WO2005014819A1

WO2005014819A1 - Esophageal cancer antigen and utilization thereof

Info

Publication number: WO2005014819A1
Application number: PCT/JP2004/011736
Authority: WO
Inventors: Tetsuya Nakatsura; Yoshihiro Yoshitake; Yusuke Nakamura; Yasuharu Nishimura
Original assignee: Kumamoto Technology & Industry Foundation
Priority date: 2003-08-11
Filing date: 2004-08-10
Publication date: 2005-02-17
Also published as: JPWO2005014819A1; JP4557886B2

Abstract

It is intended to provide a human esophageal cancer antigen which is applicable to the diagnosis and treatment of various cancers such as esophageal cancer and tumors; a gene encoding the same; an anticancer vaccine using the same, and so on. Namely, an antigen comprising a protein having the amino acid sequence represented by SEQ ID NO:1; a peptide comprising a part of the above protein and having an immune inducing activity; an anticancer vaccine containing the peptide; the base sequence represented by SEQ ID NO:2 or a sequence complementary thereto, and a DNA containing a part of such a sequence or the whole thereof; and an anticancer vaccine containing the DNA. The above-described antigen protein has the potential to relate to the grade of malignancy in esophageal cancer and an RNAi inhibiting its expression (for example, 5’-CAACAGCACUCCUGGAAUC-3’ (SEQ ID NO:17)) is usable in treating esophageal cancer or the like showing overexpression of the antigen protein.

Description

Specification

Antigen of esophageal cancer and its application

The present invention relates to a novel human esophageal cancer antigen useful for the diagnosis and immunotherapy of various cancers including esophageal cancer, and uses thereof. More specifically, the present invention relates to a protein specific to human cancer, a partial peptide thereof, a DNA encoding the same, an antibody having the above protein as an epitope, a killer T cell, a cancer vaccine, a cancer diagnostic probe, and a cancer diagnostic. Drugs Prevention and treatment of cancer. Background art

At present, cancers, which are the leading cause of death, still cannot treat many advanced cancers, despite advances in their mechanisms, diagnostics, and treatments. To improve this, new early diagnostics and treatments need to be developed.

Immunotherapy has long been expected as a treatment for cancer, and various attempts have been made, but it has not yet been shown to have a sufficient antitumor effect. Especially for esophageal cancer, radiation therapy and surgery are mainly used as treatments, but the locational burden on the patient is immeasurable due to its location. Therefore, it is desired to establish a treatment method with as few side effects and invasiveness as possible. Conventionally, immunotherapy for cancer has been performed mainly by non-specific immunotherapy, but in recent years it has been revealed that T cells play an important role in tumor rejection in vivo, and cytotoxic T cells Efforts are being focused on isolating T cell-recognizing tumor antigens that can induce (CTL: Cytotoxic T Lymphocyte) and determining MHC class I-restricted epitopes.

Traditionally, many tumor antigens have been isolated by cDNA expression cloning using CTL.However, since tumor cell lines and CTL establishment are required, It has been difficult to isolate tumor antigens. Disclosure of the invention

An object of the present invention is to provide a human esophageal cancer antigen applicable to diagnosis and treatment of various cancers and tumors, a gene encoding the same, an anticancer vaccine using the same, an antibody, and a CTL that specifically reacts with the above antigen. (Cytotoxic T cells). A second object of the present invention is to provide an effective antigen-specific immunotherapy using the above antigen or cytotoxic T cell.

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, using a cDNA microarray array method, a protein excellent in the effect of immunotherapy and many peptides constituting the protein have been demonstrated. O successful isolation of the antigen, which led to the completion of the present invention o

That is, according to the present invention, any one of the following proteins (A) and (B) is provided.

(A) a protein having the amino acid sequence of SEQ ID NO: 1;

(B) a protein having an amino acid sequence including substitution, deletion, insertion and / or addition of one or several amino acids in the amino acid sequence of SEQ ID NO: 1 and having immunity-inducing activity;

According to another aspect of the present invention, there is provided a peptide comprising a part of the above-described protein of the present invention and having immunity-inducing activity. The peptide of the present invention is preferably a peptide capable of activating a cytotoxic T cell recognizing a cancer antigen protein.

According to still another aspect of the present invention, there is provided a peptide comprising an amino acid sequence according to any one of the following (1) to (10).

Leu— Tyr— Pro— Pro— Pro— Pro—Hi— Thr— Leu (1)

Gly-Tyr-Ser-Val-Pro-Pro-Pro-Gly-Phe (2)

Ala— Tyr— Tyr— Gly— Arg— Ser— Val— Asp— Phe (3)

Glu—Phe—Tyr—Arg—Glu—Gin—Arg—Arg—Leu (4)

Arg—Tyr—Arg—Glu—Val—Pro—Pro—Pro—Tyr (5)

Gly— Tyr— Thr— Lys— Leu— Arg— Lys— Gin— Leu (6)

Gly— Tyr— Leu— Val— Ser— Pro— Pro— Gin— Gin— lie (7)

t E done paper (-i! i Lys— Phe— Leu— Arg— Gin— Ala— Val— Asn— Asn— Phe (8) Val— Phe— Val— Pro— Val— Pro— Pro— Pro— Pro— Leu (9)

Glu-Phe-Thr-Asn- Asp—Phe—Ala-Lys-Glu-Leu (10)

According to still another aspect of the present invention, there is provided a mammalian DNA encoding the above-described protein of the present invention. Preferably, the mammal is a human or a mouse. According to still another aspect of the present invention, there is provided any one of the following DNAs (a), (b) and (c).

(a) DNA having the nucleotide sequence of SEQ ID NO: 2.

(b) DNA that hybridizes with DNA having the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having immunity-inducing activity.

(c) a DNA having a partial sequence of the DNA of the above (a) or (b) and encoding a protein having immunity-inducing activity.

According to still another aspect of the present invention, there is provided an antibody against the above-described protein or peptide of the present invention. According to still another aspect of the present invention, there is provided a cytotoxic T cell induced by in vitro stimulation using the above-described protein or peptide of the present invention. According to still another aspect of the present invention, there is provided a cancer vaccine comprising the above-described protein or peptide of the present invention. According to still another aspect of the present invention, there is provided a cancer vaccine comprising the above-described DNA of the present invention or a recombinant virus or bacterium containing the DNA. The cancer vaccine of the present invention preferably further comprises an adjuvant.

According to still another aspect of the present invention, there is provided a cancer diagnostic probe comprising the above-described DNA of the present invention. According to still another aspect of the present invention, there is provided a cancer diagnostic agent comprising the above-described probe for cancer diagnosis and / or antibody of the present invention. According to still another aspect of the present invention, there is provided a prophylactic / therapeutic agent for cancer comprising the above-mentioned protein, peptide, antibody, and Z or cytotoxic T cell of the present invention. Preferably, the cancer is esophageal cancer, brain tumor, malignant melanoma, chronic myeloid leukemia, acute myeloid leukemia, lymphoma, head and neck cancer, kidney cancer, prostate cancer, lung cancer, thyroid cancer, breast cancer, gastric cancer, colon cancer, Teng cancer , Biliary tract cancer, It is liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, or sarcoma.

According to still another aspect of the present invention, there is provided a nucleic acid capable of suppressing the expression of the above-described protein of the present invention by an RNAi phenomenon. Preferably, the nucleic acid is siRNA, shRNA or an expression vector thereof. As a specific example, an RNA having the sequence of SEQ ID NO: 17 or an expression vector capable of expressing the same is provided. According to still another aspect of the present invention, there is provided an antitumor agent comprising the nucleic acid or RNA described above or an expression vector capable of expressing the same. Brief Description of Drawings

FIG. 1 is a graph showing the relative ratio of PP-RP expression between the expression levels of cancerous and non-cancerous parts in 26 cases of esophageal cancer patients.

FIG. 2 is a graph showing relative values of the amount expressed in other organs when the expression amount of PP-RP in normal organs is set to 1. A of FIG. 2 shows the relative ratio of PP-RP gene expression in the cancerous / non-cancerous part of 26 esophageal cancer patients. B in Figure 2. 2 shows the expression of the PP-RP gene in various normal organs.

FIG. 3 shows the results of Northern plot analysis on 12 normal cells and an esophageal cancer cell line.

FIG. 4 shows the results of analyzing the expression of PP-RP mRNA in various cancer cell lines by RT-PCR.

FIG. 5 shows the results of immunostaining of normal and esophageal cancer tissues using goat polyclonal anti-RBQ-l (Santa Cruz).

FIG. 6 shows the results of observing changes in cells in which the PP-RP gene was introduced into the NIH3T3 cell line. 1 [1113 C3 cell line? ? When -1? Is transfected, a pile-up image of the cells is observed.

FIG. 7 shows the state of the injection site of a mouse two weeks after injection of cells obtained by transfecting the NI H3T3 cell line with PP-RP into a nude mouse. Three mice were injected with 10 ⁶ cells. Cells transfected with PP-RP into the NI H3T3 cell line Trout was formed in one mouse.

FIG. 8 is a graph showing the results of ⁵¹ Cr release assay for each peptide of the CTL strain obtained by inducing with a mixture of 10 kinds of peptide vaccines derived from PP-RP protein. CTLs obtained by induction with a mixture of 10 types of peptide vaccines derived from PP-RP protein specifically recognize peptide 7.

FIG. 9 is a graph showing the results of measuring the cytotoxic activity of a CTL strain obtained by inducing with PP-RP peptide 2 against a C1R-A2402 cancer cell line by ⁵¹ Cr release assay. CTLs from esophageal cancer patients induced with PP-RP-derived p420-428GYSVPPPGF peptide can peptide-specifically damage cancer cell lines.

FIG. 10 is a graph showing the results obtained by measuring the cytotoxic activity of the CTL strain obtained by inducing with PP-RP peptide 2 against TE9 and TE11 by ⁵¹ Cr release assay. ? ? One? CTLs of esophageal cancer patients induced by the derived 420-428GYSVPPPGF peptide damage the cancer cell line in a PP-RP antigen-specific manner.

FIG. 11 is a graph showing the results of measuring the cytotoxic activity of a CTL strain obtained by inducing with PP-RP peptide 3 against a C 1 R-A2402 cancer cell line by ⁵¹ Cr release assay. PP-RP-derived 634-642 AYYGRS VDF peptide-induced CTLs from esophageal cancer patients can peptide-specifically damage cancer cell lines.

FIG. 12 is a graph showing the results of measuring the cytotoxicity of CTL lines obtained by inducing with PP-RP peptide 3 against TE9 and TE11 by ⁵¹ Cr release assay. PP-RP-derived p634-642 AYYGRS VDF peptide-induced CTLs of esophageal cancer patients damage PP-RP antigen-specific cancer cell lines.

Figure 13 A is PP- RP Bae CTL lines obtained by induced peptide 3, C 1 R- A 2402, and SK- the He p 1 cancer cell line ⁵¹ Cr release cytotoxicity against It is a graph which shows the result measured by Suatsushi. FIG. 13B is a graph showing the results obtained by measuring the cytotoxic activity of CTL lines obtained by inducing with PP-RP peptide 3 on TE13 by ⁵¹ Cr release assay. Figure 13 C is PP- RP peptide CTL lines obtained by induced 3, the TE 9, TE 1 1, TE 13 Oyo cytotoxic activity against Pi SK_He 1, the results of measurement by ⁵¹ Cr release mediation Si It is a graph shown.

TE 1 1: HLA—A24,? ? Expression of -1? ++

TE 13: Expression of HLA—A24, PP—RP ++

TE 9: HLA-A33, PP— RP expression + +

SK-He1: HLA—A24, PP—RP expression—

CTLs of esophageal cancer patients induced by PP-RP-derived p634-642 AYYGRSVDF peptide specifically recognize the peptide and damage cancer cell lines expressing HLA-A24-restricted PP-RP.

Fig. 14A shows the results of measuring the cytotoxic activity of the CTL strain obtained by inducing PP-RP peptide 9 against C1R-A2402 and SK-Hep1 cancer cell lines using ⁵¹ Cr release assay. FIG. Figure 148? 1 is a graph showing the results obtained by measuring the cytotoxic activity of a CTL strain obtained by inducing -1 peptide 9 against TE13 by ⁵¹ Cr release assay. Fig. 14〇 Graph showing the results of CTL lines obtained by induced in one P peptide 9, the TE 9, TE 11, TE 1 3 Oyo Pi SK- cytotoxic activity to the He p 1, was determined by ⁵¹ Cr release mediation Si It is.

TE11: HLA-A24, PP—RP expression + +

TE13: Expression of HLA-A24, PP-RP ++

TE 9: Expression of HLA-A33, PP-RP + +

SK-He 1: HLA_A24, PP— expression of RP

PP— RP-derived p379-388VFVPVPPPP L-peptide-induced CTL in esophageal cancer patients specifically recognizes the peptide, and HLA—A24-restricted PP— Damages cancer cell lines expressing RP.

FIG. 15A is a graph showing the results obtained by measuring the cytotoxic activity of the CTL lines obtained by inducing with PP-RP peptide 10 on C1R-A2402 and SK-He1 cancer cell lines by using ⁵¹ Cr release assay. It is. FIG. 15B is a graph showing the results obtained by measuring the cytotoxic activity against TE13 of the CTL strain obtained by inducing with PP-RP peptide 10, using ⁵¹ Cr release assay. Figure 1 5 C is the CTL lines obtained by induced in PP- RP peptide 10, a TE 9, TE 1 1, TE 1 3 and cytotoxic activity against SK_He p 1, was measured with a ⁵ r release mediation Si It is a graph which shows a result.

TE11: Expression of HLA-A24, PP-RP ++

TE 13: Expression of HLA-A24, PP-RP + +

TE 9: Expression of HLA-A33 _N- purified-1 + +

SK-He 1: HLA_A24, PP—RP expression—

PP— RP-derived p 484-493 EF TND F AKE L peptide-induced CTL in esophageal cancer patients specifically recognizes the peptide, and: «Entry into 24 restricted? ? -Damages cancer cell lines expressing RP.

FIG. 16 shows the results of measuring the cell growth rate when PP-RP was knocked down by RNAi in the esophageal cancer cell line TE13 overexpressing PP-RP.

FIG. 17 is a survival curve showing that PP-RP is a prognostic factor. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

(1) Protein and peptide of the present invention

The protein collected from the esophageal cancer of the present invention is any of the following (A) or (B):

(A) a protein having the amino acid sequence of SEQ ID NO: 1 (hereinafter also referred to as “PP-RP”); (B) a protein having an amino acid sequence of SEQ ID NO: 1 including substitution, deletion, insertion, Z or addition of one or several amino acids, and having immunity-inducing activity;

As used herein, the term "protein having immunity-inducing activity" refers to a protein having an activity of inducing an immune response such as antibody production and cellular immunity.

(Killer T cell CTL) and a protein having T cell inducing activity that stimulates are particularly preferred.

PP— RP (proliferation potential related protein) is a 1616 amino acid residue protein (5376 bp of gene base), called RBBP 6 (retinoblastoma binding protein 6) 5926 b P 1 792 amino acid residue Is a split mutant a. This PP-RP is a nucleoprotein that has a domain that binds directly to the Rb protein and can also bind to DNA.

The protein was hardly expressed in normal human organs other than placenta and testis by Northern blotting. The PP-RP gene was transformed when expressed in NIH / 3T3 cells. Moreover, when these cells are transplanted into nude mice, they survive and form tumors, which may be related to canceration.

PP-RP, which is an esophageal cancer antigen of the present invention, can be detected, for example, from a cancer cell collected from an esophageal cancer patient by cDNA microarray analysis as described in the Examples below.

A protein having an amino acid sequence similar to that of the human protein PP-RP also exists in mice. This protein was named P 2 P—R. This P 2 P_R is a nuclear protein that binds to p53 and the Rb protein, and its expression level increases in the M phase.

The cDNA microarray method according to the present invention is, as is known, for example, mRNA is separately prepared from a tissue extracted from a cancer patient into a cancerous part and a non-cancerous part, and a fluorescently labeled cDNA is prepared therefrom. On a slide glass on which at least about 50%, preferably at least 60%, and particularly preferably at least about 70% of the genes are spotted, hybridized, and the signal was acquired with a scanner to obtain the gene. This is a method for analyzing offspring expression.

`` One or several amino acids in the amino acid sequence of SEQ ID NO: 1 including substitution, deletion, insertion and / or addition of one or several amino acids '' Although the range is not particularly limited, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and particularly preferably 1 to 3 means.

The method for obtaining and producing the protein of the present invention is not particularly limited, and may be any of a naturally-derived protein, a chemically synthesized protein, and a recombinant protein produced by a genetic recombination technique. Recombinant proteins are preferred in that they can be produced in large quantities with relatively easy operation.

When a naturally-occurring protein is obtained, it can be isolated from cells or tissues expressing the protein by an appropriate combination of methods for isolating and purifying the protein. When obtaining a chemically synthesized protein, for example, the protein of the present invention can be obtained according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t_butyloxycarbonyl method). Can be synthesized. The protein of the present invention can also be synthesized using various commercially available peptide synthesizers.

In order to produce the protein of the present invention as a recombinant protein, a DNA having a nucleotide sequence encoding the protein (for example, the nucleotide sequence of SEQ ID NO: 2) or a mutant or homologue thereof is obtained and preferably used. By introducing the protein into an expression system, the protein of the present invention can be produced.

The expression vector is preferably any vector that can replicate autonomously in the host cell or can be integrated into the chromosome of the host cell, and contains a promoter at a position capable of expressing the gene of the present invention. Is used. In addition, a transformant having a gene encoding the protein of the present invention can be prepared by introducing the above expression vector into a host. The host may be any of bacteria, yeast, animal cells, and insect cells, and the expression vector may be introduced into the host by a known method appropriate for each host. In the present invention, the transformant having the gene of the present invention prepared as described above is cultured, the protein of the present invention is produced and accumulated in a culture, and the protein of the present invention is collected from the culture. Thus, the recombinant protein can be isolated.

When the transformant of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the microorganisms. However, either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant. The culture conditions may be the same as those usually used for culturing the microorganism. After culturing, the protein of the present invention may be isolated and purified by a conventional protein isolation and purification method.

In addition, in the amino acid sequence of SEQ ID NO: 1, a protein having an amino acid sequence containing substitution, deletion, insertion and Z or addition of one or several amino acids is a DNA encoding the amino acid sequence of SEQ ID NO: 1. Those skilled in the art can appropriately produce or obtain the sequence based on information on the nucleotide sequence described in SEQ ID NO: 2 showing one example of the sequence.

That is, in the amino acid sequence of SEQ ID NO: 1, a gene (mutant gene) having a base sequence encoding a protein having an amino acid sequence including substitution, deletion, insertion, and Z or addition of one or several amino acids is included. It can also be made by any method known to those skilled in the art, such as, chemical synthesis, genetic engineering techniques or mutagenesis. Specifically, mutant DNAs can be obtained by using DNAs having the nucleotide sequence of SEQ ID NO: 2 and introducing mutations into these DNAs.

For example, the method can be carried out using a method in which a DNA having the base sequence of SEQ ID NO: 2 is brought into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, or a genetic engineering technique. Site-directed mutagenesis method which is one of genetic engineering technique is useful because it is a method capable of introducing a specific mutation into a specific position, Molecular Cloning:. A laboratory Mannual , 2 nd Ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989 (hereinafter abbreviated as Molecular Clawing second edition), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current 'protocols' in 'molecular' biologic).

The present invention further relates to a peptide comprising a part of the above-mentioned protein of the present invention and having immunity-inducing activity. The peptide of the present invention is preferably one that can activate cytotoxic T cells that recognize a cancer antigen protein. Specific examples of such peptides include those having any one of the following amino acid sequences.

Leu——Tyr——Pro—Pro——Pro—Pro—His——Thr——Leu, 丄)

Gly-Tyr-Ser-Val-Pro-Pro-Pro-Gly-Phe (2)

Ala-Tyr-Tyr-Gly-Arg-Ser-Val-Asp-Phe (3)

Glu—Phe—Tyr—Arg—Glu—Gin—Arg—Arg—Leu (4)

Arg—Tyr—Arg—Glu—Val—Pro—Pro—Pro—Tyr (5)

Gly— Tyr— Thr— Lys— Leu— Arg— Lys— Gin— Leu (6)

Gly-Tyr-Leu-Val-Ser-Pro-Pro-Gln-Gln-Ile (7)

Lys— Phe— Leu— Arg— Gin— Ala— Val— Asn—Asn— Phe (8)

Val-Phe-Val-Pro-Val-Pro-Pro-Pro-Pro-Leu (9)

Glu— Phe— Thr— Asn— Asp— Phe— Ala— Lys— Glu— Leu (10)

The peptide of the present invention can be synthesized according to a method used in ordinary peptide chemistry. The commonly used synthesis methods are, for example, Peptide Synthesis, Interscience, New ΥΟΓ, 1966; The Proteins. Vol 2, Academic Press inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; 1985; Pharmaceutical Development, Vol. 14 Peptide Synthesis, Hirokawa Shoten, 1991, etc., and International Publication W099 / 67288. Specifically, it can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butynoleoxycarbonyl method). The peptides of the present invention can also be synthesized using various types of commercially available peptide synthesizers. The peptide of the present invention has a binding motif for HLA-A24. Selection of such a peptide having a binding motif for HLA-A24 can be performed by, for example, (J. Immunol.,

11 Corrected form (Rule 91) 152. 3913. 1994; J. Immunol., 155. 4307. 1994). Alternatively, the binding affinities of various peptides with HLA antigens can be determined using software available on the Internet, such as those described in Parker KC, J. Immunol., 152, 1994. It can also be calculated in silico. For example, it can be measured as described in (J. Immunol., 152. 1994; Int. J Cancer., 80, 1999. Nukaya,).

(2) DNA of the present invention

The DNA of the present invention is a DNA encoding the protein of the present invention described in (1) above, and is preferably any one of the following DNAs (a), (b) and (c).

(a) DNA having the nucleotide sequence of SEQ ID NO: 2.

(c) DNA having a partial sequence of the DNA of (a) or (b) and encoding a protein having immunity-inducing activity.

The term "hybridize under stringent conditions" refers to the use of DNA as a probe and colony hybridization, plaque hybridization, Southern blot hybridization, or the like. Means the nucleotide sequence of the obtained DNA, for example, using a filter on which DNA or a fragment of the DNA derived from a colony or plaque is immobilized, in the presence of 0.7 to 1.OM NaCl at 65 ° C. After hybridization with C, wash the filter with 0.1 to 2 XSSC solution (1 XSSC solution is 15 OmM sodium chloride and 15 nxM sodium citrate) under 65 conditions. And the like which can be identified by the above method. Hybridization can be carried out in accordance with the method described in, for example, "Molecular Development, Second Edition".

Examples of DNA that hybridizes under stringent conditions include DNΑ having a certain degree of homology with the nucleotide sequence of DNΑ used as a probe. For example, a DNA having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, particularly preferably 95% or more, and most preferably 98% or more. .

The method for obtaining DNA of the present invention is not particularly limited. Appropriate probes and primers are prepared based on the amino acid sequence and base sequence information shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing in this specification, and cDNAs for humans or the like are prepared using them. The DNA of the present invention can be isolated by screening the library. The cDNA library is preferably produced from a cell, organ or tissue expressing the DNA of the present invention.

DNA having the nucleotide sequence shown in SEQ ID NO: 2 can also be obtained by the PCR method. Using a human chromosomal DNA or cDNA library as type I, PCR is performed using a pair of primers designed to amplify the nucleotide sequence described in SEQ ID NO: 2. PCR reaction conditions can be appropriately set. For example, the reaction process consisting of 30 seconds at 94 ° C (denaturation), 30 seconds to 1 minute at 55 ° C (annealing), and 2 minutes at 72 (extension) is defined as one cycle. And a reaction at 72 ° C. for 7 minutes. Next, the amplified DNA fragment can be cloned into an appropriate vector that can be amplified in a host such as E. coli.

Operations such as the preparation of probes or primers described above, construction of a cDNA library, screening of a cDNA library, and cloning of a target gene are known to those skilled in the art.For example, Molecular Cloning, Second Edition, Current 'Protocols It can be performed according to the method described in “In Molecular Biology”.

(3) Antibodies and cytotoxic cells of the present invention

The present invention relates to an antibody that recognizes part or all of the protein or peptide of the present invention as an epitope (antigen), and a cytotoxicity (killer) induced by in vitro stimulation using the protein or peptide. ) Also related to T cells (CTL). In general, CTL shows stronger antitumor activity than antibodies.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and its production can be performed by a conventional method.

For example, a polyclonal antibody can be obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood. For example, mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, and birds can be immunized. Methods of immunization are known to those of skill in the art, for example, administering the antigen, for example, 2-3 times at 7 to 30 day intervals. The dose may be, for example, about 0.05 to 2 mg per antigen. The administration route is also not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected. The antigen can be used by dissolving it in an appropriate buffer, for example, an appropriate buffer containing a commonly used adjuvant such as complete Freund's adjuvant or aluminum hydroxide.

If the antibody titer increases after the immunized mammal has been bred for a certain period of time, booster immunization can be performed using, for example, 100 μg to 100 μg of the antigen. One to two months after the last administration, blood is collected from the immunized mammal, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene dalicol, gel filtration chromatography, ion exchange chromatography. Separation and purification by a conventional method such as chromatography such as affinity chromatography can provide a polyclonal antibody recognizing the protein of the present invention as a polyclonal antiserum.

On the other hand, a monoclonal antibody can be obtained by preparing a hybridoma. For example, a hybridoma can be obtained by cell fusion between an antibody-producing cell and a Myeoma cell line. The hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.

Antibody-producing cells include spleen cells, lymph node cells, and B phosphorus from immunized animals. Use a ball or the like. As the antigen, the protein of the present invention or its partial peptide is used. Mice, rats, and the like can be used as immunized animals, and administration of the antigen to these animals is performed by a conventional method. For example, a suspension of an adjuvant, such as complete Freund's adjuvant or incomplete Freund's adjuvant, and the protein or peptide of the present invention, which is an antigen, is administered several times to an animal intravenously, subcutaneously, intradermally, or intraperitoneally. To immunize animals. For example, spleen cells are obtained as antibody-producing cells from the immunized animal, and these are fused with myeloma cells by a known method to produce a hybridoma.

Examples of myeloma cell lines used for cell fusion include P3X63Ag8, P3U1 strain, and Sp2 / 0 strain in mice. For cell fusion, a fusion promoter such as polyethylene glycol or Sendai virus is used. For selection of hybridomas after cell fusion, hypoxanthine 'aminopterin' thymidine (HAT) medium is used according to a conventional method. The hybridoma obtained by cell fusion is cloned by a limiting dilution method or the like. If necessary, screening can be performed by enzyme immunoassay using the protein of the present invention to obtain a cell line that produces a monoclonal antibody that specifically recognizes the protein of the present invention. To produce the desired monoclonal antibody from the thus obtained hybridoma, the hybridoma is cultured by a conventional cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. Just fine. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, and the like can be used in appropriate combination.

Also, fragments of the above antibodies are within the scope of the present invention. Antibody fragments include F (ab ') 2 fragments, Fab' fragments and the like.

Furthermore, labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention prepared as described above can be labeled and used. The types of antibody labeling and labeling methods are known to those skilled in the art. For example, horseradish peroxidase or Is an enzyme label such as alkaline phosphatase, a fluorescent label such as FITC (fluorescein isothiosinate) or TRITC (teloramethylrhodamine B isothiosinate), or a coloring substance such as colloidal metal latex Labeling, affinity labeling such as biotin, and isotope labeling such as ¹²⁵ I. Analysis or measurement of the protein or peptide of the present invention (that is, a cancer antigen) using the labeled antibody of the present invention can be performed by an enzyme antibody method, an immunohistochemical staining method, an immunoplot method, a direct fluorescent antibody method or an indirect fluorescent antibody method. The method can be performed according to a method known to those skilled in the art.

The present invention also relates to an activated T cell induced by in vitro stimulation with the protein or pepsid of the present invention. For example, when peripheral blood lymphocytes or tumor-infiltrating lymphocytes are stimulated in vitro with the protein or peptide of the present invention, tumor-reactive activated T cells are induced, and the activated τ cells are effectively used for adoptive immunotherapy. be able to. In addition, the protein or peptide of the present invention can be expressed in vitro or in vitro in dendritic cells, which are powerful antigen-presenting cells, to induce immunity by administering the antigen-expressing dendritic cells.

(4) The cancer vaccine of the present invention

Since the DNA, protein and peptide of the present invention can induce T cells that can damage cancer cell lines in an antigen-specific manner, they can be expected as therapeutic or preventive agents for cancer. For example, bacteria of BCG bacteria transformed with the DNA of the present invention into an appropriate vector and transformed with the recombinant DNA, or viruses such as vaccinia virus with the DNA of the present invention integrated in the genome, are used for treatment of human cancer. . Can be used effectively as a live vaccine for prevention. The dose and method of administration of the cancer vaccine are the same as those for normal vaccination and BCG lectin.

That is, the DNA of the present invention (as it is or in the form of a plasmid DNA incorporated into an expression vector), a recombinant virus or a recombinant bacterium containing the DNA as it is or dispersed in an adjuvant can be used as a cancer vaccine containing humans. It can be administered to animals. Similarly, the peptide of the present invention is dispersed with adjuvant. Can be administered as a cancer vaccine.

Examples of the adjuvant that can be used in the present invention include incomplete Freund's adjuvant, BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS), alum adjuvant, and silica adjuvant. It is preferable to use Freund's incomplete adjuvant (FIA) because of its performance.

(5) The cancer diagnostic probe, cancer diagnostic agent, cancer prevention and therapeutic agent of the present invention (DNA probe and diagnostic agent)

The DNA of the present invention can be used as a diagnostic probe by extracting DNAs of various human cancers and examining their homology. In addition, the probe and the antibody can be used as a cancer diagnostic agent.

That is, the present invention relates to a cancer diagnostic probe containing all or a part of the antisense chain of DNA or RNA encoding the protein of the present invention. The present invention further relates to a cancer diagnostic comprising the above-mentioned probe for cancer diagnosis or an antibody against the protein of the present invention. The cancer diagnostic probe of the present invention is all or part of the antisense strand of DNA (cDNA) or RNA encoding the protein of the present invention, and has a length sufficient to be a probe (at least 20 bases or more). Those with are preferred. For example, cancer can be diagnosed by detecting mRNA of the protein (cancer antigen) of the present invention obtained from a specimen using the antisense strand. Samples used for detection include, but are not limited to, genomic DNA, RNA or cDNA obtained from a biopsy of a subject's cells, for example, blood, urine, saliva, tissue, etc. Not something. When such a sample is used, a sample amplified by PCR or the like may be used.

The type of cancer referred to in the present invention is not particularly limited, and specific examples include esophageal cancer, brain tumor, malignant melanoma, chronic myeloid leukemia, acute myeloid leukemia, lymphoma, head and neck cancer, kidney cancer, prostate cancer, and lung cancer. , Thyroid, breast, gastric, colon, knee, biliary tract, liver, gallbladder, testicular, uterine, ovarian, or sarcoma, but are not limited to these. It is not something to be done.

(Immunoprevention and therapeutic drugs consisting of proteins and peptides)

Cancer Patients The immune response to cancer cells has been more active than expected, and they have found that IgG antibodies are produced against a wide variety of proteins. Examples of the antibody that specifically binds to the protein or a peptide that is a part thereof of the present invention include immunospecific antibodies such as a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a single-chain antibody, and a humanized antibody. These can be specifically prepared. These can be prepared by a conventional method using the above-mentioned protein such as the esophageal cancer antigen PP-RP or a part thereof as an antigen, and are used for diagnosis of esophageal cancer using them. be able to.

The protein or peptide of the present invention can also induce cancer cell-specific cytotoxic T cells as T cell epitopes, and is therefore useful as an agent for preventing or treating human cancer. Further, the antibody of the present invention is useful as a diagnostic agent for cancer. In actual use, the protein, peptide or antibody of the present invention may be used as an injectable solution as it is, or together with a pharmaceutically acceptable carrier, and Z or a diluent, and, if necessary, the following adjuvants. It may be administered by administration, or may be administered by percutaneous absorption through mucous membranes by spraying or the like. In addition, as the carrier here, for example, human serum albumin and the like can be mentioned. Examples of the diluent include PBS, distilled water and the like.

The dose of the protein, peptide or antibody of the present invention per adult can be, for example, from 0.01 mg to 100 mg per dose, but is limited to this range. is not. The form of the preparation is not particularly limited, and may be freeze-dried or granulated with excipients such as sugar.

Examples of adjuvants for enhancing cytotoxic T cell-inducing activity that can be added to the agent of the present invention include cell components such as BCG bacteria, ISC0M described in nature, vol. 344, p873 (1990), J. Immunol, vol. 148, pl438 (1992), such as saponin QS-21, ribosome, and aluminum hydroxide. In addition, immunostimulants such as lentinan, schizophyllan, and picibanil are used as adjuvants. You can also. In addition, cytokines that enhance the proliferation and differentiation of T cells, such as IL-2, IL_4, IL-12, IL-11, IL-6, and TNF, can also be used as adjuvants.

In addition, the antigen peptide is added in vitro to cells collected from a patient or cells having the same HLA pap type, and the antigen is presented. Toxic T cells can also be induced. In addition, by adding the peptide to peripheral blood lymphocytes of the patient and culturing it in a test tube, cytotoxic T cells can be induced in the test tube and then returned to the blood vessel of the patient. Such treatment by cell transfer has already been performed as a cancer treatment method, and is a method well known to those skilled in the art.

(Immunotherapy antigen)

In order to be a target antigen for specific anti-JB tumor immunotherapy, it is necessary that the antigen be a recognition antigen for cytotoxic T cells (killer T cells / CTL). The antigen of the present invention increased killer T cell-inducing activity in the mouth of HLA-A24, which is common in Japanese. Thus, injection of the antigen of the present invention into the body induces and activates CTL, and as a result, an antitumor effect can be expected. When stimulated with the antigen of the present invention, activated T cells are induced in vitro, and the activated T cells can be effectively used for adoptive immunotherapy by injecting the activated T cells into the body.

(5) RNAi of the present invention

The present invention further relates to a nucleic acid capable of suppressing the expression of the protein of the present invention by the RNAi phenomenon. Examples of such a nucleic acid include si RNA, sh RNA and their expression vectors, and specific examples include RNA having the sequence of SEQ ID NO: 17 and an expression vector capable of expressing the same. These nucleic acids (such as RNA or its expression vector) can suppress the growth rate of esophageal cancer cells overexpressing the protein (PP-RP) of the present invention, as shown in the following Examples. It is useful as an antitumor agent.

The administration method of the antitumor agent of the present invention is not particularly limited, and oral administration, parenteral administration (for example, Examples include intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, topical administration to the affected area, and dermal administration), and direct administration to the affected area.

When the antitumor agent of the present invention is used as a pharmaceutical composition, a pharmaceutically acceptable additive can be added as necessary. Specific examples of pharmaceutically acceptable excipients include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles. Diluents, carriers, excipients and Z or pharmaceutical adjuvants, but are not limited thereto.

The dose of the nucleic acid as an active ingredient of the antitumor agent of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the severity of the disease, the age, weight, sex, or history of the patient. it can. The dose of the nucleic acid as an active ingredient is not particularly limited, but it is, for example, about 0.1 ng to about 10 Omg Zkg, preferably about 1 ng to about 1 Omg at a time. Hereinafter, the antigen of the present invention, its production method, and effects will be described with reference to examples, but the present invention is not limited to these examples. Example

Example 1: Screening of PP-RP by cDNA microarray method

Cancer tissues were collected from 26 esophageal cancer patients with informed consent and consent. The collected cancer tissues were stored at 180 ° C. The cancer tissue sample was collected by a laser-microphone beam microdissection system to collect only the cancerous part or non-cancerous part, prepare mRNA, and then create fluorescently labeled cDNA. The gene was analyzed by placing it on a slide glass on which 23040 genes, 70% of all genes, were spotted, hybridizing, capturing signals with a scanner, and analyzing gene expression. As a result, in 21 out of 26 esophageal cancer patients, the relative ratio of the expression level of the cancerous part to the non-cancerous part was 5 times or more in the expression of the PP-RP gene. In addition, the gene, PP-RP, which is hardly expressed outside the normal placenta, Identified. (Figure 1, Figure 2) Example 2: Northern blot analysis

(Test method)

The expression of PP-RP gene in 12 normal tissues and esophageal cancer and head and neck cancer cell lines was confirmed by Northern blot analysis. The Northern plot method was performed by the following method. That is, in order to confirm the expression of the PP-RP gene in normal tissues, a human tissue poly (A) ⁺ RNA blot (human 12-lane MTN blot, Clontech) was used. In addition, membranes were prepared by hand using total RA of normal esophagus, normal testis, normal placenta, and cancer cell lines.

The PCR products obtained with the primers of 5'-TGCTGTTGTGATTCCCTGCTG-3, (SEQ ID NO: 13) and Antisense 5, -AGGAAACTGAGGAGAAAACTG-3, (SEQ ID NO: 14) were hybridized as PP-RP probes to these membranes. .

(Result)

The results are shown in Figure 3. As is clear from this figure,? Kei-shaku? Was found to be expressed only in the placenta in cancer cell lines and 12 normal tissues. Example 3: Expression analysis of PP-RP mRNA in various cancer cell lines by RT-PCR

Upper primmer: 5, -TGCTGTTGTGATTCCCTGCTG-3, (SEQ ID NO: 15), and

Lower primmer: 5'-AGGAAACTGAGGAGAAAACTG-3 '(SEQ ID NO: 16)

Was used to analyze the expression of PP-RP mRNA in various cancer cell lines. PCR was performed at 94 ° C for 1 minute, 58 ° C for 1 minute, and 72 ° C for 1.5 minutes for 30 cycles. Fig. 4 shows the results. As can be seen from the results in Fig. 4, PP-RP expression was observed not only in esophageal cancer but also in various cancer cell lines. Example 4: Immunostaining

Nichirei's Histofine 'Simple Simple Sting Method. The antibody used was Normal and esophageal cancer tissues were immunostained with goat polyclonal anti-RBQ-1 (Santa Cruz). Fig. 5 shows the results. As can be seen from the results in Fig. 5, in normal tissues, expression was observed in the testis and placenta, but not in spleen, lymph nodes, brain, kidney, lung, liver, and esophagus tissue (A ~! Expression is not observed in normal esophageal tissue, but expression is observed in esophageal cancer (S-V). Its localization was around the chromosome (W, X). Example 5: Gene transfer into NIH3T3 cells

(Test method)

A vector with a 3056 bp to 4942 bp portion (ORF 92 bp to 4942 bp) of the PP-RP gene was purchased from Takara Holding Co., Ltd. This and the neo-resistance gene expression vector pCI-neo were simultaneously transfected using Ribofectamine ™ 2000. Cells were selected by Neo and cloned by limiting dilution. The cells were inoculated once subcutaneously into nude mice to verify whether a bulge was formed.

(Test results)

The results are shown in Figs. As is apparent from the figure, swelling was observed in the transfected cells, indicating that PP-RP has an oncogene-like function. Example 6: Screening of peptides constituting PP-RP

Originally, considering that various proteins are presented in vivo on antigen presenting cells after being degraded into 9-mer peptides, HLA-A24 accounts for 60% of the total Japanese population. A 9- or 10-mer peptide derived from PP-RP having a binding motif was synthesized. Select peptides common to human PP-RP and mouse P2P-R predicted to bind to both HLA-A24 and ^Kd using HLA-peptide binding prediction from the PP-RP sequence, 9 or 1 An automatic peptide synthesizer converts the following 10 types of peptides consisting of 0 amino acids (PSSM8, manufactured by Shimadzu Corporation) by the Fmoc (9-fluorenylmethyloxycarbonyl) method. The synthesized peptides were converted into pure peptides using high performance liquid chromatography.

Leu—Tyr—Pro—Pro—Pro—Pro—His—Thr—Leu (1) (SEQ ID NO: 3) Gly-Tyr-Ser-Val-Pro-Pro-Pro-Gly—Phe (2) (SEQ ID NO: 4) Ala— Tyr— Tyr-Gdy— Arg— Ser— Val— Asp-Phe (3) (SEQ ID NO: 5) Glu— Phe— Tyr— Arg— Glu— Gin— Arg— Arg— Leu (4) (SEQ ID NO: 6) Arg—Tyr—Arg—Glu—Val—Pro—Pro—Pro—Tyr (5) (SEQ ID NO: 7) Gly—Tyr—Thr—Lys—Leu—Arg—Lys—Gin—Leu (6) (SEQ ID NO: 8) Gly-Tyr-Leu-Val-Ser-Pro-Pro-Gln-Gln-Ile (7) (SEQ ID NO: 9) Lys-Phe-Leu-Arg-Gln-Ala-Val-Asn-Asn-Phe (8) ( SEQ ID NO: 10) Va 1-Phe-Val-Pro-Val-Pro-Pro-Pro-Pro-Leu (9) (Distribution No. 1 1) Glu-P e-Thr-Asn-Asp-Phe-Ala- Lys-Glu-Leu (10) (SEQ ID NO: 12) Example 7: ⁵¹ Cr.

(Test method)

Using dendritic cells derived from peripheral blood monocytes as antigen-presenting cells, and using T cell lines obtained by stimulating CD8-positive T cells with synthetic PP-RP peptide, their peptide specificity and cancer cell injury Activity was measured. The target cells were C1R-A2402 cells with or without peptide addition, TE11 (HLA-A24, expression of PP-RP ++), TE13 (expression of HLA-A24, PP-RP ++), TE9 (HLA-A24). A33, PP-RP expression ++), SK-Hepl (HLA-A24, PP-RP expression-), and PP-RP siRNA (small interfering RNA) expression vector pSilencer ™ siRNA expression vector (Ambion (registered) (Trademark)), a cell line called TE13 shPP-RP in which the expression of PP-RP was suppressed and a cell line called TE13 shGFP were prepared as a control, and a CTL line induced by the above method was added as an effector to the cell line. Was measured. As the peptides, a mixture of the above 10 peptides was used.

(Test results)

23 Corrected S Paper (Edition ίί91> Fig. 8 shows the results. Example 8:

The procedure was performed in the same manner as in Example 7 except that peptide 2 was used as the antigen peptide. The test results are shown in FIGS. 9 and 10. Example 9:

The procedure was performed in the same manner as in Example 7, except that peptide 3 was used as the antigen peptide. The test results are shown in Fig. 11 to Fig. 13 A, B and C. Example 10:

The procedure was performed in the same manner as in Example 7 except that peptide 9 was used as the antigen peptide. The test results are shown in Figures 14A, B, and C. Example 11:

The procedure was performed in the same manner as in Example 7, except that peptide 10 was used as the antigen peptide. The test results are shown in Figures 15A, B, and C. Example 12: Growth rate

PP-RP was knocked down using RAi5, -GAACAGCACUCCUGGAAUC-3 '(SEQ ID NO: 17) from an esophageal cancer cell line TE13 which highly expresses PP-RP. In fact, Western blotting confirmed that the expression level of PP-RP protein was reduced in TE13-shPP-RP cells in which PP-RP was knocked down compared to TE13 cells. The cells were 1x10 ^s / plate at 1 ° /. When cultured in serum, the cell growth rate was significantly (p-slow) in TE13-shPP-RP cells compared to TE13 (Fig. 16). In addition to suggesting that it may be related to the degree of RNAi 5, -GAACAGCACUCCUGGAAUC-3 '(SEQ ID NO: 17) It also suggests that it can be used to treat road cancer. Example 13: PP-RP expression level and prognosis

After surgery for esophageal cancer, R0 indicates no cancer remains, R2 indicates gross cancer remains, and R1 indicates conditions other than R2. The lifetime was examined. The results are shown in Table 1 below.

Table 1: Rl clinicopathologic features of 15 patients with esophageal cancer Gender Age 7Γ in cancer tissue, mouth a Survival time b T M histopathology Residual tumor d

(Years) PP-RP gene (months) Evaluation c

Relative expression ratio

Male 43> 1000 died 24 IVG 2 R 1 Male 60> 1000 died 16 IVG 2 R 1 Puta 55> 1000 died 16 IVG 1 R 1 Male 56> 1000 died 12 IVG 2 R 1 Male 60> 1000 died 4 IVG 1 R 1 Male 61 142.1 Survival> 62 IVG 1 R 1 Field 61 48.6 Survival> 98 IV G2 R 1 Paddy 54 11 Survival> 122 IIIG 2 R 1 Strength 50 10.5 Survival> 34 IVG 1 R 1 Strength 58 10.2 Death 19 IVG 2 R 1 Field 67 6.3 Survival> 30 IV G2 R 1 Field

另 67 6 survival> 30 I V G 2 R 1 field

Power 63 5 death 1 1 I V G 2 R 1 field

另 57 2.2 Survival> 30 I V G 2 R 1 field

另 55 2.1 Death 20 I V G 2 R 1 Note a: Eight patients died of esophageal cancer-related causes.

b: Follow-up months were calculated from the period from surgery to death or last follow-up date.

c: Gl, G2 and G3 indicate that the degree of SCC differentiation is sufficient, moderate or weak, respectively.

d: Rl indicates neither RO nor R2, R0 indicates no residual tumor, and R2 indicates macroscopic residual JI swelling. As can be seen from the results in Table 1, the expression level of PP-RP was very high (> 1000 times in non-cancerous part compared to non-cancerous part). The survival time was significantly (p 05) significantly shorter than that of PP-RP (142.1 times). Example 14: PP-RP as a prognostic factor

After surgery for esophageal cancer, R0 if no cancer remained, R2 if there was gross cancer, and R1 if the condition other than R2 was R1, PP-RP in 15 cases of R1 The expression level was very high (> 1000-fold) in 5 cases, all died within 25 months, whereas 7 out of 10 other cases (142.1-fold increase in PP-RP expression) Confirmed that they survived for more than 30 months. Figure 17 shows the survival curves prepared by Kaplan-Meier method for each group. A Breslow-Gehan-Wilcoxon test revealed a significant difference between the two groups (ft 05). These results suggested that the expression level of PP-RP may be used as a prognostic predictor of esophageal cancer. Industrial applicability

The antigen protein and antigen peptide of the present invention, or DpD encoding the protein or peptide of the present invention, can be used as an excellent anticancer vaccine with few side effects such as self-damage. In addition, antibodies can be used as diagnostics. Killer T cells stimulated and activated by the antigen of the present invention can be used as anticancer agents. In addition, the antigenic protein of the present invention may be related to the malignancy of esophageal cancer. Further, it can be used for the treatment of esophageal cancer and the like which highly express the antigen protein of the present invention.

Claims

The scope of the claims

1. Any of the following proteins (A) or (B):

(A) a protein having the amino acid sequence of SEQ ID NO: 1;

(B) a protein having the amino acid sequence of SEQ ID NO: 1 having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, and / or Z-adducts, and having immunity-inducing activity;

2. A peptide comprising a part of the protein according to claim 1 and having immunity-inducing activity.

3. The peptide according to claim 2, which is capable of activating cytotoxic T cells recognizing a cancer antigen protein.

4. The peptide according to claim 2 or 3, comprising an amino acid sequence according to any one of the following (1) to (10).

Leu— Tyr— Pro— Pro— Pro— Pro— His— Thr— Leu

Gly-Tyr-Ser-Val-Pro-Pro-Pro-Gly-Phe (2)

Ala— Tyr— Tyr— Gly— Arg— Ser— Val— Asp— Phe (3)

Glu—Phe—Tyr—Arg—Glu—Gin—Arg—Arg—Leu (4)

Arg—Tyr—Arg—Glu—Val—Pro—Pro—Pro—Tyr (5)

Gly— Tyr— Thr— Lys— Leu— Arg— Lys— Gin— Leu (6)

Gly-Tyr-Leu-Val— Ser-Pro-Pro-Gln-Gln-Ile (7)

Lys— Phe— Leu— Arg— Gin— Ala— Val— Asn—Asn— Phe (8)

Va 1— Phe— Val— Pro— Val— Pro— Pro— Pro— Pro— Leu (9)

Glu-Phe-Thr-Asn-Asp-Phe-Ala-Lys-Glu-Leu (10)

5. A mammalian DNA encoding the protein of claim 1.

6. The D according to claim 5, wherein the mammal is a human or a mouse.

7. DNA of any of the following (a), (b) or (c):

28 Corrected Form (Rule 91) (a) DNA having the nucleotide sequence of SEQ ID NO: 2.

(b) a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having immunity-inducing activity.

(c) DNA encoding a protein having the partial sequence of DNA of (a) or (b) above and having immunity-inducing activity.

8. An antibody against the protein according to claim 1 or the peptide according to any of claims 2 to 4.

9. A cytotoxic T cell induced by in vitro stimulation using the protein according to claim 1 or the peptide according to any one of claims 2 to 4.

10. A cancer vaccine comprising the protein according to claim 1 or the peptide according to any one of claims 2 to 4.

1 1. The cancer vaccine according to claim 10, further comprising an adjuvant.

1 2. A cancer vaccine comprising the DNA of claim 5 or a recombinant virus or a bacterium containing the DNA.

13. The cancer vaccine according to claim 12, further comprising an adjuvant.

14. A cancer diagnostic probe comprising the DNA according to any one of claims 5 to 7.

15. A cancer diagnostic agent comprising the cancer diagnostic probe according to claim 14 and Z or the antibody according to claim 8.

16.Prevention of cancer comprising the protein according to claim 1, the peptide according to any one of claims 2 to 4, the antibody according to claim 8, and / or the cytotoxic T cell according to claim 9. Therapeutic drugs.

1 7. Cancer is esophageal cancer, brain tumor, malignant melanoma, chronic myeloid leukemia, acute myeloid leukemia, lymphoma, head and neck cancer, kidney cancer, prostate cancer, lung cancer, thyroid cancer, breast cancer, gastric cancer, colon cancer, 17. The probe or agent according to claim 14, which is cancer, biliary tract cancer, liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, or sarcoma.

18. A nucleic acid capable of suppressing the expression of the protein according to claim 1 by the RNAi phenomenon.

1 9. Claim 1 which is siRNA, shRNA or an expression vector thereof. 9. The nucleic acid according to 8.

20. An RNA having the sequence of SEQ ID NO: 17, or an expression vector capable of expressing the same.

21. An antitumor agent comprising the nucleic acid according to claim 18 or 19, or the RNA according to claim 20 or an expression vector capable of expressing the same.