspect of the present invention. In the above (<NUM>), cancer diagnosis is conducted preferably using WT1 specific CTLs induced by above-mentioned method. Examples of the WT1 protein or WT1 peptide include the WT<NUM> peptide. the WT1<NUM> peptide and a modified peptide thereof, and preferably the WT1<NUM> peptide and the WT1<NUM> peptide.

According to the present invention, an exemplary in vitro method cancer diagnosis for HLA-A*<NUM>-positive persons comprises a step of detecting or quantifying the WT1 protein or WT1 peptide, an antibody or WT1-specific CTLs thereagainst in a sample from an HLA-A*-<NUM>-positive person, and a step of comparing the amount of said protein or partial peptide thereof, or said antibody or said WT1-specific CTLs thereagainst with that in the case where cancer is not developed.

In a cancer patient sample (for example, blood), the WT1 peptide and/or WT1 protein released from cancer cells is present, and the immunological response against a cancer antigen is enhanced. That is, the cancer patient sample has an increased amount of an antibody against the WT1 peptide or WT1 protein, WT1-specific CTLs, etc. For this reason, when the amount of the WT1 peptide or WT1 protein the antibody or the WT1-specific CTLs thereagainst in the sample is increased. compared with that in the case where cancer is not developed, cancer may have been developed. The amount of the antibody can be measured by the ELISA method, for example, The WT1-specific CTLs can be detected by a method using WT1 multimers such as MHC tetramers described below.

Alternatively, cancer diagnosis can also be performed by incubating the above-mentioned CTLs, dendritic cells or antibody together with a sample from an HLA-A*<NUM>-positive subject, or administering the above-mentioned CTLs, dendritic cells or antibody into an HLA-A*<NUM>-positive subject; and then determining the position, region, amount, etc. of the CTLs, dendritic cells or antibody. Since CTLs and dendritic cells have a property to gather around cancer cells, cancer diagnosis can be performed by administering the CTLs or dendritic cells into the subject, and examining the position or region thereof. A method of cancer diagnosis for HLA-A*<NUM>-positive persons, comprising a step of administering WT1-specific CTLs or dendritic cells induced by the above-mentioned method into an HLA-A*<NUM>-positive subject, and a step of determining the position or region of the CTLs or dendritic cells in the HLA-A*<NUM>-positive subject is also disclosed herein.

Cancer diagnosis can also be performed by incubating CTLs or dendritic cells together with a sample from an HLA-A*<NUM>-positive subject to allow them to react, adding an antibody against the CTLs or dendritic cells, continuing incubation, and detecting or quantifying an antibody-bound complex of the cancer cell and CTLs, antibody-bound dendritic cells, etc. via a label etc. bound to the antibody. When the amount of the antibody-bound complex of the cancer cell and CTLs or the antibody-bound dendritic cells is increased compared with that in the case where cancer is not developed, cancer may have been developed. The above-mentioned CTLs, dendritic cells or antibody may be labeled. The labeling enables the diagnosis to be efficiently performed. Examples of the sample from an HLA-A*<NUM>-positive subject include biological specimens obtained from HLA-A*<NUM>-positive persons, such as urine, blood, tissue extract fluid, saliva, tear and other body fluids, and blood is preferable.

Examples of the method of cancer diagnosis for HLA-A*<NUM>-positive persons using the above-mentioned WT1 protein or WT1 peptide include the MHC tetramer assay, the MHC pentamer assay and the MHC dextramer assay, each of which uses the WT1 peptide as an antigen. For example, in the MHC tetramer assay or MHC pentamer assay using the WT1<NUM> peptide or WT1<NUM> peptide as an antigen peptide, WT1-specific CTLs in HLA-A*<NUM>-positive persons can be detected by use of an MHC/WT1<NUM> peptide complex or an MHC/WT1<NUM> peptide complex as a probe. Since cancer patients show high expression of WT1-specific CTLs, cancer can be diagnosed by measuring the expression of WT1-specific CTLs in HLA-A*<NUM>-positive persons. Since cancer patients manifest an enhanced immunological response against cancer antigens, cancer can be diagnosed also by examining immunological response against the WT1 protein or WT1 peptide in HLA-A*<NUM>-positive persons. Examples of the method of examining immunological response include a method involving measuring an antibody against the WT1 protein or WT1 peptide by ELISA. Such a method of cancer diagnosis for HLA-A*<NUM>-positive persons using a protein product of the tumor suppressor gene WT1 or a partial peptide thereof is also one aspect of the present invention. The MHC tetramer assay and MHC pentamer assay can be performed by a known method using a commercially available kit, for example, "WT1 tetramer" (Medical & Biological Laboratories, Co.

Cancer diagnosis for HLA-A*<NUM>-positive persons can also be performed by a method comprising a step of reacting a sample from an HLA-A*<NUM>-positive subject with an antibody against the following: the WT1 protein or WT1 peptide, WT1-specific CTLs induced by the above-mentioned method or dendritic cells induced by the above-mentioned, and a step of detecting or quantifying a complex of the antibody with the WT1 protein or WT1 peptide, or a complex of the antibody with WT1-specific CTLs or dendritic cells. When the amount of the complex of the antibody with the WT1 protein or WT1 peptide, or the complex of the antibody with WT1-specific CTLs or dendritic cells is increased compared with that in the case where cancer is not developed, cancer may have been developed.

Examples of the antibody against dendritic cells include an antibody which recognizes a WT1 peptide/HLA-A*<NUM> complex. Since such an antibody can recognize the WT1 peptide and an HLA-A*<NUM> molecule, the antibody can recognize dendritic cells having the WT1 peptide presented via HLA Class I.

An antibody which recognizes a complex of WT1 peptide/HLA-A*<NUM>/TCR (T cell antigen receptor) of CTLs can also be used as the antibody against dendritic cells. Such an antibody can recognize a complex of a dendritic cell and a CTL, and a complex of a cancer cell and a CTL.

Cancer diagnosis can be performed by incubating such an antibody together with a sample from an HLA-A*<NUM>-positive subject to allow them to form a complex, and detecting or quantifying an antibody-bound complex of the cancer cell and CTLs , antibody-bound dendritic cells presenting the WT1 peptide, or the like via the fluorescence emitted by the antibody. When the amount of the antibody-bound complex of the cancer cell and CTLs, the antibody-bound dendritic cells presenting the WT peptide, or the like is increased compared with that in the case where cancer is not developed, cancer may have been developed.

A method of cancer treatment or prevention, comprising administering a composition containing the WT1 protein or WT1 peptide into an HLA-A*<NUM>-positive person, is also disclosed herein. The composition comprising the WT1 protein or WT1 peptide and preferable embodiments thereof are the same as described regarding the above-mentioned cancer vaccine composition.

Use of the WT1 protein or WT1 peptide for cancer treatment or prevention in HLA-A*<NUM>-positive persons, and use thereof for production of a cancer vaccine composition used for cancer treatment or prevention in HLA-A*<NUM>-positive persons is also one aspect of the present invention. The WT1 protein or WT1 peptide and preferable embodiments thereof are the same as described regarding the above-mentioned cancer vaccine composition.

A cancer vaccine composition for HLA-A*<NUM>-positive persons , comprising a modified peptide of the WT1<NUM> peptide (SEQ ID NO: <NUM>) or the WT1<NUM> peptide (SEQ ID NO: <NUM>), either of which is a partial peptide of a protein product of the tumor suppressor gene WT1, the modified peptide being immunogenic in HLA-A*<NUM>-positive persons, is also one aspect of the present invention.

Examples of a modified WT1<NUM> peptide or a modified WT1<NUM> peptide include peptides comprising deletion, substitution or addition of one or several amino acids of the above-mentioned WT1<NUM> peptide or WT1<NUM> peptide. The modified WT1<NUM> peptide is preferably a peptide comprising the same amino acid residues at positions <NUM> to <NUM> from the N terminus as the WT1<NUM> peptide has at the corresponding positions. As such a modified peptide, preferred are the above-mentioned peptides of SEQ ID NOS: <NUM> to <NUM>, <NUM> and <NUM>, <NUM> to <NUM> and <NUM> to <NUM>. The WT1<NUM>P9L peptide (SLGEQQYSL; SEQ ID NO: <NUM>) is also preferred. The modified WT1<NUM> peptide is preferably a peptide comprising the same amino acid residues at positions <NUM> to <NUM> from the N terminus as the WT1<NUM> peptide has at the corresponding positions. For example, preferred are the above-mentioned peptides of SEQ ID NOS: <NUM> to <NUM> and <NUM> to <NUM>.

In yet another preferable embodiment of the present invention, the above-mentioned peptides of SEQ ID NOS: <NUM> to <NUM> and <NUM> to <NUM> may be used as a modified WT1<NUM> peptide, and the above-mentioned peptides of SEQ ID NOS: <NUM> to <NUM> and <NUM> to <NUM> may be used as a modified WT1<NUM> peptide. Among the modified peptides of SEQ ID NOS: <NUM> to <NUM>, the peptides except the WT1<NUM>P1D peptide, the WT1<NUM>P1E peptide, the WT1<NUM>P1H peptide, the WT1<NUM>P1P peptide and the WT1<NUM>P2Q peptide; and the WT1<NUM>P1D peptide, the WT1<NUM>P1E peptide, the WT1<NUM>P1P peptide, the WT1<NUM>P2A peptide and the WT1<NUM>P2Q peptide are preferred.

Inter alia, the modified WT1<NUM> peptide is preferably the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide or the WT1<NUM>P3M peptide, and more preferably the WT1<NUM>P1F peptide or the WT1<NUM>P2M peptide. The modified WT1<NUM> peptide is preferably the WT1<NUM>P1F peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide, and more preferably the WT1<NUM>P1F peptide or the WT1<NUM>P2L peptide.

The amount for use of the modified WT1<NUM> peptide or WT1<NUM> peptide which is immunogenic in HLA-A*<NUM>-positive persons is the same as that of the WT1 peptide in the above-mentioned cancer vaccine composition for HLA-A*<NUM>-positive persons. The other ingredients of the cancer vaccine composition for HLA-A*<NUM>-positive persons and preferable embodiments thereof are the same as those of the above-mentioned vaccine composition for HLA-A*<NUM>-positive persons.

DNA and RNA encoding the above-mentioned modified WT1<NUM> peptide or WT1<NUM> peptide which is immunogenic in HLA-A*<NUM>-positive persons can also be used as an active ingredient of the cancer vaccine composition for HLA-A*<NUM>-positive persons. Such a cancer vaccine composition for HLA-A*<NUM>-positive persons is also one aspect of the present invention.

The other ingredients than the above-mentioned DNA and RNA in the cancer vaccine composition for HLA-A*<NUM>-positive persons and preferable embodiments thereof are the same as those of the above-mentioned cancer vaccine composition for HLA-A*<NUM>-positive persons.

WT1-specific CTLs can be induced from PBMCs derived from an HLA-A*<NUM>-positive person by culturing the PBMCs in the presence of the modified WT1<NUM> peptide or WT1<NUM> peptide which is immunogenic in the above-mentioned HLA-A*<NUM>-positive person. Such a method of inducing WT1-specific CTLs is also disclosed herein.

Preferable examples of the modified WT1<NUM> peptide or WT1<NUM> peptide which is immunogenic in HLA-A*<NUM>-positive persons are the same as used for the above-mentioned cancer vaccine composition for HLA-A*<NUM>-positive persons.

Dendritic cells that present the modified WT1<NUM> peptide or WT1<NUM> peptide can be induced from immature dendritic cells derived from an HLA-A*<NUM>-positive person by culturing the immature dendritic cells in the presence of the modified peptide which is immunogenic in the above-mentioned HLA-A*<NUM>-positive person. Such a method for inducing dendritic cells that present the modified WT1<NUM> peptide or WT1<NUM> peptide is also disclosed herein. Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*<NUM>-positive persons.

Cancers in HLA-A*<NUM>-positive persons can be diagnosed by use of the above-mentioned modified WT1<NUM> peptide or modified WT1<NUM> peptide immunogenic in HLA-A*<NUM>-positive persons, an antibody thereagainst, WT1-specific CTLs induced by the modified peptide or dendritic cells induced by the modified peptide. Such a method of cancer diagnosis for HLA-A*<NUM>-positive persons is also disclosed herein.

The method of cancer diagnosis for HLA-A*<NUM>-positive persons and preferable embodiments thereof are the same as the above-mentioned method of cancer diagnosis for HLA-A*<NUM>-positive persons and preferable embodiments thereof.

Examples of the method of cancer diagnosis for HLA-A*<NUM>-positive persons include the MHC tetramer assay, the MHC pentamer assay and the MHC dextramer assay, each of which uses the modified WT1<NUM> peptide or modified WT1<NUM> peptide immunogenic in HLA-A*<NUM>-positive persons as an antigen. Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*<NUM>-positive persons.

Cancers in HLA-A*<NUM>-positive persons can be diagnosed by use of an antibody against the following: the above-mentioned modified WT1<NUM> peptide or modified WT1<NUM> peptide immunogenic in HLA-A*<NUM>-positive persons, WT1-specific CTLs induced by the modified peptide or dendritic cells induced by the modified peptide. Such a method of cancer diagnosis for HLA-A*<NUM>-positive persons is also disclosed herein.

Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*<NUM>-positive persons. The method of cancer diagnosis for HLA-A*<NUM>-positive persons and preferable embodiments thereof are the same as the above-mentioned method of cancer diagnosis for HLA-A*<NUM>-positive persons and preferable embodiments thereof.

A method of cancer treatment or prevention, comprising administering an HLA-A*<NUM>-positive person a cancer vaccine composition containing the following peptide:.

Preferable examples of the modified peptide are the same as used for the above-mentioned cancer vaccine composition for HLA-A*<NUM>-positive persons. The cancer vaccine composition and preferable embodiments thereof are the same as described regarding the above-mentioned vaccine composition for HLA-A*<NUM>-positive persons.

The present invention relates to use of the following peptide:.

Hereinafter, the present invention will be illustrated in more detail by way of examples, but is not limited thereto. Abbreviations in Examples indicate the following meanings. Synthetic peptides were purchased from SIGMA GENOSYS JAPAN.

HLA molecules capable of binding with the WT1<NUM> peptide (SEQ ID NO: <NUM>) were predicted using the NetMHC2. <NUM> Server-prediction program.

As a result, the HLA-A*<NUM>-restricted WT1<NUM> peptide capable of inducing WT1-specific CTLs was ranked high in terms of binding affinity to an HLA-A*<NUM> molecule in the NetMHC2. <NUM> Server-prediction program (http://www. dk/services/NetMHC-<NUM>/).

First, PBMCs were isolated from peripheral blood of each of HLA-A*<NUM> healthy blood donors (three persons) by Ficoll-Hypaque density gradient centrifugation. Then, CD14-positive cells were selected from the PBMCs using anti-human CD14 Magnetic Particles-DM (manufactured by Becton, Dickinson and company (BD)). In this case, it was considered that a large number of CD14-positive cells are present in the monocyte population. The selected CD14-positive cells were cultured in an X-VIVO15 medium (manufactured by BioWhittaker, Walkersville, MD) supplemented with <NUM> v/v% human AB serum, <NUM> IU/mL GM-CSF (manufactured by Pepro Tech INC, Rocky Hill, NJ) and <NUM> IU/mL IL-<NUM> (manufactured by Pepro Tech INC) to prepare DCs.

The DCs prepared in the above (<NUM>) were cultured at <NUM> for <NUM> day, and then a maturation cytokine cocktail containing <NUM> ng/mL TNFα (tumor necrosis factor-α; Pepro Tech INC, Rocky Hill, NJ), <NUM> ng/mL IL-β. <NUM> IU/mL IL-<NUM> and <NUM>µg/mL PGE2 was added to culture wells containing the DCs. After <NUM> hour-culture at <NUM>, autologous mature DCs were obtained.

The autologous mature DCs were pulsed with the WT1<NUM> peptide, irradiated with 30Gy of radiation, and co-cultured with CD8-positive T cell-enriched PBMCs obtained from the HLA-A*<NUM>-positive healthy blood donor. The pulsing of the DCs with the WT1<NUM> peptide was performed by culturing the DCs in the presence of <NUM>µg/mL of the WT1<NUM> peptide at <NUM> for <NUM> minutes. The CD8-positive T cells were enriched from PBMCs of the HLA-A*<NUM>-positive healthy blood donor using CD8 MicroBeads and MS column (manufactured by Miltenyi Biotec GmbH).

From the second stimulation, autologous PBMCs which had been pulsed with the peptide and then irradiated with radiation were used as selective stimulator cells. Two days after the second stimulation, recombinant IL-<NUM> (provided by Shionogi & Co. ) and IL-<NUM> (manufactured. by Pepro Tech INC) were added to the culture medium at the concentrations of <NUM> IU/mL and <NUM> ng/mL, respectively. After the 4th stimulation, the cells were cultured for <NUM> days at <NUM> and then the resulting cells (CTLs) were collected by centrifugation using a centrifuge. The cytotoxic activity of these cells (CTLs) against target cells was examined by a <NUM>Cr release cytotoxicity test.

The cytotoxicity test was performed by a <NUM>Cr release cytotoxicity test. The <NUM>Cr release cytotoxicity test was performed as follows. First, target cells (<NUM>×<NUM><NUM> cells/mL) were incubated in the presence of <NUM>µL of <NUM>Cr (specific activity: <NUM> mCi/ml) in RPMI1640 (manufactured by NIHON PHARMACEUTICAL CO. ) supplemented with <NUM>% fetal bovine serum at <NUM> for <NUM> hours to label the target cells with <NUM>Cr. Then, the <NUM>Cr-labeled target cells were added to wells of <NUM> round-bottom well plates containing various numbers of CTLs obtained in the above (<NUM>) (suspended in <NUM>µL of an assay medium), mixed with the CTLs and then incubated at <NUM> for <NUM> hours. These cells were mixed so that the E/T ratio (cell number ratio) was <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM> or <NUM>:<NUM>, with the proviso that CTLs and the <NUM>Cr-labeled target cells are expressed as "E" and "T", respectively. After the completion of incubation, <NUM>µL of the supernatant was collected from each well. The amount of <NUM>Cr release from the labeled cells was determined, and the specific lysis (%) based on the <NUM>Cr release was calculated. The specific lysis (%) was calculated in the following manner.

In the formula, the amount of spontaneous release refers to the amount of fluorescence of culture supernatant in the wells containing target cells only, and the maximum release refers to the amount of fluorescence of culture medium in which the target cells have been completely lysed by treatment with <NUM> mass % Triton X-<NUM>.

The target cells to be used were B-LCLs, K562 cells, JY cells, and KH88 cells, which will be described in detail below. KH88 cells are the same as KH88OF8 cells used in the following Example <NUM>.

B-LCLs, which were established by EB virus-mediated transformation of peripheral blood B lymphocytes obtained from an HLA-A*<NUM>-positive blood donor, do not express WT1.

K562 cells, which were established from a patient with chronic myelogenous leukemia in blastic crisis, are a WT1-expressing, non-HLA class I-expressing cell line. The present inventor was not able to obtain a WT1-expressing, HLA-A*<NUM>-positive wild-type leukemia cell line. For this reason, 0206K562 cells, which were prepared by transformation of K562 cells with HLA-A*<NUM> genes, were also used. The FACS analysis using an anti-HLA-A2 antibody (cloneBB7. <NUM>; manufactured by BD Biosciences Pharmingen) showed that the 0206K562 cells transformed with HLA-A*<NUM> genes express HLA-A*<NUM> molecules on the cell surfaces.

The western blot analysis showed that B-LCL cells transformed with the WT1 gene express WT1. B-LCL cells transformed with a mock vector were used as a control.

JY cells are a non-WT1-expressing, HLA-A*<NUM>-negative B cell line established by EB virus-mediated transformation.

KH88 cells are a WT1-expressing, HLA-A*<NUM>-negative leukemia cell line.

Each cell line was cultured in a RPMI1640 culture medium supplemented with <NUM> v/v% heat-inactivated fetal bovine serum, <NUM> IU/mL penicillin and <NUM>/mL streptomycin.

Anti-human CD14, CD86, CD80, CD83 and HLA-DR mAbs were purchased from BD. Concentration and maturation of DCs were confirmed by analysis of cell surface antigens using the monoclonal antibodies (mAbs) listed above. Samples were analyzed with a flow cytometer (FACS Calibur; manufactured by BD) using CellQest software.

It was examined whether WT1<NUM> peptide-specific CTLs can be prepared from PBMCs of HLA-A*<NUM>-positive blood donors. The WT1<NUM> peptide-specific cytotoxic activity was examined using the CTLs obtained by repeatedly stimulating the CD8-positive T cell-enriched PBMCs from the HLA-A*<NUM>-positive healthy blood donor with WT1<NUM> peptide-pulsed autologous DCs or PBMCs. The CTLs showed a stronger cytotoxic activity against WT1<NUM> peptide-pulsed autologous B-LCL cells than against non-WT1<NUM> peptide-pulsed B-LCL cells (<FIG>). In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CTLs obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed triangle represents the cells pulsed with <NUM>µg/mL of the WT1<NUM> peptide, and the closed square represents the cells not pulsed with the WT1<NUM> peptide. The same cytotoxic activity as above was shown also by the CTLs similarly prepared from the PBMCs isolated from the two different HLA-A*<NUM>-positive healthy blood donors (<FIG>). In <FIG>, the closed triangle represents the cells pulsed with <NUM>µg/mL of the WT1<NUM> peptide, and the closed square represents the cells not pulsed with the WT1<NUM> peptide. These results show that each cytotoxic activity is specific to the WT1<NUM> peptide.

The cytotoxic activity of the CTLs increased in parallel with the concentration of the WT1<NUM> peptide used to pulse the DCs or PBMCs with, and reached the plateau at the peptide concentration of <NUM>µg/mL (<FIG>). The half maximum concentration of the WT1<NUM> peptide for specific lysis (half-maximal lysis value) was about <NUM>×<NUM>-<NUM> µg/mL. This shows that the affinity of TCRs (T cell antigen receptors) of the CTLs to a WT1<NUM> peptide/HLA-A*<NUM> complex was relatively high. This result strongly suggests that CTLs induced with the WT1<NUM> peptide can recognize the WT1<NUM> peptide.

In the same manner as above, the cytotoxic activity against various target cells endogenously expressing WT1 was examined using the CTLs obtained by stimulating the CD8-positive T cell-enriched PBMCs from the HLA-A*<NUM>-positive blood donor with WT1<NUM> peptide-pulsed DCs or PBMCs. The results are shown in <FIG>. The respective cytotoxic activities for the target cells shown in <FIG> were determined at the same time. <FIG> shows the cytotoxic activity of WT1<NUM> peptide-specific CTLs against B-LCLs transformed with the WT1 gene (WT1-expressing, HLA-A*<NUM>-positive; closed triangle), or B-LCLs transformed with a mock vector (non-WT1-expressing, HLA-A*<NUM>-positive; closed square). <FIG> shows that the cytotoxic activity of WT1<NUM> peptide-specific CTLs against 0206K562 cells (WT1-expressing, HLA-A*<NUM>-positive; closed square), K562 cells (WT1-expressing, HLA-A*<NUM>-negative; open square), KH88 cells (WT1-expressing, HLA-A*<NUM>-negative; closed circle), or JY cells (non-WT1-expressing, HLA-A*<NUM>-negative; closed triangle).

The CTLs showed a stronger cytotoxic activity against the B-LCLs transformed with WT1 (WT1-expressing, HLA-A*<NUM>-positive) than against the B-LCLs transformed with a mock vector (non-WT1-expressing, HLA-A*<NUM>-positive) (<FIG>). Further, as shown in <FIG>, the CTLs showed a stronger cytotoxic activity against the 0206K562 cells transformed with HLA-A*<NUM> (WT1-expressing, HLA-A*<NUM>-positive) than against the K562 cells (WT1-expressing, HLA-A*<NUM>-negative), the KH88 cells (WT1-expressing, HLA-A*<NUM>-negative), or the JY cells (non-WT1-expressing, HLA-A*<NUM>-negative). In other words, the CTLs showed a significant cytotoxic activity against WT1-expressing, HLA-A*<NUM>-positive target leukemia cells , but no cytotoxic activity against non-WT1-expressing and/or HLA-A*<NUM>-negative cells. This result demonstrates that WT1<NUM> peptide-specific CTLs prepared in vitro show the cytotoxic activity against tumor cells endogenously expressing WT1 like leukemia cells and being HLA-A*<NUM>-positive. The result in <FIG> strongly suggests that the cytotoxic activity of WT1<NUM> peptide-specific CTLs was restricted by HLA-A class I. This is based on the fact that a stronger cytotoxic activity was observed against 0206K562 cells than against K562 cells.

The above results demonstrate that the above-mentioned cultured CTLs are WT1<NUM> peptide-specific CTLs.

The results of each figure are typical data, and basically reproducible with some variation.

It was examined whether the cytotoxic activity of the WT1<NUM> peptide-specific CTLs obtained in Example <NUM> was restricted by HLA class I. The <NUM>Cr release cytotoxicity test was performed in the presence or absence of mAbs against HLA class I or HLA class II. Autologous β-LCLs were used as a target cell. In this experiment, the E/T ratio was <NUM>:<NUM>.

The results are shown in <FIG>. Fig. 4a shows the results of the test that was performed using B-LCLs (non-WT1<NUM>-expressing, HLA-A*<NUM>-positive) as a target cell in the absence of mAbs against HLA class I (anti-HLA class I mAbs) and mAbs against HLA class II (anti-HLA class II mAbs). Fig. 4b shows the results of the test that was performed using WT1<NUM> peptide-pulsed B-LCLs (WT1<NUM>-expressing, HLA-A*<NUM>-positive) as a target cell in the absence of anti-HLA class I mAbs and in the presence of anti-HLA class II mAbs. Fig. 4c shows the results of the test that was performed using WT1<NUM> peptide-pulsed B-LCLs (WT1<NUM>-expressing, HLA-A*<NUM>-positive) as a target cell in the presence of anti-HLA class I mAbs and in the absence of anti-HLA class II mAbs. Fig. 4d shows the results of the test that was performed using WT1<NUM> peptide-pulsed B-LCLs (WT1<NUM>-expressing, HLA-A*<NUM>-positive) as a target cell in the absence of anti-HLA class I mAbs and anti-HLA class II mAbs.

As shown in <FIG>, the cytotoxic activity of the WT1<NUM> peptide-specific CTLs was completely inhibited by addition of an anti-HLA class I antibody, not an anti-HLA class II antibody. The result shows that the cytotoxic activity of WT1<NUM> peptide-specific CTLs was restricted by HLA class I as expected.

The cytotoxicity test against WT1-expressing, HLA-A*<NUM>-positive tumor cells was performed in vitro using the WT1<NUM> peptide-specific CTLs obtained in Example <NUM>. The cytotoxicity test was performed according to the <NUM>Cr release cytotoxicity test described in Example <NUM>. As a result, the WT1<NUM> peptide-specific CTLs showed the cytotoxic activity against WTl-expressing tumor cells (data not shown).

WT1<NUM> peptide-specific CTLs were prepared in the same manner as in Example <NUM> (<NUM>) except that the WT1<NUM> peptide (SEQ ID NO: <NUM>) was used instead of the WT1<NUM> peptide. The cytotoxicity test was performed using these CTLs in the same manner as in Example <NUM>, to determine the WT1<NUM> peptide-specific cytotoxic activity. <FIG> shows the cytotoxic activity of WT1<NUM> peptide-specific CTLs induced from PBMCs of the same HLA-A*<NUM>-positive healthy blood donor as in <FIG>. In <FIG>, the closed triangle represents cells pulsed with <NUM>µg/mL of the WT1<NUM> peptide, and the closed square represents cells not pulsed with the WT1<NUM> peptide. The CTLs showed a stronger cytotoxic activity against the WT1<NUM> peptide-pulsed autologous B-LCL cells than against the non-WT1<NUM> peptide-pulsed B-LCL cells (<FIG>). This result shows that the cytotoxic activity of the CTLs is specific to the WT1<NUM> peptide.

In the same manner as in Example <NUM>, the cytotoxic activity against various target cells endogenously expressing WT1 was examined using the CTLs prepared by stimulating the CD8-positive T cell-enriched PBMCs from the HLA-A*<NUM>-positive blood donors with WT1<NUM> peptide-pulsed DCs or PBMCs. The cytotoxic activity against each target cell is shown in <FIG>. The target cells in <FIG> are 0206K562 cells (WT1-expressing, HLA-A*<NUM>-positive; closed square), K562 cells (WT1-expressing, HLA-A*<NUM>-negative; open square), KH88 cells (WT1-expressing, HLA-A*<NUM>-negative; closed circle), and JY cells (non-WT1-expressing, HLA-A*<NUM>-negative; closed triangle). <FIG> shows the cytotoxic activity of WT1<NUM> peptide-specific CTLs induced from PBMCs of the same HLA-A*<NUM>-positive healthy blood donor as in <FIG>. <FIG> shows the cytotoxic activity of WT1<NUM> peptide-specific CTLs induced from PBMCs of the same HLA-A*<NUM>-positive healthy blood donor as in <FIG>.

Like WT1<NUM> peptide-specific CTLs, the WT1<NUM> peptide-specific CTLs showed a significant cytotoxic activity against WT1-expressing, HLA-A*<NUM>-positive target leukemia cells, but no cytotoxic activity against non-WTl-expressing and/or HLA-A*<NUM>-negative cells. This result demonstrates that WT1<NUM> peptide-specific CTLs prepared in vitro show the cytotoxic activity against tumor cells endogenously expressing WT1 like leukemia cells and being HLA-A*<NUM>-positive. The results of <FIG> strongly suggest that the cytotoxic activity of WT1<NUM> peptide-specific CTLs was restricted by HLA-A class I. This is based on the fact that a stronger cytotoxic activity was observed against 0206K562 cells than against K562 cells.

The above results demonstrate that the obtained CTLs are WT1<NUM> peptide-specific CTLs.

The following cancer vaccine compositions <NUM> to <NUM> were prepared. These are only examples of the cancer vaccine composition of the present invention.

The above-mentioned ingredients were mixed and the mixture was named cancer vaccine composition <NUM>.

Cancer vaccine compositions <NUM> to <NUM> were prepared in the same manner as in the above-mentioned cancer vaccine compositions <NUM> to <NUM> except that the WT1<NUM> peptide was used instead of the WT1<NUM> peptide.

As for the WT1<NUM> peptide, the WT1<NUM> peptide, and modified peptides comprising substitution of an amino acid residue at position <NUM>, <NUM>, <NUM> or <NUM> from the N terminus of the WT1<NUM> peptide or the WT1<NUM> peptide, the affinity to HLA-A*<NUM> molecules was analyzed by use of the NetMHC2. <NUM> Server-prediction program. The analysis results of modified WT1<NUM> peptides and modified WT1<NUM> peptides are shown in Tables <NUM> and <NUM>, respectively. The smaller value (the peptide has a binding ability at a lower concentration) indicates the higher affinity.

As for the WT1<NUM> peptide, the WT1<NUM> peptide, and modified peptides comprising substitution of an amino acid residue at position <NUM>, <NUM>, <NUM> or <NUM> from the N terminus of the WT1<NUM> peptide or the WT1<NUM> peptide, the affinity to HLA-A*<NUM> molecules was analyzed by use of the NetMHC2. <NUM> Server-prediction program. The analysis results of modified WT1<NUM> peptides and modified WT1<NUM> peptides are shown in Tables <NUM> and <NUM>, respectively. The smaller value indicates the higher affinity.

In view of the results of Example <NUM>, the WT1<NUM>P1F peptide (SEQ ID NO: <NUM>), the WT1<NUM>P2L peptide (SEQ ID NO: <NUM>), the WT1<NUM>P3M peptide (SEQ ID NO: <NUM>) and the WT1<NUM>P9V peptide (SEQ ID NO: <NUM>) were selected as modified WT1<NUM> peptides to be tested, and the following experiments were conducted to screen for modified WT1<NUM> peptides capable of inducing CTLs having a high cytotoxic activity. The reagents, media, experimental methods, etc. used in Examples <NUM> to <NUM> were the same as in Example <NUM>, unless otherwise specified. In Examples <NUM> to <NUM>, culture was performed at <NUM>, unless otherwise specified.

From a healthy human donor showing expression of HLA-A*<NUM> molecules (HLA-A*<NUM>-positive healthy blood donor), PBMCs were isolated, and CD14-positive cells were separated from the PBMCs by use of anti-human CD14 Magnetic Particles-DM. A culture medium was prepared by adding <NUM> IU/mL GM-CSF and <NUM> IU/mL IL-<NUM> to an X-VIVO15 medium supplemented with <NUM> v/v% human AB serum, and the CD14-positive cells were cultured in the culture medium for <NUM> day.

To the above culture, a maturation cytokine cocktail containing <NUM> ng/mL TNFα, <NUM> ng/mL IL-β, <NUM> ID/mL IL-<NUM> and <NUM>µg/mL PGE2 was added. After additional one day culture, autologous mature DCs were obtained.

The autologous mature DCs were pulsed with <NUM>µg/mL of a WT1<NUM> modified peptide obtained in Example <NUM> (the WT1<NUM>P1F peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide), cultured for <NUM> hours, and irradiated with 35Gy of radiation. The thus-obtained cells were used as stimulator cells for CTL induction.

The PBMCs (<NUM>×<NUM><NUM> cells/well) serving as responder cells and the above-mentioned DCs (<NUM>×<NUM><NUM> cells/well) were co-cultured in a <NUM>-well plate. One week later, re-stimulation was given by addition of T2 cells which had been pulsed with the peptide and irradiated with 75Gy of radiation. Three days after re-stimulation, <NUM> lU/mL of IL-<NUM> was added. The same re-stimulation was repeated another <NUM> times by addition of the peptide-pulsed, irradiated T2 cells, and then CD8-positive cells in the responder cells were enriched.

As for the CD8-positive T cells, the reactivity on an HLA-A*<NUM> tetramer bound to the WT1<NUM> peptide was analyzed by a flow cytometer, and the cytotoxic activity against various target cells was examined.

The target cells to be used were K562 cells, 0206K562 cells, JY cells, KH88OF8 cells, TF-<NUM> cells and THP-<NUM> cells, which are shown in Table <NUM>. The features of these cells are shown in Table <NUM>. A B-lymphoblastoid cell line (B-LCL) established by EB viral infection from the blood of an HLA-A*<NUM>-positive donor was also used as a target cell.

<FIG> shows the results of flow cytometric analysis of PBMCs from the HLA-A*<NUM>-positive donor <NUM> which were stimulated with different modified WT1<NUM> peptides and then stained with a PE (Phycoerythrin) -labeled HLA-A*<NUM> tetramer bound to the WT1<NUM> peptide (Medical & Biological Laboratories, Co. ), and an APC-Cy7-labeled anti-CD8 antibody (APC-Cy7: Allophycocyanin-Cyanine-<NUM>). When the PBMCs are stained with the above-mentioned tetramer and anti-CD8 antibody, CTLs induced by stimulation with the modified peptide bind to the tetramer and the anti-CD8 antibody, and thereby, fluorescence emitted by the tetramer and fluorescence emitted by the anti-CD8 antibody can be detected, respectively. In <FIG>, the vertical axis represents the intensity of fluorescence emitted by the HLA-A*<NUM> tetramer, and the horizontal axis represents the intensity of fluorescence emitted by the anti-CD8 antibody. Each box in <FIG> shows the frequency (%) of induced, HLA-A*<NUM>-restricted CTLs capable of recognizing the WT1<NUM> peptide. <FIG> shows the analysis result of PBMCs which were not stimulated with any WT1<NUM> modified peptide and stained with the above-mentioned tetramer and anti-CD8 antibody (background). <FIG> shows the analysis result of PBMCs which were stimulated with the WT1<NUM>P1F peptide and stained. <FIG> shows the analysis result of PBMCs which were stimulated with the WT1<NUM>P2L peptide and stained. <FIG> shows the analysis result of PBMCs which were stimulated with the WT1<NUM>P3M peptide and stained, <FIG> shows the analysis result of PBMCs which were stimulated with the WT1<NUM>P9V peptide and stained.

The frequency of the above-mentioned CTLs induced by stimulation of PBMCs with the WT1<NUM>P1F peptide was <NUM>% (<FIG>). The frequency of the above-mentioned CTLs induced by stimulation of PBMCs with the WT1<NUM>P2L peptide was <NUM>% (<FIG>). The CTLs induced separately with these peptides were HLA tetramer-positive, CD8-positive and capable of binding to the HLA-A*<NUM> tetramer bound to the WT1<NUM> peptide. These results show that stimulation of PBMCs with the modified WT1<NUM> peptide induced CTLs which can recognize the wild-type peptide (WT1<NUM> peptide).

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the donor <NUM> with the WT1<NUM>P1F peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT112CP1F peptide were used as a target cell.

JY cells are HLA-A*<NUM>-positive and WT1-negative. The CTLs showed a stronger cytotoxic activity against the WT1<NUM>P1F peptide-pulsed JY cells than against the non-WT1<NUM>P1F peptide-pulsed JY cells. This result shows that CTLs which are specific to the peptide used for the above-mentioned stimulation and restricted by HLA-A*<NUM> were induced.

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the donor <NUM> with the WT1<NUM>P2L peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In <FIG>, the closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1<NUM>P2L peptide were used as a target cell. In <FIG>, the closed diamond represents the group in which TF-<NUM> cells were used as a target cell, the closed square represents the group in which THP-<NUM> cells were used as a target cell, the closed triangle represents the group in which KH88OF8 cells were used as a target cell, and the cross represents the group in which B-LCL cells were used as a target cell.

The induced CTLs showed a stronger cytotoxic activity against the WT1<NUM>P2L peptide-pulsed JY cells than against the non-WT1<NUM>P2L peptide-pulsed JY cells (<FIG>). The induced CTLs showed a stronger cytotoxic activity against the TF-<NUM> cells and THP-<NUM> cells, both of which are HLA-A*<NUM>-positive and WT1-positive, than against the KH88OF8 cells, which are HLA-A*<NUM>-negative and WT1-positive, and the B-LCL cells, which are HLA-A*<NUM>-positive and WT1-negative (<FIG>). As is clear from the results, the CTLs induced by stimulation with the WT1<NUM>P2L peptide are restricted by HLA-A*<NUM>, and capable of destroying cancer cells endogenously expressing WT1.

<FIG> shows the results of flow cytometric analysis of PBMCs from the HLA-A*<NUM>-positive donor <NUM> which were stimulated with the WT1<NUM>P2L peptide and then stained with the PE-labeled HLA-A*<NUM> tetramer bound to the WT1<NUM> peptide, and the APC-Cy7-labeled anti-CD8 antibody. Namely, <FIG> shows the results of the flow cytometric analysis of induced CTLs which were stained with the HLA tetramer bound to the WT1<NUM> peptide, and the anti-CD8 antibody. The vertical axis represents the intensity of fluorescence emitted by the HLA-A*<NUM> tetramer, and the horizontal axis represents the intensity of fluorescence emitted by the anti-CD8 antibody.

The cells in the upper right area of <FIG> are induced CTLs which are restricted by HLA-A*<NUM> and can recognize the WT1<NUM> peptide. <NUM>% of lymphocytes of the PBMCs stimulated with the WT1<NUM>P2L peptide were HLA tetramer-positive, CD8-positive CTLs which are capable of binding to the tetramer of HLA-A*<NUM> bound to the WT1<NUM> peptide. This result shows that stimulation of PBMCs with the modified peptide induced CD8-positive CTLs which can recognize the wild-type peptide.

<FIG> shows the measurement results of the cytotoxic activity of the CTLs induced by stimulation of PBMCs from the donor <NUM> with the WT1<NUM>P2L peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In <FIG>, the closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1<NUM> peptide were used as a target cell. In Fig. lib, the closed diamond represents the group in which JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1<NUM>P2L peptide were used as a target cell.

The induced CTLs showed a stronger cytotoxic activity against the WT1<NUM> peptide-pulsed JY cells than against the non-WT1<NUM> peptide-pulsed JY cells (<FIG>). The induced CTLs also showed a stronger cytotoxic activity against the WT1<NUM>P2L peptide-pulsed JY cells than against the non-WT1<NUM>P2L peptide-pulsed JY cells (<FIG>). As is clear from the results, the CTLs induced by stimulation with the WT1<NUM>P2L peptide can recognize both of the WT1<NUM>P2L peptide and the wild-type WT1<NUM> peptide.

In view of the results of Example <NUM>, the WT1<NUM>P1F peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide and the WT1<NUM>P9V peptide were selected as modified WT1<NUM> peptides to be tested, and the following experiments were conducted to screen for modified WT1<NUM> peptides capable of inducing CTLs having a high cytotoxic activity.

To the above culture, a maturation cytokine cocktail containing <NUM> ng/mL TNFα, <NUM> ng/mL IL-β, <NUM> IU/mL IL-<NUM> and <NUM>µg/mL PGE2 was added. After additional one day culture, autologous mature DCs were obtained.

CD8-positive T cell-enriched PBMCs (<NUM>×<NUM><NUM> cells/well) and the above-mentioned DCs (<NUM>×<NUM><NUM> cells/well) were co-cultured in a <NUM>-well plate. Ten days later, re-stimulation was given by addition of PBMCs which had been pulsed with the peptide and irradiated with 35Gy of radiation. Two days after re-stimulation, <NUM> IU/mL of IL-<NUM> and <NUM> ng/mL of IL-<NUM> were added. After the same re-stimulation was repeated another <NUM> times, CD8-positive T cells were enriched. The CD8-positive T cells were examined for the cytotoxic activity against various target cells.

The target cells to be used were B-LCLs established by EB viral infection from the blood of an HLA-A*<NUM>-positive donor, K562 cells and 0206K562 cells.

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*<NUM>-positive donor <NUM> with different peptides. <FIG> shows the cytotoxic activity of CTLs induced by stimulation with the WT1<NUM>P2L peptide. <FIG> shows the cytotoxic activity of CTLs induced by stimulation with the WT1<NUM>P3M peptide. <FIG> shows the cytotoxic activity of CTLs induced by stimulation with the WT1<NUM>P9V peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which autologous B-LCL cells were used as a target cell, and the closed square represents the group in which autologous B-LCL cells pulsed with the same modified WT1<NUM> peptide as used for the above-mentioned stimulation were used as a target cell.

The CTLs induced by stimulation with the WT1<NUM>P2L peptide showed a stronger cytotoxic activity against the WT1<NUM>P2L peptide-pulsed autologous B-LCL cells, which are HLA-A*<NUM>-positive and WT1-negative, than against the non-WT1<NUM>P2L peptide-pulsed autologous B-LCL cells (<FIG>). The CTLs induced by stimulation with the WT1<NUM>P3M peptide showed a stronger cytotoxic activity against the WT1<NUM>P3M peptide-pulsed autologous B-LCL cells, which are HLA-A*<NUM>-positive and WT1-negative, than against the non-WT1<NUM>P3M peptide-pulsed autologous B-LCL cells (<FIG>). The CTLs induced by stimulation with the WT1<NUM>P9V peptide showed a stronger cytotoxic activity against the WT1<NUM>P9V peptide-pulsed autologous B-LCL cells, which are HLA-A*<NUM>-positive and WT1-negative, than against the non-WT1<NUM>P9V peptide-pulsed autologous B-LCL cells (<FIG>).

These results show that CTLs which are specific to the peptide used for the above-mentioned stimulation and restricted by HLA-A*<NUM> were induced.

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*<NUM>-positive donor <NUM> with the WT1<NUM>P9V peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which autologous B-LCL cells were used as a target cell, and the closed square represents the group in which WT1 gene-transfected autologous B-LCL cells were used as a target cell.

In <FIG>, the CTLs induced by stimulation with the WT1<NUM>F9V peptide showed a stronger cytotoxic activity against autologous B-LCL cells made to be WT1-positive by transfection of the WT1 gene into B-LCL cells, which were originally HLA-A*<NUM>-positive and WT1-negative, than against the non-WT1 gene-transfected autologous B-LCL cells (<FIG>). As is clear from the result, the CTLs induced by stimulation with the WT1<NUM>P9V peptide are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously.

The CTLs induced by stimulation with the WT1<NUM>P2L peptide showed a stronger cytotoxic activity against the WT1<NUM>P2L peptide-pulsed autologous B-LCL cells, which are HLA-A*<NUM>-positive and WT1-negative, than against the non-WT1<NUM>P2L peptide-pulsed autologous B-LCL cells (<FIG>). The CTLs induced by stimulation with the WT1<NUM>P3M peptide showed a stronger cytotoxic activity against the WT1<NUM>P3M peptide-pulsed autologous B-LCL cells, which are HLA-A*<NUM>-positive and WT1-negative, than against the non-WT1<NUM>P3M peptide-pulsed autologous B-LCL cells (<FIG>). The CTLs induced by stimulation with the WT1<NUM>P9V peptide showed a stronger cytotoxic activity against the WT1<NUM>P9V peptide-pulsed autologous B-LCL cells, which are HLA-A*<NUM>-positive and WT1-negative, than against the non-WT1<NUM>F9V peptide-pulsed autologous B-LCL cells (<FIG>). These results show that CTLs which are specific to the peptide used for the above-mentioned stimulation and restricted by HLA-A*<NUM> were induced.

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*<NUM>-positive donor <NUM> with different peptides. The target cells to be used were HLA-A*<NUM>-negative, WT1-positive K562 cells, and K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*<NUM> gene thereinto (0206K562 cells). <FIG> shows the cytotoxic activity of CTLs induced by stimulation with the WT1<NUM>P2L peptide. <FIG> shows the cytotoxic activity of CTLs induced by stimulation with the WT1<NUM>P3M peptide. <FIG> shows the cytotoxic activity of CTLs induced by stimulation with the WT1<NUM>P9V peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which K562 cells were used as a target cell, and the closed square represents the group in which 0206K562 cells, i.e. , K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*<NUM> gene thereinto, were used as a target cell.

In <FIG>, the CTLs induced by stimulation with the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide showed a stronger cytotoxic activity against the 0206K562 cells than against the K562 cells, in each case. As is clear from the results, the CTLs induced by stimulation with any of these modified peptides are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously.

In view of the results of Example <NUM>, the WT1<NUM>P1F peptide (SEQ ID NO: <NUM>), the WT1<NUM>P2M peptide (SEQ ID NO: <NUM>) and the WT1<NUM>P3M peptide (SEQ ID NO: <NUM>) were selected as modified WT1<NUM> peptides to be tested, and the following experiments were conducted to screen for modified WT1<NUM> peptides capable of inducing CTLs having a high cytotoxic activity.

From a healthy human donor showing expression of HLA-A*<NUM> molecules (HLA-A*<NUM>-positive healthy blood donor), PBMCs were isolated, and CD14-positive cells were separated from the PBMCs using anti-human CD14 Magnetic Particles-DM. A culture medium was prepared by adding <NUM> IU/mL GM-CSF and <NUM> IU/mL IL-<NUM> to an X-VIVO15 medium supplemented with <NUM> v/v% human AB serum, and the CD14-positive cells were cultured in the culture medium for <NUM> day.

The autologous mature DCs were pulsed with <NUM>µg/mL of a modified WT1<NUM> peptide obtained in Example <NUM> (the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide or the WT1<NUM>P3M peptide), cultured for <NUM> hours, and irradiated with 35Gy of radiation. The thus-obtained cells were used as stimulator cells for CTL induction.

The PBMCs (<NUM>×<NUM><NUM> cells/well) serving as responder cells and the above-mentioned DCs (<NUM>×<NUM><NUM> cells/well) were co-cultured in a <NUM>-well plate. One week later, re-stimulation was given by addition of T2 cells which had been pulsed with the peptide and irradiated with 75Gy of radiation. Three days after re-stimulation, <NUM> IU/mL of IL-<NUM> was added. The same re-stimulation was repeated another <NUM> times by addition of the peptide-pulsed, irradiated T2 cells, and then CD8-positive cells in the responder cells were enriched. The CD8-positive T cells were examined for the cytotoxic activity against target cells, i.e., JY cells here.

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*<NUM>-positive donor with the WT1<NUM>P1F peptide (<FIG>) or the WT1<NUM>P2M peptide (<FIG>). In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). The closed diamond represents the group in which JY cells were used as a target cell, the closed triangle represents the group in which WT1<NUM> peptide-pulsed JY cells were used as a target cell, and the closed square represents the group in which JY cells pulsed with the WT1<NUM> modified peptide (WT1<NUM>P1F peptide) that was used for the above-mentioned stimulation were used as a target cell. JY cells are HLA-A*<NUM>-positive and WT1-negative.

The CTLs induced by stimulation with the WT1<NUM>P1F peptide showed an equal cytotoxic activity against the WT1<NUM> peptide-pulsed JY cells and the WT1<NUM>P1F peptide-pulsed JY cells, and the activity was stronger than that against the non-peptide-pulsed JY cells (<FIG>). The CTLs induced by stimulation with the WT1<NUM>P2M peptide showed an equal cytotoxic activity against the WT1<NUM> peptide-pulsed JY cells and the WT1<NUM>P2M peptide-pulsed JY cells, and the activity was stronger than that against the non-peptide-pulsed JY cells (<FIG>). As is clear from the results, the CTLs induced by stimulation with the modified peptide can recognize both of the modified peptide and the wild-type WT1<NUM> peptide.

In view of the results of Example <NUM>, the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide and the WT1<NUM>P3M peptide were selected as modified WT1<NUM> peptides to be tested, and the following experiments were conducted to screen for modified WT1<NUM> peptides capable of inducing CTLs having a high cytotoxic activity.

The autologous mature DCs were pulsed with a modified WT1<NUM> peptide obtained in Example <NUM> (the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide or the WT1<NUM>P3M peptide) , cultured for <NUM> hours, and irradiated with 35Gy of radiation. The thus-obtained cells were used as stimulator cells for CTL induction.

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*<NUM>-positive donor with the WT1<NUM>P1F peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In <FIG>, the closed diamond represents the group in which B-LCL cells were used as a target cell, the closed square represents the group in which WT1<NUM> peptide-pulsed B-LCL cells were used as a target cell, and the closed triangle represents the group in which WT1<NUM>P1F peptide-pulsed B-LCL cells were used as a target cell. In <FIG>, the closed diamond represents the group in which K562 cells were used as a target cell, and the closed square represents the group in which 0206K562 cells, i.e., K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*<NUM> gene thereinto, were used as a target cell.

The CTLs induced by stimulation with the WT1<NUM>P1F peptide showed an equal cytotoxic activity against the WT1<NUM> peptide-pulsed B-LCL cells and the WT1<NUM>P1F peptide-pulsed B-LCL cells, and the activity was stronger than that against the non-peptide-pulsed B-LCL cells (<FIG>). When HLA-A*<NUM>-negative, WT1-positive K562 cells, and K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*<NUM> gene thereinto (0206K562 cells) were used as a target cell, the CTLs induced by stimulation with the WT1<NUM>P1F peptide showed a stronger cytotoxic activity against the 0206K562 cells than against the K562 cells (<FIG>). As is clear from the results, the CTLs induced by stimulation with the WT1<NUM>P1F peptide can recognize both of the WT1<NUM>P1F peptide and the wild-type WT1<NUM> peptide. Similarly, it was found that the CTLs are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously.

<FIG> shows the measurement results of the cytotoxic activity of CTLs induced by stimulation of PBMCs from the HLA-A*<NUM>-positive donor with the WT1<NUM>P2M peptide. In <FIG>, the vertical axis represents the cytotoxic activity, and the horizontal axis represents the ratio of CD8-positive T cells obtained by peptide stimulation (effector: E) relative to target cells (target: T) (E/T ratio). In <FIG>, the closed diamond represents the group in which B-LCL cells were used as a target cell, the closed square represents the group in which WT1<NUM> peptide-pulsed B-LCL cells were used as a target cell, and the closed triangle represents the group in which WT1<NUM>P2M peptide-pulsed B-LCL cells were used as a target cell. In <FIG>, the closed diamond represents the group in which K562 cells were used as a target cell, and the closed square represents the group in which 0206K562 cells, i.e., K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*<NUM> gene thereinto, were used as a target cell.

The CTLs induced by stimulation with the WT1<NUM>P2M peptide showed an equal cytotoxic activity against the WT1<NUM> peptide-pulsed B-LCL cells and the WT1<NUM>P2M peptide-pulsed B-LCL cells, and the activity was stronger than that against the non-peptide-pulsed B-LCL cells (<FIG>). When HLA-A*<NUM>-negative, WT1-positive K562 cells, and K562 cells made to endogenously present WT1 antigen peptides by transfection of the HLA-A*<NUM> gene thereinto (0206K562 cells) were used as a target cell, the CTLs induced by stimulation with the WT1<NUM>P2M peptide showed a stronger cytotoxic activity against the 0206K562 cells than against the K562 cells (<FIG>). As is clear from the results, the CTLs induced by stimulation with the WT1<NUM>P2M peptide can recognize both of the WT1<NUM>P2M peptide and the wild-type WT1<NUM> peptide. Similarly, it was found that the CTLs are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously.

As is clear from the results of Examples <NUM> to <NUM>, the CTLs induced by stimulation with the modified WT1<NUM> peptide, i.e., the WT1<NUM>P1F peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide, are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously. Inter alia, the WT1<NUM>P9V peptide, the WT1<NUM>P2L peptide and the WT1<NUM>P3M peptide were highly effective.

As is clear from the above results, the CTLs induced by stimulation with the modified WT1<NUM> peptide, i. , the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide or the WT1<NUM>P3M peptide, are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously. Inter alia, the WT1<NUM>P2M peptide and the WT1<NUM>P1F peptide were highly effective.

Therefore, it was shown that these modified peptides are effective in treatment and prevention of cancers accompanied by increased expression of the WT1 gene in HLA-A*<NUM>-positive persons.

As is clear from the above results, the CTLs induced by stimulation with the modified WT1<NUM> peptide, i. , the WT1<NUM>P1F peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P3M peptide or the WT1<NUM>P9V peptide, are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously. Inter alia, the WT1<NUM>P1F peptide and the WT1<NUM>P2L peptide were highly effective.

As is clear from the above results, the CTLs induced by stimulation with the modified WT1<NUM> peptide, i. , the WT1<NUM>P1F peptide, the WT1<NUM>P2M peptide or the WT1<NUM>P3M peptide, are restricted by HLA-A*<NUM>, and show the cytotoxic activity by recognizing the wild-type WT1<NUM> peptide presented endogenously. Inter alia, the WT1<NUM>P2M peptide and the WT1<NUM>P1F peptide were highly effective. Therefore, it was shown that these modified peptides are effective in treatment and prevention of cancers accompanied by increased expression of the WT1 gene in HLA-A*<NUM>-positive persons.

<NUM> of an Fmoc-Val-Alko-resin (Alko is p-alkoxybenzyl alcohol) (manufactured by WATANABE CHEMICAL INDUSTRIES, LTD; <NUM> mmol/g) was placed into the reaction vessel of the ACT496 solid-phase synthesizer manufactured by Advanced ChemTech, washed with DMF (N,N'-dimethylformamide) (Step <NUM>), treated with a <NUM>% solution of piperidine in DMF (<NUM> minutes x <NUM> time, and <NUM> minutes × <NUM> time) to remove the Fmoc group (Step <NUM>), and was again washed with DMF (Step <NUM>) to give an H-Val-Alko-resin. To this reaction vessel, <NUM> of NMP (N-methylpyrrolidinone) and a solution of <NUM> (<NUM> mmol) of DIPCI (N,N'-diisopropylcarbodiimide) in <NUM> of NMP, and then a solution of <NUM> (<NUM> mmol) of Fmoc-Ser(tBu)-OH and <NUM> (<NUM> mmol) of HOBT (<NUM>-hydroxybenzotriazol) monohydrate in <NUM> of NMP were added. Coupling reaction was performed at room temperature for <NUM> minutes (Step <NUM>). Additional coupling reaction was performed using the same amounts of Fmoc-Ser(tBu) -OH, HOBT monohydrate and DIPCI as above (Step <NUM>). The resulting resin was washed with DMF (Step <NUM>), deprotected (Step <NUM>) and washed again (Step <NUM>) to give an H-Ser(tBu)-Val-Alko-resin. Then, couplings were successively performed by repeating Steps <NUM> to <NUM> using Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH and Fmoc-Ser(tBu)-OH. The resulting peptide resin was collected from the reaction vessel, washed with ether and then dried in vacuo to give <NUM> of an H-Ser(tBu)-Val-Gly-Glu(OtBu)-Gln(Trt)-Gln(Trt)-Tyr(tBu)-Ser (tBu)-Val-Alko-resin. The outline of the synthesis process mentioned above is shown in Table <NUM>.

To <NUM> of the H-Ser(tHu)-Va1-Gly-Glu(OtBu)-Gln(Trt)-Gln(Trt)-Tyr(tBu)-Ser (tBu)-Val-Alko-resin was added <NUM> of a mixed solution of trifluoroacetic acid/water/triisopropylsilane (<NUM>/<NUM>/<NUM> (volume ratio)). The mixture was stirred at room temperature for <NUM> hours. The resin was filtered off and the resulting filtrate was added to ice-cold diethyl ether. The resulting precipitate was collected with a glass filter. The residue was washed with diethyl ether and dried in vacuo to give <NUM> of a crude peptide.

<NUM> of the obtained crude peptide was dissolved in a <NUM>% aqueous acetic acid solution, and purified by reverse phase liquid chromatography.

After equilibrated with the second eluent at a concentration of <NUM>%, the column was loaded with the crude peptide solution. After that, the concentration of the second eluent was allowed to increase to <NUM>% over <NUM> minutes and subsequently to <NUM>% over <NUM> minutes. Fractions containing the objective compound were collected, acetonitrile was evaporated off in vacuo and then the residue was freeze-dried. Thus, <NUM> of the objective WT1<NUM>P2V peptide (SVGEQQYSV; SEQ ID NO: <NUM>; H-Ser-Val-Gly-Glu-Gln-Gln-Tyr-Ser-Val-OH) was obtained.

The conditions used for HPLC analysis and mass spectrometry of the purified peptide. are as follows.

The amino acid sequence was checked sequentially from the N terminal Ser to the C terminal Val.

The peptides shown in Tables <NUM> and <NUM> were synthesized in the same manner as above.

The peptides shown in Tables <NUM> to <NUM> were similarly synthesized, but the conditions used for HPLC analysis and mass spectrometry of the purified peptides are as follows.

(Evaluation of modified peptides on the activity of inducing specific immune cells using HLA-A*<NUM>-expressing transgenic mice, and confirmation of cross reactivity of induced specific immune cells to the wild-type peptide).

As for the WT1<NUM> peptide, the WT1<NUM> peptide, and modified peptides thereof (peptides comprising substitution of one or two amino acid residues at position <NUM>, <NUM>, <NUM> and/or <NUM> from the N terminus of the WT1<NUM> peptide or the WT1<NUM> peptide), the affinity against HLA-A*<NUM> molecules was analyzed using the known method in the technical field, i.e., the method mediated by the following four computer databases:
BIMAS (http://www. mpiib-berlin. de/MAPPP/binding.

(http://www. mpiib-berlin. de/MAPPP/binding.

(http://immunax. edu/Tools/rankpep. html), and NetMHC3. <NUM> (http://www. dk/services/NetMHC/). The analysis results of the WT1<NUM> peptide and its modified peptides are shown in Tables <NUM> to <NUM> and <NUM>. The analysis results of the WT1<NUM> peptide and its modified peptides are shown in Tables <NUM> to <NUM> and <NUM> to <NUM>. The predicted affinity is shown in scores.

A modified peptide which was predicted to have an equal or higher affinity compared with the wild-type peptide (the WT1<NUM> peptide or the WT1<NUM> peptide) in at least one of the databases was selected as a sample to be tested in the following (<NUM>) to (<NUM>), in addition to wild-type peptides.

A peptide synthesized and freeze-dried in Example <NUM> was prepared at the concentration of <NUM>/mL in DMSO (manufactured by Nacalai Tesque, Inc. After that, <NUM>µL of the prepared DMSO solution of the peptide was mixed with <NUM>µL of distilled water for injection (manufactured by Otsuka Pharmaceutical Factory, Inc. Next, <NUM>µL of the mixture was mixed with <NUM>µL of the Freund's incomplete adjuvant (Montanide ISA-<NUM>) using a glass syringe to prepare a water-in-oil emulsion. An HLA-A*<NUM>-expressing transgenic mouse (strain name: HLA-A2+HLA-DR1+/Iaβ° β2m, EMMA ID number EM: <NUM>) was immunized by subcutaneous administration of <NUM>µL of the preparation (water-in-oil emulsion) into the base of the tail. The evaluation of each peptide was performed using <NUM> or <NUM> mice.

The spleen was isolated <NUM> days after immunization. The spleen was smashed by rubbing against the frothed part of a slide glass and then subjected to hemolysis treatment with ACK Lysing Buffer (manufactured by Lonza Co. ) to prepare splenic cells. In this experiment, CTM (Complete T-cell Medium: RPMI-<NUM> medium (manufactured by Invitrogen Corporation) supplemented by <NUM>%FBS, <NUM> HEPES, <NUM> L-glutamine, <NUM> sodium pyruvate, <NUM> MEM non-essential amino acid, <NUM>%MEM vitamin and <NUM> <NUM>-mercaptoethanol with the proviso that these concentrations were all final concentrations) was used as the medium for the splenic cells, and the cell suspension was prepared at the concentration of <NUM>×<NUM><NUM> cells/mL.

Whether the administered peptide has the activity of inducing WT1-specific immune cells was examined by the ELISPOT method using IFNγ as an index. The method was performed according to the attached manual. After the CTM was added in a volume of <NUM>µL/well into plates for ELISPOT (manufactured by BD Japan. catalog No. <NUM>), the splenic cell suspension was plated therein in a volume of <NUM>µL (<NUM>×<NUM><NUM> cells/well). Further, the administered peptide or the wild-type peptide was added thereto in a volume of <NUM>µL/well (peptide final concentration: <NUM>µg/mL). This assay method is known as one of the substitute methods that enable prediction of cytotoxic activity (<NPL>).

The evaluation results of the activity of inducing specific cell-mediated immunity are shown in <FIG> and <FIG> for the modified WT1<NUM> peptides, and in <FIG> and <NUM> to <NUM> for the modified WT1<NUM> peptides. In each of <FIG>, the vertical axis represents the number of antigen peptide-specific responsive cells in <NUM>×<NUM><NUM> splenic cells (spots/<NUM>×<NUM><NUM> cells), and the horizontal axis represents the individual mouse (<NUM> or <NUM> mice) used for evaluation. The white bar represents the number of the specific immune cells responded under no stimulation with antigen peptides. The gray bar represents the number of the specific immune cells responded to stimulation with the wild-type peptide. The black bar represents the number of the specific immune cells responded to stimulation with the administered (modified) peptide.

<FIG> show the respective activities of inducing specific cell-mediated immunity regarding the following peptides.

As shown in these results, when the modified peptides except the WT1<NUM>P1D peptide, the WT1<NUM>P1E peptide, the WT1<NUM>P1H peptide, the WT1<NUM>P1P peptide and the WT1<NUM>P2Q peptide; the WT1<NUM>P1D peptide, the WT1<NUM>P1E peptide, the WT1<NUM>P1P peptide, the WT1<NUM>P2A peptide and the WT1<NUM>P2Q peptide were administered into the mice, specific immune cells were remarkably induced in an efficient manner.

Next, the specific immune cells induced by stimulation of the modified peptide were analyzed for the cross reactivity to the wild-type peptide (Tables <NUM> to <NUM>). The results show that particularly the WT1<NUM>P1A peptide, the WT1<NUM>P1I peptide, the WT1<NUM>P1L peptide, the WT1<NUM>P1M peptide, the WT1<NUM>P1N peptide, the WT1<NUM>P1Q peptide, the WT1<NUM>P1T peptide, the WT1<NUM>P1V peptide, the WT1<NUM>P2V peptide, the WT1<NUM>P2M peptide, the WT1<NUM>P2I peptide, the WT1<NUM>P3A peptide, the WT1<NUM>P3F peptide, the WT1<NUM>. P3P peptide, the WT1<NUM>P3S peptide, the WT1<NUM>P3V peptide and the WT1<NUM>P9L peptide among the WT1<NUM> modified peptides; and the WT1<NUM>P1S peptide, the WT1<NUM>P2I peptide, the WT1<NUM>P2L peptide, the WT1<NUM>P2V peptide, the WT1<NUM>P3W peptide, the WT1<NUM>P9I peptide, the WT1<NUM>P9M peptide and the WT1<NUM>F9V peptide among the WT1<NUM> modified peptides, can induce specific immune cells that can efficiently recognize both of the modified peptide and the wild-type peptide.

Claim 1:
A partial peptide, a composition containing the partial peptide, or a cancer vaccine composition comprising DNA or RNA encoding the partial peptide, for use in cancer treatment or prevention in HLA-A*<NUM>-positive persons, wherein the partial peptide is
(a) a modified peptide of the WT1<NUM> peptide consisting of the sequence Ser Leu Gly Glu Gln Gln Tyr Ser Val of SEQ ID NO: <NUM> comprising substitution of one or two amino acid residues in the amino acid sequence of the WT1<NUM> peptide and the same amino acid residues at positions <NUM> to <NUM> from the N terminus as the WT1<NUM> peptide has at the corresponding positions, which is immunogenic in HLA-A*<NUM>-positive persons, or
(b) a modified peptide of the WT1<NUM> peptide consisting of the sequence Arg Met Phe Pro Asn Ala Pro Tyr Leu of SEQ ID NO: <NUM> comprising substitution of one or two amino acid residues in the amino acid sequence of the WT1<NUM> peptide and the same amino acid residues at positions <NUM> to <NUM> from the N terminus as the WT1<NUM> peptide has at the corresponding positions, which is immunogenic in HLA-A*<NUM>-positive persons.